Truncation of GSK‐3β in Cardiac Mitochondria is the Master Switch of the mPTP

Glycogen Synthase Kinase‐3β (GSK‐3β) is proposed as one of the key molecules that regulate cell death during ischemia reperfusion (IR) injury in the heart, largely via modulating mitochondria permeability transition pore (mPTP) activity, where both Ca 2+ and oxidative stress are at peak levels. However, the exact mechanism of how GSK‐3β controls cardiomyocyte survival and death has remained elusive. In non‐cardiac cells, μ‐Calpain activated by high levels of Ca 2+ and ROS can truncate GSK‐3β at both the N and C termini to generate a constitutively‐active GSK‐3β by removing the inhibitory phosphorylation sites. Therefore, we hypothesize that truncation of GSK‐3β in cardiac mitochondria can modulate mPTP function via the phosphorylation and binding affinity of mPTP component proteins. Using mitochondrial fractionation and proteinase‐K digestion, we found that GSK‐3β is truncated by μ‐Calpain in the mitochondria matrix. Using co‐immunoprecipitation (IP) we determined that truncated GSK‐3β associates with and phosphorylates cyclophilin D (mCypD). In addition, P‐mCypD increases the binding affinity to ATP‐Synthase, a component of the mPTP pore. Overexpression of a mitochondrial targeted Calpain inhibitory peptide based on the Calpastatin inhibitory domain in H9c2 cardiac myoblasts significantly blocked ionomycin‐induced cell death, as measured by Caspase 3/7 activation, to a greater extent than pretreatment with cyclosporine A, a potent mPTP inhibitor. In conclusion, Calpain‐mediated truncation of GSK‐3β in the mitochondrial matrix leads to phosphorylation of mCypD, allowing P‐mCypD to bind to and open the mPTP pore complex in cardiac cells. Funding Source: NIH Grant 5T32AA007463‐28