Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis.

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

Protein kinase D activation induces mitochondrial fragmentation and dysfunction in cardiomyocytes.

KEY POINTS: Abnormal mitochondrial morphology and function in cardiomyocytes are frequently observed under persistent Gq protein-coupled receptor (Gq PCR) stimulation. Cardiac signalling mechanisms for regulating mitochondrial morphology and function under pathophysiological conditions in the heart are still poorly understood. We demonstrate that a downstream kinase of Gq PCR, protein kinase D (PKD) induces mitochondrial fragmentation via phosphorylation of dynamin-like protein 1 (DLP1), a mitochondrial fission protein. The fragmented mitochondria enhance reactive oxygen species generation and permeability transition pore opening in mitochondria, which initiate apoptotic signalling activation. This study identifies a novel PKD-specific substrate in cardiac mitochondria and uncovers the role of PKD on cardiac mitochondria, with special emphasis on the molecular mechanism(s) underlying mitochondrial injury with abnormal mitochondrial morphology under persistent Gq PCR stimulation. These findings provide new insights into the molecular basis of cardiac mitochondrial physiology and pathophysiology, linking Gq PCR signalling with the regulation of mitochondrial morphology and function. ABSTRACT: Regulation of mitochondrial morphology is crucial for the maintenance of physiological functions in many cell types including cardiomyocytes. Small and fragmented mitochondria are frequently observed in pathological conditions, but it is still unclear which cardiac signalling pathway is responsible for regulating the abnormal mitochondrial morphology in cardiomyocytes. Here we demonstrate that a downstream kinase of Gq protein-coupled receptor (Gq PCR) signalling, protein kinase D (PKD), mediates pathophysiological modifications in mitochondrial morphology and function, which consequently contribute to the activation of apoptotic signalling. We show that Gq PCR stimulation induced by α1 -adrenergic stimulation mediates mitochondrial fragmentation in a fission- and PKD-dependent manner in H9c2 cardiac myoblasts and rat neonatal cardiomyocytes. Upon Gq PCR stimulation, PKD translocates from the cytoplasm to the outer mitochondrial membrane (OMM) and phosphorylates a mitochondrial fission protein, dynamin-like protein 1 (DLP1), at S637. PKD-dependent phosphorylation of DLP1 initiates DLP1 association with the OMM, which then enhances mitochondrial fragmentation, mitochondrial superoxide generation, mitochondrial permeability transition pore opening and apoptotic signalling. Finally, we demonstrate that DLP1 phosphorylation at S637 by PKD occurs in vivo using ventricular tissues from transgenic mice with cardiac-specific overexpression of constitutively active Gαq protein. In conclusion, Gq PCR-PKD signalling induces mitochondrial fragmentation and dysfunction via PKD-dependent DLP1 phosphorylation in cardiomyocytes. This study is the first to identify a novel PKD-specific substrate, DLP1 in mitochondria, as well as the functional role of PKD in cardiac mitochondria. Elucidation of these molecular mechanisms by which PKD-dependent enhanced fission mediates cardiac mitochondrial injury will provide novel insight into the relationship among mitochondrial form, function and Gq PCR signalling.