Patent Application
20110003844
This invention relates to modulation of cardiac contractility and cardiomyopathic phenotypes, prevention and treatment of the same, and transgenic mice related to the same.
Heart failure afflicts an estimated 5 million Americans, with approximately 400,000 new individuals diagnosed each year at an annual cost of over $20 billion (Lloyd-Jones et al. (2002) Circulation 106:3068-3072). The predominant therapeutic strategy employed over the past two decades has been based on pharmacological manipulation of cardiac contractility (Remme, W. J. (2001) Cardiovasc. Drugs Ther. 15:375-377; Felker et al (2001) Am. J. Heart 142:393-401; Packer, M. (2001) Am. J. Med. 110 Suppl 7A:81S-94S). Heart failure may be characterized by a progressive loss in contractility, ventricular chamber dilation, increased peripheral vascular resistance, and/or dysregulated fluid homeostasis. Positive inotropic agents were initially employed as a means of enhancing cardiac pump function, yet are now only utilized to acutely bridge patients in severe heart failure since they worsen long-term survival (Felker et al (2001) Am. J. Heart 142:393-401). More recently, pharmacological blockade of β-adrenergic receptors has emerged as the favored treatment for heart failure, although it remains uncertain whether or not β-blockers benefit the myocardium by diminishing cardiac contractility (short-term) or augmenting it (long-term) (Packer, M. (2001) Am. J. Med. 110 Suppl 7A:81S-94S; Bristow M. W. (2000) Circulation 101:558-569; Bouzamondo et al. (2001) Fundam. Clin. Pharmacol. 15:95-109). Two other features associated with the failing human heart are a dysregulation in calcium homeostasis and an increase in neuroendocrine stimulatory agents that signal through both Gαq- and Gαs-coupled receptors.
A variety of human diseases and conditions manifested by cardiac abnormalities or cardiac dysfunction may lead to heart failure. Heart failure is a physiological condition in which the heart fails to pump enough blood to meet the circulatory requirements of the body. The study of such diseases and conditions in genetically diverse humans is difficult and unpredictable. Therefore, there is a need for a model system that facilitates the identification of potential therapeutic agents of cardiomyopathy.
When stimulated by an array of neuro-humoral factors or when faced with an increase in ventricular wall tension, the myocardium undergoes an adaptive hypertrophic response. Cardiac hypertrophy is an adaptive response of the heart to many forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias, endocrine disorders, and/or genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to heart failure, and sudden death.
The causes and effects of cardiac hypertrophy have been documented, but the underlying mechanisms that couple hypertrophic signals initiated at the cell membrane to the reprogramming of cardiomyocyte gene expression remain poorly understood. Elucidation of these mechanisms is a central issue in cardiovascular biology and is important for designing new strategies for prevention or treatment of cardiac hypertrophy and heart failure.
Studies have implicated intracellular Ca2+ as a signal for cardiac hypertrophy. In response to myocyte stretch or increased loads on working heart preparations, intracellular Ca2+ concentrations increase (Marban et al. (1987) Proc. Natl. Acad. Sci. USA 84:6005-6009; Bustamante et al. (1991) J. Cardovasc. Pharmacol. 17:S110-S113; and Hongo et al. (1995) Am. J. Physiol. 269:C690-C697), consistent with a role of Ca2+ in coordinating physiological responses with enhanced cardiac output.
Hypertrophic stimuli result in reprogramming of gene expression in the adult myocardium, such that genes encoding fetal protein isoforms like β-myosin heavy chain (MHC) and α-skeletal actin are up-regulated, whereas the corresponding adult isoforms α-MHC and α-cardiac actin, are down-regulated. The natriuretic peptides, atrial natriuretic factor and b-type natriuretic peptide, which decrease blood pressure by vasodilation and natriuresis, are also rapidly up-regulated in the heart in response to hypertrophic signals. (Komuro and Yazaki (1993) Ann. Rev. Physiol. 55:55-75). The mechanisms involved in coordinately regulating these cardiac genes during hypertrophy are unknown.
A number of signaling molecules have been characterized as important transducers of this disease response sequelae, including, but not limited to, specific G-protein isoforms, low molecular weight GTPases (Ras, RhoA, Rac), mitogen-activated protein kinases (MAPK), protein kinase C (PKC), calcineurin, gp130-STAT, insulin-like growth factor-I receptor, fibroblast growth factor, and transforming growth factorβ. For example, binding of the cell surface receptors for AngII, PE, and ET-1 leads to activation of phospholipase C, resulting in the production of diacylglycerol and inositol triphosphate, mobilization of intracellular Ca2+, and activation of protein kinase C. The extent to which these signaling pathways interact during cardiac hypertrophy is unknown (Molkentin et al. (2001) Annu. Rev. Physiol. 63:391-426).
The protein kinase C (PKC) family of calcium and/or lipid-activated serine-threonine kinases functions downstream of nearly all membrane-associated signal transduction pathways (Molkentin et al. (2001) Annu. Rev. Physiol. 63:391-426). Approximately 12 different isozymes comprise the PKC family, which are broadly classified by their activation characteristics. The conventional PKC isozymes (PKCα, βI, βII, and γ) are calcium- and lipid-activated, while the novel isozymes (ε, θ, η, and δ) and atypical isozymes (ζ, ι, υ, and μ) are calcium independent but activated by distinct lipids (Dempsey et al. (2000) Am. J. Physiol. Lung Mol. Physiol. 279:247-251). Once activated, PKC isozymes translocate to discrete subcellular locations through direct interactions with docking proteins termed RACKs (Receptor for Activated C Kinases), which permit specific substrate recognition and subsequent signal transduction (Mochly-Rosen, D (1995) Science 268:247-251).
Reports have associated PKC activation with hypertrophy, dilated cardiomyopathy, ischemic injury, or mitogen stimulation (DeWindt et al. (2000) J. Biol. Chem. 275:13571-13579; Gu & Bishop (1994) Circ. Res. 75:926-931; Jalili et al. (1999) Am. J. Physiol. 277:H2298-H12304; Takeishi et al (1999) Am. J. Physiol. 276:H53-H62). For example, hemodynamic pressure overload stimulation in rodents promotes translocation of PKCα, β, γ, ε, and θ. In diverse cultured cardiomyocytes, agonists and stress stimuli are also potent activators of PKC isozyme translocation. Isozyme-specific peptide inhibitors have been employed in cultured cardiomyocytes and in transgenic mice to afford greater specificity of PKC inhibition. Specifically, overexpression of a PKCβ C2 domain peptide in cardiomyocytes blocked phorbol ester-mediated calcium channel activity (Zhang et al. (1997) Circ. Res. 80:720-729), while a PKCε inhibitory or activating peptide affected inotropy and ischemia-induced cellular injury (Gray et al. (1997) J. Biol. Chem. 272:30945-30951; Johnson et al. (1996) J. Biol. Chem. 271:24962-24966; Dorn et al. (1999) Proc. Natl. Acad. Sci USA. 96:12798-12803). Additionally, adenovirus-mediated gene transfer of PKCε into cultured adult rabbit ventricular myocytes augmented basal myocyte contractility and calcium transients. The results in myocytes suggest that PKCε functions to enhance cardiac contractile performance (Baudet et al. (2001) Cardiovasc. Res. 50:486-494).
Phorbol esters exert acute biologic effects on metazoan cells, mostly consistent with immediate activation of multiple PKC isozymes. In cardiac myocytes, PMA is a potent inducer of many PKC isozymes including, but not limited to, PKCα, β, δ, and ε translocation and activation (Braz et al. (2002) J. Biol. Chem. 156:905-919). Thus acute PMA administration may be used to examine the immediate, but non-specific effects of PKC translocation on alterations in cardiac inotropy and contractility. Acute phorbol ester administration has been used to assess the hypothesis that PKC isozymes regulate, in part, the contractile performance of the whole heart or isolated myocytes. For example, using isolated chicken ventricular myocytes, PMA treatment produced a concentration and time-dependent decrease in the amplitude of cell shortening, reaching a maximum of a 54% decrease at 1 μM drug (Leatherman et al. (1987) Am. J. Physiol. 253:H205-209). Consistent with this effect, PMA produced a decrease in intracellular calcium concentration and the rate of calcium reuptake. In contrast, PMA pretreatment of papillary muscles from the heart potentiated alpha 1-adenoceptor-mediated positive inotropy, demonstrating the non-selective effects of using PMA (Otani et al. (1988) Circ. Res. 62:8-17). An analysis performed in isolated ventricular myocytes from adult rats showed that PMA has an acute negative contractile effect (Capogrossi et al. (1990) Circ. Res. 66:1143-1155). These authors used steady field stimulation of adult myocytes at 1 Hz in 1 mM calcium, after which PMA (10−7 M) was applied resulting in a decrease in twitch amplitude to approximately 60% of control. In single cardiac myocytes myofilament responsiveness to calcium was not affected by PMA, but that the negative inotropic effect was due to diminished amplitude of the calcium transient. In contrast to this report, a separate study using a slightly different phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), showed significant increases in cell shortening and an increase in the rate of change in cell length during relaxation, suggesting enhanced contractility by activation of PKC isozyme(s) (MacLeod et al. (1991) J. Physiol. 444:481-498). A more elaborate study in both isolated cardiac myocytes and whole guinea-pig hearts showed a significant positive inotropic response with 10−12 M PMA, but a negative inotropic response at concentrations higher than 10−10 M PMA (Ward and Moffat (1992) J. Mol. Cell Cardiol. 24:937-948). The results discussed above suggest that phorbol ester-mediated alterations in cardiac contractility are complex.
