Balancing functions of annexin A6 maintain equilibrium between hypertrophy and apoptosis in cardiomyocytes

Pathological cardiac hypertrophy is a major risk factor associated with heart failure, a state concomitant with increased cell death. However, the mechanism governing progression of hypertrophy to apoptosis at the single-cell level remains elusive. Here, we demonstrate annexin A6 (Anxa6), a calcium (Ca(2+))-dependent phospholipid-binding protein critically regulates the transition of chronic hypertrophied cardiomyocytes to apoptosis. Treatment of the H9c2(2-1) cardiomyocytes with hypertrophic agonists upregulates and relocalizes Anxa6 with increased cytosolic punctate appearance. Live cell imaging revealed that chronic exposure to hypertrophic agonists such as phenylephrine (PE) compromises the mitochondrial membrane potential (ΔΨ(m)) and morphological dynamics. Such chronic hypertrophic induction also activated the caspases 9 and 3 and induced cleavage of the poly-(ADP-ribose) polymerase 1 (Parp1), which are the typical downstream events in the mitochondrial pathways of apoptosis. An increased rate of apoptosis was evident in the hypertrophied cardiomyocytes after 48–72 h of treatment with the hypertrophic agonists. Anxa6 was progressively associated with the mitochondrial fraction under chronic hypertrophic stimulation, and Anxa6 knockdown severely abrogated mitochondrial network and dynamics. Ectopically expressed Anxa6 protected the mitochondrial morphology and dynamics under PE treatment, and also increased the cellular susceptibility to apoptosis. Biochemical analysis showed that Anxa6 interacts with Parp1 and its 89 kDa cleaved product in a Ca(2+)-dependent manner through the N-terminal residues (1–28). Furthermore, expression of Anxa6(S13E), a mutant dominant negative with respect to Parp1 binding, served as an enhancer of mitochondrial dynamics, even under chronic PE treatment. Chemical inhibition of Parp1 activity released the cellular vulnerability to apoptosis in Anxa6-expressing stable cell lines, thereby shifting the equilibrium away from cell death. Taken together, the present study depicts a dual regulatory function of Anxa6 that is crucial for balancing hypertrophy with apoptosis in cardiomyocytes.