Patent Application
20120237588
The present invention is directed towards methods of improving cardiac function and/or methods for attenuating and/or prevention of cardiac remodeling in an individual's heart. The invention is also directed to methods of enhancing stem cell survival.
Heart disease is a major cause of death and disability. For example, ischemic heart disease causes approximately one third of all deaths in men and approximately one quarter of all deaths in women. This detriment reflects lack of effective therapies, targeted to the underlying biological processes within diseased and/or ischemic cardiomyocytes. In the heart, transient ischemia followed by reperfusion (ischemia/reperfusion, I/R) induces necrosis and apoptosis, leading to myocardial dysfunction. Preservation of cardiac function, attenuation and/or prevention of cardiac remodeling and reduction of the infracted area after I/R depends on critical adaptive responses. Accordingly, it would be advantageous to develop therapies for improving cardiac function, attenuating and/or preventing of cardiac remodeling, and/or reducing or preventing I/R injury in the heart.
Recent experimental results have suggested the possibility of regenerating damaged myocardium using stem cells, such as adult bone-marrow-derived mesenchymal stem cells (MSCs). However, a major limitation to the efficacy of stem cell therapy is the poor viability of the transplanted cells. Indeed, the functional improvement from stem cell therapy has been quite modest. A high level of engrafted cell death occurs within four days after grafting into injured hearts. Furthermore, regenerated tissue from stem cells does not survive repeated bouts of ischemia. Therefore, cytoprotection for one week is critical for improving the efficiency of cell therapy, and genetic modification of stem or progenitor cells may represent an important strategic advancement in regenerative medicine.
The present invention is directed to methods of improving cardiac function in an individual's heart. The methods comprise administering to the individual an effective amount of Heat-Shock Protein Hsp20 or an agent that increases the level of and/or the activity of Hsp20 to improve the function in the heart.
The present invention is also direct to methods of attenuating and/or preventing cardiac remodeling in an individual's heart. The methods comprise administering to the individual an effective amount of Heat-Shock Protein Hsp20 or an agent that increases the level of and/or the activity of Hsp20 to attenuate and/or prevent cardiac remodeling.
The present invention is further directed to methods of enhancing stem cell survival. The methods comprise modifying a stem cell with Hsp20 to enhance the survival of the stem cells.
The following Detailed Description may be more fully understood in view of the Figures, in which:
Heat-shock protein (Hsp) synthesis arises transiently as a tool to protect cellular homeostasis after exposure to heat and a wide spectrum of stressful and potentially deleterious stimuli. Hsps have been implicated as mediators of myocardial protection, particularly in experimental models of ischemia and reperfusion injury. Hsps have also been shown to render cardioprotection from stress-induced injury. The cardioprotective effects of Hsp 70 have been shown in isolated adult feline cardiomyocytes, rabbit hearts after adenovirus-mediated gene transfer, and transgenic (TG) mouse hearts after global or regional ischemia. Recently, protection during myocardial ischemia has also been shown for the small heat-shock proteins Hsp27 and αβ-crystallin; however, little is known about the role of another small Hsp, Hsp20, which shares considerable sequence homology with Hsp27 and αβ-crystallin, in cardioprotection against ischemic injury.
The small Hsp, Hsp20, was initially identified as a member of the crystallin Hsp family from skeletal muscle. It provides resistance to heat treatment in Chinese hamster ovary cells, and its expression levels are elevated on heat pretreatment of swine carotid artery, insulin exposure of skeletal muscle, and β-agonist stimulation of cardiomyocytes. Interestingly, Hsp20 has been shown to regulate vasodilation and suppress platelet aggregation. Moreover, the recent studies of the inventors have indicated that adenoviral gene transfer of Hsp20 in isolated cardiomyocytes improved contractile function and protected against β-agonist-mediated apoptosis.
Increasing evidence has shown that several Hsps have antiapoptotic roles, and overexpression of Hsp27, αβ-crystallin, Hsp32 (HO-1), and Hsp70 in the heart can attenuate I/R injury and improve cardiac function; however, the Hsp20 studied here is different from the other Hsps in its unique protein kinase A/protein kinase G phosphorylation site RRAS (phosphorylation of the Ser-16 site significantly increases the contractility in cardiomyocytes; unpublished data), regulatory activities of vasorelaxation, and platelet aggregation. Furtheimore, previous studies have shown that Hsp20 is translocated to actin filaments on stress, suggesting its cytoskeletal stabilizing function in cardiomyocytes. Taken together, these properties of Hsp20 suggest that it may benefit the ischemic heart at multiple levels.
