Cardiac GRK2 Ablation Induces a Novel Ca²⁺ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Abstract

Background

G-protein coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. GRK2 ablation ameliorates heart failure development but the cellular mechanisms have not been elucidated and are the aim of this study.

Methods and Results

Myocyte contractility, Ca²⁺ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type (WT) and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed WT and GRK2KO hearts, myocyte contractions and Ca²⁺ transients were similar but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca²⁺ content due to increased sodium-Ca²⁺ exchanger (NCX) activity and inhibited SR Ca²⁺ ATPase by local PKA mediated activation of PDE4 resulting in hypophosphorylated phospholamban. This Ca²⁺ handling phenotype is explained by a higher fractional SR Ca²⁺ release induced by increased L-type Ca²⁺ channel currents (I_Ca,L). After β-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca²⁺ transients, which were not mediated through cardiac L-type Ca²⁺ channels (LTCCs), but through an increased SR Ca²⁺. Interestingly, post-MI GRK2KO mice showed better cardiac function than post-MI control mice, which is explained by an improved Ca²⁺ handling phenotype. The SR Ca²⁺ content was better maintained in post-MI GRK2KO myocytes than in post-MI control myocytes, due to better maintained I_Ca,L density and no increase in NCX in GRK2KO myocytes. An LTCC blocker, verapamil, reversed some beneficial effects of GRK2KO.

Conclusions

These data argue for novel differential regulation of I_Ca,L and SR load by GRK2. GRK2 ablation represents a novel beneficial Ca²⁺ handling phenotype resisting adverse remodeling after MI.

Introduction

Alterations in myocyte contractility and Ca²⁺ cycling are hallmarks of the progression of heart failure (HF). In failing cardiac myocytes, the SR Ca²⁺ content is decreased and the Ca²⁺ transient is decreased and prolonged, resulting in depressed myocyte contractility¹. This is generally considered to be a consequence of increased sodium– Ca²⁺ exchanger (NCX) activity, reduced sarcoplasmic (SR) reticulum Ca²⁺ ATPase (SERCA2a) expression and activity (probably due to decreased phospholamban (PLB) phosphorylation), and increased PLB/SERCA2a ratio, as well as an augmented open probability of the ryanodine receptor (RyR), causing SR Ca²⁺ leak ^{2, 3}. Alterations of the L-type Ca²⁺ channel (LTCC), the trigger for the Ca²⁺ induced-SR Ca²⁺ release, also contribute to the pathophysiological changes in Ca²⁺ homeostasis in failing myocytes⁴. In addition to these changes causing dysfunctional contractile performance, the rise of diastolic Ca²⁺ may increase the risk of arrhythmias and induce pathological cardiac remodeling⁵.

The increased activity of the sympathetic nervous system associated with HF is a compensation to normalize cardiac function by enhancing Ca²⁺ cycling and maximize contractile force through the β-adrenergic signaling pathway. In acute HF these changes can improve systemic perfusion, whereas in chronic HF the augmentation in catecholamines is associated with mortality⁶ and results in a down-regulation of β-adrenergic receptors (βARs) promoted by upregulated G protein-coupled receptor kinase 2 (GRK2). GRK2 is the primary GRK in the heart and a prototype regulator of βAR signaling⁷. We have previously identified GRK2 as a culprit in the progression of HF and GRK2 inhibition (by expression of its c-terminal domain, called βARKct) or gene silencing has rescued disparate models of HF^8-10. Our recent study indicates the benefits of βARKct could be related to enhanced myocyte contractility by increasing LTCC currents and its responsiveness to β-adrenergic agonists¹¹. However, the exact underlying cellular mechanisms for these beneficial effects in HF after βARKct expression or GRK2 ablation are not clearly defined. It is especially important to define these mechanisms since in light of the recent success of βAR blocker therapy in clinical HF management, the results with βARKct and GRK2 silencing appear paradox, as the major function of GRK2 in cardiac myocytes is to dampen βAR signaling, similar to βAR blockade. We have found that βARKct expression can cause a molecular remodeling of the cardiac βAR system with receptor up-regulation and improved βAR signaling, and a recent study with chronic AAV-mediated βARKct expression in a rat HF model showed that myocardial βAR changes are probably down-stream of neurohormonal lowering including reduction in sympathetic nervous system activity¹². In this regard, the role of GRK2 inhibition must mechanistically go beyond resensitizing βARs and fully understanding GRK2-dependent signaling pathways might enlighten novel therapeutic targets.

