Caveolin-3 and Caveolae regulate ventricular repolarization

Abstract

Rationale: Flask-shaped invaginations of the cardiomyocyte sarcolemma called caveolae require the structural protein caveolin-3 (Cav-3) and host a variety of ion channels, transporters, and signaling molecules. Reduced Cav-3 expression has been reported in models of heart failure, and variants in CAV3 have been associated with the inherited long-QT arrhythmia syndrome. Yet, it remains unclear whether alterations in Cav-3 levels alone are sufficient to drive aberrant repolarization and increased arrhythmia risk.

Objective: To determine the impact of cardiac-specific Cav-3 ablation on the electrophysiological properties of the adult mouse heart.

Methods and results: Cardiac-specific, inducible Cav3 homozygous knockout (Cav-3KO) mice demonstrated a marked reduction in Cav-3 expression by Western blot and loss of caveolae by electron microscopy. However, there was no change in macroscopic cardiac structure or contractile function. The QT_c interval was increased in Cav-3KO mice, and there was an increased propensity for ventricular arrhythmias. Ventricular myocytes isolated from Cav-3KO mice exhibited a prolonged action potential duration (APD) that was due to reductions in outward potassium currents (I_to, I_ss) and changes in inward currents including slowed inactivation of I_Ca,L and increased I_Na,L. Mathematical modeling demonstrated that the changes in the studied ionic currents were adequate to explain the prolongation of the mouse ventricular action potential. Results from human iPSC-derived cardiomyocytes showed that shRNA knockdown of Cav-3 similarly prolonged APD.

Conclusion: We demonstrate that Cav-3 and caveolae regulate cardiac repolarization and arrhythmia risk via the integrated modulation of multiple ionic currents.

Keywords: Action potential; Arrhythmia; Cardiac repolarization; Caveolae; Ion channels.

Fig. 1.. Cardiac-specific Cav3 knockout mice exhibit reduced Cav-3 expression and decreased cardiomyocyte caveolae but have normal macroscopic cardiac structure and contractile function.

(A) Schematic representation of Tamoxifen (Tam)-inducible activation of αMHC-MerCreMer for cardiac-specific Cav-3 knockout mouse generation and timeline for experiments. (B) Immunoblot and quantification of relative Cav-3 expression to loading control actin from left ventricle of Control (n = 6), Cav-3Het (n = 5), and Cav-3KO (n = 5) mice (one-way ANOVA with post hoc Tukey method). (C) Transmission electron micrographs of left ventricle sections (scale bar 500 nm). Morphological caveolae are denoted by arrow heads. (D) Caveolae per micron membrane from Control (n = 242 images), Cav-3Het (n = 342 images), and Cav-3KO (n = 437 images) mice from 3 animals per group. (E) Assessment of cardiac function by echocardiographic parameters before and after Tam treatment in Control and Cav-3KO mice (3–5 animals per group, n.s. by 2-tailed paired t-test before and after Tam). (F) Heart weight/body weight (HW/BW) ratios (Control n = 6, Cav-3KO n = 8, n.s. by 2-tailed unpaired t-test). (G) Histological assessment of mid-LV transverse section Control and Cav-3KO hearts stained with Masson’s trichrome and H&E. Scale bars 100 μm.

Fig. 2.. Cav-3KO mice exhibit prolonged QTc interval and increased susceptibility to ventricular arrhythmia.

(A, B) Representative single ECG complex, measured heart rate, PR interval, and QTc interval before and after Tam treatment for Con and Cav-3KO mice (n = 7–8 for QTc measurement and n = 7–8 for HR, statistical comparisons by two-tailed paired t-tests), with Cav-3KO mice showing significant QTc prolongation post-Tam. (C) Representative traces of ventricular tachycardia (VT) induced with ventricular burst pacing from Con and Cav-3KO mice enlarged on right (scale bar 1 sec). (D) Comparison of induced VT incidence in mice (P < 0.001, chi-square statistic is 10.37). Tam treatment was for 1 week and after another 2 weeks animals were studied.

Fig. 3.. Loss of Cav-3 expression in ventricular myocytes prolongs APD and reduces I_to and I_ss current densities.

