Nitric oxide (NO) and protein S-nitrosylation (SNO) have been shown to play important roles in ischemic preconditioning (IPC)-induced acute cardioprotection. Our previous study in mouse embryonic fibroblasts showed that cysteine 202 of cyclophilin D (CyPD) is necessary for redox stress-induced activation of the mitochondrial permeability transition pore (mPTP). To further investigate the essential function of this cysteine residue in situ, we used CRISPR to develop a knock-in mouse model (C57BL/6N stain), where CyPD cysteine 202 was mutated to a serine (C202S-KI). The amount of total CyPD expressed in the CyPD C202S-KI did not differ compared to the wild-type (WT). However, the CyPD C202S-KI mouse hearts elicit a significant cardioprotective effect against ischemia-reperfusion (I/R) injury in the Langendorff perfused heart model. After 20 min of global ischemia followed by 90 min of reperfusion, the post-ischemic recovery of rate pressure product (RPP= heart rate x LVDP) was 45.04.2% in CyPD WT and 59.64.0% in CyPD C202S-KI mice. Myocardial infarct size was decreased in CyPD C202S-KI mouse hearts versus CyPD WT mice (24.54.7% vs 49.82.7%). Isolated heart mitochondria from CyPD C202-KI mice had a higher calcium retention capacity compared to CyPD WT mice (140.020.82 vs 213.316.67 umol Ca+2/g protein). However, in contrast to CyPD knockout mice which exhibit more pronounced cardiac hypertrophy in response to pressure overload stimulation than control mice, CyPD C202S-KI mice developed a comparable level of hypertrophy to their WT littermate in an angiotensin II-induced hypertrophy model delivered by implanted osmotic minipumps. In conclusion, these results show that mutated CyPD C202S affords cardioprotection against I/R injury, suggesting that the redox-modification of cysteine 202 might play an important role in the regulation of CyPD and its downstream targets such as mPTP. We also find S-nitrosylation of C144 of TRIM-72 in preconditioning. TRIM72 regulates membrane repair and metabolism in the heart. Our previous study identified S-nitrosylation (SNO) of Cysteine 144 (C144) on TRIM72 and suggested that it protects against ischemia reperfusion (I/R) injury. To further investigate the function of C144 we generated a mouse with C144 mutated to serine (KI). As expected, langendorff perfused KI hearts were protected against I/R injury. Post-ischemic recovery of RPP was higher (626% vs 373%, n=5) and infarct size was less (274% vs 544%, n=5) in KI. We further characterized the KI mice and found that they had improved insulin sensitivity compared to WT. Glucose tolerance was improved in KI (1917 vs 24322 mmol/L blood glucose 30 min post glucose injection, n=5) and insulin induced a greater decline in blood glucose with 30% lower blood glucose at 30 min. Because TRIM72 overexpression has been reported to cause cardiac hypertrophy, we examined whether there was a difference in hypertrophy in KI vs WT. KI did not show a difference in hypertrophy as measured by heart weight/tibia length (20% increase, n=6) or %EF (472% vs 474%, n=5) after 4 wk of Angiotensin II (1.5 mg/kg BW). These data suggest that post translational modification of C144 is important in the regulation of specific functions of TRIM72 and in TRIM72 regulation of insulin sensitivity and I/R injury.
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