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 PTP. Another common post-translational modification of cysteines is S-acylation. S-acylation is the covalent attachment of a fatty acid to a protein thiol group, and has been shown to affect the localization of proteins or their catalytic activity. CypD C202 matches an S-acylation site motif found commonly in soluble proteins. TRIM72 is a membrane repair protein that protects against ischemia reperfusion (I/R) injury. We previously identified Cys144 (C144) on TRIM72 as a site of S-nitrosylation. To study the importance of C144, we generated a knock-in mouse with C144 mutated to a serine (TRIM72 C144S). We subjected ex vivo perfused mouse hearts to 20 min of ischemia followed by 90 min of reperfusion and observed less injury in TRIM72 C144S compared to WT hearts. Infarct size was smaller (54 vs 27% infarct size) and cardiac functional recovery (37 vs 62% RPP) was higher for the TRIM72 C144S mouse hearts. We also demonstrated that TRIM72 C144S hearts were protected against I/R injury in the in vivo LAD model. As TRIM72 has been reported to be released from muscle we tested whether C144 is involved in TRIM72 release. After I/R there was significantly less TRIM72 in the perfusate normalized to total released protein from the TRIM72 C144S compared to WT hearts, suggesting that C144 of TRIM72 regulates myocardial TRIM72 release in I/R injury. In addition to TRIM72s protective role in I/R injury, TRIM72 has also been implicated in cardiac hypertrophy and insulin resistance, and secreted TRIM72 has recently been shown to impair insulin sensitivity. However, insulin sensitivity (measured by glucose and insulin tolerance) of TRIM72 C144S mice was not impaired. Further, whole body metabolism, as measured using metabolic cages, was not different in WT vs TRIM72 C144S mice and we did not observe enhanced cardiac hypertrophy in the TRIM72 C144S mice. In agreement, protein levels of the TRIM72 ubiquitination targets insulin receptor beta, IRS1 and focal adhesion kinase was similar between WT and TRIM72 C144S hearts. Overall, these data indicate that mutation of TRIM72 C144 is protective during I/R and reduces myocardial TRIM72 release without impairing insulin sensitivity or enhancing the development of hypertrophy. We have also studied the role or relaxin and nitric oxide signaling in reducing hypertrophy. Relaxin-2 is a peptide hormone that mediates multiple pleiotropic cardiovascular effects including anti-fibrotic, angiogenic, vasodilatory, anti-apoptotic and anti-inflammatory effects in vitro and in vivo. We developed RELAX10, a fusion protein comprised of human relaxin-2 hormone and the Fc of a human antibody, to test the hypothesis that extended exposure of the relaxin-2 peptide could reduce cardiac hypertrophy and fibrosis. RELAX10 demonstrated the same specificity and similar in vitro activity as the relaxin-2 peptide. The terminal half-life of RELAX10 was 7 days in mouse and 3.75 days in rat following subcutaneous administration. We evaluated whether treatment with RELAX10 could prevent and reverse isoproterenol-induced cardiac hypertrophy and fibrosis in mice. Isoproterenol administration in mice resulted in an increased cardiac hypertrophy and fibrosis compared to vehicle. Co-administration with RELAX10 significantly attenuated the cardiac hypertrophy and fibrosis as compared to untreated animals. Isoproterenol administration significantly increased TGF1/Smad-induced fibrotic signaling, which was attenuated by RELAX10. We found that RELAX10 also significantly increased AKT/eNOS signaling and protein S-nitrosylation. In the reversal study, RELAX10 treated animals showed significantly reduced cardiac hypertrophy and collagen levels. RELAX10 treated animals also showed changes in signaling with AKT/eNOS increased and TGF1/Smad signaling reduced. These findings support a potential role for RELAX10 in the treatment of heart failure.
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