Transgenic mice have been generated with altered PKC isozyme expression in the heart. Overexpression of either wild-type or a constitutively active deletion mutant of PKCβ in a mouse heart was reported to induce cardiomyopathy (Wakasaki et al. (1997) Proc. Natl. Acad. Sci. USA 94:9320-9325; Bowman et al (1997) J. Clin. Invest. 100:2189-2195) but more recent investigation has suggested that lower levels of expression or adult onset PKCβ activation benefits ischemic recovery (Tiang et al. (1999) Proc. Natl. Acad. Sci. 96:13536-13541; Huang et al. (2001) Am. J. Physiol. Cell. Physiol. 280:C1114-C1120). Three groups have also reported transgenic mice with altered PKCε or PKCδ activity in the heart. Expression of a PKCε or PKCδ activating peptide in the mouse heart was associated with a physiologic activation of each isozyme and a mild hypertrophic response (Mochly-Rosen et al. (2000) supra; Chen et al. (2001) Proc. Natl. Acad. Sci. 98:11114-11119) Similarly, overexpression of an activated mutant PKCε cDNA in the mouse heart was reported to induce significant cardiac hypertrophy (Takeishi et al. (2000) Circ. Res. 86:1218-1223), but such a result is likely dependent on the absolute levels of PKCε overexpression and activity (Pass et al. (2001) Am. J. Physiol. Heart Circ. Physiol. 280:H946-H955). While a number of studies have demonstrated associations between various PKC isozymes and cardiac hypertrophy or ischemic injury, the necessary and sufficient functions of specific PKC isozymes are still debated. For example, while transgenic overexpression of PKCβ, δ, or ε in the mouse heart can initiate cardiac hypertrophy, gene targeting for these 3 isoforms did not overtly affect the heart, nor were PKCβ null mice defective in their ability to mount a hypertrophic response (Roman et al. (2001) Am. J. Physiol. Heart Circ. Physiol. 280:H2264-H2270). Collectively these results highlight the confusion in the art as to the PKC isozymes' roles as regulators of cardiac contractility. PKCα knock out mice have also been generated by Legites et al. Mol. Endocrinol. 16, 847-858) showing that PKCα enhances insulin signaling through PI3K.
PKCα is the predominant PKC isoform expressed in the small and large mammal heart, yet little is understood of its function in this organ (Pass et al. (2001) supra; Ping et al. (1997) Circ. Res. 81:404-414). While a number of correlative studies have been published showing associations between PKCα activation and cardiac hypertrophy or heart failure, almost no causal or mechanistic data have been reported. Gain- and loss-of function analysis of PKC isozyme function using cultured neonatal cardiac myocytes and recombinant adenoviruses expressing either wild-type or dominant negative mutants of PKCα, η, δ, and ε has been performed. It was reported that PKCα regulates the hypertrophic growth of cultured neonatal myocytes in part through ERK1/2, but its role in cardiac contractility is unknown (Braz et al. (2002) supra). Similarly, antisense phosphorothioate oligonucleotides against PKCα in cultured neonatal cardiac myocytes reduced hypertrophic gene expression following agonist stimulation (Kerkela et al. (2002) Mol. Pharmacol. 62:1482-1491). However, none of these observations include a mechanical assessment of PKCα's in vivo.
Little is understood of the role that various PKC isoforms play in potentially regulating cardiac contractility. PKC isoforms are known to directly phosphorylate sarcomeric proteins such as cTnI, which has been reported to affect the rate of maximal ATPase activity due to actin-myosin interactions (de Tombe & Solaro. (2000) Ann. Biomed. Eng. 28:991-1001). However, it remains unclear if PKC-mediated phosphorylation of contractile proteins significantly alters cardiac performance, in contrast to the well characterized effects of PKA.
Thus, a mechanistic assessment of PKCα's in vivo role is desirable. It is of importance to develop methods of modulating PKCα activity in cardiac tissue. It is also important to develop a model transgenic system for identifying PKCα modulating and anti-cardiomyopathic compounds and studying cardiomyopathies.
Treatment of heart failure in humans is based, in part, on the underlying causes, if known, and other factors including the severity of the disease, existing medications and other coinciding risk factors (for example, coronary artery disease, hypertension, valvular defects or hyperlipidemia). Advanced heart failure in patients may consist of both acute and chronic presentations, which may require varying treatments. Thus, current heart failure strategies target either acute decompensated heart failure (ADHF) or the chronic remodeling effects of heart failure. Treatment of ADHF is an unmet medical need, serving as the primary diagnosis for approximately 1 million hospital admissions per year in the United States and is the secondary diagnosis for another 2 million hospitalizations (DiDomenico R J et al. (2004) Ann Pharmacother. 38:649-660). ADHF is marked by functional deficits due to acute injury to the heart, e.g., myocardial infarction, arrhythmia, or may be precipitated by complications of chronic heart failure, e.g., progressive LV remodeling, cardiomegaly and myocyte loss (Cleland J G et al. (2001) Prog. Cardiovasc. Dis. 43:433-455). In either case, the patient requires immediate intervention for successful outcomes. Current treatments for ADHF depend largely on symptoms upon presentation to the emergency room, but may include inotropes (such as dobutamine and milrinone), intravenous diuretics (such as furosemide) and/or vasodilators (such as Nesiritide® in order to improve myocardial performance and maintain sufficient cardiac output (DiDomenico R J et al. supra). The goal of treatments using these drugs is to enhance or restore cardiac contraction and relaxation acutely and provide symptomatic improvement. In addition to ADHF, the drugs mentioned above may be administered in any setting of cardiac dysfunction (such as left ventricular dysfunction as a result of sepsis) when it is deemed medically necessary for survival, regardless of the etiology. In chronic heart failure, the drug regime is distinct and commonly includes agents such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, diuretics and/or β-adrenergic receptor blockers. These drugs are not administered for ADHF and some may in fact be counter-productive in this setting (e.g., β-adrenergic receptor blockers). While these drugs provide little or no immediate improvement in cardiac contraction or relaxation, they have been demonstrated to improve survival and cardiac remodeling in heart failure patients (Aronow W S. (2003) Heart Dis. 5:279-294). Therefore, there is a need to identify novel targets and their modulators to provide sufficient acute and chronic benefits in heart failure.
The inventions are based on the novel discovery that PKCα regulates cardiac contractility and cardiomyopathy and therefore both acute decompensated heart failure (ADHF) and chronic heart failure. Accordingly, it is believed that modulation of PKCα activity may provide therapeutic means for enhancing cardiac inotropy and ventricular performance. Transgenic animals of the invention are useful in identifying compounds for prophylaxis and treatment of disorders modulated by cardiac contractility, and cardiomyopathy including cardiac hypertrophy. These animals are also useful for investigative purposes, for examining signal transduction pathways involved in response to hypertrophic signals. The invention also details a process for measuring PKCα activation in vivo to screen for pharmacologic modulators of PKCα activity.
Compositions of the invention include transgenic mice, transgenic cells and transgenic tissues. In an embodiment, transgenic mice, cells and tissues of the invention comprise an expression cassette comprising a cardiac tissue-preferred regulatory sequences operably linked to a PKCα nucleotide sequence (SEQ ID NO: 1, NCBI Accession No. X04796) or a fragment or variant thereof. A variant is set forth in SEQ ID NO: 7, and encodes a polypeptide (SEQ ID NO: 8) exhibiting a dominant-negative effect. The cell expressing the expression cassette exhibits altered PKCα expression or activity. In another aspect, the transgenic mouse of the invention exhibits altered cardiac contractility. In another aspect, a mouse of the invention exhibits altered susceptibility to cardiomyopathy.
In another aspect, transgenic mice, transgenic cells and transgenic tissue comprise at least one disrupted PKCα gene. In one aspect, the disruption is sufficient to decrease or eliminate PKCα expression levels. In another aspect, a PKCα null mouse of the invention exhibits altered cardiac contractility. In another aspect, a mouse of the invention exhibits an altered susceptibility to cardiomyopathy.
In one aspect, methods of identifying compounds that modulate cardiac contractility are disclosed, comprising: providing a first and a second cell, tissue, or mouse, expressing a PKCα gene; administering a compound of interest to said first cell; incubating both the first and second cells for a suitable, predefined period of time; measuring the activity of PKCα in said first and said second cell; and identifying those compounds that modulate the activity of PKCα in said first cell compared to activity in said second cell as modulators of cardiac contractility.
In another aspect, methods of identifying compounds that modulate cardiomyopathy are disclosed, comprising: providing a first and a second cell expressing PKCα protein; administering a compound of interest to said first cell; incubating both the first and second cells for a suitable, predefined period of time; measuring the activity of PKCα in said first and said second cell; and identifying those compounds that modulate the activity of PKCα in said first cell compared to activity in said second cell as modulators of cardiomyopathy.
In another aspect, methods of identifying compounds that modulate PKCα activity are disclosed, comprising: providing a first and a second cell expressing PKCα protein; administering a compound of interest to said first cell; incubating both the first and second cells for a suitable, predefined period of time; measuring the activity of PKCα in said first and said second cell; and identifying those compounds that modulate the activity of PKCα in said first cell compared to activity in said second cell as modulators of PKCα activity.
For the above-described assays to identify compounds that modulate cardiac contractility, cardiomyopathy, and PKCα activity; any cell expressing suitable levels of PKCα protein could be used, e.g., standard laboratory-derived cell lines, cardiomyocyte cell lines, or any animal-derived primary cells, or tissues. Cells, and tissues from transgenic, or knock out mice; the transgenic animals themselves; and the dominant negative mutants of the invention are suitable for the purpose.