To further define the functional significance of Hsp20 in vivo and (its potential protective mechanisms, the inventors generated a TG mouse model with cardiac-specific overexpression of Hsp20. The findings demonstrate that increased Hsp20 expression in the heart improves cardiac function. Accordingly, the inventors have determined methods for improving cardiac function in an individual's heart. The methods comprise administering to the individual an effective amount of Hsp20 or an agent that increases the level of and/or the activity of Hsp20 to improve cardiac function in the heart. The inventors have further determined that these methods may also be employed to improve the survival rate of not only the individual, but also the survival rate of a tissue and/or a cell. As used herein, “individual”, is intended to refer to an animal, including but not limited to humans, mammals, and rodents.
One skilled in the art will appreciate the various means in which the cardiac function of an individual's heart may be improved using the methods of the present invention. In one embodiment, cardiac function is improved by attenuating and/or reducing hypertrophy in the individual's heart. In another embodiment, cardiac function is improved by enhancing cardiac contractility in the individual's heart. In yet another embodiment, cardiac function is improved by attenuating and/or preventing the development or the time course of heart failure in an individual.
One skilled in the art will appreciate that the Hsp20 administered to the individual may comprise full-length Hsp20, a Hsp20 fragment or a combination thereof. As used herein, “Hsp20 fragment” refers to any fragment of the full-length Hsp20 that has the same activity as full-length Hsp20, or is a chemical or structural analogue of the full-length Hsp20. Agents that increase the levels of and/or the activity of Hsp20 may also be employed in the present invention to improve cardiac function. One skilled in the art will appreciate the various agents that increase the level and/or the activity of Hsp20, any of which may be employed herein. One skilled in the art will also appreciate the various effective amounts of Hsp20 or an agent that increases the level of and/or the activity of Hsp20 which may be administered to an individual for improving cardiac function and/or attenuating and/or preventing cardiac remodeling in the heart, any of which may be employed herein.
Hsp20 and/or an agent that increases the level of and/or the activity of Hsp20 may be administered by any methods known to one of ordinary skill in the art. In one embodiment, the administration of Hsp20 and/or an agent that increases the level of and/or the activity of Hsp20 comprises intravenous, intrademial, subcutaneous, oral, transdermal, transmucosal, or a combination thereof. In another embodiment, for nucleic acid delivery of Hsp20 and/or an agent that increases the level of and/or the activity of Hsp20 the administration comprises a viral vector, liposome, a non-viral delivery system or a combination thereof.
As illustrated in the Example, cardiac function may also be improved by reducing and/or preventing ischemia and reperfusion (I/R) injury in the individual's heart by administering Hsp20 or an agent that increases the level of and/or the activity of Hsp20. In one embodiment, cardiac function of the individual is improved after the I/R injury as compared with an individual not administered Hsp20 or an agent that increases the level of and/or the activity of Hsp20. In another embodiment, the infarction size is reduced after the I/R injury as compared to an individual not administered Hsp20 or an agent that increases the levels of and/or the activity of Hsp20. In yet another embodiment, there is less damage to structural components of the heart after I/R injury as compared to an individual not administered Hsp20 or an agent that increases the level of and/or the activity of Hsp20.
Recently, Hsp60 and αβ-crystallin have also been shown to be complexed with the proapoptotic protein Bax. Under stress conditions, these Hsps and Bax dissociate, whereupon Bax translocates to the mitochondria to participate in apoptosis, suggesting a role for Hsps upstream of caspase activation; however, Hsp70 is found to protect cells from death induced by enforced expression of caspase-3, suggesting that protection by Hsp70 may occur downstream of caspase activation. In addition, Hsp70 has been reported to interfere with apoptosis by a direct interaction with Apaf-1. Moreover, Hsp27 has been reported to directly bind to cytosolic cytochrome c and sequester it from Apaf-1. Thus, different Hsps may act via different mechanisms to prevent cell death. The data presented in the Example demonstrates that Hsp20 prevents I/R-induced apoptosis, possibly through the Bax-caspase pathway. While not wishing to be bound by theory, it is believed that apoptosis-related proteins, such as Bcl-2 and Bax, are stabilized after the I/R injury and carpase-3 activity is decreased after the I/R injury.
