Patent Application
20110166066
The present invention relates generally to the field of biochemistry and medicine relates to the polypeptide catestatin and its variants, and particularly to any and all portions the wild-type catestatin (“WT-CST”) and naturally occurring variants that act as a cardio-depressant agent and/or are able to treat hypertensive individuals. Variation of Gly364 to Ser within the catestatin domain is designated as Gly364Ser (“Gly364Ser”), and variation of Pro370 to Leu within the catestatin domain is designated as Pro370Leu (“Pro370Leu”).
Throughout this application various publications are referenced, many referenced by numbers in parenthesis. Full citations for these publications are provided later in this application. All of the disclosures of these publications are hereby incorporated by reference, in their entirety, in this application. Citation of these documents is not intended as an admission that any of the material is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the Applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
Chromogranin A (CgA), an index member of the chromogranin/secretogranin protein family, is a secretory pro-protein (1-3) that gives rise to several peptides of biological importance including the dysglycemic hormone pancreastatin (4), vasodilator vasostatin (5), and the catecholamine release inhibitory peptide catestatin (CST: human CgA352-372, bovine CgA344-364) (6) (7) (8) (9) (10, 11). CgA is co-stored with catecholamines and its co-release documents exocytosis as the mechanism of physiologic catecholamine release in humans. The basal plasma CgA level not only correlates with sympathetic tone, but also shows highest heritability. Plasma CgA levels increase in patients with essential hypertension. The serum levels of CgA are also elevated in patients with heart failure. CST is decreased not only in patients with essential hypertension but also in normotensive subjects with a family history of hypertension (12). People with such a family history demonstrate increased epinephrine secretion in addition to diminished CST (12), implicating an inhibitory effect of catestatin on chromaffin cells in vivo. Consistent with human studies, genetic ablation of Chga gene results in high blood pressure in mice (13). High blood pressure in Chga-/- mice can be rescued either by pre-treatment with CST or introduction of the human gene in the Chga-/- background utilizing bacterial artificial chromosome (BAC) transgenic technology. Furthermore, low CST levels predict augmented adrenergic responses to stressors, suggesting that a reduction in CST may increase the risk of hypertension.
To understand human genetic variation at CHGA and its CST fragment, O'Connor's group systematically searched for polymorphism at the CHGA locus, and reported 3 naturally occurring amino acid substitution variants in the catestatin region (9). Although two of the catestatin variants (Pro370Leu & Arg374Gln) were reported to be relatively rare (minor allele frequencies 0.3-0.6%), one variant, Gly364Ser (S352SMKLSFRARGYS364FRGPGPQL372: SEQ ID NO. 1; position in the mature CgA protein) showed an allele frequency of ˜3-4%. These CST variants displayed differential potencies toward inhibition of nicotinic cholinergic agonist-evoked catecholamine secretion from sympathochromaffin cells in vitro, in the following rank order of potency Pro370Leu>wild-type (WT)>Gly364Ser>Arg374Gln (11). In vivo, human carriers of the 364Ser allele had profound alterations in autonomic activity, in both the parasympathetic and sympathetic branches, and may be protected against the future development of hypertension, especially in males (14).
The working heart and the arterial system are such close functional complements that the analysis of their dynamic interaction represents an obligatory step in the integral understanding of cardiovascular homeostasis both under normal and abnormal (hypertension) conditions. Nothing is known regarding the influence of catestatin on the pumping heart. Therefore, we looked at the effects of catestatin and its naturally occurring variants on the inotropic and lusitropic functions using the Langendorff perfused rat heart. The results provide for the first time an insight into the role of catestatin as a cardiac modulator, adding new aspects to the anti-hypertensive activity of this peptide.
CST is the most potent endogenous nicotinic cholinergic antagonist. The structure-function relationship of this peptide has been established and human studies indicate that CST is low, not only in hypertensive individuals, but also in individuals with family history positive for hypertension. Pre-treatment with CST lowers the high blood pressure in CgA null mice to normal levels. It appears from the recent human data that the Gly364Ser variant of catestatin prevents humans from developing hypertension. The present invention demonstrates that catestatin acts on the heart and dramatically reduces the cardiac contractility. These effects are mediated via the nitric oxide/cyclic GMP pathway.
The present invention relates generally to the polypeptide catestatin and its variants, and particularly to any and all portions the wild-type catestatin (“WT-Cts”) and naturally occurring variants (“Pro370Leu” and “Gly364Ser”) that act as a cardio-depressant agent and/or are able to treat hypertensive individuals. The amino acid sequences of these polypeptides are as follow:
The present invention also includes the nucleic acid sequences of the DNA and RNA molecules, which ultimately encode for these polypeptides. The invention teaches a method to decrease cardiac contractility (by >60%) using an endogenous peptide catestatin and its naturally occurring variants. CST and its naturally occurring human variants (Gly364Ser or Pro370Leu) act as myocardial modulators in the mammalian to almost abolish isoproterenol or endothelin-induced changes in cardiac parameters. The invention can be used as a cardio-depressant agent and to treat hypertensive individuals.
CST is decreased not only in patients with essential hypertension but also in normotensive subjects with a family history of hypertension. Genetic ablation of Chga gene resulted in high blood pressure in mice and pre-treatment with catestatin rescued mice from such high blood pressure indicating a role of CST in the development of hypertension.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a term is provided in the singular, the inventor also contemplates the plural of that term. The nomenclature used herein and the procedures described below are those well known and commonly employed in the art. All of the disclosures of these publications are hereby incorporated by reference, in their entirety, in this application.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. Various changes and departures may be made to the present invention without departing from the spirit and scope thereof. Accordingly, it is not intended that the invention be limited to that specifically described in the specification or as illustrated in the drawings, but only as set forth in the claims.
