Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes

Abstract

T-type Ca(2+) channels (TTCCs) are expressed in the developing heart, are not present in the adult ventricle, and are reexpressed in cardiac diseases involving cardiac dysfunction and premature, arrhythmogenic death. The goal of this study was to determine the functional role of increased Ca(2+) influx through reexpressed TTCCs in the adult heart. A mouse line with cardiac-specific, conditional expression of the alpha1G-TTCC was used to increase Ca(2+) influx through TTCCs. alpha1G hearts had mild increases in contractility but no cardiac histopathology or premature death. This contrasts with the pathological phenotype of a previously studied mouse with increased Ca(2+) influx through the L-type Ca(2+) channel (LTCC) secondary to overexpression of its beta2a subunit. Although alpha1G and beta2a myocytes had similar increases in Ca(2+) influx, alpha1G myocytes had smaller increases in contraction magnitude, and, unlike beta2a myocytes, there were no increases in sarcoplasmic reticulum Ca(2+) loading. Ca(2+) influx through TTCCs also did not induce normal sarcoplasmic reticulum Ca(2+) release. alpha1G myocytes had changes in LTCC, SERCA2a, and phospholamban abundance, which appear to be adaptations that help maintain Ca(2+) homeostasis. Immunostaining suggested that the majority of alpha1G-TTCCs were on the surface membrane. Osmotic shock, which selectively eliminates T-tubules, induced a greater reduction in L- versus TTCC currents. These studies suggest that T- and LTCCs are in different portions of the sarcolemma (surface membrane versus T-tubules) and that Ca(2+) influx through these channels induce different effects on myocyte contractility and lead to distinct cardiac phenotypes.

Figure 1. Properties of mice with α1G-TTCC overexpression

A, Schematic of the bitransgenic system for cardiac-specific α1G overexpression. tTA is the tetracycline-controlled transactivator system. B, Histology of α1G mice was not different than control. C, Representative M-mode echo recording in control and α1G mice. D, Fractional shortening (FS) and ejection fraction (EF) were greater in α1G vs control mice at comparable heart rates. *P<0.05.

Figure 2. Ca²⁺ influx in α1G and β2α myocytes

A, Representative I_Ca,T (I_Ca,L from −50 mV holding potential was subtracted from total Ca²⁺ current recorded from −90 mV holding potential) in control and α1G VMs. B, Voltage–current relationship of I_Ca,T (subtraction as in A) in control (n=10) and α1G (n=15) myocytes. C, Time course of I_Ca,T recovery from inactivation (n=7) and representative example of I_Ca,T recovery from inactivation (inset). D, Representative example of total Ca²⁺ currents under AP voltage clamp in control, β2a, and α1G VMs at 0.5 Hz. E, Integral of total Ca²⁺ current (without drugs) at different pacing frequencies in control (n=6), β2a (n=8), and α1G VMs (n=6). F, Representative example of [Ca²⁺]_i transients at baseline and after thapsigargin application (1 μmol/L) at 1 mmol/L and 3 mmol/L bath Ca²⁺. G, Peak [Ca²⁺]_i transient and rise rate of the [Ca²⁺]_i transient after thapsigargin treatment (in 3 mmol/L Ca) in control (n=20), β2a (n=20), and α1G VMs (n=25).

Figure 3. I_Ca,L density in control, α1G, and β2a VMs

A, Representative examples of I_Ca,L in control, α1G, and β2a VMs. B and C, Voltage–current relationships (B) and peak I_Ca,L (C) in control (n=11), α1G (n=15), and β2a VMs (n=10). D and E, Voltage dependence of I_Ca,L activation (D) and average half-activation potential (V_0.5) (E) in control (n=11), α1G (n=15), and β2a VMs (n=8). *P<0.05, **P<0.01.

Figure 4. APs, contractions, and cytosolic [Ca²⁺]_i transients are altered in α1G VMs

A, Representative example of APs in control and α1G VMs. B, Average AP durations (APDs) at 50%, 70%, and 90% repolarization in control (n=9) and α1G (n=11) VMs. C and E, Representative example of [Ca²⁺]_i transients (C) and contractions (E) from control, β2a, and α1G VMs. D, Peak [Ca²⁺]_i transients and ô (Tau) of control (n=10), β2a (n=19), and α1G VMs (n=17). F, Average fractional shortening (FS) in control (n=10), β2a (n=19), and α1G VMs (n=17). *P<0.05, **P<0.01. See text for discussion.

Figure 5. SR Ca²⁺ content and NCX current are increased in β2a but not in α1G VMs

A, Representative example of caffeine-induced [Ca²⁺]_i transients in control, β2a, and α1G VMs. B, Average data of peak caffeine-induced [Ca²⁺]_i transients and ô of decay in control (n=15), β2a (n=13), and α1G VMs (n=17). C, Representative example of I_NCX from control and α1G VMs. D, Peak I_NCX at +60 mV and −80 mV in control (n=16) and α1G VMs (n=17).

Figure 6. Western blot analysis of Ca²⁺ regulatory proteins in control and α1G hearts

A, Representative Western blots of Ca²⁺ regulatory proteins from control (n=7) and α1G (n=6) hearts. B, Analysis of Ca²⁺ regulatory protein abundance normalized to GAPDH. RyR and PLB phosphorylation levels normalized to total RyR and PLB, respectively. R.U. indicates relative units. *P<0.05, **P<0.01.

Figure 7. I_Ca,T is less effective than I_Ca,L in inducing SR Ca²⁺ release (n=10)

A, Representative example of peak I_Ca,L and I_Ca,T and their corresponding [Ca²⁺]_i transients and contractions. B, Average I_Ca,L (at + 10 mV) and I_Ca,T (at −40 mV), as measured in 1 mmol/L bath Ca²⁺. C and D, Peak [Ca²⁺]_i transients (C) and peak contractions (D) triggered by I_Ca,L and I_Ca,T. E, EC coupling gain, determined as the ratio of peak [Ca²⁺]_i transients/peak current for I_Ca,L and I_Ca,T. F, Maximum rate of rise of the [Ca²⁺]_i transient induced by I_Ca,L and I_Ca,T. G, Maximum rate of contraction attributable to I_Ca,L and I_Ca,T. **P<0.01.

Figure 8. TTCC localization to surface sarcolemma away from the junctional SR

A, TTCC staining pattern (red) in α1G VMs at the level of the nucleus (blue) using an α1G-specific antibody. B, Background α1G staining pattern (red) in control VMs. C, LTCC staining pattern (red) using an α1C-specific antibody. D, RyR staining pattern using a RyR2-specific antibody. E and F, Membrane staining with di-8-ANEPPS (green) showing extensive T-tubular system in normal α1G VMs (E) and lack of T-tubules in detubulated α1G VMs (F) at the level of the nucleus (blue). G, Representative example of peak I_Ca,L and I_Ca,T in detubulated α1G VMs. H, Comparison of peak I_Ca,L and I_Ca,T before (n=15) and after (n=7) detubulation in α1G VMs. **P<0.01.