Modulators of PKCα activity include inhibitors or activators of the various PKCα activities, including, but not limited to, the enzymatic activity; the translocation activity; and the binding to various RACKs.
In another aspect, the compounds identified using above-described methods could be further validated using assays that utilize various cell culture, cultured tissues, or animal models of cardiac contractility, or cardiomyopathy as described herein.
In another embodiment, the invention provides a method of preferentially modulating PKCα activity in cardiac tissue. The method comprises providing a transgenic mouse comprising a stably incorporated expression cassette in the genome of at least one cell. The stably incorporated expression cassette comprises a cardiac preferred regulatory sequence operably linked to the PKCα nucleotide sequence set forth in SEQ ID NO: 1 or fragment or variant thereof. Variants of interest include, but are not limited to, dominant negative mutations such as the site directed mutant having the nucleotide sequence set forth in SEQ ID NO: 7. The invention further comprises determining the PKCα expression levels in the cardiac tissue of the mouse. In an aspect of the method, the mouse exhibits altered cardiac contractility. In another aspect, the mouse exhibits an altered susceptibility to cardiomyopathy.
In an embodiment, the invention provides a method of modulating PKCα expression in a mouse. The method comprises providing a transgenic mouse comprising at least one disrupted PKCα gene in the genome of at least one cell. The invention further comprises determining the PKCα expression levels in the mouse.
In an embodiment, the invention provides a method of treating or preventing an acute heart failure resulting from abnormal cardiac contractility in an animal. The method comprises the step of administering a PKCα modulating compound to the animal. In an aspect of the invention, the PKCα modulating compound is administered to the animal's cardiac tissue. In an aspect of the invention, the PKCα modulating compound is a PKCα inhibitor. In an aspect of the invention, the method increases the animal's cardiac contractility. Suitable animals include, but are not limited to, mice, guinea pigs, hamsters, humans, rabbits, dogs, pigs, goats, cows, rats, monkeys, chimpanzees, sheep, and zebrafish.
In an additional embodiment, the invention provides a method of treating or preventing a cardiomyopathy in an animal. The method comprises the step of administering a PKCα modulating compound to the animal. In an aspect of the invention, the PKCα modulating compound is administered to the animal's cardiac tissue. In an aspect of the invention, the PKCα modulating compound is a PKCα inhibitor or agonist. In an aspect of the invention, the method decreases the animal's susceptibility to cardiomyopathy. Suitable animals include, but are not limited to, mice, guinea pigs, hamsters, humans, rabbits, dogs, pigs, goats, cows, rats, monkeys, chimpanzees, sheep, and zebrafish.
PKCα inhibitors that may be used in the treatment of cardiac contractility or cardiomyopathy include, but are not limited to, nucleic acids, antibodies, small molecules, activator and inhibitor peptides, and Ro-32-0432, LY333531 and Ro-31-8220.
The invention also provides kits for performing a method of identifying a PKCα modulating compound. In an aspect of the invention a kit for identifying a PKCα modulating compound comprises a PKCα indicator polypeptide. In an aspect of the invention a kit for identifying a PKCα modulating compound comprises a cell comprising a PKCα indicator polypeptide.
Oryctolagus cuniculus
Mus musculus
Homo sapiens
Oryctolagus cuniculus
Mus musculus
Mus musculus
Mus musculus
Mus musculus
Mus musculus
Mus musculus
The present invention provides for modulation of cardiac contractility in acute heart failure, and cardiomyopathy in heart failure in general. Compositions of the invention include transgenic animals comprising either a PKCα nucleotide sequence or animals with a disruption in a PKCα nucleotide sequence. The invention further comprises cells and tissues isolated from these mice. The invention provides methods of modulating PKCα activity, PKCα expression levels, cardiac contractility, and susceptibility to cardiomyopathies, and acute heart failure. The invention provides kits for performing the methods of identifying PKCα modulating compounds.
The invention relates to compositions and methods drawn to PKCα gene (SEQ ID NO: 1). In an embodiment, an animal is stably transformed with an expression cassette comprising a cardiac-preferred regulatory sequences operably linked to a PKCα nucleotide sequence. In an embodiment, an animal of the invention is stably transformed with an expression cassette comprising a cardiac-preferred regulatory sequences operably linked to a fragment or variant of the PKCα nucleotide sequence such as the dominant negative variant set forth in SEQ ID NO: 7. In another embodiment, an animal of the invention is stably transformed with an isolated nucleic acid molecule that disrupts the native PKCα nucleotide sequence such that PKCα expression levels are decreased. In one aspect, the cardiac-preferred regulatory sequences are cardiac-preferred promoter sequences.
In an embodiment, the genome of a germ-line cell of a transgenic animal comprises the nucleotide sequence of interest. A transgenic cell is a cell isolated from a transgenic animal of the invention comprising at least one expression cassette or disruption cassette. Transgenic tissue, e.g. cardiac tissue, is tissue comprising transgenic cells.
In embodiments involving disruption cassettes, the nucleotide sequence of interest may be flanked by nucleotide sequences that naturally occur in the genomic DNA of the cell into which the nucleic acid molecule is transformed.
Fragments and variants of the PKCα nucleotide sequence and protein encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence exhibit a PKCα activity. Alternatively, fragments of a nucleotide sequence are useful as hybridization probes. A biologically active portion of a PKCα can be prepared by isolating a portion of one of the PKCα nucleotide sequences of the invention, expressing the encoded portion of the PKCα protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the PKCα protein.
One of skill in the art would also recognize that PKCα genes and proteins from a species other than those listed in the sequence listing, particularly mammalian species, would be useful in the present invention. One of skill in the art would further recognize that by using probes from the known species' sequences, cDNA or genomic sequences homologous to the known sequence could be obtained from the same or alternate species by known cloning methods. Such PKCα homologs and orthologs are included in the definition of PKCα gene and proteins of the invention.
Thus, a fragment of a protein kinase C-α nucleotide sequence may encode a biologically active portion of a protein kinase C-α (PKCα) or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a PKCα can be prepared by isolating a portion of one of the PKCα nucleotide sequences of the invention, expressing the encoded portion of the PKCα protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the PKCα protein. Nucleic acid molecules that are fragments of a protein kinase C-α nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, or 3524 nucleotides, or up to the number of nucleotides present in a full-length protein kinase C-α nucleotide sequence disclosed herein, or that contain additional sequences from the PKCα genomic locus alone or in combination with the sequence discussed above.
By “variants” are intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the PKCα polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques that are known in the art. In order to isolate orthologs and other variants generally stringent hybridization conditions are utilized mainly dictated by specific sequence, sequence length, GC content and other parameters. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis. Variants may also contain additional sequences from the genomic locus alone or in combination with other sequences.
Variant proteins may be derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids; deletion or addition of one or more amino acids; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention may or may not retain biological activity. Such variants may result from, for example, genetic polymorphism or from human manipulation. An exemplary variant PKCα protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 7. The variant protein encoded by the nucleotide sequence set forth in SEQ ID NO: 7 exhibits dominant negative effects.
The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. For example, amino acid sequence variants of the PKCα proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. /54:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).
Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different PKCα coding sequences can be manipulated to create a new PKCα possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the PKCα gene of the invention and other known PKCα genes to obtain a new gene coding for a protein with an altered property of interest e.g. a dominant negative mutation (Ohba et al. (1998) Mol. Cell. Biol. 18:51199-51207, Matsumoto et al. (2001) J. Biol. Chem. 276:14400-14406). Strategies for such DNA shuffling are known in the art.
The “percentage of sequence identity” or “sequence identity” is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical residue (e.g., nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
Percentage sequence identity can be calculated by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482-485 (1981); or by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443-445 (1970); either manually or by computerized implementations of these algorithms (GAP & BESTFIT in the GCG Wisconsin Software Package, Genetics Computer Group).
A preferred method for determining homology or sequence identity is by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268 and Altschul, (1993) J. Mol. Evol. 36, 290-300), which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are generally set at the default-scoring matrix BLOSUM62 for blastp, blastx, tblastn, and tblastx (Henikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919).
As described herein, PKCα genes and proteins, their allelic and other variants (e.g. splice variants), their homologs and orthologs from other species and various fragments and mutants will exhibit sequence variations. Typically, these sequences may exhibit at least about 75% sequence identity, preferably at least about 80% sequence identity, more preferably at least about 90% sequence identity and more preferably at least about 95% sequence identity to the genes and proteins of the invention.
The PKCα sequences of the invention are provided in expression cassettes for expression in the animal of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to a PKCα sequence of the invention. By “operably linked” is intended the transcription and translation of the heterologous nucleotide sequence is under the influence of the regulatory sequences. In this manner, the nucleotide sequences for the PKCα nucleotide sequences of the invention may be provided in expression cassettes along with cardiac tissue-preferred promoters for expression in the animal of interest, more particularly in the heart of the animal.
Such an expression cassette is provided with at least one restriction site for insertion of the nucleotide sequence to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
The expression cassette will include in the 5′-to-3′ direction of transcription, a transcriptional and translational initiation region, and a heterologous nucleotide sequence of interest. In addition to containing sites for transcription initiation and control, expression cassettes can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome-binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
The expression cassette comprising the PKCα sequence of the present invention operably linked to a promoter nucleotide sequence may also contain at least one additional nucleotide sequence for a gene to be co-transformed into the organism. Alternatively, the additional sequence(s) can be provided on another expression cassette.
The regulatory sequences to which the polynucleotides described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
It is recognized that a PKCα nucleotide sequence of the invention can be operably linked to any cardiac tissue preferred promoter and expressed in cardiac tissue. By “cardiac tissue” is intended any tissue obtained from the heart, including but not limited to, tissues developmentally related to the heart such as the pulmonary myocardium.