In addition to improving cardiac function, the inventors have deteimined methods of attenuating and/or preventing cardiac remodeling in an individual heart. The methods comprise administering to the individual an effective amount of Hsp20 or an agent that increases the levels of and/or the activity of Hsp20 to attenuate and/or prevent cardiac remodeling in the individual. Examples of cardiac remodeling include, but are not limited to, hypertrophy, heart failure progression or a combination thereof. In one embodiment, administration of Hsp 20 or an agent that increases the level of and/or the activity of Hsp20 improves the maintenance of muscle integrity, for example, during I/R injury, as evidenced in the Example by the presence of less damaged myofibril in the TG hearts after an I/R injury. While not wishing to be bound by theory, because there is strong evidence that cytoskeletal injury plays a crucial role in the pathogenesis of myocardial ischemic injury, it is plausible that Hsp20 translocates from the soluble fraction of cardiomyocytes to the insoluble fraction after I/R, leading to protection of the collapsed intermediate filament network or cytoskeletal protein damage.
These cardioprotective effects may also be associated with increased Hsp20 phosphorylation, consistent with previous reports on enhanced interaction of phosphorylated Hsp20 with actin and actinin, which further stabilizes the microfilaments. In addition, while not wishing to be bound by theory, the inventors believe that other mechanisms may include ASK1, P13K-Akt and protein phosphatase 1. Accordingly, the inventors' findings demonstrate that increased expression of Hsp20 and its accompanied phosphorylation protects the heart, leading to restoration of cardiac function and reduced infarction.
Cardiac function may be improved and/or cardiac remodeling may be attenuated and/or prevented in an individual that has or is at risk of developing a heart disorder. As used herein, “heart disorder” refers to a structural or functional abnormality of the heart that impairs its normal functioning. One skilled in the art will appreciate the various types of heart disorders. For example, heart disorder includes, but is not limited to, heart failure, ischemia, myocardial infarction, congestive heart failure or any combination of heart disorders. In one embodiment, the heart of the individual that has or is at risk of developing a heart disorder has full functional recovery after administration of Hsp20 or an agent that increases the level of and/or the activity of Hsp20. In another embodiment, the integrity of the muscle of the heart of the individual that has or is at risk of developing a heart disorder is maintained after administration of Hsp20 or an agent that increases the level of and/or the activity of Hsp20.
While not wishing to be bound by theory, the inventors also believe that stem cells, including but not limited to adult stem cells, embryonic stem cells, bone-marrow-derived stem cells, such as mesenchymal stem cells (MSCs), or combinations thereof, modified with Hsp20 will become more resistant to apoptosis in vitro and in vivo. When injected into infarcted hearts, stem cells modified with Hsp20 will limit ventricular remodeling and improve cardiac function, leading to more efficient healing compared with control stem cells, suggesting an ideal combination of cell and gene therapy for myocardial repair and regeneration. Clinical benefits of administering Hsp20 may also be employed with using adenoviral or adenovirusassociated viral gene transfer in vivo or engraft Hsp20-modified stem cells after myocardial infarction may further elucidate the potential clinical benefits of Hsp20
To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, the inventors generate a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold).
TG mice are generated by using mouse cardiac Hsp20 cDNA under the control of the α-myosin heavy chain promoter (αMHCp) (
Heart homogenates are analyzed by standard Western blotting to compare Hsp20, Hsp25, αβ-crystallin, Bcl-2, Bax, actin, and α-actin levels. Binding of the primary antibody is detected by peroxidaseconjugated secondary antibodies and enhanced chemiluminescence (Amersham), and bands are quantified with densitometry. Antibodies Hsp20 (Research Diagnostics Inc), Hsp25 (Affinity BioReagents Inc), αβ-crystallin (Calbiochem), Bcl-2(C-2), Bax (B-9), actin, and α-actin (Santa Cruz Biotechnology Inc).
Association of Hsp20 with Bax and Bcl-2 is studied using Dynabeads Protein G (Dynal Biotech) according to the manufacturer's instructions. Briefly, cardiac homogenate from wild-type (WT) heart is precleared by incubating with the beads for 1 hour at 4° C. to minimize nonspecific binding. Fresh beads are washed with 0.1 mol/L sodium phosphate buffer and then coated with anti-Hsp20 or anti-Bax antibody. The bound antibody is crosslinked to the beads using 20 mmol/L dimethyl pimelidate in a 0.2 mol/L triethanolamine solution. The precleared homogenate is then added to the crosslinked beads, and binding is mediated at 4° C. for 1 hour. Finally, the proteins are eluted off (using 0.1 mol/L citrate), and their identity is determined by immunoblotting.
