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International audience; Diabetic cardiomyopathy (DCM) is a leading complication in type 2 diabetes patients. Recently, we have shown that the reticulum-mitochondria Ca2+ uncoupling is an early and reversible trigger of the cardiac dysfunction in a diet-induced mouse model of DCM. Metformin is a first-line antidiabetic drug with recognized cardioprotective effect in myocardial infarction. Whether metformin could prevent the progression of DCM remains not well understood. We therefore investigated the effect of a chronic 6-week metformin treatment on the reticulum-mitochondria Ca2+ coupling and the cardiac function in our high-fat high-sucrose diet (HFHSD) mouse model of DCM. Although metformin rescued the glycemic regulation in the HFHSD mice, it did not preserve the reticulum-mitochondria Ca2+ coupling either structurally or functionally. Metformin also did not prevent the progression towards cardiac dysfunction, i.e., cardiac hypertrophy and strain dysfunction. In summary, despite its cardioprotective role, metformin is not sufficient to delay the progression to early DCM.

The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death.

Introduction Type 2 diabetic cardiomyopathy has been linked to Ca2+ signaling alterations, notably a decreased mitochondrial Ca2+ uptake. Recent discovery of Ca2+ microdomains between cardiac mitochondria and reticulum launched a new investigation avenue for cardiometabolic diseases. Objective Hereby, we aimed to investigate if the mitochondrial Ca2+ mishandling is due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2+ uniporter in the diabetic mouse heart. Methods and results Mice fed for 16 weeks with a high fat high sucrose diet (HFHSD) displayed a cardiac insulin resistance combined to cardiac fibrosis, hypertrophy and contractile dysfunction (decreased strain rate) versus standard diet-fed mice (SD). Fractionation of HFHSD mice hearts revealed a decreased protein content of their mitochondria associated-reticulum membranes, together with a lower expression of tethering proteins quantified by mass spectrometry. On functional terms, reduced IP3R-VDAC proximity and co-immunoprecipitation were associated with a reduced IP3-stimulated Ca2+ transfer to mitochondria in isolated HFHSD cardiomyocytes, yet no changes in mitochondrial calcium uniporter protein expression and function were detected. Moreover, HFHSD cardiomyocytes displayed no significant changes in the reticular Ca2+ release level and the amplitude of cytosolic Ca2+ transients. Eight weeks of diet reversal restored the cardiac insulin signaling, MAM content, Ca2+ transfer and cardiac function in HFHSD mice. Conclusion Therefore, our data indicate that disruption of the cardiac reticulum-mitochondria interactions is an early and reversible event impairing mitochondrial Ca2+ handling and contributing to the cardiac contractile dysfunction in the diabetic mouse heart, which could be counteracted by diet reversal.

Cardiovascular diseases associated with metabolic syndrome remain frequent and still disabling for patients. The uncovering of Ca2+ microdomains between mitochondria and reticulum (SR) in the heart has launched a new area of investigation for cardiometabolic diseases. Here, we sought to determine the structural and functional role of the SR-mitochondria interactions in the mice heart during diabetes. We hypothesized that an alteration of the SR-mitochondria interactions would impair the Ca2+ signaling between the two organelles, thus leading to contractile dysfunction. Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat-high-sucrose diet (HFHSD:20% proteins, 36% lipids) for 16 weeks. In the HFHSD group, half of the mice were subjected to metformin gavage, an antidiabetic agent, for the last 4 weeks. Cardiac mitochondria associated membranes (MAM) isolation showed an increased protein amount of MAM/pure mitochondria in the HFHSD group vs. SD mice, which was prevented by metformin treatment (SD: 2.0 ± 0.4, HFHSD: 5.5 ± 0.9, HFHSD + MET: 2.8 ± 0.4, n = 5). After 70 min hypoxia-2 hrs reoxygenation, HFHSD isolated cardiomyocytes displayed higher propidium-iodide positive cells compared to SD and HFHSD + MET groups (60 ± 1 vs. SD: 46 ± 3 and HFHSD + MET: 50 ± 2%, n = 5). Interestingly, histamine-stimulated Ca2+ transfer to mitochondria was reduced by 50% in HFHSD ± MET cardiomyocytes. Concomitantly, IP3R-VDAC interactions assessed by proximity ligation assay were 30–40% reduced in HFHSD ± MET cardiomyocytes. Hence, our data indicate a role for the SR-mitochondria interactions during diabetes in the heart in hypoxia-reoxygenation-induced cell death and in the Ca2+ signaling dysfunction. Indeed, reduced formation of the IP3R-VDAC Ca2+ channeling complex and of the IP3R-stimulated Ca2+ transfer to mitochondria in the HFHSD ± MET cardiomyocytes suggest an inappropriate thickness of the extended MAM (too close or too far) for the IP3R complex formation.

Introduction Diabetic cardiomyopathy has been linked to Ca2 + signaling alterations, notably a decreased mitochondrial Ca2 + uptake. Uncovering the changes occurring at Ca2 + microdomains between reticulum and mitochondria in the heart has launched a new area of investigation for cardiometabolic diseases. Objective We here aimed to study if the impairment of mitochondrial Ca2 + handling could be due to a dysregulation of the reticulum-mitochondria interactions or of the mitochondrial Ca2 + uniporter in the diabetic mice heart. Methods Mice were either fed with a standard diet (SD: 16.9% proteins, 4.3% lipids) or a high-fat high sucrose diet (HFHSD: 20% proteins, 36% lipids) for 16 weeks. Cardiac mitochondria associated membranes (MAM) composition was analyzed by proteomics and immunoblotting. Proximity ligation assay, calcium imaging and hypoxia/reoxygenation were performed on isolated cardiomyocytes. Results Our HFHSD mice displayed a cardiac insulin resistance and hypertrophy. Decreased MAM/pure mitochondria content in the HFHSD vs SD heart was observed, with an elevated level of proteins involved in lipid metabolism and a decrease in tethering proteins. Decreased IP3R-VDAC proximity upon HFHSD was concomitant to a reduced IP3R-stimulated Ca2 + transfer to mitochondria, with no changes in mitochondrial calcium uniporter protein expression and function. Additionally, decreased amplitude of cytosolic Ca2 + transients was seen in the HFHSD cardiomyocytes, with no significant changes in the reticular Ca2 + release level. Besides, increased cell death susceptibility was measured after both in vitro hypoxia/reoxygenation and in vivo ischemia/reperfusion under HFHSD. Conclusion Our data, therefore, indicate that decreased reticulum-mitochondria interactions trigger an impaired mitochondrial Ca2 + handling in the diabetic mice heart, with no alteration of the mitochondrial Ca2 + uniporter, while the enhanced susceptibility to cell death could be referred to lipid toxicity.