Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Ischemia/reperfusion (I/R) injury results from damage to the myocardium following blood restoration after a prolonged period of myocardial ischemia, as seen clinically after therapeutic interventions such as thrombolysis and CABG. Oxidative stress, a state of imbalance between ROS generation and intracellular antioxidant molecules, has been proposed as an underlying cause in the pathogenesis of I/R injury. Manganese superoxide dismutase (MnSOD) is a mitochondrial antioxidant enzyme that scavenges these locally produced ROS and may have cardioprotective effects against I/R injury. Over‐accumulation of ROS may produce 4‐hydroxynonenal (4‐HNE), a biomarker of oxidative stress that can also modulate cellular signaling and cause apoptosis. In this study, we investigated the role of MnSOD in AIFm2‐induced apoptosis during I/R injury of the heart. Methods The generation of heart specific MnSOD knockout mice was done by backcrossing SOD2fl/fl mice for more than 10 generations to the C57Bl/6 genetic background. These mice were then crossed with C57Bl/6 mice expressing Cre recombinase under the control of the Myh6 promoter. Immunoblot was performed by using primary antibodies against AIFm2 and MnSOD with GADPH as a loading control. For IHC, heart sections of 5 micrometers were stained with H&E or Masson’s trichrome for light microscopy. For immunogold electron microscopy, left ventricular tissue was sectioned, fixed, and imaged by using a Hitachi TEM. Oxygen consumption and extracellular acidification rate of the neonatal cardiomyocytes were determined by using the XF‐24 analyzer. Results When compared to the MnSOD flox/flox mice sham group, the MnSOD flox/flox mice following 24 hours after reperfusion showed significant reduction in MnSOD mRNA expression and a 10‐fold decrease in MnSOD protein expression. IHC of the heart 24 hours post‐reperfusion shows decreased MnSOD compared to a normal heart. The mice lacking cardiac specific MnSOD (Myh6‐SOD2Delta) have a shorter life span and dilated cardiomyopathy, evidenced by the sarcomere disruption and mitochondrial damage shown by H&E and TEM. A typical TTE of Myh6‐SOD2Delta mice revealed significantly reduced EF and a 50% reduction in fractional shortening. Myh6‐SOD2Delta mice showed increased ECAR values and decreased OCR values, showing an energy metabolism shift from mitochondrial respiration to glycolysis. Western blot analysis showed significant increase in AIFm2 expression in ischemic human heart when compared to normal heart and in mice 24 hours post‐reperfusion compared to the sham group. Further analysis revealed AIFm2 localization in the mitochondria and its translocation mediated by 4‐HNE following reperfusion in I/R injury. Conclusion Our study shows that deficiency in cardiac specific MnSOD leads to I/R‐induced cardiac dysfunction, which is associated with AIFm2 mediated myocardial apoptosis. The study also demonstrates that upon reperfusion, 4‐HNE levels increase which then promotes AIFm2 translocation from the mitochondria to the nucleus. Support or Funding Information NIH‐HL141998; HL14199801S1, AA023610; AA025744; AA02574402S1; COBRE1P20GM121307; AAA‐FGAP‐5148Mitochondrial MnSOD regulation in SOD fl/fl mice following ischemia reperfusion injury. (A) qPCR was used to measure expression of MnSOD in heart apex following ischemia for 60 minutes followed by reperfusion for 24h. (B) Immunoblot analysis of MnSOD expression in mice heart following I/R injury. (C) Immunoblot analysis of MnSOD at different time intervals following I/R injury. GAPDH was used as a loading control. MnSOD expression was normalized to GAPDH staining (n = 3 animals per time point) and graphed as mean ± SD in arbitrary units in which the density of MnSOD in sham operated mice samples were set to 1. Mean ± SD, * P<0.05. (D) Immunohistochemistry for MnSOD in myocardial infarct (24h).Cardiac dysfunction exhibited in Myh6‐SOD2Δ mice. (A) Kaplan‐Meier analysis of survival probabilities for the SOD2fl (n=) vs the Myh6‐SOD2Δ (n =) animals (left) and the hearts of the 4‐month‐old Myh6‐SOD2Δ mice are significantly larger than the heart of the SOD2fl (right). (B) Histological observation after haematoxylin and eosin staining of hearts (LV) from Myh6‐SOD2Δ as compared to their SOD2fl littermates (c) Transmission electron micrograph of the ~4 months (LV) from Myh6‐SOD2Δ as compared to their SOD2fl littermates. Myh6‐SOD2Δ myocardium showed distorted (ID) intercalated disc with damaged mitochondria (Mi) (→). Mitochondria of Myh6‐SOD2Δ showed disorganization of cristae (C), vacuole formation. Bar denotes 500 nm.Cardiac function in vivo. A and B: representative M‐mode echocardiograms of SOD2 flox/flox and Myh6‐SOD2Δ mice. Myh6‐SOD2Δ mice exhibited a decrease in the LV Ejection fraction and fractional shortening percentage compared with control mice. Values are means SE; n 6 mice in SOD2 flox/flox and Myh6‐SOD2Δ.Effect of MnSOD and PMA on mitochondrial respiration and ROS generation. (A) oxygen consumption rate (OCR, pmol/min/10 5 cells) was determined with Seahorse analyzer for OXPHOS activity. (B) Extracellular acidification rate was measured by XF24 analyzer for glycolysis activity. (C) We used the XF24 Seahorse analyzer to determine the effect of PMA on respiration in pluripotent stem cell‐derived cardiomyocytes (iPSC‐CM). (D) Superoxide radical produced in cardiomyocyte were analyzed with a dihydroethidium fluorescence probe. All Data are representative of 4 experiments. * P<0.05 as compared to normal; * P<0.05 as compared to PMA stimulated cells.Effect if Ischemic reperfusion injury on AIfm2. (A) Protein levels of AIFm2 after I/R condition in human heart (B) Protein levels of AIFm2 in mice heart (C). Immunoblot analysis of AIFm2 levels in mitochondria (i), cytosol (ii) and nucleus (iii) of cardiac tissue of mice during reperfusion compared to sham group. IgG is an internal loading control for human heart (n=). Results are presented as mean ± SEM. *P<0.05; **P<0.00. (i) Representative Immunogold electron micrographs showing the localization of AIFm2 in cardiomyocytes. Electron dense beads indicate positive staining for AIFm2 protein (arrows).(D) Identification of AIFm2 as a mitochondrial resident target of ROS. (i)Left ventricular tissues from saline treated SOD2+/+ mice demonstrate strong labeling of AIFm2 in mitochondria (M) and low labeling but not in myofilaments (Myo) whereas mice undergoing I/R shows strong labeling in myofilaments (Myo), ii) H9C2 cells were transfected with GFP‐tagged AIFm2‐expressing lentivirus, and ~72 h later were co‐stained with MitoTracker to identify the mitochondria and DAPI to show the nucleus. Localization of AIFm2 was visualized by indirect immunofluorescence microscopy.