It is recognized that to increase transcription levels or to alter tissue specificity, enhancers and/or tissue-preference elements may be utilized in combination with the promoter. For example, quantitative or tissue specificity upstream elements from other cardiac-preferred promoters may be combined with the α-MHC promoter region used to generate the PKCα overexpressing mice to augment cardiac-preferred transcription. Such elements have been characterized, for example, the murine TIMP-4 promoter, A and B-type natriuretic peptide promoters, human cardiac troponin I promoter, mouse S100A1 promoter, salmon cardiac peptide promoter, GATA response element, inducible cardiac preferred promoters, rabbit β-myosin promoter, and mouse α-myosin heavy chain promoter (Rahkonen, et al. (2002) Biochim Biophys Acta 1577:45-52; Thuerauf and Glembotski (1997) J. Biol. Chem. 272:7464-7472; LaPointe et al (1996) Hypertension 27:715-722; Grepin et al. (1994) Mol. Cell Biol. 14:3115-29; Dellow, et al. (2001) Cardiovasc. Res.50:3-6; Kiewitz, et al. (2000) Biochim Biophys Acta 1498:207-19; Majalahti-Palviainen, et al (2000) Endocrinology 141:731-740; Charron et al. (1999) Molecular & Cellular Biology 19:4355-4365; Genbank 071441; U.S. Provisional Patent Application No. 60/393,525 and 60/454,947; and U.S. patent application Ser. No. 10/613,728).
A variety of cardiac tissue preferred promoter elements have been described in the literature and can be used in the present invention. These include, but are not limited to, tissue preferred elements from the following genes: myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, and cardiac α-actin. See, e.g. Franz et al (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol Cell Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051.
In other embodiments, the coding region is operably linked to an inducible regulatory element or elements. A variety of inducible promoter systems has been described in the literature and can be used in the present invention. A known and useful conditional system is the binary, tetracycline-based system, which has been used in both cells and animals to reversibly induce expression by the addition or removal of tetracycline or its analogues. Another example of such a binary system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.
Another class of promoter elements are those which activate transcription of an operably linked nucleotide sequence of interest in response to hypoxic conditions. These include promoter elements regulated at least in part by hypoxia inducible factor-1. Hypoxia response elements include, but are not limited to, the erythropoietin hypoxia response enhancer element (HREE1), the muscle pyruvate kinase HRE; the β-enolase HRE; and endothelin-1 HRE element, and chimeric nucleotide sequence comprising these sequences. See Bunn and Poynton (1996) Physiol. Rev. 76:839-885; Dachs and Stratford (1996) Br. J. Cancer 74:S126-S132; Guillemon and Krasnow (1997) Cell 89:9-12; Firth et al. (1994) Proc. Natl. Acad. Sci. 91:6496-6500; Jiang et al. (1997) Cancer Res. 57:5328-5335; U.S. Pat. No. 5,834,306).
In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
Where appropriate, the PKCα nucleotide sequence of the present invention and any additional nucleotide sequence(s) may be optimized for increased expression in the transformed animal. That is, these nucleotide sequences can be synthesized using species preferred codons for improved expression, such as mouse-preferred codons for improved expression in mice. Methods are available in the art for synthesizing species-preferred nucleotide sequences. See, for example, Wada et al. (1992) Nucleic Acids Res. 20 (Suppl.), 2111-2118; Butkus et al. (1998) Clin Exp Pharmacol Physiol Suppl. 25:S28-33; and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The G-C content of the heterologous nucleotide sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
In those instances where it is desirable to have the expressed product of the heterologous PKCα nucleotide sequence directed to a particular organelle, particularly the mitochondria, the nucleus, the endoplasmic reticulum, or the Golgi apparatus; or secreted at the cell's surface or extracellularly; the expression cassette may further comprise a coding sequence for a transit peptide. Such transit peptides are known in the art and include, but are not limited to, the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, and the like.
Disruption cassettes are used to interrupt and/or remove a sequence of interest from the genome of an animal cell in order to generate a “knock-out,” “deletion,” or “null” mutant. By “targeting vector” and “disruption cassette” is intended an isolated nucleic acid molecule comprising a 5′ flanking region, a disruption region, and a 3′ flanking region. Disruption cassettes and methods of their use are known in the art. See Doetschman et al. (1987) Nature 330:576-578; Doetschman et al. (1988) Proc. Natl. Acad. Sci 85:8583-87; Schwartz et al (1991) Proc. Natl. Acad. Sci.88:10416-20; Oliver et al (1997) Proc. Natl. Acad. Sci. 94:14730-14735; Nagy et al Ed. (2003) Manipulating the Mouse Embryo Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
Reporter genes or selectable marker genes may be included in the expression cassettes. Examples of suitable reporter genes known in the art can be found in, for example, Ausubel et al. (2002) Current Protocols in Molecular Biology. John Wiley & Sons, New York, N.Y. Selectable marker genes for selection of transformed cells or tissues can include genes that confer antibiotic resistance. Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as GUS (β-glucuronidase), fluorescence proteins (e.g. GFP), CAT; and luciferase.
Delivery vehicles suitable for incorporation of a polynucleotide for introduction into a host cell include, but are not limited to, viral vectors and non-viral vectors (Verma and Somia (1997) Nature 389:239-242).
A variety of non-viral vehicles for delivery of a polynucleotide are known in the art and are encompassed in the present invention. An isolated nucleic acid molecule can be delivered to a cell as naked DNA (WO 97/40163). Alternatively, a polynucleotide can be delivered to a cell associated in a variety of ways with a variety of substances (forms of delivery) including, but not limited to, cationic lipids; biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; protein transduction domains, and bacteria. A delivery vehicle can be a microparticle. Mixtures or conjugates of these various substances can also be used as delivery vehicles. A polynucleotide can be associated non-covalently or covalently with these forms of delivery. Liposomes can be targeted to a particular cell type, e.g., to a cardiomyocyte.
Viral vectors include, but are not limited to, DNA viral vectors such as those based on adenoviruses, herpes simplex virus, poxvirus such as vaccinia virus, and parvoviruses, including adeno-associated virus; and RNA viral vectors, including but not limited to, the retroviral vectors. Retroviral vectors include murine leukemia virus, and lentiviruses such as human immunodeficiency virus. See Naldini et al. (1996) Science 272:263-267.
Non-viral delivery vehicles comprising a polynucleotide can be introduced into host cells and/or target cells by any suitable method known in the art, such as transfection by the calcium phosphate coprecipitation technique; electroporation; electropermeablization; liposome-mediated transfection; ballistic transfection; biolistic processes including microparticle bombardment, jet injection, and needle and syringe injection, or by microinjection. Numerous methods of transfection are known to the skilled artisan.
Viral delivery vectors can be introduced into cells by infection. Alternatively, viral vectors can be incorporated into any of the non-viral delivery vectors described above for delivery into cells. For example, viral vectors can be mixed with cationic lipids (Hodgson and Solaiman (1996) Nature Biotechnol. 14:339-342); or lamellar liposomes (Wilson et al. (1977) Proc. Natl. Acad. Sci. 74:3471-3475; and Faller et al. (1984) J. Virol. 49:269-272).
For in vivo delivery, the vector can be introduced into an individual or organism by any method known to the skilled artisan.
Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. In one embodiment, the animal cell can be a fertilized oocyte or embryonic stem cell that can be used to produce a transgenic animal comprising at least one stably transformed expression cassette comprising the nucleotide sequence of interest. Alternatively, the host cell can be a stem cell or other early tissue precursor that gives rise to a specific subset of cells and can be used to produce transgenic tissues in an animal. See also Thomas et al., (1987) Cell 51:503 for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced gene has recombined with the genome are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the recombined DNA by germ line transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos. WO 90/11354; WO 91/01140; and WO 93/04169.
Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; 4,873,191; 6,201,165 and in Nagy et al Ed. (2003) Manipulating the Mouse Embryo Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to a pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
Other examples of transgenic animals include non-human primates, sheep, dogs, pigs, guinea pigs, hamsters, cows, goats, rabbits, and rats. Methods for providing transgenic rabbits are described in Marian et al. (1999) J. Clin. Invest. 104:1683-1692 and James et al. (2000) Circulation 101:1715-1721.
By “PKCα activity” is intended any activity exhibited by the wild-type PKCα described herein. Such activities include, but are not limited to, kinase activity, receptor of activated C kinase (RACK) binding activity, expression, translocation from the cytosolic fraction to the particulate fraction, and translocation to the sarcolemma. Modulation of PKCα activity includes but is not limited to modulation of a PKCα activity such as kinase activity, RACK binding, or modulation of PKCα expression levels or cellular distribution.
Methods of assaying kinase activity are known in the art and include, but are not limited to, immunoprecipitation with antibodies to phosphor-peptides; fluorescence polarization; filter binding assays with radioisotopes, scintillation proximity assays, 96 well assays with conjugated antibodies; time resolved fluorescent assays, thin layer chromatography; immunoprecipitation and immune complex assays; non-trichloroacetic acid phosphoamino acid determinations; and protein kinase assays. See Braz et al. (2002) J. Cell Biol. 156:905-919; Ping et al. (1999) Am. J. Physiol. 276:H1468-H1481; U.S. Patent Application No:20030036106; U.S. Pat. No. 5,447,860; Walker, John, ed. (2002) Protein Protocols on CD-ROM v. 2; and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, (Greene Publishing and Wiley-Interscience, New York).
