Transient Receptor Potential Vanilloid Type 1 Protects Against Pressure Overload–Induced Cardiac Hypertrophy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes

FIGURE 1.. Pressure overload increased TRPV1 expression in myocardium and cardiomyocytes. TRPV1 protein and mRNA expression in myocardium following TAC (A and C). PE-treated neonatal rat cardiomyocytes (B and D) were detected by immunoblotting and reverse transcription polymerase chain reaction. n = 6 per group. *P < 0.05 compared with sham or control.

FIGURE 2.. TRPV1 activation alleviated pressure overload–induced myocardial hypertrophy. A, Histological analysis of heart sections from normal diet (Cont)-fed or capsaicin diet (CAP)-fed mice with or without TAC (scale bar = 1 mm). Heart weight to body weight ratio in the indicated groups. B, Heart cross-sections were stained with wheat germ agglutinin and the average cross-sectional areas of cardiomyocytes were quantified (scale bar = 20 μm). C, The echocardiographic assessment of cardiac function from normal diet-fed or CAP-fed mice. The animal experiments were performed 8 weeks after TAC surgery, n = 8 mice per group. *P < 0.05 compared with sham-cont, #P < 0.05 compared with TAC-cont group.

FIGURE 3.. TRPV1 activation protected against PE-induced cardiomyocyte hypertrophy in vitro. A, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 μm). B, The ANP, BNP, β-MHC protein expression in primary cardiomyocytes treated with capsaicin and PE (n = 5). C, Quantitative polymerase chain reaction analyses of the mRNA levels of ANP, BNP, β-MHC in the indicated groups. n = 5 per group. *P < 0.05 compared with control, #P < 0.05 compared with PE group.

FIGURE 4.. TRPV1 activation attenuated mitochondrial dysfunction in cardiomyocytes. A, The mitochondrial morphology was observed by transmission electron microscopy (scale bar = 200 nm). B, MMP was assessed using a JC-1 kit. The JC-1 aggregate/JC-1 monomer ratio was measured for MMP. (n = 6, scale bar = 10 μm). C, ATP levels in cultured cardiomyocytes (n = 6). *P < 0.05 compared with control, #P < 0.05 compared with PE group.

FIGURE 5.. TRPV1 activation promoted MAM formation in cardiomyocytes. A, Representative transmission electron microscopy image indicating the ER and mitochondrial contacts in cardiomyocytes. Mitochondria and ER were delimitated using the free hand selection tool of ImageJ (NIH). Masks were generated with corresponding object identification (scale bar = 200 nm). Quantification of the ER adjacent to the mitochondria in cardiomyocytes was normalized by total ER length (n = 6). B, Representative confocal images showing the association between ER (ER-Tracker Green) and mitochondria (MitoTracker Red) in cardiomyocytes (scale bar = 10 μm). Quantitation of ER-mitochondria contacts using Pearson's coefficient (n = 5). C, The interaction between voltage-dependent anion channels and IP3R in cardiomyocytes was determined by IP and WB (n = 4). The bar chart shows levels of voltage-dependent anion channels bound to IP3R in the immunoprecipitations following quantification of signals from immunoblots. Voltage-dependent anion channels signals were normalized to GAPDH signals. *P < 0.05 compared with control, #P < 0.05 compared with PE group.

FIGURE 6.. Disruption of MAM by siMFN2 abolished capsaicin-mediated mitochondria protection. A, MMP was assessed using a JC-1 kit. The JC-1 aggregate/JC-1 monomer ratio was measured for MMP (n = 6, scale bar = 10 μm). B, Effects of capsaicin on ROS production in PE-treated cardiomyocytes using MitoSOX red staining (n = 5, scale bar = 10 μm). C, ATP levels in cultured cardiomyocytes (n = 6). D, Representative images and analysis of the cell surface area of neonatal rat cardiomyocytes stained with cTnT (green) and DAPI (blue) (n = 5, scale bar = 10 μm). *P < 0.05 compared with control, #P < 0.05 compared with PE group, $P < 0.05 compared with PE + CAP group.

FIGURE 7.. TRPV1 activation promotes MAM formation via AMPK/MFN2 pathway. A, The p-AMPK, AMPK, MFN2 protein expression in primary cardiomyocytes treated with capsaicin and CC (n = 5). B, Representative confocal images showing the association between ER (ER-Tracker Green) and mitochondria (MitoTracker Red) in cardiomyocytes (scale bar = 10 μm). Quantitation of ER-mitochondria contacts using the Pearson's coefficient (n = 5). *P < 0.05 compared with control, #P < 0.05 compared with PE group, $P < 0.05 compared with PE + CAP group.