Administration of metformin rescues age-related vulnerability to ischemic insults through mitochondrial energy metabolism

Abstract

Ischemic heart disease (IHD) is the leading cause of death on a global scale. Despite significant advances in the reperfusion treatment of acute myocardial infarction, there is still a significant early mortality rate among the elderly, as angioplasty-achieved reperfusion can exacerbate myocardial damage, leading to severe ischemia/reperfusion (I/R) injury and induce fatal arrhythmias. Mitochondria are a key mediator of ischemic insults; a transient blockade of the electron transport chain (ETC) at complex I during reperfusion can reduce myocardial infarct caused by ischemic insults. The reversible, transient modulation of complex I during early reperfusion is limited by the available of clinically tractable agents. We employed the novel use of acute, high dose metformin to modulate complex I activity during early reperfusion to decrease cardiac injury in the high-risk aged heart. Young (3-6 months) and aged (22-24 months) male and female C57BL/6 J mice were subjected to in vivo regional ischemia for 45 min, followed by metformin (2 mM, i. v.) injection 5 min prior to reperfusion for 24 h. The cardiac functions were measured with echocardiography. A Seahorse XF24 Analyzer was used to ascertain mitochondrial function. Cardiomyocyte sarcomere shortening and calcium transients were measured using the IonOptix Calcium and Contractility System. The results demonstrated that administration of acute, high dose metformin at the onset of reperfusion significantly limited cardiac damage and rescued cardiac dysfunction caused by I/R in both young and aged mice. Importantly, metformin treatment improves contractile functions of isolated cardiomyocytes and maintains mitochondrial integrity under I/R stress conditions. Thus, acute metformin administration at the onset of reperfusion has potential as a mitochondrial-based therapeutic to mitigate reperfusion injury and reduce infarct size in the elderly heart attack patient who remains at greater mortality risk despite reperfusion alone.

Keywords: Aging; Ischemic insults; Metformin; Mitochondria integrity.

Figure 1.

Experimental Design. Male and female, young and aged C57BL/6J wild type mice underwent a left anterior descending artery (LAD) ligation for 45 minutes followed by 24 hours of reperfusion. 2 mmol/L of metformin were given via jugular vein at 5 min before reperfusion. Echo-cardiograms were performed before and after ischemia-reperfusion (I/R) surgery. After 24 hours of reperfusion, heart tissues were collected for TTC and Evans blue staining, cardiomyocyte function test, and mitochondria stress test.

Figure 2.

Metformin ameliorates ischemia-reperfusion (I/R) injury. (A) Echocardiography was performed to measure cardiac function. Metformin improved I/R caused left ventricular dysfunction, as shown by the decreased ejection fraction (EF; left) and fractional shortening (FS; right). Values are means ± SEM, n = 10 for each group. (B) Myocardial infarction was determined by TTC-Evans Blue staining. Ratio of the infarct size to the area at risk (AAR; left), and ratio of the AAR to the total myocardial area (right). Values are means ± SEM, n = 6 for each group.

Figure 3.

Metformin rescued the impaired cardiomyocyte contractility caused by I/R injury. (A) The contractile properties of isolated cardiomyocytes from young/aged, under sham operations and I/R stress conditions, with and without metformin treatment. Values are means ± SEM. Biological replicates n = 3 for each group. (B) The transient calcium signal response of the isolated cardiomyocytes from young/aged, under sham operations and I/R stress conditions, with and without metformin treatment. Values are means ± SEM. Biological replicates N = 3 for each group.

Figure 4.

Metformin maintains mitochondrial integrity in response to ischemic stress. (A) Mitochondrial stress assay examined oxygen consumption rate (OCR) in the isolated cardiomyocytes from young and aged hearts. (B) Quantitative analysis of mitochondrial function. Basal respiration and maximal respiration are shown in bar charts. Values are means ± SEM. Biological replicates N = 3 for each group.