Inhibition of myocardial reperfusion injury by ischemic postconditioning requires sirtuin 3-mediated deacetylation of cyclophilin D.

RATIONALE: How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP. OBJECTIVE: The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD. METHODS AND RESULTS: Using hypoxia-reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871 ± 266 vs 1193 ± 263 nmoles Ca(2+)/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compared to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice. CONCLUSIONS: Our study suggests that the increased acetylation of CyPD following myocardial ischemia-reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion.

Enhanced activity of the myocardial Na+/H+ exchanger contributes to left ventricular hypertrophy in the Goto-Kakizaki rat model of type 2 diabetes: critical role of Akt.

AIMS/HYPOTHESIS: Diabetes mellitus is a strong risk factor for the development of heart failure, and left ventricular (LV) hypertrophy has been detected in a significant proportion of diabetic patients. Because several studies have suggested that the Na(+)/H(+) exchanger (NHE1) plays a part in the molecular mechanisms involved in cardiac hypertrophy, we investigated its activity and its role in LV myocytes from the Goto-Kakizaki (GK) rat model of type 2 diabetes. MATERIALS AND METHODS: Fluorometric measurements were used to assess sarcolemmal NHE1 activity in isolated myocytes. NHE1 levels and the possible molecular pathways driving and/or related to NHE1 activity were investigated in relation to the diabetic LV phenotype. RESULTS: Enhanced NHE1 activity was associated with LV myocyte hypertrophy. This occurred in the absence of any change in NHE1 protein levels; however, activation of several molecular pathways related to NHE1 activity was demonstrated. Thus, phosphorylation of the extracellular signal-regulated protein kinase (Erk), of the protein kinase Akt (also known as protein kinase B) and of the Ca(2+)/calmodulin-dependent kinase II was increased in GK LV myocytes. Intracellular Ca(2+) levels were also increased. Chronic treatment (10-12 weeks) with the NHE1 inhibitor cariporide normalised NHE1 activity, decreased [Formula: see text] levels and reduced LV myocyte hypertrophy. Moreover, among the various activated pathways, cariporide treatment markedly reduced Akt activity only. CONCLUSIONS/INTERPRETATION: These findings indicate that activation of the Akt pathway represents a likely mechanism mediating the hypertrophic effect of increased NHE1 activity in the GK model of type 2 diabetes.

Intracellular sodium increase and susceptibility to ischaemia in hearts from type 2 diabetic db/db mice.

AIMS/HYPOTHESIS: An important determinant of sensitivity to ischaemia is altered ion homeostasis, especially disturbances in intracellular Na(+) (Na(i)(+)) handling. As no study has so far investigated this in type 2 diabetes, we examined susceptibility to ischaemia-reperfusion in isolated hearts from diabetic db/db and control db/+ mice and determined whether and to what extent the amount of (Na(i)(+)) increase during a transient period of ischaemia could contribute to functional alterations upon reperfusion. METHODS: Isovolumic hearts were exposed to 30-min global ischaemia and then reperfused. (23)Na nuclear magnetic resonance (NMR) spectroscopy was used to monitor[Formula: see text] and (31)P NMR spectroscopy to monitor intracellular pH (pH(i)). RESULTS: A higher duration of ventricular tachycardia and the degeneration of ventricular tachycardia into ventricular fibrillation were observed upon reperfusion in db/db hearts. The recovery of left ventricular developed pressure was reduced. The increase in[Formula: see text] induced by ischaemia was higher in db/db hearts than in control hearts, and the rate of pH(i) recovery was increased during reperfusion. The inhibition of Na(+)/H(+) exchange by cariporide significantly reduced (Na(i)(+)) gain at the end of ischaemia. This was associated with a lower incidence of ventricular tachycardia in both heart groups, and with an inhibition of the degeneration of ventricular tachycardia into ventricular fibrillation in db/db hearts. CONCLUSIONS/INTERPRETATION: These findings strongly support the hypothesis that increased (Na(i)(+)) plays a causative role in the enhanced sensitivity to ischaemia observed in db/db diabetic hearts.

Changes in intracellular sodium and pH during ischaemia-reperfusion are attenuated by trimetazidine. Comparison between low- and zero-flow ischaemia.

OBJECTIVE: The aim of this study was to investigate whether trimetazidine (TMZ; 10(-6)M), which has been shown to inhibit fatty acid oxidation, reduces the ionic imbalance induced by ischaemia and reperfusion, especially through an attenuation in intracellular changes in H(+) and Na(+). METHODS: Isovolumic rat hearts receiving 5.5 mM glucose and 1.2 mM palmitate as metabolic substrates were exposed to zero-flow ischaemia (TI) or low-flow ischaemia (LFI - coronary flow decreased by an average of 90%) (30 min at 37 degrees C) and then reperfused. 23Na nuclear magnetic resonance (NMR) spectroscopy was used to monitor intracellular Na(+) (Na(+)(i)) and 31P NMR spectroscopy was used to monitor intracellular pH (pH(i)). RESULTS: During LFI the major effect of TMZ was a significant reduction in intracellular acidosis, whereas during TI the main effect of TMZ was a significant reduction in Na(+)(i) gain. In addition, the further gain in Na(+)(i) that occurred during the first minutes of reperfusion following TI, and to a far lesser extent following LFI, was suppressed in TMZ-treated hearts and also suppressed when hearts were perfused without fatty acid. In both LFI and TI, TMZ-induced attenuation of ionic imbalance was associated with a significantly improved recovery of ventricular function on reperfusion, as assessed by a lower increase in diastolic pressure and an increased recovery of developed pressure. CONCLUSION: Our data provide evidence that specific myocardial metabolic modulation plays a significant role in reducing ionic imbalance during ischaemia and reperfusion.