Exercise mitigates calpain induced Purkinje cell loss in diabetes

Calpain-1 is a ubiquitous calcium dependent cysteine protease and found in cytoplasm as well as mitochondria. We have earlier reported that active calpain-1 is translocated from cytosol to mitochondria and activates MMP9. Calpain-1 activation is detrimental to the heart in several different ways, but there is little evidence that it can degrade Purkinje cell protein (PCP-4) and impair contractility in diabetes. Our hypothesis is that in diabetes, PCP-4 is degraded by calpain-1, causing contractile dysfunction that can be mitigated by exercise. To test this hypothesis, we recruited four groups of mice, 1) db/+ control, 2) db/+ with exercise, 3) db/db, 4) db/db with exercise. The mice were exercised on treadmill for 8 weeks as per American Veterinary Research Guidelines. Adding calcium to isolated cardiomyocytes caused them to lose shape and die. Compared with live myocytes, we observed high calpain-1 levels as well as significantly lower levels of PCP-4 and increased levels of calmodulin and calmodulin kinase II (CaMKII) in dead myocytes. We used the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) plasmid to knock down calpain-1 in HL-1 myocytes which restored the levels of PCP-4 along with calmodulin and CaMKII. In vivo, we found upregulated levels of calpain-1 in db/db mice (diabetic) as compared to db/+ which were mitigated in the exercised mice. Conclusively our data strongly suggests that in diabetes there is high induction of calpain-1 with degrades PCP-4, a protein important for contractility and exercise can mitigate this.