To date, little is known about how GRK2 specifically alters cardiac myocyte function and Ca²⁺ cycling in normal and failing cardiac myocytes. The present study was designed to define the role of GRK2 and GRK2-dependent signaling in excitation-contraction coupling (ECC) in normal and diseased hearts. We used our previously characterized cardiac-specific GRK2 knockout mice^{10, 13} to study myocyte ECC coupling and Ca²⁺ homeostasis in cardiac myocytes from mice with or without post-MI ischemic HF. We demonstrate for the first time that loss of GRK2 induces a distinct Ca²⁺ handling phenotype: myocyte contractility and Ca²⁺ handling are normal even though the SR Ca²⁺ content is reduced because there is an increased LTCC activities and resulting increases in I_Ca,L with a compensatory increase in NCX activity. This Ca²⁺ handling phenotype brought about by GRK2 ablation is resistant to adverse Ca²⁺ handling remodeling after MI and leads to better cardiac function in GRK2 KO mice post MI.

Materials and Methods

Conditional mice bearing floxed GRK2 alleles were described previously^{10, 13}. GRK2KO (αMHC-Cre × GRK2fl/fl) and WT (GRK2fl/fl) mice were maintained on a C57BL6 genetic background. All animal procedures and experiments were performed in accordance with the guidelines of the IACUC of Thomas Jefferson University. GRK2KO and WT mice were 8-10 weeks of age when entering the study. Unstressed normal mice and mice with coronary artery ligation (myocardial infarction (MI)) or sham-operation were studied. MI was induced by ligating the left anterior descending coronary artery at 2-3 mm below its origin as described previously^{10, 14} and animals were studied 28 days post-MI or sham-operation. For the verapamil study, mice were treated with verapamil starting 14 days post MI or sham-operation until the end of the study period (42 days post MI). Verapamil treated mice received oral supplementation of verapamil (Sigma-Aldrich, St. Louis, Missouri, USA) as described previously¹⁵ and were evaluated by echocardiography (ECHO). Cardiac myocytes were isolated cultured from animals. Myocyte Ca²⁺ transients and contractions, SR Ca²⁺ load, LTCC currents (I_Ca,L), and I_NCX were measured. Quantitative real-time polymerase chain reaction and Western blot analysis were done for gene expression assessment. Detailed description of experimental procedures is available in the expanded online-only data supplement.

Data are expressed as mean±SEM. An unpaired two tailed t-test or a one-way ANOVA and a two-way ANOVA (linear mixed effects model) were performed with SAS 9.3 for between-group comparisons followed by a post-hoc Bonferroni adjustment. For all tests, a P value < 0.05 was considered significant.

Results

Myocyte GRK2 ablation enhances adrenergic responsiveness of cellular contractility and Ca²⁺ transients

Our previous study has shown that GRK2 KO mice have comparable cardiac function as control mice at baseline (Supplemental Table 1) but their cardiac function responds to β-adrenergic stimulation better¹⁰. Here we determine the cellular mechanisms for this observation. Myocyte contraction and intracellular Ca²⁺ transients were recorded from WT and GRK2KO myocytes under baseline conditions and after βAR stimulation. Myocytes from both lines revealed a similar fractional shortening when paced at both 0.5Hz and 2Hz (Figure 1A-C). However, after isoproterenol (ISO), myocytes from GRK2 KO mice showed a significantly greater increase in fractional shortening (Figure 1A-C, Supplemental Table 2). In a good agreement, as shown in Figure 1D, at baseline, the characteristics and amplitude of the Ca²⁺ transients (as βFura-2 340/380nm ratio) were similar in both groups of myocytes. Stimulation with ISO mediated an anticipated increase in the amplitude of the Fura-2 ratio in WT cardiac myocytes and a greater increase in GRK2KO cardiac myocytes (Figure 1D-F, Supplemental Table 2). We observed no differences in the Ca²⁺ transient decay time at baseline and after ISO between groups.

