Effect of Ischemic Post-Conditioning and Nicotinamide Mononucleotide on Autophagy activation and cardioprotection following Myocardial Ischemia-Reperfusion Injury in Old Male Rats

Abstract

Cardiovascular diseases are one of the leading causes of death in aging. Due to the ineffectiveness of single therapies in aging, in this study, the effect of nicotinamide mononucleotide (NMN), as an autophagy activator, and ischemic post-conditioning (IPostC; as a cardioprotective intervention) at the beginning of reperfusion on cardioprotection, cardiac function, mitochondrial function, and expression of autophagy genes and miR-499 were evaluated following ischemia-reperfusion injury in the aged rats. Materials and Methods: Sixty old male rats were randomly divided into 5 groups of 12/each; including Sham, control (IR), NMN, IPostC, and combination therapy (NMN+IPostC). First, the NMN and IPostC+NMN groups were treated with intraperitoneal injections of NMN at a dose of 100 mg/kg every other day for 28 days. Then, under deep anesthesia, rats’ cardiac tissue was excised and mounted on the Langdorff setup and regional ischemia was induced for 30 min by ligation of the left anterior descending coronary artery (LAD), and reperfusion was induced for 60 min by reopening the artery. In the IPostC and combination groups, at the very beginning of reperfusion the IPostC protocol was applied by six cycles of 10 s intermittent ischemia/reperfusion. Hemodynamic factors were recorded during ischemia-reperfusion. Infarct size, mitochondrial reactive oxygen species (ROS), mitochondrial membrane depolarization, lactate dehydrogenase (LDH) and histopathological changes in the necrotic region of the cardiac tissue were evaluated. In addition, the expression of autophagy genes including LC3, Mfn1, p62, FOXO1, and miR-499 expression was tested in the necrotic region by real-time PCR. Results: The combination of IPostC and NMN therapies resulted in improved heart function, a significant reduction in LDH and infarct size compared to the control (IR-received) group (P<0.001). The mitochondrial ROS levels significantly decreased in all treated groups compared to the control group (P<0.001). Furthermore, mitochondrial membrane potential (P<0.001) in the control group significantly decreased in the control group as compared to the Shan group, and treatment with IPostC (P<0.01) and NMN (P<0.001) prevented the depolarization of the mitochondrial membrane. The effects of combination therapy in reducing mitochondrial ROS, improving mitochondrial membrane potential were greater than alone treatments. In addition, IPostC and NMN increased the expression of autophagy genes and miR-499 expression both alone and in combination (P<0.001). Combination therapy also improved histopathological changes following ischemia-reperfusion injury compared with the control group.