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

Abstract

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 (Ca2+)-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 (ΔΨm) 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 Ca2+-dependent manner through the N-terminal residues (1–28). Furthermore, expression of Anxa6S13E, 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.

Epithelial-to-mesenchymal transition drives a pro-metastatic Golgi compaction process through scaffolding protein PAQR11

“…Confocal imaging was performed on a TCS-SP8 platform (Leica Microsystems) equipped with ×63/1.4 NA oil, ×63/1.2 NA water, or ×40/1.2 NA oil objectives; 405/488/561/633 nm laser lines; and photomultiplier and hybrid (Leica HyD) detector systems. Highresolution images were acquired as described elsewhere (57,66). For faster time-lapse imaging, an 8K resonant scanner was used.…”

“…FRAP experiments were carried out as described previously (57,58), with some modifications. Recovery was calculated within a circular region of interest (ROI) 1 μm in diameter.…”

Targeting Peroxisome Proliferator Activated Receptor α (PPAR α) for the Prevention of Mitochondrial Impairment and Hypertrophy in Cardiomyocytes

“…Previously, we have also shown that PPARα agonist bezafibrate inhibited thyroid hormone induced cardiac hypertrophy via prevention of mitochondrial dysfunction suggesting cardioprotective function of the fibrates [20]. Recently, we have also reported that the dynamic state is very important for maintaining the functional state of mitochondria by using single cell fluorescence microscopy in H9c2 cardiomyocytes [21].…”

The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

“…The FAs and lipids form an integral part of the Golgi associated vesicle trafficking pathways and owing to the structural complexity of the Golgi apparatus and trans-Golgi network [65], the localization of proteins interacting with FAs and other lipids may appear condensed in the perinuclear compartment. Also, the pool of mitochondria is concentrated in the perinuclear compartment [66], thus explaining the relative abundance of IFABP in the perinuclear compartment. Our FCS measurements showed the presence of a lower D value for these clusters along with D's in the similar range as observed in the diffused cytoplasmic IFABPs.…”

“…Cells transfected with wild type IFABP-GFPconstructs were transduced with CellLight Mitochondria-RFP (Molecular Probes Inc., Eugene, OR, USA) as described elsewhere [40], washed with 1X DPBS and imaged using the imaging medium (20 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4 along with 1% glucose) for studying mitochondrial dynamics. For JC-1 staining, cells were stained using the JC-1 Mitochondrial Membrane Potential Assay Kit according to manufacturer's protocol (Cayman chemical, Ann Arbor, MI,).…”