Figure 1.

The loss of GRK2 in myocytes enhances the responsiveness of myocyte contraction and Ca²⁺ transients after bAR stimulation. A, Representative tracings of single myocyte contractions under basal conditions and stimulation with isoproterenol at 0.5 Hz. Averaged myocyte fractional shortening at 0.5 Hz (B) and 2.0 Hz (C) stimulation frequencies under basal conditions and stimulation with isoproterenol. D, Representative intracellular Ca²⁺ transients measured with Fura-2 [340/380nm ratio] under basal conditions and stimulation with isoproterenol at 0.5 Hz from the same cell as in A. Averaged Fura-2 ratio amplitude at 0.5 Hz (E) and 2.0 Hz (F) stimulation frequencies under basal conditions and isoproterenol. For measurements represented by B, C, E and F; a total of 48-63 cardiac myocytes from 3 different hearts were measured for each group; two-way ANOVA was used for B, C, E and F.

GRK2 silencing in myocytes enhances ECC efficiency

Myocyte contractility and Ca²⁺ transients are determined by Ca²⁺ release from the SR¹⁶. We measured SR Ca²⁺ content by caffeine spritz at baseline or after ISO stimulation. Figure 2A shows representative tracings of cytosolic Ca²⁺ transients (measured with indo-1 AM) induced by a rapid application of caffeine (caffeine-spritz) after 4 field stimulations in WT and GRK2KO cardiac myocytes to measure the SR Ca²⁺ load. At baseline SR Ca²⁺ load is less in GRK2 myocytes than in control myocytes (Figure 2B); in response to ISO, the SR Ca²⁺ load in cardiac myocytes from both WT and GRK2KO mice was significantly increased but GRK2KO myocytes had a greater increase (Figure 2B). Interestingly, a significantly higher fractional release of Ca²⁺ was observed in GRK2KO cardiac myocytes compared to WT myocytes at both baseline and after ISO (Figure 2C, Supplemental Table 2).

Figure 2.

GRK2 silencing in cardiac myocytes alters SR Ca²⁺ content and its regulation. A, Representative tracings of the caffeine-induced intracellular Ca²⁺ transients determined with Indo-1 in a control myocyte and a GRK2KO myocyte. B, Caffeine-induced peak intracellular Ca²⁺ amplitudes under baseline conditions and isoproterenol stimulation. C, Fractional release calculated as the ratio of peak Ca²⁺ concentration induced by field stimulation to peak Ca²⁺ concentration induced by caffeine. For measurements represented by figures B-C; a total of 16-29 cardiac myocytes from 3 different hearts were analyzed per group; two-way ANOVA with post hoc student t-test for B and C.

Loss of myocyte GRK2 enhances I_Ca,L by a local PKA-dependent mechanism but blunts its βAR responsiveness

To explore the underlying cellular mechanisms for the reduced SR Ca²⁺ content while enhanced EC coupling efficiency in GRK2 KO myocytes, we measured the LTCC current (I_Ca,L) in GRK2 KO and control myocytes because I_Ca,L serves as both the trigger of Ca²⁺ release from the SR and the source for loading the SR.¹⁶. Peak I_Ca,L density at baseline was significantly greater in GRK2KO compared to WT cardiac myocytes (Figure 3A and B, Supplemental Table 2). When stimulated with a saturating dose of ISO (10^-6M), peak I_Ca,L was increased to the same level in cardiac myocytes from both mouse lines (Figure 3A and B), suggesting that the LTCC density was similar in both groups⁴. The voltage-dependency of channel activation was shifted to more negative voltages in GRK2KO myocytes at baseline and ISO stimulation caused a significant leftward shift of activation in WT myocytes but not in GRK2KO myocytes (Figure 3C and D). These results imply that the LTCC in GRK2KO myocytes could be in such a high activity state that it loses responsiveness to β-adrenergic stimulation.

