Rem-GTPase regulates cardiac myocyte L-type calcium current

Abstract

Rationale: The L-type calcium channels (LTCC) are critical for maintaining Ca(2+)-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested.

Objective: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca(2+) current (I(Ca,L)) via LTCC in murine cardiomyocytes.

Methods and results: Rem knockout mice (Rem(-/-)) were engineered, and I(Ca,L) and Ca(2+) -handling properties were assessed. Rem(-/-) ventricular cardiomyocytes displayed increased I(Ca,L) density. I(Ca,L) activation was shifted positive on the voltage axis, and β-adrenergic stimulation normalized this shift compared with wild-type I(Ca,L). Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem(-/-) . Cell shortening was not significantly different. Increased I(Ca,L) density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I(Ca,L) density, Rem(-/-) cardiomyocyte Ca(2+) twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem(-/-) LTCC can account for the paradoxical decrease of Ca(2+) transients.

Conclusions: This is the first demonstration that loss of an RGK protein influences I(Ca,L) in vivo in cardiac myocytes.

Figure 1. Rem knockout mouse. (A) Schematic representation of the strategy used to generate the Rem knockout mouse. The coding region of Exon 2 was targeted and replaced by a LacZ expressing cassette, followed by the neomycin gene flanked by FRT sites. Arrows indicate the location of genotyping primers (a, b, c, and d). (B) Genomic DNA was isolated from mouse tail biopsies, and subjected to PCR analysis using the oligonucleotide primer pairs indicated in panel A. A 348 base pair band represents the mutant allele (lower panel; amplification using primer pair c/d), while a 500 base pair band represents the wild-type allele (upper panel; amplification using primer pair a/b). (C) Total protein was isolated from hearts obtained from Rem mutant or WT-littermates, and subjected to anti-Rem immunoblot analysis. Wild-type Rem expressed in HEK293 cells was included as a positive control. Note that Rem protein levels are completely eliminated in Rem^−/− cardiac muscle, while reduced in Rem^+/− animals.

Figure 2. I_Ca,L density for wild-type and Rem^−/− cardiac ventricular myocytes. (A) Representative original traces from wild-type and Rem^−/− mice. (B) Average I_Ca,L-V relationships for wild-type and Rem^−/− cardiac myocytes. (C) The voltage dependence of steady-state activation and steady-state inactivation of I_Ca,L for wild-type and Rem^−/− cardiac myocytes. V_½ of steady-state activation is significantly shifted +4mV for Rem^−/− compared with wild type. V_½ was obtained from fitting the current-voltage data as in panel B to a modified Boltzmann distribution of the form: I(V) = G_max*(V-E_rev)/(1+exp(V_½ - V)/k), where G_max is maximal conductance, E_rev is reversal potential, V_½ is activation midpoint potential, and k is the slope factor. The voltage dependence of steady-state inactivation of I_Ca,L was measured by a double-pulse protocol. The overlap of steady-state activation and steady-state inactivation curves (cross-hatched) illustrates decrease of window I_Ca,L in Rem^−/− compared with wild-type cardiac myocytes.

Figure 3. Isoproterenol normalizes the shift of activation V_½ for Rem^−/− compared with wild type. (A) Activation curves for wild-type I_Ca,L in control bath and 10nM isoproterenol (V_½ for control and isoproterenol were -7.2 ± 0.3mV and -11.7 ± 0.3mV, respectively; p < 10⁻³; n = 12). (B) Activation curves for Rem^−/− I_Ca,L in control bath, and following 10 nM isoproterenol (V_½ for control and isoproterenol were -3.3 ± 0.2mV and -9.4 ± 0.3mV, respectively; p < 10⁻⁴; n = 24).

Figure 4. I_NCX in wild-type and Rem^−/− ventricular myocytes. Upper panel shows voltage protocol. Outward I_NCX measured at +40 mV, and inward I_NCX measured at -80mV as indicated on hyperpolarizing ramp. Mean I_NCX is greater in wild type compared with control for (A) outward and (B) inward current. *p < 0.05

Figure 5. Ca-transients in intact ventricular myocytes. One Hz pacing reveals decreased twitch amplitude, without an effect on SR load in Rem^−/− cardiomyocytes. (A) Representative single twitch Ca²⁺-transient from myocytes 1 paced at 1Hz. (B) Rem^−/− twitch Ca-amplitude is significantly smaller than that in wild type. SR Ca-load was assessed by rapid application of 10mM caffeine. SR Ca load is not significantly different between Rem^−/− and wild type. n = 25 and 19, Rem^−/−, and wild type, respectively; *p < 0.01 control; *p < 0.05 in isoproterenol. (C) Decay kinetics of twitch Ca transients are not significantly different between Rem^−/− and wild type. (D) Simultaneous measure of fractional cell shortening revealed no significant different between Rem^−/− and wild type in either control or isoproterenol bath solution. (E and F) Computer simulations capture the decreased Ca transient amplitude in response to a +4mV shift of I_Ca,L steady-state activation. (E) Superimposed steady-state inactivation curve normalized to maximal conductance, and steady-state activation curve. For the activation curve, Gmax = 0.45 pS/pF for Rem^−/− and 0.35pS/pF for wt. Inset shows activation curves expanded for voltage ranging from -40 to -10mV. (F) Simulated Ca²⁺-transients shows smaller peak Ca²⁺-transient amplitude in Rem^−/− compared with wt.

Figure 6. Analysis of Ca_V1.2 and phospholamban phosphorylation in wt and Rem^−/−. (A) Protein extracts from wild-type and Rem^−/− hearts were subjected to western blotting and probed with antibodies recognizing Ca_V1.2 distal carboxyl terminus (DCT) and DCT-Ser1928^P. Size marker (horizontal line) is 37kDa. n = 4 Rem^−/− and n = 4 wild type. (B) Protein extract was not boiled prior to gel loading to preserve multimeric PLB structure. Six bands for PLN are resolvable; Ser16^P, and Thr17^P are confined to upper two bands. (C) Bar graph summarizes fractional phosphorylation. *p < 0.002; n = 4 Rem^−/− and n = 4 wild type. PLB phosphorylation was not significantly different. n = 6. (D) Ca_V1.2 expression is downregulated in Rem^−/− vs. wt heart; 74+/− 4% decrease in Rem^−/− vs. wild type; n = 3 Rem^−/− and n = 3 wild type; p < 0.01.