(A) Representative traces of ventricular action potentials from Con, Cav-3Het and Cav-3KO mice simulated at a rates of 5 Hz at 37 ± 2 °C. (B) Comparison of action potential traces, mean APD50, mean APD90, and resting membrane potential (RMP) from Con (n = 9), Cav-3Het (n = 13), and Cav-3KO (n = 13) myocytes from 4–5 mice per group (one-way ANOVA with post hoc Tukey method) (C) Representative traces of I_K and I-V relationships with respect to different test potentials from Con (n = 14) and Cav-3KO (n = 14) myocytes from 4 animals per group (*P<0.05 by two tailed unpaired t-test). Voltage protocols is shown in panel inset. (D) Mean I_to, I_Kslow, and I_ss determined by bi-exponential curve fit for the decay phase of I_K at +40 mV test pulse (two tailed unpaired t-test for comparisons). (E) Representative traces of I_K1 and I-V relationships for I_K1 from Con (n = 16) and Cav-3KO (n = 9) myocytes from 3 animals per group. (F) Representative Western blots showing K_v4.2, K_v4.3, K_v2.1, K_v1.4, and K_ir2.1 protein levels from left ventricular lysates prepared from Con and Cav-3KO mice. Right panel, is summary data of relative K⁺ channel expression normalized to GAPDH (n = 4).

Fig. 4.. Increased I_Na,L and reduced I_Ca,L with delayed inactivation in Cav-3KO ventricular myocytes.

(A) Representative traces of I_Na and I-V relationships from Con (n = 7, 3 mice) and Cav-3KO (n = 13, 5 mice) myocytes. (B) Representative traces of I_Na,L from Con and Cav-3KO myocytes and average integrals of TTX-sensitive I_Na,L measured between 50 and 300 ms from Con (n = 10, 3 mice) and Cav-3KO (n = 11, 3 mice) myocytes. (C) Representative traces of I_Ca,L and I-V relationships from Con (n = 11, 4 mice) and Cav-3KO (n = 17, 4 mice) myocytes (*p < 0.001). (D) Representative traces of I_Ca,L normalized to peak, indicating delayed I_Ca,L inactivation. The mean residual current at 50, 100, and 200 ms from peak during 0 mV test pulse. (E) Steady state inactivation and activation plots for Con and Cav-3KO myocytes. Mean fit parameters to inactivation data for Con (n = 9) and Cav-3KO (n=16), respectively, are V_1/2 = −23.3 ± 1.1 mV and = −19.7 ± 0.67 mV (P ≤ 0.05), k = 4.5 ± 0.2 and 4.8 ± 0.2, (n.s.). Mean fit parameters for activation data for Con (n = 8) and Cav-3 (n=15), respectively, are V_1/2 = − 10.97 ± 0.6 mV and − 8.73± 0.51 mV (n.s.), k = 4.86 ± 0.2 and 5.13 ± 0.26, (n.s.). (F) Representative Western blot analysis of Na_v1.5, Ca_v1.2, and GAPDH expression in Con and Cav-3KO heart lysates. Right panel, relative Na_v1.5, Ca_v1.2 protein expression levels normalized to GAPDH (n = 4, repeated 3 times). Voltage protocols are as shown in figure insets. Statistical comparisons use two-tailed, unpaired t-test. n.s.=no significant difference.

Fig. 5.. Numerical simulations of action potentials from Morrotti et al. mouse ventricular cell model recapitulate the prolonged action potential duration with loss of Cav-3.

(A) Summary of Cav-3KO/Con peak current ratios for I_K,slow, I_to, I_Na,L, I_ss, and I_Ca,L currents measured experimentally and employed for mathematical modeling. (B) Mathematical modeling accurately reproduced the time dependence of relative I_Ca,L current observed experimentally in Cav-3KO. WT in black and Cav-3KO in red. Experiments left and simulations right. Top: time dependence of current ratio at 50, 100 and 200 ms. Middle: peak IV-curve, Bottom: steady state activation, dss, and inactivation, fss). (C) Top: simulated action potential traces with loss of Cav-3 showing longer APD. Bottom: Superimposed traces of simulated I_Ca,L, I_K,slow, I_ss I_Na,L, I_to, for Cav-3KO and Con myocytes. (D) Mean APD50 and mean APD90 from simulated Con and Cav-3KO myocyte action potentials. Free running action potential (AP) simulations showed dynamic changes in currents at 1 Hz steady state pacing, and a prolonging effect of Cav-3KO on the APD50 and APD90. Con in black and Cav-3KO traces in red.

Fig. 6.. shRNA-mediated Cav-3 knockdown in hiPSC-CMs prolongs APD.

(A) Representative transmission electron micrograph of hiPSC-CMs with morphological caveolae denoted with arrow heads (scale bar 500 μm). (B) Representative Western blot showing loss of Cav-3 protein in lentiviral Cav-3 shRNA infected hiPSC-CMs. (C) Representative traces of action potentials from scrambled shRNA and Cav-3 shRNA infected hiPSC-CMs. (D) Comparison of MDP, APD50, and APD90 from Con and Cav-3 shRNA infected hiPSC-CMs (n = 10 cells from 4 different infections).