Chromogranin A (CgA), an index member of the chromogranin/secretogranin protein family, is a secretory pro-protein (1, 2, 15) that is endo-proteolytically processed to give rise to several peptides of biological importance including the dysglycemic hormone pancreastatin (4), vasodilator vasostatin (5), and the catecholamine release inhibitory peptide CST (6, 8, 10, 11, 16). CgA is co-stored with catecholamines and its co-release documents exocytosis as the mechanism of physiologic catecholamine release in humans (17). CST and pancreastatin have been postulated as important counter-regulatory hormones in “zero steady-state error” homeostasis (i.e. the perfect equilibrium generated by the balance between two counter regulatory hormones (18)), a role now extended also to vasostatin 1 (VS-1) in cardiac biology ((15), and references therein). The importance of CgA in cardiovascular homeostasis in man is documented by its increased plasma levels in various diseases, such as neuroendocrine tumors (18) and chronic heart failure (19) and its colocalization with BNP and over-expression in human dilated and hypertrophic cardiomyopathy (20). Basal plasma levels of CgA correlate with sympathetic tone (21) showing a high heritability (22). Plasma level of Cts peptide (˜1.5 nM) decreases in patients with essential hypertension, the complex chronic disorder with a poorly understood pathogenesis, and also in normotensive subjects with a family history of hypertension and increased epinephrine secretion (12); this implicates that CST is an inhibitor of chromaffin cell catecholamine secretion in vivo. Genetic ablation of the CgA gene results in high blood pressure in mice which can be rescued either by pre-treatment with CST peptide or by the introduction of the human CgA gene in the CgA null background (13). Accordingly, decreased CST levels may predict augmented adrenergic responses to stressors and increased risk of hypertension.
To understand human genetic variation at the level of the CgA gene, O'Connor's group at UCSD systematically searched for polymorphisms at the CHGA locus, and reported 3 naturally occurring amino acid substitution variants within the region of CST (9). Although two of the Cts variants (Pro370Leu & Arg374Gln) were reported to be relatively rare (minor allele frequencies 0.3-0.6%, respectively), one variant, Gly364Ser (S352SMKLSFRARGYS364FRGPGPQL372; position in the mature CgA protein) showed an allele frequency of ˜3-4%. These variants displayed differential potencies toward inhibition of nicotinic cholinergic agonist-evoked catecholamine secretion from sympathochromaffin cells in vitro with the following rank order of potency Pro370Leu>wildtype (WT)>Gly364Ser>Arg374Gln (11). In vivo, human carriers of the 364Ser allele had profound alterations in autonomic activity, in both the parasympathetic and sympathetic branches, and may be protected against the future development of hypertension, especially evident in males (14). On the basis of this remarkable vasoreactivity of Cts, we reasoned that its human variants could also act as cardiotropic agents exerting differential effects on the heart.
The working heart and the arterial system are such close functional complements that the analysis of their dynamic interaction represents an obligatory step in the integral understanding of cardiovascular homeostasis both under normal and abnormal (hypertension) conditions. There have been no studies investigating the direct action of CST on isolated rat heart preparations, which being independent from extrinsic neuronal and endocrine influences is an ideal model for analyzing the direct cardiac effects of a substance. Using the Langendorff perfused rat heart as a mammalian cardiac paradigm, we show that CST and its naturally occurring human variants directly influence both inotropic and lusitropic functions. In addition to signaling analyses addressed to determine the possible mechanisms of action of WT-CST, we demonstrate that CST inhibits the positive inotropic actions of both isoproterenol (ISO) and endothelin-1 (ET-1). The results provide a novel insight into the role of CST as an endocrine/paracrine cardiac modulator and inhibitor of β-adrenergic and ET-1 actions on the heart, adding new aspects on the structure-function relationship of CST variants and their anti-hypertensive potential.
Animals—Male Wistar rats (Morini, Bologna, Italy S.P.A.) weighing 180-250 g were housed (three per cage) in a ventilated cage rack system under standard conditions. Animals had food and water access ad libitum. The investigation conforms to the Guide for the Care and Use of Laboratory Animals, according to NIH Publication No. 85-23, revised 1996.
Drugs—WT-Cts, the pro370leu variant (P370L-Cts) and gly364ser variant of Cts (G364S-Cts) were synthesized by the solid-phase method, using FMOC protection chemistry (8). Peptides were purified to >95% homogeneity by preparative reverse-phase HPLC (RP-HPLC) on C-18 silica colyhhtrgumns. Authenticity and purity of the peptides was further verified by analytical chromatography (RP-HPLC) and by electrospray-ionization or MALDI mass spectrometry. Isoproterenol hydrochloride (ISO), ET-1, the NO scavenger PTIO [2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide], inhibitors of either nitric oxide synthase (NG-monomethyl-L-arginine, L-NMMA) and (N5-(1-Imino-3-butenyl)-L-ornithine, L-VNIO) or guanylate cyclase (ODQ) or protein kinase G (KT5823) were purchased from Sigma Chemical Company (St. Louis, Mo., USA). All drug-containing solutions were freshly prepared before experimentation.
Isolated heart preparation—Rats were anesthetized with ethyl carbamate (2 g/Kg rat, i.p.), the hearts rapidly excised and then transferred in ice-cold buffered Krebs-Henseleit solution (KHs). As previously described (23), the aorta was immediately cannulated with a glass cannula and connected to Langendorff apparatus to start perfusion at constant flow-rate (12 ml/min). Briefly, the apex of the left ventricle (LV) was pierced to avoid fluid accumulation. A water-filled latex balloon, connected to a pressure transducer (BLPR, WRI, Inc. USA), was inserted through mitral valve into the LV allowing isovolumic contractions and continuous mechanical parameters recording. Another pressure transducer located just above the aorta recorded coronary pressure (CP). The perfusion solution consisted of a modified non re-circulating KHs containing (in mM) NaCl 113, KCl 4.7, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.2, glucose 11, mannitol 1.1, Na-pyruvate 5 (pH 7.4; 37° C.; 95% O2-5% CO2). Hemodynamic parameters were assessed using a PowerLab data acquisition system and analysed using Chart software (both purchased by ADInstruments, Basile, Italy).
Basal conditions—Heart performance was evaluated from the LV pressure (LVP, in mmHg) which is an index of contractile activity, the rate-pressure product (RPP: HR×LVP, in 104 mmHg×beats/min) which is an index of cardiac work (24), the maximal value of the first derivative of LVP (25) (mmHg/sec) which is an index of the maximal rate of LV contraction, the time to Peak Tension of isometric twitch (Ttp) which is an assessment of inotropism. Lusitropism was determined by calculating the maximal rate of LVP decline [−(LVdP/dT)max] (mmHg/sec), the half time relaxation (HTR) (sec), which is the time required for tension to fall from the peak to 50% and T/−t ratio obtained by +(LVdP/dT)max/−(LVdP/dT)max (26). Mean CP was calculated by averaging values obtained during several cardiac cycles (23).