Figure 3.

GRK2 silencing in myocytes increases basal I_Ca,L by enhancing PKA-dependent augmentation of LTCC channel activity but reduces its responsiveness to b-adrenergic stimulation. A, Current-voltage relationships of I_Ca,L (pA/pF) under basal conditions and isoproterenol stimulation. B, Peak I_Ca,L under basal conditions and isoproterenol stimulation. C, Voltage dependence of I_Ca,L activation at basal conditions and under isoproterenol stimulation. D, Voltage potentials at 50% I_Ca,L activation in WT and KO myocytes before and after ISO. E, Charge movement measurements in control and KO myocytes at baseline, showing no difference in available LTCCs on the surface membrane in KO and WT myocytes. F, The effect of H89 on the current-voltage relationships of I_Ca,L in sham WT control and KO myocytes. H89 significantly decreased I_Ca,L at most tested voltages in KO myocytes but not in WT myocytes. G, Effects of H89 on maximum I_Ca,L in WT and KO myocytes. H & I, Effects of H89 on V0.5 of I_Ca,L activation in WT and KO myocytes. J, Examples of single channel recordings of the LTCC in KO and WT myocytes before and after the treatment of H89. Sweeps were selected in an increment of 20, i.e., the sweeps shown were sweep 1, 20, 40, etc. The downward deflections were the openings of the channel. At the bottom of the raw sweeps were the ensemble average current of all 400 sweeps recorded. The availability (K), open probability in active sweeps (L) and the overall open probability (NPo shown in M) were significantly increased in KO myocytes but were normalized by H89. H89 did not have significant effects on these parameters of single LTCC activities in WT myocytes. N, Western blot of immunoprecipated total a1c and phosphorylated a1c at ser1928. O, Phosphorylation level of a1c in unstressed WT and GRK2KO hearts. n=5 animals/group, two-way ANOVA with post hoc t-test was used for comparison for A-M in this figure; t-test was used for O.

To explore the underlying mechanisms for these changes of I_Ca,L properties, we tested whether the increased I_Ca,L was due to increased available channels on the surface membrane and/or the increase of channel activities at single channel levels. Charge movement was used to quantitate the amount of available channels on the surface membrane of KO and control WT myocytes. Figure 3E shows that there was no significant difference in the charge movements induced by various depolarizing voltages, indicating that there was no significant alteration of LTCC density on the membrane and the increased whole-cell I_Ca,L could be due to the increased single channel activities. Immunoprecipitation of α1c, the pore forming subunit of the LTCC, from the same amount of proteins from GRK2KO and control hearts showed no difference in α1c expression (Figure 3N). These data suggest that the LTCC in GRK2KO myocytes must have higher than normal activity, which was supported by single channel recording of LTCC activities (Figure 3J-M). The availability (Figure 3K) and the open probabilities (Figure 3L and M) were significantly increased in GRK2KO myocytes, which could fully explain the increase in whole cell I_Ca,L in KO myocytes.

Enhanced phosphorylation of the LTCC by protein kinase A (PKA) may result in increased LTCC activity^{4, 17}. Previously we have shown that βARs carries constitutive activation to activate PKA locally to phosphorylate the LTCC¹⁸. Here we tested if high LTCC activity was mediated by a PKA dependent mechanism. H89, a PKA specific inhibitor, was used and it normalized the current density and voltage-dependent activation of the LTCC in KO myocytes but had no significant effect on LTCCs in WT myocytes (Figure 3F-I). The single channel study further confirmed that the heightened LTCC activity in the KO myocytes was due to PKA activation because H89 also normalized the increased channel activity in KO myocytes (Figure 3J-M). The phosphorylation of α1c at Ser1928, a PKA site, was shown to be more in GRK2KO hearts than in control hearts (Figure 3N and O).

