Divergent Effects of Cyclophilin-D Inhibition on the Female Rat Heart: Acute Versus Chronic Post-Myocardial Infarction

Background/Aims: The mitochondrial permeability transition pore opening plays a critical role in the pathogenesis of myocardial infarction. Inhibition of cyclophilin-D (CyP-D), a key regulator of the mitochondrial permeability transition pore, has been shown to exert cardioprotective effects against ischemia-reperfusion injury on various animal models, mostly in males. However, failure of recent clinical trials requires a detailed elucidation of the cardioprotective efficacy of CyP-D inhibition. The aim of this study was to examine whether cardioprotective effects of sanglifehrin A, a potent inhibitor of CyP-D, on post-infarcted hearts depends on reperfusion. Methods: Acute or chronic myocardial infarction was induced by coronary artery ligation with/without subsequent reperfusion for 2 and 28 days in female Sprague-Dawley rats. Cardiac function was estimated by echocardiography. Oxygen consumption rates, ROS production, permeability transition pore opening, protein carbonylation and respiratory supercomplexes were analyzed in isolated cardiac mitochondria. Results: Sanglifehrin A significantly improved cardiac function of reperfused hearts at 2 days but failed to protect after 28 days. No protection was observed in non-reperfused post-infarcted hearts. The respiratory control index of mitochondria was significantly reduced in reperfused infarcted hearts at 2-days with no effect at 28-days post-infarction on reperfused and non-reperfused hearts. Likewise, only a minor increase in reactive oxygen species production was observed at 2-days in non-reperfused post-infarcted hearts. Conclusion: This study demonstrates that CyP-D inhibition exerts cardioprotective effects in reperfused but not in non-reperfused infarcted hearts of female rats, and the effects are observed only during acute post-infarction injury.

Abstract 071: Cytokine-angiotensin II Interplay in Cyclophilin D-mediated Vascular Oxidative Stress and Hypertension

Vascular inflammation and oxidative stress interact in a feed-forward fashion to promote vascular disease and hypertension. We hypothesized that angiotensin II and inflammatory cytokines encountered in hypertension co-operatively induce superoxide (O 2 • - ) production by mitochondrial complex I and that efforts to reduce complex I O 2 • - will reduce hypertension. Treatment of human aortic endothelial cells in culture with angiotensin II (10 nM), IL17A (10 nM) and TNFα (1 nM), factors known to contribute to the hypertensive milieu, co-operatively induced mitochondrial O 2 • - from 340 to 958 pmol/mg protein as measured by HPLC and MitoSOX. This response was abolished by the complex I inhibitor rotenone. We further tested a potential role of Cyclophilin D (CypD), the redox sensitive regulatory subunit of the mitochondrial transition pore in complex I O 2 • - production. Both the specific CypD inhibitor Sanglifehrin A and knockdown of CypD by siRNA prevented endothelial cell O 2 • - production in response to Ang/IL17/TNF. We also found that this cytokine-angiotensin II milieu induced S-glutathionylation of CypD and that scavenging mitochondrial H 2 O 2 with mitoEbselen prevents this and eliminates CypD dependent complex I O 2 • - production. We further studied the functional role of oxidative stress induced by Ang/IL17/TNF in isometric tension studies of mouse aortic rings. Twenty-four hour treatment of organoid cultured vessels with AngII/IL17/TNF reduced endothelium-dependent vasodilatation to acetylcholine and this was prevented by knockdown of CypD and was not observed in vessels of mice with overexpression of mitochondrial SOD or mitochondrial catalase. The in vivo role of CypD in regulation of vascular O 2 • - and blood pressure was further studied in mice infused with angiotensin II (490 ng/kg/min). Treatment with Sanglifehrin A after the onset of hypertension reduced blood pressure from 162 to 133 mmHg (P<0.01), reduced vascular O 2 • - and improved endothelium-dependent vasodilation. These studies have defined a novel role of Cyclophilin D as a cause of vascular dysfunction and hypertension and have provided a new target for treatment of this disease.