Catestatin stimulated preparations—Repetitive exposure of each heart to a single concentration (33 nM) of WT-Cts revealed absence of desensitization (data not shown). Thus, concentration-response curves were generated by perfusing cardiac preparations with KHs supplemented with increasing concentrations of WT-Cts (from 11 to 200 nM) for 10 min.
Adrenergic and cholinergic receptors involvement—To obtain information on the involvement of β1/β2, α-adrenergic receptors (ARs) and cholinergic receptors (AchR) on the inotropic and lusitropic effects induced by WT-Cts, cardiac preparations, stabilized for 20 min with KHs, were perfused with Nadolol (10 nM) or Phentolamine (100 nM) or Atropine (10 nM) for 10 min and then washed-out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of Cts WT (110 nM) plus Nadolol (10 nM) or Phentolamine (100 nM) or Atropine (10 nM) for an additional 10 min.
Involvement of inhibitory G-protein (Gi/o)—To evaluate the involvement of inhibitory G-proteins in the cardiac action of WT-Cts, hearts were preincubated for 60 min with KHs enriched with pertussis toxin (PTx: 0.01 nM) and then exposed for 10 min to WT-Cts (110 nM). PTx catalyzes ADP-ribosylation of Gi/o alpha-subunit, uncoupling Gi-membrane receptor interaction.
Cardiac signaling—Hearts were perfused with or without 110 nM WT-Cts as before. At the end of experiment hearts were snap frozen under liquid nitrogen and homogenized with 1 ml of ice cold 0.2 M sucrose, Tris maleate (pH 7.0) buffer supplemented with 2 mM EDTA, pH 8.0, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 0.1 mM 3-isobutyl-1-methylxanthine. SR membrane fractions were isolated from cytosolic proteins as described before (27). Protein content was determined by Bradford assay (BIO-RAD) and 100 μg of cytosolic protein was subjected to SDS-PAGE immunoblot analysis for phosphorylated-ERK, total ERK (P-ERK and T-ERK; Santa Cruz, Calif.). Two hundred μg cytosolic protein was used for immunoblot detection of phosphorylated-protein kinase B (Akt)-Ser473 (P-Akt), total-Akt (T-Akt), phosphorylated GSK-3 (P-GSK-3-Ser9) and actin (Cell signaling Technology, MA). For total phospholamban (T-PLN) and phosphorylated PLN (P-PLN-Ser16) level assessment, 10 μg of SR membranes were subjected to electrophoresis and the immunoblots were probed with anti-mouse PLN antibody (Affinity Bioreagents, CO) and anti-P-PLN-Ser16 (Badrilla, UK).
NO-pathway inhibitor stimulated preparations—Hearts were stabilized for 20 min with KHs, perfused with 110 nM WT-Cts for 10 min and then the peptide was washed-out with KHs. After returning to control conditions, each heart was perfused with KHs containing either the NO scavenger PTIO, the non-specific NOS inhibitor L-NMMA, a soluble guanylate cyclase inhibitor (ODQ) or a protein kinase G blocker (KT5823). Subsequently, the hearts were exposed to the specific signaling inhibitor plus 110 nM of WT-Cts.
Isoproterenol stimulated preparations—Cardiac preparations were stabilized for 20 min with KHs and then perfused with 5 nM ISO for 10 min. ISO was washed-out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of WT-Cts (110 nM), P370L-Cts (33 nM) or G364S-Cts (200 nM) with 5 nM ISO for a further 10 min.
To further describe the antagonistic action of either WT-Cts (33 nM), P370L-Cts (110 nM) or G364S-Cts (200 nM) against ISO-dependent stimulation, dose-response curves were generated by perfusing the heart preparations with KHs enriched with increasing concentrations of ISO (0.1 nM to 1 μM) alone. These curves were then compared to those obtained by exposing other cardiac preparations to the same perfusion medium containing increasing concentrations of ISO (0.1 nM to 1 μM) plus a single concentration of either WT-Cts (33 nM) or P370L-Cts (110 nM) or G364S-Cts (200 nM).
ET-1 stimulated preparations—Hearts, stabilized for 20 min with KHs, were perfused for 10 min with ET-1 (1 nM) and then washed-out with KHs. After returning to control conditions, each heart was perfused with KHs containing a single concentration of either WT-Cts (33 nM), P370L-Cts (110 nM) or G364S-Cts (200 nM) plus 1 nM ET-1 for a further 10 min.
Statistics—Data are expressed as the mean±SEM. Curve fitting was accomplished in the program Kaleidagraph (Synergy Software, Reading, Pa.). Peptide EC50 and IC50 values were interpolated as the concentration that achieved 50% stimulation and inhibition, respectively. For analysis of phosphorylated proteins, levels were quantified using Bio-Rad QuantifyOne and volumes of phosphorylated proteins were divided by the levels of non-phosphorylated proteins to calculate fold activation. Stimulation of protein activity was expressed as fold increase over vehicle-treated control. The means and S.E.M. were calculated for each treatment. Multiple comparisons were made using either one-way ANOVA followed by Bonferroni's post-hoc test or two-way ANOVA. Statistical significance was concluded at p<0.05. Statistics were computed with the program InStat (GraphPad Software, Inc., San Diego, Calif.).
Basal conditions—After 20 minutes of stabilization, the following basal recordings were measured: LVP=89±3 mmHg, heart rate=280±7 beats/min, RPP=2.5±0.1 104 mmHg beats/min, CP=63±3 mmHg, +(LVdP/dT)max=2492±129 (mmHg/sec), T/−t=0.08±0.01 (sec), −(LVdP/dT)max=1663±70 (mmHg/sec), HTR=0.05±0.01 (sec) and T/−t or +(LVdP/dT)max/−LVdP/dT)max=1.49±1.84 (mmHg/sec). Endurance and stability of the preparation, analyzed by measuring performance variables every 10 min, showed that the heart preparation is stable for up to 180 min on the perfusion apparatus.
