Caveolin-3 KO disrupts t-tubule structure and decreases t-tubular ICa density in mouse ventricular myocytes

Abstract

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca²⁺ channel current ( I_Ca) density. This decrease in density occurred predominantly in the t-tubules, with no change in total I_Ca, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca²⁺ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on I_Ca in KO myocytes. However, inhibition of PKA using H-89 decreased I_Ca at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na⁺/Ca²⁺ exchanger current or Ca²⁺ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular I_Ca density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca²⁺ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.

Keywords: calcium current; calcium release; calcium transient; caveolin-3.

Fig. 1.

Caveolin-3 (Cav-3) and in vivo cardiac function. A: exemplar Western blots of Cav-3 (18 kDa) and GAPDH (37 kDa) in homogenates from wild-type (WT) and Cav-3 knockout (KO) mice (left) and mean densitometry data for Cav-3 Western blots (N = 5 animals in each group in duplicate; right). B: exemplar echocardiogram recordings from WT (left) and Cav-3 KO (right) mice. C–F: in vivo measurements of left ventricular cardiac function by echocardiography (WT: N = 6 and Cav-3 KO: N = 7). C: fractional shortening (in %). D: ejection fraction (in %). E: left ventricular internal diameter at diastole (in mm). F: left ventricular internal diameter at systole (in mm). *P < 0.05; **P < 0.01.

Fig. 2.

Morphology of isolated myocytes. A: cell width (top) and length (bottom) measured from bright-field images of myocytes from wild-type (WT) and caveolin-3 (Cav-3) knockout (KO) mice [WT n cells/N hearts (n/N): 100/4 and Cav-3 KO n/N: 30/4]. B: confocal images of t-tubules and surface sarcolemma stained with di-8-ANEPPs from representative WT (top) and Cav-3 KO (bottom) myocytes. C: t-tubule power (top) and density (bottom; WT n/N: 20/4 and Cav-3 KO n/N: 20/4). D: t-tubule orientation from the z-disk plane. E and F: Western blots of junctophilin-2 (JPH-2; 95 kDa; E) and bridging integrator-1 (BIN-1; 51 kDa; F) and GAPDH (37 kDa) (left) with corresponding mean data (N = 5 animals in each group in duplicate; right). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Membrane capacitance and Ca²⁺ current (I_Ca). A: cell capacitance (in pF) of intact and detubulated (DT) myocytes used for electrophysiology from wild-type (WT) mice [intact n cells/N hearts (n/N): 35/13 and DT n/N = 38/13] and caveolin-3 (Cav-3) knockout (KO) mice (intact n/N: 39/17 and DT n/N: 43/14). B: exemplar records of I_Ca recorded at 0 mV from intact and DT WT and Cav-3 KO myocytes. C and D: mean I_Ca density-voltage relations from intact (C; WT n/N = 24/7 and Cav-3 KO n/N = 26/11) and DT (D; WT n/N = 28/9 and Cav-3 KO n/N = 31/9) WT and Cav-3 KO myocytes. E and F: mean I_Ca density (E) and absolute I_Ca (F) at 0 mV in intact and DT (“surface”) cells and calculated at the t-tubule membrane (“t-tubule”) for WT (open columns) and Cav-3 KO (solid columns) myocytes. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Ca²⁺ current (I_Ca) in the presence of H-89. A: Western blots of the L-type Ca²⁺ channel (LTCC; 160 kDa) and GAPDH (37 kDa; left) and mean densitometry data (N = 5 animals in each group in duplicate; right). B and C: I_Ca density-voltage relations recorded in the presence of H-89 from intact [B; wild type (WT) n cells/N hearts (n/N): 16/9 and caveolin-3 (Cav-3) knockout (KO) n/N: 8/5] and detubulated (DT; C; WT n/N: 14/7 and Cav-3 KO n/N: 11/3) WT and Cav-3 KO myocytes. D and E: mean I_Ca density (D) and absolute I_Ca (E) at 0 mV in the presence of H-89 in intact and DT (“surface”) cells and calculated at the t-tubule membrane (“t-tubule”) for WT (open columns) and Cav-3 KO (solid columns) myocytes. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Intracellular Ca²⁺ and Na⁺/Ca²⁺ exchanger current (I_NCX) during application of caffeine. A and B: exemplar records of the rise of intracellular Ca²⁺ (top) caused by application of 10 mM caffeine to intact (A) and detubulated (DT; B) wild-type (WT) and knockout (KO) myocytes and the accompanying inward currents (I_NCX; bottom). C: mean amplitude of the caffeine-induced rise of intracellular Ca²⁺ [intact: WT n cells/N hearts (n/N): 11/6 and Cav-3 KO n/N = 13/6; DT: WT n/N = 10/4, Cav-3 KO n/N = 12/3]. D: distribution of I_NCX density, calculated as previously described (26).

Fig. 6.

Systolic Ca²⁺ transients and local Ca²⁺ release. A: representative Ca²⁺ transients recorded from wild-type (WT) and caveolin-3 (Cav-3) knockout (KO) myocytes. B: Ca²⁺ transient amplitude, time to peak, and time to half decay (T₅₀) measured from Ca²⁺ transients of WT [n cells/N hearts (n/N): 12/3] and Cav-3 KO (n/N = 14/3) cells. C: representative optical measurements of the rising phase of the Ca²⁺ transient and associated average t-tubular di-4-AN(F)EPPTEA signal. AP, action potential. D: mean latency and heterogeneity of sarcoplasmic reticulum Ca²⁺ release in WT (n/N = 10/3) and Cav-3 KO (n/N = 26/5) myocytes.