VMP1 forms a Ca ²⁺ release channel essential for postnatal heartbeat

Figures

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  Fig. 1.. VMP1 is up-regulated in the postnatal heart, and its deficiency causes sudden cardiac death in mice. (A) VMP1 expression in the indicated tissues of adult (8-week-old) C57BL/6 mice was examined by immunoblotting, with WDR24 used as the loading control. Representative blots from five independent experiments are shown. (B to D) VMP1 expression in the hearts of C57BL/6 mice at the indicated developmental stages was examined by immunoblotting. E20 refers to embryonic day 20. Representative blots from three independent experiments are shown. (E) Survival curve of control and Myh6CreVmp1fl/fl mice (n = 38 mice per group). (F) Representative H&E staining of longitudinal heart sections from control and Myh6CreVmp1fl/fl mice at postnatal day 11. (G) Heart weight, body weight, and heart weight/body weight ratio in control and Myh6CreVmp1fl/fl mice at postnatal day 11 (n = 9 mice per group). (H) Survival curve of control (n = 55) and Myh6CreERT2Vmp1fl/fl mice (n = 30) following tamoxifen (TAM) injection. (I) Representative H&E staining of longitudinal heart sections from control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection. (J) Heart weight, body weight, and heart…

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  Fig. 2.. VMP1 deficiency causes severe arrhythmias and heart failure. (A) Representative ECG recordings from control and Myh6CreVmp1fl/fl mice at postnatal day 11. (B) Representative ECG recordings from control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection. (C) Representative MRI images of the short axis of the heart in control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection. The heart is outlined with a white dashed line. (D) Percentage of ejection fraction measured by MRI (n = 3 mice per group). (E) Echocardiographic analysis of control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection. Top panel: M-mode parasternal short-axis view at the midventricular level, showing the end-diastolic and end-systolic dimensions of the left ventricle. Bottom panel: Pulsed Doppler imaging at the mitral valve inflow, displaying the early-diastolic and late-diastolic mitral blood flow velocities. (F to H) Scatterplots of echocardiographic analysis showing left ventricular end-diastolic internal diameter (LVID;d) and end-systolic internal diameter (LVID;s) (F), diastolic and systolic left ventricular volumes (G), and percentage of fractional shortening…

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  Fig. 3.. VMP1-deficient cardiomyocytes exhibit increased SR Ca2+ content and aberrant AP. (A) Schematic diagram of cardiomyocyte isolation, calcium imaging, and AP detection. (B) Representative average traces of Ca2+ imaging of cardiomyocytes isolated from control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection. (C) Changes in the ratio of fluorescence intensities (340/380) in cardiomyocytes from control and Myh6CreERT2Vmp1fl/fl mice at 21 days post–tamoxifen injection in response to caffeine stimulation (n > 100 cells). (D) Representative fluorescence surface plot of spontaneous Ca2+ waves of cardiomyocytes isolated from control and Myh6CreERT2Vmp1fl/fl mice. (E) Quantification of the percentage (n = 5), occurrence frequency (n ≥ 30), and speed (n ≥ 16) of spontaneous Ca2+ waves in cardiomyocytes isolated from control and Myh6CreERT2Vmp1fl/fl mice. (F) Representative fluorescence surface plot of spontaneous Ca2+ sparks of cardiomyocytes isolated from control and Myh6CreERT2Vmp1fl/fl mice. (G) Quantification of the Ca2+ spark of cardiomyocytes isolated from control and Myh6CreERT2Vmp1fl/fl mice (n ≥ 15). (H) Representative AP traces of cardiomyocytes from control and…

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  Fig. 4.. VMP1 forms a Ca2+-permeable channel. (A) Single-channel current recordings of VMP1 and VMP1-D272N at the indicated potentials in a 500 mM:50 mM NaCl solution. Left: Schematic of the recording solutions on both sides. Right: Representative single-channel currents of VMP1 and VMP1-D272N. C denotes the closed state, while O denotes the open state. (B) Current histogram of the traces in (A). Curves were fitted by Gaussian fit. (C) I-V curves of VMP1 and VMP1-D272N in a 500 mM:50 mM NaCl solution (n ≥ 3). (D) Open probability of VMP1 and VMP1-D272N in a 500 mM:50 mM NaCl solution (−50 mV). (E) Single-channel current recordings of VMP1 and VMP1-D272N at the indicated potentials in a Na+/Ca2+ solution. Left: Schematic of the recording solutions in the cis and trans sides. Right: Representative single-channel currents of VMP1 and VMP1-D272N. (F) Current histogram of the traces in (E). Curves were fitted by Gaussian fit. (G) I-V curves of VMP1 and VMP1-D272N in a Na+/Ca2+ solution (n ≥ 3). (H) Open probability of VMP1 and VMP1-D272N in a Na+/Ca2+ solution (−50 mV). (I) Single-channel current recordings of VMP1 in a mixed Na+/Ca2+ solution with different Ca2+ gradients. (J) Current…

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  Fig. 5.. D272 of VMP1 is essential for cardiac function in vivo and up-regulation of VMP1 in patients with heart failure. (A) Survival curve of control and Myh6CreERT2Vmp1fl/D272N mice following tamoxifen injection (n = 6 mice per group). (B) Representative images of hearts and H&E staining of longitudinal heart sections from control and Myh6CreERT2Vmp1fl/D272N mice at 21 days post–tamoxifen injection. (C) Heart weight/body weight ratio in control mice (n = 4) and Myh6CreERT2Vmp1fl/D272N mice (n = 3) at 21 days post–tamoxifen injection. The control group data are shared with Fig. 1J. (D) Representative ECG recordings from control and Myh6CreERT2Vmp1fl/D272N mice at 21 days post–tamoxifen injection. (E) Representative MRI images of the short axis of the heart in control and Myh6CreERT2Vmp1fl/D272N mice at 21 days post–tamoxifen injection. The heart is outlined with a white dashed line. (F) Percentage of ejection fraction measured by MRI (n = 3 mice per group). (G) Representative immunohistochemistry images of human left ventricle tissues stained with an anti-VMP1 antibody, captured at 40× magnification. (H) Quantification of VMP1 expression levels by ImageJ. (I) Working model of VMP1…