Loss of GRK2 in myocytes increases Na⁺/Ca²⁺ exchanger expression

Since there is a decreased SR Ca²⁺ content with increased I_Ca,L in GRK2KO myocytes, there should be increased Ca²⁺ efflux out of GRK2KO myocytes. In ventricular myocytes, the major route of Ca²⁺ efflux is through the sodium-Ca²⁺ exchanger (NCX). One way to measure NCX activity is to examine the decay rate of caffeine-induced Ca²⁺ transients that can be fit by a single exponential decay equation¹⁹. The tau value was significantly smaller in the GRK2 KO myocytes than in control myocytes and ISO did not change these values (Figure 4A), suggesting that the NCX activity at baseline is increased in GRK2KO myocytes compared to WT myocytes, which was confirmed by direct measurement of NCX current (Figure 4B) and NCX protein expression (Figure 4C and D).

Figure 4.

GRK2 silencing in cardiac myocytes increased NCX expression, reduced PLB phosphorylation by PKA dependent PDE4 activation and enhanced responsiveness to ISO. A, Tau values of caffeine-induced intracellular Ca²⁺ transient decay, indicating increased NCX activity in GRK2KO myocytes; n=16-29 cardiac myocytes from 3 different hearts. B, NCX currents at baseline in control (WT) and GRK2KO myocytes; n=4 animals/group, two-way repeated ANOVA with post-hoc student t-test. C, Western blot of NCX proteins in WT and GRK2KO hearts. D, NCX expression normalized to GAPDH. E and F, SERCA expression in WT and GRK2KO hearts. G and H, PLB expression and phosphorylation at Ser16 (PKA site) and Thr17 (CaMK II site) in WT and GRK2KO hearts; n=5 animals/group D, F, and H; t-test for D, F, and H. I and J, PLB phosphorylation at Ser16 in response to different concentrations of ISO; n=5animals/group; data were analyzed by regression with repeated measures for J. K and L, Western blotting for pSer16-PLB and total PLB in WT and GRK2KO hearts stimulated with or without the selective PDE4 inhibitor rolipram; a total of 3-5 hearts were analyzed for each group; one-way ANOVA for L.

Loss of myocytes GRK2 decreases basal phospholamban (PLB) phosphorylation but increases its responsiveness to βAR stimulation

The loading of the SR with Ca²⁺ depends on the competition between the extrusion of Ca²⁺ out of the cell (mainly through NCX) and the resequestration of Ca²⁺ into SR by SERCA which is regulated by PLB¹⁶. Dephosphorylated PLB exerts a tonic inhibition on SERCA activity. For these reasons, we determined the expression level of SERCA and PLB as well as the phosphorylation level of PLB. The expression of SERCA and PLB was not significantly altered by silencing GRK2 (Figure 4E-H). However, the phosphorylation of PLB at Ser16, a PKA site, was significantly reduced in GRK2KO hearts but the phosphorylation of PLB at Thr17 site was not altered (Figure 4G and H). When myocytes were stimulated with the βAR agonist, ISO, a robust increase in the phosphorylation of PLB at Ser16 sites was observed in both WT and GRK2KO cardiac myocytes (Figure 4I and J). However, a significant leftward shift of the dose response curve was found in GRK2KO myocytes compared to WT cells (Figure 4J), demonstrating higher βAR sensitivity in cells isolated from unstressed GRK2KO mice.

To further clarify the mechanism responsible for the hypophosphorylation of PLB we examined whether PDE 4, which is activated by PKA, could play a mechanistic role. A PDE 4 specific inhibitor rolipram (10mg/kg BW, i.p.) was injected into unstressed WT and GRK2KO mice for 2 hours to allow it to take effect. Then the hearts were snap-frozen in liquid nitrogen and Western blotting for phospho-PLB and total PLB was performed. Interestingly, rolipram blunted the hypophosphorylation of PLB in GRK2KO myocytes (Figures 4K and L), which indicates that the proposed higher PKA activity in GRK2KO myocytes results in an increased local PDE4 activity causing hypophosphorylation of PLB.

