The broad objective of this program is to perform preclinical experimentation on animal models of myocardial ischemia and subsequent CHF to elucidate the mechanisms of its development and to evaluate the potential of different therapeutic modalities to limit the extent of myocardial damage and to prevent or attenuate the development of CHF. I. Beta-2 adrenergic receptors in treatment of CHF. The role of beta-adrenergic receptor (AR) subtype signaling in the development of CHF is clearly important. It is widely accepted now that beta-1AR activation is associated with development of CHF. Recent data indicate that stimulation of beta-1AR is proapoptotic, thus, the use of beta-1AR antagonists became a recommended therapy for CHF. The possible role of beta-2AR agonists remains debatable; however, the consensus is that similar to beta-1AR, activation of beta-2AR during CHF is harmful. Recent research in LCS using single myocytes indicated that the beta-2AR agonist, fenoterol, possesses a unique cardioprotective property, and, in fact, is antiapoptotic. In several previously published studies we have reported the positive therapeutic effect of chronic treatment with a combination of the beta-2AR agonist, fenoterol and beta-1AR blocker, metaprolol in the rat model of post MI CHF. Proposed treatment was equal to a currently standard therapy for CHF (combination of beta-1AR blocker and ACE-inhibitor) with respect to mortality and exceeded the standard therapy with respect to cardiac remodeling. II. Fenoterol exists as four stereoisomers. Combination of two of enantiomers, RR and SS, are a racemic mixture which represents an actual drug that had been used clinically for treatment of asthma and for our experiments cited above. In order to refine the therapeutic effectiveness of fenoterol in the CHF model, researchers in LCS in collaboration with BDD, NIA (Dr. Wainer) have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. The hypothesis had been tested that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). The R,R-isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R-isomers. Importantly, while R,R-fenoterol and R,R-methoxyfenoterol preferentially activate Gs signaling, their S,R-isomers were able to activate both Gs and Gi proteins as evidenced by the robust pertussis toxin-sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on Gs, Gi2 and Gi3 proteins. The inefficient Gi signaling with the R,R-isomers is not caused by the inability of the R,R-isomers to trigger the PKA-mediated phosphorylation of the beta2AR, since the R,R-isomers also markedly increased phosphorylation of the receptor at serine262 by PKA. It also has been reported that in experiments in isolated rat cardiomyocytes the R-enantiomer of fenoterol exhibits more potent effects on cardiomyocyte contractility and better Gs protein selectivity than other stereoisomers, and had been proposed as a promising new drug. We used the rat experimental model of post-MI CHF to compare the efficacy of the R- and L-enantiomers of fenoterol with its racemic mixture, whose efficacy had been demonstrated in our previous experiments. Racemic mixture of fenoterol (F) and one of its enantiomers, right (RF) or left (LF), were given to rats in drinking water as described above, starting 2 weeks after induction of MI. The LV remodeling and function were assessed during the next 5 months via serial Echo. All the effects of fenoterol described above, attenuation of LV remodeling and functional decline and arrest of MI expansion, were replicated in the present experiment, including the loss of effectiveness after 3 months as a monotherapy. Both groups treated with right or left enantiomers of fenoterol were indistinguishable from the untreated group. Thus, the therapeutic potential of the R-enantiomer of fenoterol reported on the basis of experiments on single cardiomyocyte level was not confirmed in our whole animal experiments. III. Non-erythropoietic derivatives of Erythropoietin. A pyroglutamate helix B surface peptide (HBSP), a small peptide, had been engineered on the basis of erythropoietin molecule to effect tissue protection without erythropoiesis. In experiments in the rat model of MI induced by a permanent ligation of a coronary artery, we demonstrated strong cardioprotective effects of HBSP. Taking advantage of the fact that multiple injections of HBSP do not result in the elevation of hematocrit, we tested the idea that repeated injections of HBSP can attenuate the post-MI LV remodeling. The experiment was conducted using our usual experimental design: Two weeks after coronary ligation early remodeling was evaluated by Echo and rats divided into experimental and control groups of similar average MI size. HBSP (60 ug/kg) or saline (1 mL/kg) was injected twice a week for 6 weeks. Reduction of EF and expansion of MI size observed in control rats were arrested in HBSP-treated animals. Since relatively short-term treatment with HBSP showed beneficial effects on post-MI LV remodeling in the rat model of permanent coronary occlusion, we initiated a year-long experiment injecting post-MI rats twice a week with HBSP. Progression of LV remodeling, LV function and MI expansion is assessed via serial Echo. The primary outcome of treatment is mortality. The secondary outcome is a cardiac remodeling. Survival of HBSP-treated rats was significantly improved in comparison with untreated animals. For the 10 months, starting two weeks after left coronary artery ligation, rats received the i.p. injections of HBSP (60ug/kg; n=33) or saline (n=33) 2 times per week. Compared to control, saline-treated rats, HBSP treatment significantly reduced mortality (35% vs. 65%; p<0.05). Repeated Echo demonstrated remarkable attenuation of LV chamber dilatation (EDV: 41% vs. 86%; ESV: 44% vs. 135%; p<0.05), LV functional deterioration (EF: -4% vs. -63%; p<0.05) and MI expansion (3% vs. 38%; p<0.05) in HPSP-treated compared to untreated rats. Blood hematocrit level measured monthly was not affected by HBSP treatment. An invasive hemodynamic assessment at the end of 10-mo treatment showed better LV systolic function (PRSW: 635 vs. 404; p<0.05) and arterio-ventricular coupling (Ea/Ees: 1.20.2 vs. 2.70.7; p<0.05) in HBSP-treated compared to saline-treated rats. Histological analysis revealed less apoptosis (0.60.1% vs. 0.90.1%; p<0.05), myocardial fibrosis (2.60.2% vs. 4.10.5%; p<0.05), and myocyte hypertrophy (cell diameter: 311um vs. 371um; p<0.05) in non-infarcted myocardium among HBSP-treated rats compared to saline-treated rats. Results suggest that HBSP could be considered as a safe alternative to EPO for further clinical testing in the patients with chronic heart failure. A further pathological investigation of myocardial tissue samples revealed that HBSP treatment significantly increased LV posterior wall thickness and myocyte cell density, reduced the myocyte cell hypertrophy. A classification of myocytes based on the cell diameter showed that there are significantly higher portion (43%) of small myocytes (< 20um) in LV posterior wall in HBSP treated group than in saline-treated group (23%). It indicates a possibility of myocyte regeneration by HBSP treatment in the failing heart.
