Abstract 4367240: Rab9-dependent Mitophagy is a Comprehensive Therapeutic Target for Cardiovascular Senescence, Atherosclerosis and Sarcopenia in Postmenopausal Women

Background: Menopause is a risk factor for atherosclerosis, heart failure, and sarcopenia in women. Although these age-related diseases are interrelated, there is no comprehensive treatment. Since mitochondrial dysfunction is associated with cardiovascular diseases and sarcopenia, we hypothesized that mitophagy, which controls mitochondria quality, is a comprehensive therapeutic target to treat age-related diseases in post-menopausal women. Methods: Human umbilical vein endothelial cells, Human vascular smooth muscle cells, Rat cardiomyocytes H9C2 cells and Human skeletal muscle myoblasts were cultured under estrogen-free (E(-)) and estrogen-containing (E(+)) conditions, respectively. Mitochondrial function, mitophagy, cellular senescence and skeletal muscle cell differentiation were evaluated. We also conducted in vivo experiments using female C57BL6 mice in an ovariectomized menopausal model (OVX) and sham-operated mice (Ctr) to examine cardiovascular senescence, atherosclerosis and sarcopenia. Results: Senescence associated beta-gal and oil-red O staining, p16 and p21 expression of immunoblot, JC-1, TMRE, Mito-Sox Red and 4-HNE staining revealed that cellular and cardiovascular senescence and atherosclerosis progressed, mitochondrial function was attenuated and oxidative stress increased in both E(-) cells and OVX mice. The expression of myosin heavy chain was decreased in skeletal myoblasts of both E(-) cells and OVX mice compared to E(+) and Ctr mice, suggesting that skeletal muscle cell differentiation was suppressed. Immunoblot and immunostaining of LC3, p62, Rab9, Lamp2, Tomm20 and electric microscopic analysis revealed that these changes were caused by reduced mitophagy, which originated from Rab9-dependent alternative autophagy in a LC3/Atg7-independent manner. Enhancing Rab9 expression using a Rab9 plasmid in menopausal model cells showed induction of mitophagy, inhibition of cellular senescence and ameliorated skeletal muscle cell differentiation. Furthermore, when nicotinamide mononucleotide was administered to menopausal model mice, Rab9 was increased compared to the non-administered group, and sarcopenia was improved through increased mitophagy. Conclusion: Estrogen plays an important role in vascular, skeletal and cardiomyocyte homeostasis and skeletal myoblast differentiation through mitophagy. Rab9-dependent-mitophagy may be a comprehensive therapeutic target against menopause-associated cardiovascular diseases and sarcopenia.