Loss of myocyte GRK2 before MI prevents the development of heart failure and preserves myocytes contractility

Our previous studies have shown that cardiac specific loss of GRK2 ameliorates the development of heart failure after MI¹⁰. In vivo cardiac function as assessed by echocardiography 28 days after MI showed that although sham GRK2KO mice were indistinguishable from sham WT mice, GRK2KO mice had significantly improved post-MI cardiac function and ventricular remodeling after the loss of myocyte GRK2 (Supplemental Table 1). Since this improved post-MI cardiac function was seen in GRK2KO mice with similar infarct sizes, the beneficial effects of GRK2 deficiency probably occurs at the myocyte level. Therefore, the function of cardiac myocytes isolated from WT and GRK2KO mice at 28 days after MI were determined. The basal fractional shortening (FS) and Ca²⁺ transient amplitudes in myocytes from GRK2KO mice post MI (GRK2KO MI) were almost normal compared to GRK2KO myocytes from mice not subject to MI and greater than those of WT cardiac myocytes post-MI (WT MI) at both pacing frequencies of 0.5Hz and 2Hz (Figure 5A, B, D and E, Supplemental Table 2). Furthermore, WT MI myocytes had a blunted functional response to ISO. In contrast, cardiac myocytes from GRK2KO mice post-MI displayed significantly improved β-adrenergic responses (Figure 5A, B, D and E). These results clearly show that the loss of GRK2 in cardiac myocytes can partially prevent pathological cellular mechanical and Ca²⁺ handling remodeling after MI, and provide a potential cellular mechanism for the benefits of GRK2 lowering or inhibition in the failing heart.

Figure 5.

Loss of GRK2 in cardiac myocytes ameliorates single cell contractility and Ca²⁺ handling post-MI. Measurements were obtained at 0.5Hz (A-C) or 2Hz (D-F) under basal conditions and isoproterenol stimulation (10-8M). Fractional shortenings (A & D), Fura-2 ratio amplitudes (B & E) representing the change of intracellular Ca²⁺ transient from baseline, and tau of Ca²⁺ transient decay (C & F) were shown. For all measurements a total of 60 cardiac myocytes from 6 different hearts were measured for each group; two-way ANOVA.

GRK2KO MI myocytes have preserved SR Ca²⁺ loading and I_Ca,L

A decrease in the SR Ca²⁺ load is a contributing factor for the depressed myocyte contractility, a hallmark of HF. Although GRK2KO mice without MI having a lower SR Ca²⁺ load than WT mice without MI, the SR Ca²⁺ load in GRK2KO MI myocytes was not decreased as in WT MI myocytes (Figure 6A and B, Supplemental Table 2). Fractional Ca²⁺ release from the SR was higher in GRK2KO MI myocytes as well, explaining the preserved myocyte contractility (see above and Figure 6C). NCX-activity, as indirectly assessed by the decay constant tau of the caffeine-induced peak Ca²⁺ transient was normalized in post-MI GRK2KO myocytes, despite significant increases in NCX activity in infarcted WT mice consistent with severe heart failure (Figure 6D).

Figure 6.

Myocyte GRK2 silencing inhibits adverse Ca²⁺ handling remodeling post-MI. A, Representative tracings of the caffeine-induced intracellular Ca²⁺ transients determined with Indo-1; B, Amplitudes of Ca²⁺ transients elicited by field stimulation (0.5Hz) and caffeine-spritz; C, Fractional release calculated as the ratio of peak Ca²⁺ concentration induced by field stimulation to peak Ca²⁺ concentration induced by caffeine; D, Tau of caffeine-induced intracellular Ca²⁺ transients. n=14-23 cells from 4-5 animals/group, unpaired two-tailed t-test for B, two-way ANOVA for C and D; E, Current-voltage relationship of I_Ca,L (pA/pF) under basal conditions and isoproterenol stimulation in post-MI WT and KO myocytes; F, % increase in peak I_Ca,L by isoproterenol. G, Voltage dependence (G-V curve) of I_Ca,L activation at basal conditions and under isoproterenol stimulation. H, Voltages at 50% I_Ca,L activation; n=7 cells from 3-4 different animals/group; one-way ANOVA for E-H.