The cellular and functional responses to I/R are assessed in mice by using an isolated perfused heart model as previously described. Male adult mice (12 to 14 weeks old) are anesthetized intraperitoneally (IP) with pentobarbital sodium (50 mg/kg). Hearts are rapidly excised and mounted on a Langendorff apparatus, perfused with Krebs-Henseleit buffer (noncirculating), and stabilized for 30 minutes, and then the hearts are subjected to 45 minutes of no-flow global ischemia and 2 hours of reperfusion. A fluid-filled balloon made of plastic film is inserted into the left ventricle via the mitral valve and inflated to yield a left ventricular end-diastolic pressure of 10 mm Hg. The balloon is attached via polyethylene tubing (PESO) to a pressure transducer connected to a Heart Performing Analyzer (Micro-Med), and continuous left ventricular pressure is measured. A bipolar electrode (NuMed) is inserted into the right atrium, and atrial pacing is performed at 400 bpm with a Grass S-5 stimulator.
Pacing is stopped during ischemia and restarted at reperfusion. After reperfusion, the hearts are weighed, frozen, and cut into 2-mm-thick slices parallel to the atrioventricular groove. The slices are thawed and stained by incubation in 1% triphenyl tetrazolium chloride (TTC) solution in phosphate buffer (Na2HPO4 88 mmol/L, NaH2Pa4 1.8 mmol/L) at 37° C. for 10 to 20 minutes as previously described. The area of infarction, risk zone, and nonrisk myocardium are determined by planimetry of each slice.
In addition to cardiac function, cardiac injury is assessed by measuring lactate dehydrogenase (LDH) release. Perfusion effluent is collected every 15 minutes of preischemia and also during reperfusion. Total LDH released from the heart is determined using a CytoTox 96 assay (Promega) and expressed as units per gram of wet heart weight. For terminal dUTP nick end-labeling (TUNEL) assays, hearts are removed from the apparatus after I/R, and the atrial tissue is dissected away. The ventricles are fixed in 10% buffered formalin and later embedded in paraffin according to standard procedures, and sections 3 μm thick are obtained to perform TUNEL assays using the ApopTag Plus Peroxidase In situ Apoptosis Detection Kit (Chemicon) according to the manufacturer's instructions. TUNEL-positive myocytes are determined by randomly counting 10 fields of the midventricular section and are expressed as a percentage of the total cardiomyocyte population.25
Heart samples are Dounce-homogenized in 2.5 vol of cell lysis buffer (RIPA, in μmol/L: 1 DTT and 50 PMSF) and centrifuged at 13 000 g for 10 minutes, and supernatant (100 μg) from each heart is used in a photometric enzyme immunoassay for the quantitative determination of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) of programmed cell death using the commercial assay kit Cell Death Detection ELISAPLUS (Roche). Results are normalized to the standard provided in the kit and expressed as folds of increase over control.
To detect caspase-3 activity, 200 μg of lysate from each heart is combined with fluorogenic caspase-3 substrate, diluted to 300 mg/L in caspase assay buffer (250 mmol/L PIPES, 50 mmol/L EDTA, 2.5% CHAPS, and 125 mmol/L DTT), and measured immediately in a fluorometer. Measurements are repeated every 10 minutes for 1 hour, the slope of fluorescent units per hour is calculated, and values are compared with known standards to determine enzymatic activity.
Mice weighing 25 to 30 g are anesthetized with sodium pentobarbital (90 mg/kg IP), intubated with PE 90 tubing, and ventilated by use of a mouse miniventilator (Harvard Apparatus) with room air supplemented with oxygen. The respiratory rate is 100 to 105 breaths per minute, and PO2, PCO2, blood pH, and body temperature are maintained within normal limits throughout the procedure as previously described.28 ECG electrodes are placed subcutaneously, and data are recorded with a Digi-Med Sinus Rhythm Analyzer (Micro-Med). A lateral thoracotomy (1.5-cm incision between the second and third ribs) is performed to provide exposure of the left anterior descending coronary artery (LAD), while avoiding rib and sternal resection, retraction, and rotation of the heart. Vascular bundles in the vicinity are coagulated by use of a microcoagulator (Medical Industries). An 8-0 nylon suture is placed around the LAD at 2 to 3 mm from the tip of the left auricle, and a piece of soft silicon tubing (0.64 mm ID, 1.19 mm OD) is placed over the artery. All mice are subjected to a 30-minute coronary occlusion by tightening and tying the suture. Ischemia is confirmed by visual observation (cyanosis) and continuous ECG monitoring. After 24 hours of reperfusion, the aorta is cannulated, and the heart is perfused with 1% TTC (37° C., 60 mm Hg) as previously described. The occluder, which had been left in place, is retied, and the heart is perfused with 5% phthalo blue. Hearts are transversely cut into 5 to 6 sections, with 1 section made at the site of the ligature. Infarct sizes are determined and expressed as a percentage of the region at risk.