Inotropic and lusitropic actions of WT-Cts—To test whether WT-Cts alters basal cardiac parameters, heart preparations were exposed to increasing concentrations of WT-Cts to generate concentration-response curves. Exposure of single repeated doses of WT-Cts (33 nM) showed absence of desensitization (data not shown). WT-Cts effect on LVP reached its maximum at 5 min after administration, remaining stable for 15 min and then gradually decreased with time. Accordingly, cardiac parameters were measured at 10 min.
WT-Cts significantly increased FIR at 33 nM, reaching a maximum at 165 nM (EC50˜12.5 nM) (
CST signaling to cardiac modulation—The negative inotropic and lusitropic effects of WT-CST were abolished by inhibition of β1/β2-ARs (by nadolol), reduced by α-ARs antagonist (phentolamine) or remained unaffected by inhibition of AChR (by atropine) (
It is well established that Gi/o proteins are involved in the negative inotropism exerted by several cardiodepressive agents, including CgA-derived VS-1[24]. To evaluate Gi/o proteins involvement in CST-dependent negative inotropism, hearts were perfused with the specific inhibitor PTx, in presence of Wt-CST (110 nM). PTx abolished Wt-CST-mediated negative inotropism (LVP and +(LVdP/dT)max), and lusitropism −(LVdP/dT)max), indicating Gi involvement in CST signaling (
The NOS-NO-cGMP-PKG cascade plays a key role in the control of contractile performance in mammals (28). Accordingly, we tested NO-cGMP-PKG involvement in WT-CST-dependent cardiotropism by perfusing heart preparations with either PTIO (10 μM), L-NMMA (10 μM), ODQ (10 μM) or KT5823 (0.1 μM) in presence of CST. Antagonist concentrations were selected from preliminary dose-response curves that determined the minimum antagonist concentration that did not alter basal cardiac function. WT-CST (110 nM)-induced reduction of negative inotropism (i.e, +(LVdP/dT)max), and lusitropism (i.e., −(LVdP/dT)max) was abolished by NO removal by PTIO, NOS inhibition by L-NMMA and sGC blockade with ODQ. PKG inhibition by KT5823 failed to affect CST-induced changes in cardiac performance (
The mechanism of action of Cts is summarized in Table 1.
Intracellular Ca2+ coordinates cardiac contraction and relaxation. PLN, i.e. the 52-aminoacid transmembrane SR phosphoprotein regulating Ca2+ ATPase SERCA2a, in its dephosphorylated state inhibits Ca2+ pump activity. PLN phosphorylation alters the PLN-SERCA2a interaction, relieving Ca2+-pump inhibition and enhancing relaxation rates and contractility. Phosphorylation of Protein Kinase B (Akt), GSK-3 (glycogen synthase kinase-3) and ERK1/2 (extracellular signal regulated kinase), proteins also modulate cardiac function. Therefore, we checked whether WT-Cts signals through these proteins. WT-Cts inhibited phosphorylation of PLN at the PKA specific site, Ser16 (
Inotropic and lusitropic actions of naturally occurring human CST variants—G364S-CST caused a significant increase in RPP even at 33 nM (
P370L-CST induced a negative inotropism from 110 to 200 nM, the maximum decrease in LVP and RPP being at concentration of 200 nM (
Inotropic and lusitropic effects of the three CST variants are summarized in Table 2.
CST modulation of isoproterenol-induced cardiac changes—Cardiac preparations exposed to the β-adrenergic agonist ISO (5 nM) revealed positive inotropic and lusitropic responses that were associated with vasodilation. These modifications were indicated by an increase in LVP, RPP, +(LVdP/dT)max, −(LVdP/dT)max, HTR, T/−t and a decrease in the CP, (
To characterize the inhibitory action of either WT-CST (11, 33 or 110 nM), G364S-CST (200 nM) or P370L-CST (110 nM) toward ISO-dependent stimulation of cardiac function, heart preparations were perfused with KHs containing increasing concentrations of ISO (0.1 nM to 1 μM) either alone or in combination with one of the three CST variants.
ISO alone increased LVP significantly from at concentrations ranging from 5 nM to 1 μM (
CST blockade of ET-1 stimulated cardiac functions—To verify Cts's ability to counteract the ET-1-mediated inotropic and coronary effects, hearts were perfused with KHs containing ET-1 alone or in combination with one of the Cts variants.
Administration of ET-1 alone induced a dose-dependent biphasic effect on contractility. At a concentration of 1 nM ET-1 increased LVP and +(LVdP/dT)max, whilst at higher doses (10 nM) CST decreased both parameters, thus inducing a negative inotropic effect. CST peptides differentially affected the ET-1 induced inotropic effects. In fact, positive inotropism was blocked by WT-CST (33 nM), P370L-CST (110 nM) and G364S-CST (200 nM) (
In conclusion, since the heart and the arterial system interact as a closed-loop control system, the former being a target organ of prolonged and excessive adrenosympathogenic activity, such as hypertension with consequent hypertrophy and ischemic cardiomyopathy, it is of relevance that the recently discovered anti-hypertensive modulatory action of CST now appears associated with a direct powerful counter-excitatory cardiac action. This cardiotropism of CST may be inherently important for normal heart function, but also suggests a unique inhibitory role of the peptide under abnormal cardiac conditions characterized by adrenosympathogenic over-activation. Until now, blockade of the β-adrenergic receptors is one of the most effective pharmacologic interventions in hypertensive patients. Future studies of administration of CST to hypertensive animal models will determine whether the peptide has also any therapeutic potential for the treatment of hypertensive cardiomyopathy.
Animals. Chga knockout (KO) mice and their wild-type (WT) littermates were generated as described (13). All animals were housed on a 12 hr light dark cycle and fed a standard rodent chow. The Animal Care and Use Committee of University of California at San Diego approved all protocols for animal use and euthanasia in accordance with National Institute of Health guidelines. Both WT and KO mice (5-6 month old) in this study were from mixed (129 SvJ and C57BL/6) genetic background.
Conscious mice. Measurement of BP and HR in immobilization stress-induced telemetered mice: BP & HR were measured by telemetry as described previously (13). The experiments were initiated 10 days after the surgery. Immobilization stress was initiated by placing mice in restrainer (Braintree Scientific Inc, Braintree, Mass.) at 10:00 AM and kept for 2 hrs for continuous recording of BP and HR by telemetry followed by recovery from stress in home cages for 2 hrs. CST was injected (40 μg/g bw ip) 30 minutes before stress induction to explore the role of CST in stress response.