Methods of analyzing PKCα association with RACKs are known in the art and include, but are not limited to, ELISA, protein interactive trapping, X-ray crystallography, NMR, ultracentrifugation, immunoprecipitation, co-immunoprecipitation, cross-linking, yeast two-hybrid assays, and affinity chromatography. See for example Mochly-Rosen (1995) Biochem Soc. Trans. 23(3):596-600; Walker, John, ed. (2002) Protein Protocols on CD-ROM v. 2; and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, (Greene Publishing and Wiley-Interscience, New York).
The invention provides methods of monitoring translocation of a PKCα indicator polypeptide. By “indicator polypeptide” is intended any polypeptide suitable for monitoring subcellular location. Suitable indicator polypeptides include fusion polypeptides containing reporter genes described earlier, such as, but not limited to, fluorescent proteins (e.g. GFP), β-galactosidase, c-jun, c-myc; affinity polypeptide tags (such as His tags), radiolabeled polypeptides, biotin labeled polypeptides, antigen labeled polypeptides, and dye labeled polypeptides. Suitable indicator polypeptides include antibodies specific to the polypeptide of interest.
In an embodiment, the invention provides a method of altering PKCα expression in an animal. In an embodiment, PKCα expression is modulated throughout the animal (e.g. the disruption mutant). In an embodiment PKCα expression is modulated in a cardiac preferred manner. By “cardiac-preferred” is intended that expression of the heterologous PKCα is most abundant in cardiac tissue, while some expression may occur in other tissue types, particularly in tissues developmentally related to cardiac tissue.
Methods of determining expression levels are known in the art and include, but are not limited to, qualitative Western blot analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, RT-PCR, 2-D gel electrophoresis, microarray analysis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, positron emission tomography, Northern blotting, fluorometric assays and SAGE. See, for example, Ausubel et al, eds. (2002) Current Protocols in Molecular Biology, Wiley-Interscience, New York, N.Y.; Coligan et al (2002) Current Protocols in Protein Science, Wiley-Interscience, New York, N.Y.; and Sun et al. (2001) Gene Ther. 8:1572-1579.
It is recognized that the PKCα nucleotide sequences may be used with their native promoters to increase or decrease expression resulting in a change in phenotype in the cardiac tissue of the transformed animal.
Transgenic animals that exhibit altered cardiac preferred expression of PKCα are useful to conduct assays that identify compounds that affect cardiac function such as, but not limited to, cardiac contractility. Assays to determine cardiac contractility are known in the art and include, but are not limited to, shortening assays, peak shortening, time to peak, time to ½ maximal relaxation, contracting and relaxing rate assays, changes in cardiac chronotropy, changes in cardiac lusitropy, and gross heart contraction assays. The altered cardiac-preferred expression of the PKCα expression may result in altered susceptibility to a cardiomyopathy. In an aspect of the invention, the invention provides methods of acutely modulating cardiac contractility. In another aspect of the invention, the invention provides methods of acutely modulating a cardiomyopathy.
An acute modulation or alteration begins within 1 second; 10 seconds; 30 seconds; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days after administration of the PKCα modulating agent. The duration of the modulation ranges from short durations such as, but not limited to, nanosecond, second, and minute increments; intermediate durations such as, but not limited to, hour, day, and week increments; to long durations such as, but not limited to, month and year increments, up to and including the recipient's lifespan.
In the context of the present invention, “cardiac contractility ” or “myocardial contractility” are defined as measures of cardiac function, which may include but are not limited to cardiac output, ejection fraction, fractional shortening, cardiac work, cardiac index, chronotropy, lusitropy, velocity of circumferential fiber shortening, velocity of circumferential fiber shortening corrected for heart rate, stroke volume, rates of cardiac contraction or relaxation, the first derivatives of interventricular pressure (maximum dP/dt and minimum dP/dt), ventricular volumes, clinical evaluations of cardiac function (for example, stress echocardiography and treadmill walking) and variations or normalizations of these parameters. These parameters may be measured in humans or animals alike to assess myocardial function and assist in diagnosis and prognosis of heart disease.
A “cardiomyopathy” is any disorder or condition involving cardiac muscle tissue or cardiac dysfunction. Disorders involving cardiac muscle tissue include, but are not limited to, myocardial disease, including but not limited to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, myocardial stunning, and myocarditis; heart failure; acute heart failure; rheumatic fever; rhabdomyoma; sarcoma; congenital heart disease, including but not limited to, left-to-right shunts—late cyanosis, such as atrial septal defect, ventricular septal defect, patent ductus arteriosus, and atrioventricular septal defect, right-to-left shunts—early cyanosis, such as tetralogy of fallot, transposition of great arteries, truncus arteriosus, tricuspid atresia, and total anomalous pulmonary venous connection, obstructive congenital anomalies, such as coarctation of aorta, pulmonary stenosis and atresia, and aortic stenosis and atresia; disorders involving cardiac transplantation; arterial hypertension; peripartum cardiomyopathy; alcoholic cardiomyopathy; tachycardias; supraventricular tachycardia; bradycardia; atrial flutter; hydrops fetalis; arrhythmias; extrasystolic arrhythmia; fetal cardiac arrhythmia; endocarditis; atrial fibrillation; idiopathic dilated cardiomyopathy; Chagas' heart disease; long QT syndrome; Brugada syndrome; ischemia; hypoxia; ventricular fibrillation; ventricular tachycardia; restenosis; congestive heart failure; syncope; arrythmias; pericardial disease; myocardial infarction; unstable angina; stable angina; and angina pectoris, viral myocarditis, and non-proliferating cell disorders involving cardiac muscle tissue.
By “altered susceptibility” is intended that a transgenic animal of the invention differs from a non-transgenic animal in the extent to which the transgenic animal of the invention exhibits a cardiomyopathic phenotype. The cardiomyopathic phenotype may present during any stage of development including, but not limited to, embryonically, post-natally, in the adult, and as the animal nears end of lifespan. In an embodiment, the cardiomyopathic phenotype may be induced by external stimuli such as, but not limited to, diet, exercise, chemical treatment, or surgical procedure.
Cardiomyopathic phenotypes include, but are not limited to, hypertrophy; morphology, such as interventricular septal hypertrophy; left ventricular-end systolic maximum dP/dt or end-diastolic dimension(▭); papillary muscle dimension; left-ventricular outflow tract obstruction; midventricular hypertrophy; apical hypertrophy; asymmetrical hypertrophy; concentric enlarged ventricular mass; eccentric enlarged ventricular mass; sarcomere structure; myofibril function; receptor expression; heart rate; ventricular systolic pressure; ventricular diastolic pressure; aortic systolic pressure; aortic diastolic pressure; contractility; interstitial fibrosis; cardiomyocyte disarray; Ca2+ sensitivity; Ca2+ release; Ca2+ uptake; catecholine sensitivity; α-adrenergic sensitivity; beta-adrenergic sensitivity; dobutamine sensitivity; thyroxine sensitivity; angiotensin-converting enzyme inhibitor sensitivity; amiodarone sensitivity; lidocaine sensitivity; glycoprotein receptor antagonist sensitivity; anabolic steroid sensitivity; carnitine transport irregularities; left ventricular dilation, reduced left ventricular ejection fraction; left atrial dilatation; diuretic sensitivity; volemia; ischemia; leukocyte flow properties; the polymorphonuclear leukocyte (PMN) membrane fluidity; PMN cytosolic Ca2+ content; high interventricular septal defects, rosette inhibition effect; contractile force transmission; myocardial fiber disarray; increased chamber stiffness; impaired relaxation; small-vessel disease; dyspnea; angina; presyncope; tachycardia; syncope; lethargy; respiratory distress; ruffled fur; hunched posture; peripheral edema; ascites; hepatomegaly; edematous lung; cardiomegaly; organized thrombi formation; heart weight/body weight ratio; rate of pressure development, rate of pressure fail, cell twitch measurement and the like. See, for example, Braunwald et al. (2002) Circulation 106:1312-1316; Wigle et al. (1995) Circulation 92:1680-1692; and Pi & Walker (2000) Am. J. Physiol. Heart Circ. Physiol 279:H26-H34; hereby incorporated by reference in their entirety.
Methods for measuring cardiomyopathic phenotypes are known in the art and include, but are not limited to, trans-thoracic echocardiography, transesophageal echocardiography, exercise tests, urine/catecholamine analysis, EIAs, light microscopy, heart catheterization, dynamic electrocardiography, Langendorff hanging heart preparation, working heart preparation, MRI, multiplex RT-PCR, positron emission tomography, angiography, magnetic resonance spin echo, short-axis MRI scanning, Doppler velocity recordings, Doppler color flow imaging, stress thallium studies, cardiac ultrasound, chest X-ray, oxygen consumption test, electrophysiological studies, auscultation, scanning EM, gravimetric analysis, hematoxylin and eosin staining, skinned fiber analysis, transmission electron microscopy, immunofluorescent analysis, trichrome staining, Masson's trichrome staining, Von Kossa staining, 2-D echocardiography, cardiotocography, baseline M-mode echocardiography, and myocardial lactate production assays. See, for example, Braz et al. (2002) J. Cell. Biol. 156:905-919; Braunwald et al. (2002) Circulation 106:1312-1316; Sohal et al. (2001) Circulation Res. 89:20-25; Nagueh et al. (2000) Circulation 102:1346-1350; Sanbe et al. (2001) J. Biol. Chem. 276:32682-32686; Sanbe et al. (1999) J. Biol. Chem. 274:21085-21094; Wigle et al. (1995) Circulation 92:1680-1692; Pi & Walker (2000) Am. J. Physiol. Heart Circ. Physiol 279:H26-H34; and Wang et al. (2001) Am. J. Physiol. Heart Circ. Physiol. 269:H90-H98 hereby incorporated by reference in their entirety.
