Pathological cardiac hypertrophy is an adaptive reaction in response to pressure or volume overload. Autophagy is critical for damage caused by pathological cardiac hypertrophy. Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that is effective in activating autophagy. However, the role of VMP1 in pathological cardiac hypertrophy and its underlying mechanisms remain elusive. This study was designed to explore the potential mechanisms of VMP1 on pressure overload-induced pathological cardiac hypertrophy. In this work, abdominal aorta constriction (AAC) surgery was used to induce pathological cardiac hypertrophy in male C57BL/6 mice. H9C2 cardiomyocytes were treated with phenylephrine stimulation (PE) to induce the hypertrophic response. The in vivo results revealed that mice with AAC surgery caused pathological cardiac hypertrophy as evidenced by improved cardiac function according to multiple echocardiographic parameters. Moreover, elevated VMP1 expression was also observed in mice after AAC surgery. VMP1 knockdown aggravated changes in cardiac structure, cardiac dysfunction, and fibrosis. Meanwhile, VMP1 knockdown suppressed autophagy and endoplasmic reticulum calcium ATPase (SERCA) activity in heart tissues. H9C2 cardiomyocytes with VMP1 overexpression were used to investigate the specific mechanism of VMP1 in pathological cardiac hypertrophy, and VMP1 overexpression increased autophagic flux by upregulating SERCA activity. In conclusion, these findings revealed that VMP1 protected against pressure overload-induced pathological cardiac hypertrophy by inducing SERCA-regulated autophagic flux. Our results provide valuable insights regarding the pathophysiology of pathological cardiac hypertrophy and clues to a novel target for the treatment of pathological cardiac hypertrophy.
Clioquinol is recently considered to be the most promising drug for treating cancer and neurodegenerative diseases. However, its mode of action varies from different disease models. In this study, we found that clioquinol inhibited cell growth in human neurotypic SHSY-5Y cells, which was attributed to both S-phase cell-cycle arrest and autophagic cell death. Clioquinol increased the intracellular contents of iron and zinc as well as calcium as measured by ICP-AES. Staining of Fluo-3 confirmed an increase in the level of calcium. Analysis of the metal-binding ability of clioquinol showed that it was not a chelating agent of calcium ions and the elevation of intracellular calcium content is not achieved by clioquinol as an ionophore. CaCl2 could simulate or even aggravate the cytotoxicity of clioquinol and it increased S-phase cell cycle arrest induced by clioquinol in a concentration dependent manner. Staining of acridine orange demonstrated that autophagy induced by clioquinol was not affected by addition of calcium ions. In contrast, the intracellular calcium ion chelator BAPTA-am abolished the clioquinol-induced S phase arrest and reduced the cell death caused by clioquinol. The WB assay of cell cycle-related proteins (CDK2, p21 and p27) further confirmed that S phase arrest is positively correlated with intracellular calcium elevation, which was due to the alterations of the mRNA and protein levels of calcium pumps (SERCA and SPCA). Taken together, these data indicate that clioquinol regulates the level of intracellular calcium ions to induce S-phase cell cycle arrest in human SH-SY5Y cells. Our results demonstrate for the first time that an increase of intracellular calcium content is one of the mechanisms of clioquinol in the inhibition of human neurotypic SHSY-5Y cells.
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