Catecholamine assay following immobilization stress: Mice were anesthetized by inhalation of “Isoflurane, USP” and blood was collected from the left ventricle in potassium EDTA tubes. Isoflurane anesthesia was found to be best for measurement of plasma catecholamines with minimal stress-induced variation. Plasma catecholamine was measured by HPLC connected to an electrochemical detector (Waters 600E Multisolvent Delivery system and Waters 2465 Electrochemical Detector, MA) in a group of restraint mice (non-telemetered). Separation was performed on an Atlantis dC18 column (2.1×150 mm, 3 μm) from Waters. The mobile phase (0.15 ml/min) consisted of phosphate-citrate buffer and 2.2% N, N-Dimethylacetamide and acetonitrile at 95:5 (V/V). An internal standard 3,4-Dihydroxybenzylamine (DHBA, 1 ng) and an antioxidant sodium meta-bisulfite (final concentrations of 0.125 mM) were added to 0.25 ml of plasma. Following the addition of 15 mg of alumina (Aluminium oxide, Activity Grade: Super I, Type WA-4, Sigma-Aldrich, PA), the pH of the solution was raised to pH 8.6 by adding Tris buffer (0.96 M Tris, 50 mM EDTA, pH 8.69). Following a 30 min incubation and centrifugation (5000 rpm, 5 min), the supernatant was discarded and the beads were washed with water. The catecholamine was eluted with 80 ml of 0.1 N HCl supplemented with 0.1 mM sodium meta-bisulfite. The data were analyzed using Empower software. Catecholamine levels were normalized with the recovery of internal standard.
Unconscious mice. Surgical procedures for hemodynamic measurements: Hemodynamic evaluation in both WT and KO mice was performed under general anesthesia [ketamine (100 mg/kg) and xylazine (2.5 mg/kg)] while connected to a ventilator. LV pressure was monitored by Millar micromanometer catheter of size 1.4 French (0.46 mm; Millar Instruments, Houston, Tex.); it was inserted into the LV where phasic and mean pressure was continuously monitored (Gould, Cleveland, Ohio). A venous catheter (MicroRenathane tube MRE-033; Braintree Laboratories, Braintree, Mass.) is implanted on right femoral vein for injection of peptide and other reagents. All physiologic signals were acquired by WINDAQ (Dataq Instruments, Akron, Ohio) and analyzed by BP-Analysis software (developed for own laboratory).
Assessment of baroreflex sensitivity (BRS): BRS (in milliseconds per mmHg) was assessed by both high-pressure [phenylephrine (PE) bolus] and low-pressure [sodium nitroprusside (SNP) bolus] stimuli in unconscious mice. Although conscious animals provide optimal physiological data, a host of undesirable signals are triggered by physical activities, and sleep cycle alterations in behavioral state. Therefore, we have chosen to conduct the experiments in unconscious mice to avoid some of the pitfalls encountered in awake animals. The absolute changes of HR in response to changes in SBP induced by PE or SNP were subjected to linear regression analysis to determine BRS. To test the effect of CST on BRS in KO mice, CST was injected (40 μg/g bw iv) 30 min before the injection of PE or SNP through the catheter implanted into the femoral vein.
BRS to high-pressure stimulus. High pressure BRS was evaluated by recording diminution of HR in response to PE-induced hypertension. Changes in SBP and HR were recorded continuously for 0.5 min after a bolus injection of PE (0.005 μg/g bw iv). This dose was selected from a preliminary dose-response study (0.001-0.05 μg/g bw iv).
BRS to low-pressure stimulus. This was evaluated by recording increments in HR in response to SNP-induced fall in BP. Changes in SBP and HR were recorded continuously for 0.5 min after a bolus injection of SNP (0.05 μg/g bw iv). This dose was selected from a preliminary dose-response study (0.01-0.1 μg/g bw iv).
Statistical analysis. Statistical analyses were done as described in Example 1.
SBP. SBP increased dramatically 10 min after immobilization stress in both WT (by 25.5 mmHg) and KO (by 18.6 mmHg) mice (
In WT mice, CST had no effect on basal SBP; CST attenuated (by 7.1 mmHg) stress-induced increments in SBP (
DBP. KO mice displayed significantly higher basal DBP compared to WT mice (98.2±2.9 vs. 88.2±2.7 mmHg; p<0.025) (
In both WT and KO mice, CST had no significant effect either on basal or stress-induced increments in DBP as compared to saline-treated group (
HR. Resting HR was significantly higher in KO mice compared to WT mice (
In WT mice, CST had no effect on the basal and stress-induced increase in HR (
II. Biochemistry: Catecholamines in conscious mice. As reported previously (13), KO mice showed higher basal plasma norepinephrine (by 85%) and epinephrine (by 42%) (FIG. 13A), that returned to WT level upon CST replacement (
Effects of Intravenous PE or SNP for 0.5 Min.
SBP. In KO mice, anesthesia completely abolished heightened SBP (
HR. In WT mice, PE (0.005 μg/g bw) caused significant decrease in HR (by 26 bpm) (
Baroreflex slope. Time-dependent cumulative effects of PE (0.005 μg/g bw) and SNP (0.05 μg/g bw iv) are shown in
Genetic variation at the human CHGA locus has profound consequences for control of BP, and the Chga null mouse displayed profound hypertension and increased catecholamine secretion. We sought to understand such changes, and probed autonomic function in this model by physiological, biochemical, and pharmacological means. Our results suggest global disruption of autonomic function in this model, both parasympathetic and sympathetic. Defects in baroreceptor function may underlie the cardiovascular instability and exaggerated sympathetic outflow observed in KO animals. At least some of these abnormalities can be “rescued” by administration of the CHGA catecholamine release-inhibitory fragment CST. Since the CgA and CST mechanisms are altered in human hypertension, our results in this experimental animal model may provide insight into the pathogenesis of this common disorder.