The term “treatment” is used herein to mean that, at a minimum, administration of a compound of the present invention mitigates a disease or a disorder in a host, preferably in a mammalian subject, more preferably in humans. Thus, the term “treatment” includes: preventing an infectious disorder from occurring in a host, particularly when the host is predisposed to acquiring the disease, but has not yet been diagnosed with the disease; inhibiting the infectious disorder; and/or alleviating or reversing the infectious disorder. Insofar as the methods of the present invention are directed to preventing disorders, it is understood that the term “prevent” does not require that the disease state be completely thwarted. (See Webster's Ninth Collegiate Dictionary.) Rather, as used herein, the term preventing refers to the ability of the skilled artisan to identify a population that is susceptible to disorders, such that administration of the compounds of the present invention may occur prior to onset of a disease. The term does not imply that the disease state be completely avoided.
Identification of PKCα inhibitors for the treatment of impaired cardiac contraction and relaxation can be identified by known methodologies. PKCα inhibitors could be identified by assessing the enzymatic activity of PKCα. This may be accomplished by using a number of commercially available kits. Some of these kits use “labeled” substrates, including, but not limited to luminescent, fluorescent, radioactive or other measurable and quantifiable endpoints. Alternatively, as set forth in this invention, the PKCα protein itself could be attached to a traceable marker, including, but not limited to luminescent, fluorescent or radioactive ion or molecule in order to determine the distribution and activity of PKCα in isolation or in a cell or tissue. As PKCα has many known substrates in the cell, PKCα activity could be assessed by measuring the phosphorylation or dephosphorylation of PKCα substrates. PKCα substrates phosphorylation/dephosphorylation status may be measured using labeled or unlabeled phosphorylation site-specific antibodies, luminescent, fluorescent, radioactive biological labels or other means to assess PKCα activity against its substrates. Alternatively, the redistribution of the substrate(s) may also serve as a means of measuring its response to alterations in PKCα activity. In the case where the substrate is a kinase, phosphatase or other enzyme, the activity of the substrate may be measured by established techniques.
Identification of PKCα inhibitors that would be beneficial in humans with cardiac dysfunction may be accomplished using isolated cells or isolated tissues in which it has been determined that PKCα is present. For instance, PKCα inhibitors may be tested in isolated cells, preferably cardiomyocytes, from mammals or other organisms and determine the effect of PKCα inhibitors by measuring the percent shortening of the cell (% FS): the rates of shortening or re-lengthening (±dL/dt), by standard techniques (Chaudhri B et al. (2002) Am J Physiol Heart Circ Physiol. 283:H2450-H2457). Alternatively, muscle(s), preferably of cardiac origin, may be isolated and measurements of contractile function assess in the presence and absence of PKCα inhibitors, by standard techniques (Slack J P et al. (1997) J Biol Chem. 272:18862-18868). PKCα inhibitors may be identified as outlined in the present invention by measuring acute hemodynamics, including heart rate, blood pressure, rates of contraction and relaxation (+dP/dt, and −dP/dt), left ventricular pressure and derivations of these parameters. PKCα inhibitors may be identified by these methods in suitable, normal animals including, but not limited to, various genetic strains of mice, rats, guinea pigs, hamsters, humans, rabbits, dogs, pigs, goats, cows, monkeys, chimpanzees, sheep, hamsters and zebrafish. PKCα inhibitors could be identified by these methods in suitable, animal models of heart failure or cardiac dysfunction including, but not limited to, various genetic strains of transgenic or knockout mice, such as the MLP(−/−) KO mice, type-1 serine/threonine phosphatase overexpressing mice (PP1c), and PKCα overexpressing transgenic mice. In addition, PKCα inhibitors may be identified in spontaneous or natural models of heart failure and cardiac dysfunction due to a genetic or multiple genetic defects, including but not limited to the spontaneous hypertensive heart failure rat or the Dahl salt sensitive rat. In addition, PKCα inhibitors may be identified in surgically induced models of cardiac dysfunction including, but not limited to, myocardial infarction models, coronary microembolism model, aortic constriction model, arteriovenous fistula model or other pressure or volume overload models in rats, guinea pigs, rabbits, dogs, pigs, goats, cows, monkeys, chimpanzees, sheep, hamsters and zebrafish.
In an embodiment, a transgenic animal, tissue, or cell of the invention may be used to identify PKCα modulating compounds. A “PKCα modulating compound” is a compound that modulates a PKCα activity. PKCα modulating compounds include, but are not limited to, diacylglycerols, phosphatidylserine, Ca++; PMA, CGP54345, bisindolylmaleimide, AAP10, staurosporine, H-7 (Sigma Co.), diazoxide, DiC8, arachidonic acid, Gö-6976 (PKC and including PKCα), CGP 54345, HBDDE (also PKCγ), and Ro-32-0432 (also PKCβ). Methods for assaying PKCα activity are described elsewhere herein. Any method of assaying a PKCα activity known in the art may be used to monitor the effects of the compound of interest on a transgenic animal of the invention.
PKCα inhibitors include, but are not limited to, kinase inhibitors, protein kinase C inhibitors, and PKCα specific inhibitors. By “kinase inhibitor” is intended a compound that inhibits multiple kinases including PKCα. By “protein kinase C inhibitor” is intended a compound preferentially inhibits activities of a protein kinase C as compared to its effect on other kinases. By “PKCα specific inhibitor” is intended a compound that reduces a PKCα activity more than it reduces an activity of another kinase, including other protein kinase C isozymes. Known PKCα inhibitors include, but are not limited to, nucleic acid molecules having antisense nucleotide sequences and antisense molecules commercially available from Isis Pharmaceuticals and dominant negative mutations of PKCα, such as the lysine 368 arginine mutation (Braz et al. (2002) J. Cell. Biol. 156:905-919).
Antisense constructions complementary to at least a portion of the messenger RNA (mRNA) for a PKCα nucleotide sequence can be constructed. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequences may be made as long as the sequences hybridize to and interfere with expression of the corresponding mRNA. In this manner, antisense constructions having at least about 70%, preferably at least about 80%, more preferably at least about 85% sequence identity to the corresponding antisensed sequences may be used. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or greater may be used. Thus, antisense DNA sequences may be operably linked to a cardiac tissue-preferred promoter to reduce or inhibit expression of a native protein in cardiac tissue.
In addition to antisense technologies, gene expression can be repressed by double stranded RNA including short-hairpin RNA (shRNA), RNA interference (RNAi), short terminal RNA (stRNA), mikroRNA (miRNA) or short interfering RNA (siRNA) (Schutze N. (2004) Mol Cell Endocrinol. 213, 115-119). These RNA interfering approaches can use RNA of varying sizes, but are generally limited to 15-28 nucleotides and act by an as yet unclear mechanism. This technique has been successfully employed in vitro and in vivo as a means to inhibit gene functions (McCaffrey A P et al. (2002) Nature 418, 38-39).
Criteria evaluated for augmented contractility and heart failure progression include, but are not limited to, β-receptor number, β-receptor coupling, adenylyl cyclase activity, cAMP levels at rest, cAMP levels after forskolin administration, PKA activity, PKA protein levels, L-type calcium channel current density, SERCA2a protein levels, and phospholamban mRNA levels, or phospholamban phosphorylation of proteins.
Compounds that can be screened in accordance with the assays of the invention include but are not limited to, libraries of known compounds, including natural products, such as plant or animal extracts, synthetic chemicals, biologically active materials including proteins, peptides such as soluble peptides, including but not limited to members of random peptide libraries and combinatorial chemistry derived molecular library made of D- or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries), antibodies (including, but not limited to, polyclonal, monoclonal, chimeric, human, anti-idiotypic or single chain antibodies, and Fab, F(ab′)2 and Fab expression library fragments, and epitope-binding fragments thereof), organic and inorganic molecules.
In addition to the more traditional sources of test compounds, computer modeling and searching technologies permit the rational selection of test compounds by utilizing structural information from the ligand binding sites of proteins of the present invention. Such rational selection of test compounds can decrease the number of test compounds that must be screened in order to identify a therapeutic compound. Knowledge of the sequences of proteins of the present invention allows for the generation of models of their binding sites that can be used to screen for potential ligands. This process can be accomplished in several manners known in the art. A preferred approach involves generating a sequence alignment of the protein sequence to a template (derived from the crystal structures or NMR-based model of a similar protein(s), conversion of the amino acid structures and refining the model by molecular mechanics and visual examination. If a strong sequence alignment cannot be obtained then a model may also be generated by building models of the hydrophobic helices. Mutational data that point towards residue-residue contacts may also be used to position the helices relative to each other so that these contacts are achieved. During this process, docking of the known ligands into the binding site cavity within the helices may also be used to help position the helices by developing interactions that would stabilize the binding of the ligand. The model may be completed by refinement using molecular mechanics and loop building using standard homology modeling techniques. General information regarding modeling can be found in Schoneberg, T. et. al., Molecular and Cellular Endocrinology, 151:181-193 (1999), Flower, D., Biochimica et Biophysica Acta, 1422:207-234 (1999), and Sexton, P. M., Current Opinion in Drug Discovery and Development, 2(5):440-448 (1999).
Once the model is completed, it can be used in conjunction with one of several existing computer programs to narrow the number of compounds to be screened by the screening methods of the present invention, like the DOCK program (UCSF Molecular Design Institute, 533 Parnassus Ave, U-64, Box 0446, San Francisco, Calif. 94143-0446). In several of its variants it can screen databases of commercial and/or proprietary compounds for steric fit and rough electrostatic complementarity to the binding site. Another program that can be used is FLEXX (Tripos Inc., 1699 South Hanley Rd., St. Louis, Mo.).
