The broad objective of this program is to perform preclinical experimentation on animal models of myocardial ischemia and subsequent chronic heart failure (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.? 1. Beta-2 adrenergic receptors in treatment of CHF.? The role of beta-adrenergic receptors (AR) subtype signaling in development of CHF is clearly important but poorly understood. It is widely accepted now that beta-1 AR activation is associated with development of CHF. Recent data indicates that stimulation of beta-1 AR is proapoptotic, thus, the use of beta-1 AR antagonists became a recommended therapy for HF. The possible role of beta-2 AR agonists remains debatable, however the consensus is that similarly to beta-1 AR, activation of beta-2 AR during CHF is harmful. Recent research in LCS using single myocytes indicated that beta-2 AR agonist, fenoterol, possesses a unique cardioprotective property, and, in fact is antiapoptotic. Capitalizing on this finding we studied the effects of chronic treatment with beta-2 AR agonist, fenoterol, and beta-1 AR blocker, metaprolol, in rats starting 2 weeks after ligation of a coronary artery. Our results indicated that both, beta-2 AR agonist and beta-1 AR blocker reduced the apoptosis in myocardium, but beta-2 AR agonist was superior to beta-2 AR blocker in attenuation of left ventricular remodeling and functional decline. Moreover, beta-2 agonist treatment arrested the infarct expansion, resulting in actual decrease of the relative infarct size. These two therapies affected different aspects of cardiac function: metaprolol improved systolic cardiac performance by increasing left ventricular elastance, while fenoterol achieved the same result by reducing the arterial elastance (after-load). Metaprolol did not improve diastolic function, while fenoterol normalized it. Combined treatment with beta-1 blocker and beta-2 agonist resulted in morphometric and functional improvements similar to a treatment with beta-2 agonist alone. In additional experiments we showed that a treatment with a combination of beta -1 blocker, mataprolol and beta-2 agonist, fenoterol is clearly superior than a single therapy with beta1-blocker in reduction of cardio myocyte apoptosis, attenuation of LV remodeling, and it actually improves the LV function. ? In a year long experiment we demonstrated a significant survival benefit of combined therapy with beta-2 agonist and beta-1 blocker comparing with any single therapy alone. Moreover, a therpeutic benefit of beta-2 agonist lasted only 2 months after coronary ligation, while the effect of combined therapy lasted six months.? 2. Erythropoietin reduses the myocardial ischemic damage.? Erythropoietin (EPO), natural stimulant of erythropoiesis, recently emerged as potential antiapoptotic factor with neuroprotective properties. We have demonstrated that the antiapoptotic effects of EPO also resulted in cardioprotection. In experiments in rats we showed that single systemic administration of recombinant human EPO (3000 IU/kg) immediately after ligation of a coronary artery results in 75% reduction of the size of myocardial infarction eight weeks later. During eight weeks after induction of myocardial infarction, left ventricular remodeling and function decline in EPO treated rats was significantly attenuated and statistically was not different from that in sham operated animals. Twenty four hours after ligation of coronary artery the amount of apoptotic myocytes measured in the myocardial risk area (area immediately adjacent to the infarct site) was reduced in half in the EPO treated rats in comparison to untreated animals. In new experiments we established that single intravenous dose of 3000 IU/kg is cardioprotective up to 12 hrs after coronary ligation, but it is losing its cardioprotective properties after 24 hrs. If animals are treated with EPO immediately after coronary ligation, the treatment dose can be reduced up to 150 IU/kg (usual, FDA approved dose for the treatment of anemia) without the lose of effectiveness, however, the therapeutic window in this case is also reduced to 4 hrs. ? In clinical situation associated with acute myocardial infarction, as well as in chronic myocardial ischemia, it is necessary to extend the antiapoptotic treatment over long time. However, the erythropoietic properties of EPO preclude its repeated use in non-anemic patients, because the excessive production of the red blood cells would have an adverse effect on circulation and would increase the danger of blood clot formation. The EPO incubated with cyanide (carbomilated EPO or CEPO) reportedly retains the antiapoptotic properties, but does not stimulate erythropoiesis. In experiments similar with those described above we showed the a single intravenous application of 3000 IU/kg of CEPO immediately after coronary ligation in rats results 24 hrs later in reduction of apoptosis in the myocardial area at risk, reduction of the size of myocardial infarction 8 weeks later, and reduction of left ventricular remodeling and functional decline in comparison with untreated animals. The magnitude of the effects achieved by CEPO treatment was comparable with those of EPO. These results suggest the possibility of clinical application of CEPO, in the situations when repeated, systematic antiapoptotic treatment is required. ? In additional experiments we showed that repeated daily EPO injection do not have any added benefits comparing with a single injection immediately after coronaty ligation? 3. Cardioprotective properties of intermittent food deprivation.? Intermittent Fasting (IF), i.e., the feeding regimen when ad lib food is available only every other day, had been reported to increase the life span and to reduces the incidence of age-associated diseases including cancer, diabetes and kidney disease. Neuroprotective effects of IF against ischemic injury of the brain have also been reported. We investigated the cardioprotective effect of IF in rats using the model of experimental myocardial infarction induced by a permanent coronary ligation. After three months of IF or regular, daily, feeding ad libitum (AF), 5-mo old rats were subjected to coronary ligation or sham operation. A subset of rats was sacrificed 24 hours later to measure the size of myocardial infarction (MI) and the extent of apoptosis. The remainder of the animals were continued for 10 weeks on the same food regimen, during which time the progression of left ventricular (LV) remodeling was assessed by serial echocardiography. After ten weeks LV function was measured by pressure-volume loops analyses, and hearts were evaluated histologically. We showed that 24 hrs following coronary ligation the ischemic area of myocardium, i.e., the area at risk (AAR), was similar in both groups, but in IF rats MI size, expressed as a percent of AAR, was more than 2-fold smaller, apoptosis in the AAR was reduced by more than 4-fold, and the inflammatory response was significantly reduced. At 10-wks, late LV remodeling and MI expansion occurred in AF rats but not in IF rats, and LV pump function and arterio-ventricular coupling were superior in IF vs AF rats. The myocyte hypertrophy in areas remote from the MI was also absent in IF rats. The results indicate that Intermittent Food Deprivation protects the heart from ischemic injury in part, at least, via an enhanced anti-apoptotic mechanism.
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