Data are expressed as mean ±SEM. Statistical analysis is performed using a 2-tailed Student t test for unpaired observations and ANOVA for multiple comparisons. Values of P<0.05 are considered statistically significant.
The TG mice that carry the mouse cardiac Hsp20 cDNA under the control of the αMHC mouse promoter is generated (
Because acute expression of Hsp20 in rat cardiomyocytes is protective against apoptosis, it is examined whether increased in vivo expression of Hsp20 protects against postischemic injury. To exclude the involvement of inflammatory components on reperfusion, an isolated perfused heart preparation is used Body weights and heart weights of the mice used in these studies are similar between the TG and WT groups (Table). Hearts are stabilized for 30 minutes, and baseline function is measured. Hsp20 overexpression results in an increased contractile function under basal conditions (Table), consistent with the inventors' previous report using adenovirus-mediated Hsp20 gene transfer in cardiomyocytes. 20 Hearts are then subjected to 45 minutes of global ischemia and 2 hours of reperfusion. During reperfusion, the TG hearts exhibit significantly better functional recovery than the WT hearts (
To determine the degree of necrosis in these I/R hearts, the level of LDH released during the first hour of reperfusion after global ischemia is assessed. The total LDH is twice as high in WT hearts compared with TG hearts (
After 45 minutes of no-flow, global ischemia, followed by 120 minutes of reperfusion ex vivo, myocardial infarct size by histochemical and TTC staining is determined. Histological examination of post-ischemic/reperfused WT hearts reveal contraction bands (hypercontracted myofibers) and vacuolizations (
To elucidate the potential mechanism(s) of Hsp20 cardioprotection, Bcl-2 and Bax protein expression levels in hearts before or after I/R is assessed first, because alterations of Bcl-2 and Bax protein levels have been shown in isolated cardiomyocytes after hypoxia/reoxygenation and in hearts during I/R. Overexpression of Hsp20 does not alter the expression levels of either Bcl-2 or Bax (
Hsp20 has been shown previously to be phosphorylated under β-adrenergic stimulation of isolated cardiomyocytes. To determine whether the protective effects of Hsp20 against I/R injury in vivo may be associated with increases in its phosphorylation levels, the 2D gel electrophoresis is applied. The findings indicate that 16±1% of total Hsp20 is phosphorylated in TG hearts (
In the present study, the inventors discovered the role of Hsp20 on cardioprotection during myocardial ischemia. Interestingly, cardiac-selective overexpression of Hsp20 is associated with full functional recovery and decreased infarct size both ex vivo and in vivo on I/R injury. There are at least 2 mechanisms that underlie cardioprotection in Hsp20 TG mice from I/R injury. The first involves stabilization of the apoptosis-related proteins Bcl-2 and Bax by Hsp20 overexpression (
Importantly, Hsp20 complexed with the protein Bax (
Thus, the inventors have discovered that Hsp20 may provide a new potential therapeutic target for heart disease. Specifically, the findings demonstrate that increased Hsp20 expression in the heart improves cardiac function and/or attenuates and/or prevents cardiac remodeling.
The specific illustrations and embodiments described herein are exemplary only in nature and are not intended to be limiting of the invention defined by the claims. Further embodiments and examples will be apparent to one of ordinary skill in the art in view of this specification and are within the scope of the claimed invention.
This application claims priority under 35 U.S.C. §119 of U.S. Provisional Application Ser. No. 60/707,704 filed Aug. 12, 2005.
This invention was made, at least in part, with funds from the Federal Government, awarded through grant numbers HL-26057, HL-64018 and HL-52318. The U.S. Government therefore has certain acknowledged rights to the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60707704 | Aug 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11503706 | Aug 2006 | US |
| Child | 13272800 | US |