As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a carboxypeptidase protein or anti-carboxypeptidase antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel®, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Anti-cardiomyopathic compounds identified by the methods of this invention may be used in the treatment of humans.
The PKCα gene was targeted for deletion by standard homologous recombination in embryonic stem cells, followed by production of chimeric mice, which were bred and passed the targeted allele into the germline. The exon encoding the ATP binding cassette in PKCα was deleted resulting in a null allele with regards to protein expression. For generation of PKCα overexpressing transgenic mice, a cDNA encoding PKCα was subcloned into the murine α-myosin heavy chain promoter-containing expression vector and injected into newly fertilized oocytes. The MLP, PP1 c, and pressure-overload surgical model (TAC) were all described elsewhere (Arber et al. (1997) Cell 88:393-403; Carr et al. (2002) Mol. Cell. Biol. 22:4124-4135; and Liang et al. (2003) EMBO. J. 22:5079-5089). Males were exclusively used in all studies for consistency. All animal experiments were approved by the Institutional Animal Care and Use Committee.
Mice from all genotypes or treatment groups were anesthetized with isoflurane, and echocardiography was performed using a Hewlett Packard 5500 instrument with a 15-MHZ microprobe. Echocardiographic measurements were taken on M-mode in triplicate from four separate mice per group. The isolated ejecting mouse heart preparation used in the present study has been described in detail previously (Gulick et al. (1997) Circ. Res. 80:655-664), as was the close-chested working heart model employed here (Lorenz et al. (1997) Am. J. Physiol. 272:H1137-H1146).
Hearts were collected at the indicated times, fixed in 10% formalin containing PBS, and embedded in paraffin. Serial 5-μm heart sections from each group were analyzed. Samples were stained with hematoxylin and eosin or Masson's trichrome. Cardiac gene expression of hypertrophic molecular markers was assessed by RNA dot-blot analysis as described previously (Jones et al. (1996) J. Clin. Invest 98:1906-1917).
Ventricular myocytes were isolated from Sprague-Dawley rat hearts (Westfall et al, (1997 Methods Cell Biology 52:307-322), and plated on laminin-coated coverslips in DMEM with 5% serum for 2 hr. Media was then replaced with serum-free DMEM containing a recombinant viral vector. Serum-free DMEM was added after 1 hr, and media was changed every 2 days. About 70-85% of isolated cells are rod shaped, with 1-2×106 rod-shaped myocytes per heart. Myocytes used for shortening assays were electrically stimulated in media 199 supplemented with Penicillin/Streptomycin, 10 mM Hepes, 0.2 mg/ml albumin, and 10 mM glutathione (Westfall and Borton, (2003) J. Biol. Chem. 278:33694-33700). Myocytes were transferred to a stimulation chamber with platinum electrodes 1 day after plating, and stimulated at 0.2 Hz with a 2.5 ms pulse at a voltage producing twitches in <25% of myocytes. Media in the stimulation chamber was replaced every 12 hrs. For contractile function studies, coverslips were mounted in a thermo-controlled chamber containing M199 for sarcomere shortening measurements. Sarcomere length was measured via a variable field rate CCD video camera (Ionoptix; Milton, Mass.), and recorded with sarcomere length detection software. Myocytes were stimulated at 0.2 Hz, and sarcomere shortening was recorded for 60 sec. Measurements of peak shortening, time to peak, time to half maximal relaxation and contraction plus relaxation rates were obtained from the signal average for 10 contractions. Results from these studies were compared by a one-way analysis of variance and a post-hoc Newman-Keuls test.
Cardiac myocytes were dissociated from the ventricles of 3-month-old wildtype or non-transgenic (Ntg) and PKCα-KO mice and electrophysiological recording were performed as described before (Petrashevskaya et al. (2002) Cardiovasc. Res. 54:117-132 and Masaki et al. (1997) Am. J. Physiol. 272:-H606-H612). Briefly, the heart was subject to retrograde coronary perfusion with Ca2+-free Tyrode's solution for 10 minutes, and with Tyrode's solution (250 μM Ca2+) containing collagenase type II (Worthington; 1.0 mg/ml) supplemented with 5 mM taurine and 10 mM BDM (2,3-butane, dione-monoxamine) for 8-12 minutes at 37° C. bubbled with 95% O2 and 5% CO2. At the end of the perfusion the heart was removed and the ventricular tissues were mechanically minced in low Cl−, high K+-KB medium. The minced ventricular tissue was then gently filtered, and stored at 4° C. until electrophysiological study. Only Ca2+ tolerant cells with clear cross striations and without spontaneous contraction or significant granulation were selected for experiments.
Experiments were performed on the dissociated cardiac myocytes at 20 to 24° C. All current recordings were obtained in the whole cell, voltage-clamp configuration of the patch clamp technique by using 1.60 OD borosilicate glass electrodes (Garner Glass Company). Cell capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 25 mV hyperpolarizing test pulse (25 ms) from a holding potential of 0 mV. Resistance was within the range of 2 to 11 MΩ. Most of the data presented in these studies were obtained with electrodes having a resistance of 0.5-3 MΩ. After formation of a high resistance seal between the recording electrode and the myocyte membrane, electrode capacitance was fully compensated electronically before breaking the membrane patch. ICa currents were elicited by depolarizing voltage steps (380 ms) from −50 mV to +40 mV in 10 mV increments from a holding potential −60 mV. The recorded currents were filtered at 2 kHz through a four-pole low-bass Bessel filter and digitized at 5 kHz. The experiments were controlled using pClamp 5.6 software (Axon Instruments) and analyzed using Clampfit 6.0.3. Ca2+ currents were recorded using an external solution containing (in mM): CaCl2 1.8, tetraethyl-ammonium chloride (TEA-Cl) 135, 4-aminopyridine (4-AP) 5, glucose 10, HEPES 10, MgCl2, (pH 7.3). The pipette solution contained (mM): cesium aspartate 100, CsCl 20, MgCl2 1, Mg-ATP 2, Na2-GTP 0.5, EGTA 5, HEPES 5, (pH 7.3 with CsOH). These solutions isolated Ica from other membrane currents such as Na+ and K+ channel currents and also Ca2+ flux through the Na+/Ca2+ exchanger.
Isolation of mouse left ventricular myocytes for assessment of calcium transient measurements was carried out as described previously (Chu et al. (1996) Circ. Res. 79:1064-1076). Ca2+ transients were measured from cardiomyocytes at room temperature. Briefly, mouse hearts were excised from anesthetized (pentobarbital sodium, 70 mg/kg, i.p.) adult mice, mounted in a Langendorff perfusion apparatus, and perfused with Ca2+-free Tyrode solution at 37° C. for 3 min. The normal Tyrode solution contained 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.4. Perfusion was then switched to the same solution containing 75 units/ml type 1 collagenase (Worthington), and perfusion continued until the heart became flaccid (˜10-15 min). The left ventricular tissue was excised, minced, pipette-dissociated, and filtered through a 240-μm screen. The cell suspension was then sequentially washed in 25, 100, 200 μM and 1 mM Ca2+−Tyrode. To obtain intracellular Ca2+ signals, cells were incubated with the acetoxymethyl ester form of fura-2 (Fura-2/AM; 2 μM) for 30 min at room temperature and resuspended in 1.8 mM Ca2+-Tyrode solution. The myocyte suspension was placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200), and perfused with 1.8 mM Ca2+-Tyrode solution at room temperature (22° C.-23° C.). Myocyte contraction was field-stimulated by a Grass S5 stimulator (0.5 Hz, square waves), and the cells were alternately excited at 340 and 380 nm 5 by Delta Scan dual-beam spectrophotofluorometer (Photon Technology International). Ca2+ transients were recorded as the 340/380 nm ratio of the resulting 510 nm emissions. Baseline and amplitude, estimated by the 340/380nm ratio, and the times for 80% decay of the Ca2+ signal and tau were acquired. All data were analyzed using software from FeliX and Ionwizard.
PKC kinase reaction mixtures included 10 μM inhibitor-1, 20 mM MOPS, pH 7.2, 25 mM β-glycerol phosphate, 1 mM MgCl2, 1 mM sodium orthovanadate, 1 mM DTT, 1 mM CaCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml diacylglycerol, 100 μM ATP, and 0.6 mCi/ml [32P]ATP. PKC was isolated from rabbit heart muscle (Woodgett and Hunter, (1987) J. Biol. Chem. 262:4836-4843) and recombinant wildtype and Ser-67-Ala inhibitor-1 was isolated from E. coli (Bibb et al. (2001) J. Biol. Chem. 276:14490-14497). Reactions were conducted at 30° C. and aliquots were removed at specific time points and stopped by addition of protein sample buffer. Stoichiometries were determined by SDS-PAGE and direct quantification of radioactivity.
Primary cultures of neonatal rat cardiomyocytes were obtained by enzymatic dissociation of 1-2 day-old Sprague-Dawley rat neonates as described previously (De Windt et al. (2000) J. Biol. Chem. 275:13571-13579). Cardiomyocytes were cultured under serum-free conditions in M199 media supplemented with penicillin/streptomycin (100 U/ml) and L-glutamine (2 mmol/L).