Cardiovascular performance is controlled by the autonomic nervous system (ANS). Beat-to-beat fluctuation in the heart rate (HR) is a balanced consequence of ANS tone to the heart, both sympathetic (increasing HR) and parasympathetic (decreasing HR). A decrease in heart rate variability (HRV) is longitudinally predictive of increased cardiovascular morbidity and mortality. Thus, we examined whether both short- and long-term HRV concerning temporal and spectral features recorded from the sedated mice to identify perturbations of the autonomic system in the chromogranin A knockout (CgA-KO) mice, which has earlier been reported to have echocardiographic abnormalities and prone to hypertension. Use of CST ameliorates both systolic and diastolic BP elevations (13). Since ANS status can be reliably monitored by analyzing time and frequency-domain characteristics of HRV, we sought to use the technology to evaluate functional alterations in ANS activity of CgA-KO mice.
Animals. Six Wild-type and seven CgA-KO mice were used in this study (see example 1 for the details).
Mice were sedated by an intraperitoneal injection of a 1 μl solution per gm body weight, containing Ketamine (100 mg/ml)+Xylazine (20 mg/ml) and Acepromazine (10 mg/ml). Mice were gently strapped onto the surgical table in a fixed supine position with silk tape. Body temperature was continuously monitored on an analog thermometer, model 44TA (YSI, Ohio) using a rectal probe, and maintained between 36.5 and 38.5° C. throughout the experiment using a controlled heating box. The analog output signal fed into the chart recorder (AD Instruments, Colorado) and suitably amplified. The adequacy of sedation was regularly verified by absence of vibrissae twitching and withdrawal reflex to a noxious toe-pinch and was supplemented intraperitoneally as needed. Animals breathed ambient air spontaneously throughout the experimental run. Investigational agent, a nicotinic cholinergic receptor antagonist (8), CST was dissolved in distilled water and injected intraperitoneally at a volume of 70 μl from a 1 mM working solution.
EKG electrodes were attached to all four limbs (incl. reference electrode) via 30G stainless steel hypodermic needles (Popper & Sons, New York) so as to record standard bipolar Einthoven derivation (lead I, II and III) EKG signals. The EKG signals were bandpass-filtered between 0.1 and 3 kHz with a notch filter set at 60 Hz, and sampled at 2 kHz. The voltage output fed into a data acquisition system (PowerLab-8, AD Instruments) for eventual off-line measurements and analysis by chart and scope (Labchart 6) software. Heart rate and rhythm were also monitored continuously on an oscilloscope (Tektronix 5113, Oregon) throughout the experiment.
EKG traces were edited using the Chart® software package. High frequency noise from the EKG was removed using a 45 Hz low-pass filter to enable detection of the R-waves, whenever appropriate. A threshold electrical potential value was set for each trace and the post-threshold maximum was taken as the R-wave. Each trace was visually inspected for any undetected R-waves and these were manually inserted, while incorrectly detected R-waves or non-normal beats such as extrasystoles were deleted. Time and frequency domain analysis of HRV was performed using fast Fourier-transformation with a Hanning window. Spectral power was calculated on 50% overlapped blocks on sequences of 1024 points (beats). Time-domain parameters recorded or calculated included: mean R-R interval (in ms) [i.e., NN interval, i.e., normal-to-normal intervals]; standard deviation of all normal R-R interval (SDNN, in ms), reflecting total autonomic variability; square root mean of squared differences between adjacent normal R-R intervals (RMSSD), an index for short-term variations in inter-beat intervals, primarily of parasympathetic nature; and percentage of normal consecutive inter-beat intervals differing by >7 ms (NN7%), reflecting cardiac parasympathetic tone, were calculated directly from the sequence of interval times. NN7 is a murine-adjusted value based on PNN50 determined for humans (32). The mean HR was calculated as the mean sequence of reciprocals of the interval times. For non-linear measurements on the return map of R-R intervals (Lorenz plot), the images acquired by the chart software were imported into Image J (National Institutes of Health, Bethesda) for evaluating point dispersion of S1 and S2 (see below). The width and length of the dispersion of data in Lorenz plots reflects the level of short- and long-term beat-to-beat variability, respectively. The different frequency domain measures of HRV were computed using cut-off frequencies for power in the very low frequency (VLF), low-frequency range (LF) and high-frequency range (HF) based on human studies multiplied by a factor of ten for HR adjustment (the approximate ratio between murine and human HR) (33). The area under the curve was calculated for the very-low-frequency (VLF: up to 0.4 Hz), low-frequency (LF: 0.4 to 1.5 Hz), and high frequency (HF: 1.5 to 4.0 Hz) bands, as previously defined in the mouse species (34). Total power (variance of normal R-R intervals over temporal segment) was defined as DC 4 Hz. Spectral variability at LF and HF bandwidths was also normalized to the total spectral area. Five-minute epochs of heart rate as a function of time were used for power spectrum (power) analysis. In the present paper we report the area of the LF band in milliseconds squared, the area of the HF band in milliseconds squared and the total power between 0.0033 and 4.0 Hz (total power) in milliseconds squared. Variables not normally distributed were naturally log-transformed. In order to investigate if HF portion of the HRV is coupled to respiration, we recorded an electromyogram (EMGdi) in two mice, using fine Teflon-coated silver wire with bare tip hooked onto the costal diaphragm following a laparotomy, and output signal suitably amplified and stored for analysis.
Mice with atrial fibrillation, excessive ectopic beats or technically inadequate recordings were excluded.
Statistical analyses. Statistical analyses were carried out as described in Example 1.
Frequency domain, and effects of catestatin. At baseline, KO mice showed higher heart rate compared with the wild-type group (427±31 vs. 330±20 beats.min−1, p=0.014) (
Time domain. As shown in
Lorenz analysis. The reductions in time-domain parameters were further supported by the non-linear measurements of R-R intervals, that showed parallel decrease in the index of short-term variability (S1: axis perpendicular to the line of identity) and that of S2 axis (along the line of identity) that reflects short-to-long term variability, primarily of sympathetic nature. These values are plotted in
Fourier transform to the frequency domain. Fourier transform power spectral analysis of HRV in WT and KO mice in the resting state showed periodic components of HRV in 2 distinct peaks mostly centered around 2.4-3.2 Hz (HF), considered to index cardio-vagal activity and below 0.2 Hz (VLF) in most instances, with occasional peak at 1 Hz. Spectral peak positions were not visibly altered under the influence of CST treatment. At baseline, KO mice had reduced VLF power. Treatment with CST did not have much influence on the HRV VLF parameters (Control: 2.156±0.939; CST: 2.468±0.69 [ln ms2]) in WT mice. However, in KO mice, CST increased HRV in the VLF band (Control: 1.296±0.521; Catestatin: 2.899±1.05 [ln ms2], p=0.05).