The characterization of adenovirus-encoding wildtype or dominant negative mutants of PKCα in cardiomyocytes was described previously (Braz et al. (2002) J. Cell Biol. 156:905-919). The dominant negative PKCα cDNA consisted of a lysine to arginine mutation in the ATP binding domain at amino acid position 368. Each recombinant adenovirus was plaque purified, expanded, and titered in HEK293 cells. Typical experiments involved infection of 6 neonatal rat cardiomyocytes at a moi of 100 plaque forming units for 2 h at 37° C. in a humidified, 6% CO2 incubator. Subsequently, the cells were cultured in serum-free M199 media for an additional 24 h before analysis. Under these conditions 95% of the cells showed expression of the recombinant protein.
Soluble and particulate fractions were prepared as described previously (Braz et al. (2002) J. Cell Biol. 156:905-919). Protein samples were subjected to SDS-PAGE (10% gels), transferred to Hybond-P membrane (Amersham Pharmacia Biotech), blocked in 7% milk, and incubated with primary antibodies against PKCα, β, δ, ε, SERCA2, calsequestrin, PLB, phospho-serine-16 PLB, inhibitor-1, and PP1cα. Phospho-specific I-1 antibodies were described previously (Bibb et al. (2001) J. Biol. Chem. 276:14490-14497. Primary antibodies were incubated overnight in 3% milk at 4° C. Secondary antibodies IgG (alkaline phosphatase—conjugated anti-mouse, -rabbit, or -goat) were incubated for 1 h at room temperature in 0.5-3% milk. Chemifluorescent detection was directly performed with the Vistra ECF reagent (RPN 5785; Amersham Pharmacia Biotech) and scanned with a PhosphorImager or chemiluminescence was performed with ECL (Amersham Pharmacia Biotech) and exposed on film.
Protein extracts were generated from cardiomyocytes infected with adenovirus encoding β-galactosidase, I-1, PKCα, and PKCα-dn. Extracts were immunoprecipitated with PPIcα conjugated to agarose beads, followed by western blotting against I-1. Preparation of phosphorylated protein substrate and radioactive assay of protein phosphatases were prepared as instructed by the Protein Serine/Threonine Phosphatase (PSP) Assay System (New England BioLabs, Inc.).
Caffeine induced calcium transients were measured in a total of 37 myocytes from 4 PKCα null mice and 19 control myocytes from 3 wild type mice. After collagenase digestion, myocytes were loaded with Indo-1 AM (25 μg/2 ml) for 12 min at room temperature. Intracellular calcium transients (measured by Indo-1 fluorescence ratio) were recorded at resting state (no electrical stimulation) before and during 20 mM caffeine addition.
Hearts were isolated from four wild-type and four PKCα −/− (PKCα null) transgenic mice. The isolated hearts were infused with PMA at 9 different concentrations ranging from 8×10−11 through 8×10−7 M. Acute PMA infusion of each concentration occurred for a 7 minute period. The hearts were measured for maximal and minimal dP/dt in systole and diastole respectively. Results from one such experiment are presented in
A standard curve was used to assess the relative abundance of the PKC isozymes in healthy human hearts. Recombinant human protein PKCα, PKCβI, PKCβII, PKCγ, and PKCε generated in bacteria were purchased from a commercial vendor. Three aliquots of known concentrations were prepared.
Adult human ventricular tissue was explanted from six undiseased individuals. Whole cell protein lysates were prepared. The three standard PKC aliquots and the heart proteins were subjected to polyacrylamide gel electrophoresis on the same gel. The proteins were transferred to a membrane. The membrane was blocked and incubated with antibodies specific to the PKCα, PKCβI, PKCβII, PKCγ, and PKCε isozymes. Data from such an experiment are presented in
The relatively selective PKCα/β inhibitory compound Ro-32-0432 [2-{8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-(1-methylindol-3-yl)maleimide, HCl Salt] [3-{8-[(Dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}-4-(1-methylindol-3-yl)-1H -pyrrole-2,5-dione, HCl Salt] was used as a means of directly examining the effects of acute PKCα inhibition on cardiac function and contractility using an ex vivo working heart preparation. The working heart preparation separates the inherent pump function of the heart from potential alterations in total vascular resistance as might occur if the drug were infused in vivo.
Working adult wildtype mouse hearts were infused with vehicle control (10% DMSO) or Ro-32-0432 in 10% DMSO at concentrations ranging between 4×10−10 through 4'10−6M. Four animals were analyzed in the Ro-32-0432 group and compared with three animals in the vehicle control group. Values throughout the concentration time course (7 minutes per 10 different incremental concentrations) were summated for statistical purposes, representing an average dosage of approximately 1×10−8 M. The vehicle control and experimental groups showed heart rates of 363+/−15 and 295+/−26 beats per minute, respectively, before any treatments were begun. The average heart rate of the vehicle and drug treated groups was 351+/=3 and 292+/−6 beats per minute, respectively. Despite the lower heart rate, the Ro-32-0432 infused group showed an increase in acute contractile function measured as maximum dP/dt, and an increase in left ventricular pressure developed. The approximate 20% change in acute contractile performance in the Ro-32-0432 treated group is similar to the increase in cardiac function observed in PKCα null mice. The data from one such experiment are presented in
A PKCα indicator was prepared by operably linking a nucleotide sequence encoding PKCα to a nucleotide sequence encoding green fluorescence protein (GFP). An expression cassette comprising the PKCα-GFP nucleotide sequence was prepared. Adenovirus comprising the PKCα-GFP expression cassette was prepared.
Neonatal rat cardiomyocytes were cultured in plastic dishes and incubated until the appropriate density was reached. The cardiomyocytes were infected with an adenovirus encoding PKCα-GFP. The cultures were incubated for 24 hours. After 24 hours the cells were incubated with either DMSO alone (the vehicle treatment) or DMSO and PMA for 60 minutes. The cells were fixed and examined by confocal microscopy. The PMA stimulated cells exhibit a highly localized and punctate staining pattern whereas the vehicle only stimulated cells exhibit a relatively diffuse PKCα-GFP localization.
Selected PKCα inhibitors are evaluated in both naive rats and rats with myocardial infarction (MI) for effects on cardiac contractility and hemodynamics.
Male, Sprague-Dawley or Lewis rats weighing between 225-500 gm are anesthetized with isoflurane and an MI is induced as follows. A thoracotomy at the fourth or fifth intercostal space is done, the heart is exposed and the pericardium is opened. A 5-0 suture is placed around the left descending coronary artery 2-4 mm from its origin and permanently tied. The ribs, muscle and skin are separately closed and the animal is allowed to recover. Twenty to twenty-three weeks after surgery the animals are used to evaluate the effects of PKCα inhibitors on cardiac contractility and hemodynamics.
The effects of inhibitors on cardiac contractility and hemodynamics are evaluated in naïve and MI rats as follows. The animals are anesthetized with isoflurane. A femoral artery is isolated and cannulated for the measurement of systemic blood pressure. A jugular vein is isolated and cannulated for the intravenous infusion of inhibitor. The right carotid artery is isolated and a Millar conductance catheter is inserted to the left ventricle (LV) of the heart. The LV systolic pressure, end-diastolic pressure, +maximum dP/dt, −minimum dP/dt, and heart rate are derived from the LV pressure waveform. Mean arterial blood pressure is derived from the systemic blood pressure waveform. Data are recorded continuously and derived using computerized data acquisition software (Notocord or Powerlab).
After a period of stabilization, PKCα inhibitors are infused at the following infusion doses in naïve rats: 0.1, 0.3, 1.0, 3.0, 10, 30, 100, 300 and 1000 nmol/kg/min. The infusion of each dose is allowed to run for at least five minutes. In MI rats, the infusion doses are as follows: 10, 30, 100, 300, and 1000 nmol/kg/min for at least five minutes. Equivalent infusion volumes are administered to separate, vehicle-control naïve and MI animals. At the end of the test infusions, 5.0 μg/kg/min of dobutamine is infused.
This assay can be used for a variety of cardiomyopathic phenotypes. A PKCα nucleotide sequence of interest is cloned into an expression vector containing a cardiac tissue-preferred promoter. The expression cassette comprising the promoter, operably linked to the nucleotide sequence of interest is digested with a restriction enzyme. The restriction reaction products are electrophoresed on an agarose gel, and the expression cassette is purified from the agarose. The expression cassette is prepared for microinjection according to any method known to one skilled in the art. The expression cassette is used to provide a transgenic mouse. The presence of the transgene is confirmed using Southern blot analysis.
Two cohorts of age-matched transgenic mice are established. The diet of one cohort is supplemented with a compound of interest. The diet of the second cohort is supplemented with a placebo. The two mice cohorts are incubated for an appropriate time and the experiment is terminated. The mice are monitored for a cardiomyopathic phenotype such as hypertrophy using the left ventricle/body mass ratios described elsewhere herein. A cardiomyopathic phenotype presented by the mice of the each cohort is compared. Alternatively, the compound may be administered directly to the animals using established methodologies and technologies, including but not limited to, intra-arterial or intravenous injection of a compound by syringe or osmotic mini-pumps or other means, oral gavage, intraperitoneal injection or subcutaneous injection.
In the following figures, data are presented with the standard error of the mean, unless otherwise indicated.
Except as otherwise noted, all amounts including quantities, percentages, portions, and proportions, are understood to be modified by the word “about”, and amounts are not intended to indicate significant digits.
Except as otherwise noted, the articles “a”, “an”, and “the” mean “one or more”.
All documents cited are, in relevant part, incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope.
This application claims priority under 35 USC 119(e) to and benefit of U.S. Provisional Application No. 60/503,853, filed on Sep. 19, 2003, which is herein incorporated by reference in its entirety.
This invention was made with Government support under NIH Grant Nos. HL62927, HL26057, and HL64018. The United States Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60503853 | Sep 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10944375 | Sep 2004 | US |
| Child | 12772024 | US |