Mice deficient in endogenous peptide, CST have increased heart rate, decreased HRV and altered autonomic heart rate modulation in comparison to WT control mice. The widening of HRV value by CST suggests its potential utility in the cardiovascular area. The findings of improved HRV by CST in mice seem to corroborate our findings in modification of human autonomic activity by CST (14). At baseline, standard deviation of N-N intervals, total power, VLF and LF power of HRV were lower. Spectral analysis of HR signals in the frequency domain revealed that HR variability of KO mice were decreased in the VLF band indicating general modifications of neurohumoral This observation mends well with the findings in human subjects having hypertension associated with insulin-resistance syndrome, and hypertension has been a distinct feature in the current knock-out mice model (13) and in human genetic hypertension involving CgA (12). It also strengthens the arguments concerning increased cardiovascular risk by impairments in total and VLF power of heart rate in KO mice and the important role CGA peptide, catestatin plays in the stability of HRV.
The LF component corresponds mainly to sympathetic modulation and partially to parasympathetic modulation, whereas the HF component represents only parasympathetic modulation that was also assessed by short-term indices of Lorentz' plot (S1). The quotient of (S1/S2) was lower in the KO group in the basal stage, suggesting dampened sympatho-vagal balance and favoring parasympathetic modulation. Frequency power also showed diminished LF responses at baseline in KO group, which shows that direct sympathetic modulation of cardiac activity decreases. The finding of reduced LF power in KO mice runs contrary to usual expectation and to the proposed view of enhanced sympathetic drive triggering elevation of systolic and diastolic pressure. However, it is known that enhanced sympathetic drive may not necessarily reflect in altered LF or any other spectral power. This is even more evident in case of CHF, whereby enhanced sympathetic drive does not translate into increased LF power, as reported by Guzzetti et al. (35). Since KO mice are diseased or prone to compromise of their cardiovascular system like CHF patient, and thereby may fail to reveal its enhanced LF properties. The findings in KO mice are very similar to the unhealthy human subjects, in that the total power is reduced and the largest RR intervals are lost, as in CHF.
CST peptide and antiserum. Please see Example 2 for the details.
Cornea Neovascularisation Assay. Pellets containing 500 ng Catestatin were implanted in C57/BL/6J mice (age of 8 weeks) as described (36). On postoperative day 7 mice received an intravenous injection of 500 μg BS1 lectin conjugated to FITC (Vector Laboratories). After euthanasia, enucleated eyes were fixed in 1% paraformaldehyde, corneas dissected and examined by fluorescence microscopy.
Mouse hind-limb ischemia model. Male C57BL/6 wild-type mice at the age of 12 months were subjected to unilateral hind-limb surgery. Briefly, the left femoral artery was exposed, ligated with 5-0 silk ligatures, and excised. Mice were injected with saline or 10 μg catestatin into thigh and calf muscles after operation and every other day for weeks 1 and 2 and 2 times per week for weeks 3 and 4. All further measurements and analyses were performed by investigators blinded to the treatment of the animals.
Blood flow measurement. Blood flow measurements were performed using a laser Doppler perfusion image (LDPI) analyzer (Moor Instruments, USA) as described. To minimize data variables attributable to ambient light and temperature animals were kept on a heating plate at 37° C. before measurement and blood perfusion is expressed as the LDPI index representing the ratio of left (=operated, ischemic leg) versus right (=not-operated, not-ischemic leg) limb blood flow. A ratio of 1 before operation indicates equal blood perfusion of both legs, whereas after femoral artery excision this ratio drops to 0.26, indicating severe attenuation of leg blood supply in the operated leg.
Alkaline phosphatase staining and Immunohistochemistry. For tissue staining, mice were sacrificed and ischemic limb tissues were retrieved after 4 weeks. Specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with graded ethanol series and embedded in paraffin. Alternatively, fresh tissue was embedded in OCT compound (TISSUE-TEK®, Sakura Finetek) and snap-frozen in liquid nitrogen. Tissues were sliced into 5-μm sections. Vascular endothelial cells were identified by alkaline phosphatase staining and for assessment of artery/arteriole density sections were stained with a mouse monoclonal alpha-smooth muscle actin antibody (Pharmingen) as described (36). Adductor muscle samples of each leg were divided into 2 parts and capillaries (alkaline phosphatase positive) and arteries (alpha-smooth muscle actin positive) were counted in five sections of each part and are expressed as capillary and arteriole density per mm2.
Statistical Analysis. Statistical analyses were done as described in Example 1.
To evaluate if catestatin induces angiogenesis also in-vivo a cornea neovascularization assay was performed. Catestatin induced growth of arteries out of the limbus artery leading to a capillary network around the pellet. Also vessels reaching the limbus vein and presumably representing veins were observed (
Intra-muscular injections of catestatin peptide into the ischemic limb improved blood perfusion as measured by LDPI. Perfusion ratio of control vs. ischemic limb improved in both groups, however 4 wks after induction of ischemia LDPI ratio was 0.76±0.04 in saline injected mice and 0.94±0.03 in catestatin treated animals (n=10 mice each group, P<0.01 saline vs. cat;
Since the initial observation that the peptide catestatin, which is derived from CgA, inhibits catecholamine secretion from chromaffin cells (6) several further biological effects of this molecule have been reported. Catestatin was shown to induce histamine release from mast cells (37) and increases this factor substantially in the circulation (38). Furthermore, catestatin exerts anti-microbial activity in-vitro (39) and in cutaneous wounds where it was up-regulated in fibroblasts (40). In Langendorff-perfused rat hearts and the avascular frog heart catestatin decreased cardiac contractility induced by isoproterenol and endothelin-1 (41, 42). This mechanism was dependent on beta2-adrenergic receptors as well as Gi/o protein-nitric oxide-cGMP signaling mechanisms and these findings might indicate that catestatin is able to protect the heart against excessive sympatho-chromaffin over-activation.
We recently observed that catestatin induces directed migration of human blood monocytes and therefore might act as an inflammatory cytokine (43). It also has been shown before that the precursor of catestatin, CgA is up-regulated by hypoxia in the brain (44). As inflammation and hypoxia usually are accompanied or followed by increased blood vessel generation we tested if catestatin exerts effects on ECs in-vitro and angiogenesis in-vivo. We found that catestatin exerts several direct effects on ECs including EC migration and proliferation. Blockade by a neutralizing antibody indicates specificity of observed effects. Mechanistically, catestatin induced activation of MAPK signaling pathway in ECs and inhibition of ERK inhibited observed effects indicating that activation of MAPK in ECs mediates catestatin induced effects like reported before for other angiogenic cytokines like VEGF or bFGF. Inhibition of EC chemotaxis by pertussis toxin indicates involvement of G-proteins in this observed effect.
Beside its effects on ECs catestatin also showed effects on EPCs like EPC chemotaxis and incorporation of these cells into vascular structures in-vitro. These findings indicate that this peptide also might induce post-natal vasculogenesis. It has to be shown if catestatin also induces vasculogenesis in-vivo in animal models.
In-vivo catestatin induced angiogenesis in the mouse cornea neovascularization assay and angiogenesis and arteriogenesis in the hind-limb ischemia model. Serial measurements of blood perfusion indicates that catestatin indeed was able to increase perfusion to levels before ligation of the femoral artery yielding a significant better value compared to saline injected animals. These observations indicate that catestatin indeed is a novel angiogenic cytokine that exerts direct effects on ECs.
Notably, it has been shown that another peptide derived from CgA, vasostatin, inhibited angiogensis induced by vascular endothelial growth factor (VEGF). Vasostatin blocked VEGF-induced MAPK activation in ECs and inhibited angiogenesis exerted by this factor in-vivo in the matrigel assay. The main difference between our findings on angiogenesis induced by catestatin and the inhibitory effect of vasostatin on VEGF-induced blood vessel formation is the concentration of the respective peptide. Whereas vasostatin-induced effects were found at a concentration of 330 nM, we observed a maximal function of catestatin at 1 nM, i.e. two magnitudes of concentration lower. At higher concentrations of catestatin (100 nM) angiogenic effects were still observed in-vitro (EC migration, proliferation and in-vitro matrigel assay) indicating that CgA-derived peptides do not block angiogenesis un-selectively at higher concentrations. The biological consequences of these, in the initial view contradictory findings-inhibitory versus stimulatory effects on angiogenesis by two different peptides derived from the same molecule CgA- have to be determined. Nevertheless it is conceivable that dependent on local processing of CgA, on local concentrations of peptides and the responsiveness of the target cell, CgA-derived peptides might act as angiogenic or anti-angiogenic molecules.
It is also notable that secretoneurin, a peptide derived from another member of the chromogranin/secretogranin family of acidic storage proteins of vesicles in neuro-endocrine cells, secretogranin II, induces angiogenesis (36). Therefore, catestatin and secretoneurin might be promising novel candidates in the therapy of diseases like limb or myocardial ischemia.
CST raises blood glucose level in both CgA-knockout (CgA-KO) and wild type (WT) mice but suppress glucose level in high fat fed insulin resistant hyperglycemic mice. It possible that CST reduces blood glucose level in all diabetic models and diabetic patients. These disparate results can be reconciled by assigning a critical role of eNOS and nitric oxide (NO) on hepatic glucose metabolism. A number of publications in literature demonstrated that NO inhibits glucose production and glycogen synthesis but stimulates glycogenolysis. CST, on the other hand, reduces p-AMPK and p-eNOS signals (see our results), elevates gluconeogenesis but, may reduce glycogenolysis (effects of low NO level and AMPK activity levels).
Chronic intake of nicotine through smoking keeps catecholamine pathway active and blood pressure and pre- as well as postprandial glucose levels higher than non-smokers. Thus, repeated assault on the homeostatic mechanism with higher blood glucose level and blood pressure end up producing increased risk of metabolic disorders in the smokers. If the smokers are diabetic, their risk in cardiovascular dysfunction is further increased. In this set up, treatment with CST, which antagonizes nicotine induced cardiovascular dysfunction, will provide double benefit by reducing glucose level in diabetic patients. We believe that CST will do that by inhibition of glycogenolysis and enhancement of insulin-stimulated glycogen production in both liver and muscle.
Adult WT (C57BL/6) and CgA-KO mice with mixed genetic background (129svJ×C57BL/6) were used in this study. Both WT and KO mice were generated from the original founder carrying mixed genotype (50% 129svJ: 50% C57BL/6) and were maintained by brother/sister mating. Hypertensive CGA-KO mice will serve as a model for hyper insulin sensitivity. WT mice were fed 60% high fat diet (HFD) for 20 weeks. Body weight and blood glucose levels were monitored and glucose and insulin tolerance testa were performed to determine status of insulin resistance. After 20 weeks of HFD feeding mice were injected IP with catestatin (40 μg/gm body weight) twice daily for 7 days. On the 8th day, 12 hours fasted mice were subjected to glucose tolerance tests to determine their insulin sensitivity. Tissues of WT and CgA-KO mice are analyzed AMPK and eNOS signalling by immunoblotting.
As shown in
Treatment of insulin-resistant HFD mice with CST alone reduced blood glucose level by 15-20% as seen by glucose tolerance tests (
We have discovered that CST is a new player in the physiological regulation of glucose production and insulin clearance, the two key factors whose dysfunctions lead to metabolic syndrome. Our results suggest that CST could be a practical therapy against insulin resistance and diabetes, particularly for diabetic patients who are also smokers and have hypertension. Antinicotinic CST will reduce blood pressure and glucose levels at the same time in those patients.
The present invention claims benefit of priority to U.S. provisional patent application Ser. No. 61/126,913, filed on May 8, 2008, the entire contents of which are incorporated herein by reference in its entirety.
This invention was made with Government support under Grant No. DA011311, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2009/002865 | 5/8/2009 | WO | 00 | 2/1/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61126913 | May 2008 | US |