Improvements in intracellular Ca²⁺ transients and SR fractional Ca²⁺ release in the GRK2KO MI myocytes could result from changes in cardiac myocyte I_Ca,L. Although basal I_Ca,L amplitudes in both post-MI WT and GRK2KO myocytes were reduced compared to pre-MI values (see Figure 3A and B), peak I_Ca,L in post-MI myocytes were significantly greater with the loss of GRK2 (Figure 6E, Supplemental Table 2). When stimulated with ISO (10^-6M), I_Ca,L in GRK2KO post-MI myocytes was only insignificantly increased (12.9±6.9%, n=4) but I_Ca,L in post-MI WT myocytes was significantly increased by 84.3±18.1% (n=7) (Figure 6F). However, after ISO, I_Ca,L amplitudes in myocytes were not different between the two mouse lines (Figure 6G and H), indicating similar LTCC density. The marked enhancement in basal I_Ca,L in the GRK2KO MI myocytes might contribute to the normalization of intracellular Ca²⁺ handling and improved cardiac myocyte contractility. Of interest, the ISO stimulation caused a significant leftward shift of voltage-dependency of channel activation in WT post-MI myocytes but not in GRK2KO myocytes probably because the activation of I_Ca,L in GRK2KO MI myocytes at baseline was already shifted to the left. After ISO stimulation the voltage-dependent activation of I_Ca,L was similar in both groups (Figure 6G and H).

Loss of myocyte GRK2 inhibits adverse cellular remodeling post-MI

Cardiac myocyte size at 28 days post-MI was assessed by measurements of myocyte capacitance. Myocytes isolated from GRK2KO MI mice had significantly smaller capacitance, indicating less myocyte hypertrophy and inhibition of adverse cellular remodeling compared to WT mice post-MI (GRK2KO MI 211±19pF vs. WT MI 311±35pF vs. WT Sham 178±19pF vs. KO Sham 188±15pF; p<0.05 between GRK2KO MI and WT MI).

Beneficial effects of myocyte GRK2 silencing is suppressed by the LTCC blocker verapamil

To determine if the beneficial Ca²⁺ effects seen post-MI after myocyte GRK2 lowering is mediated through the novel changes in LTCC function, we treated WT and GRK2KO mice with verapamil from 14 days till 42 days post-MI. Interestingly, echocardiography revealed that verapamil treatment negated some of the beneficial effects of GRK2 silencing on post-MI cardiac function while the LTCC blocker had minimal effects on post-MI WT mice (Figure 7A). Furthermore, cardiac BNP mRNA expression as a molecular marker of HF was significantly lower in post-MI GRK2KO mice compared to WT MI mice but was reversed by verapamil treatment to the level seen in post-MI WT mice (Figure 7C). It could be true that the absolute amount of Ca²⁺ current blocked by verapamil is more in GRK2 KO myocytes because the total LTCC density is higher in GRK2 KO MI myocytes and thus verapamil had a stronger effect in KO MI myocytes. In conclusion, it appears that the beneficial effects offered by a loss of GRK2 in cardiomyocytes are at least in part attributable to the upregulation of I_Ca,L.

Figure 7.

Beneficial effects of myocytes GRK2 lowering are reduced by the LTCC blocker verapamil. Echocardiographic measurements of fractional shortening [FS] (A) and left ventricular end diastolic diameter [EDD] (B) at 42 days post MI or sham operation. Mice were supplemented orally with verapamil starting 14 days post MI until the end of the study period (= 42 days post MI). n=6-8/sham group, n=15-25/MI group without verapamil, n=10-13/MI group with verapamil, one-way ANOVA. C, cardiac myocyte BNP mRNA levels 42 days post MI or sham operation; n=5-6/group for sham, n=8-9/group for MI; one-way ANOVA and unpaired two-tailed t-test.

Discussion

GRK2 is an important molecule in the heart not only as a primary regulator of adrenergic signaling but also claims an important role in the development of HF^8-10. It is up-regulated during the early stage in injured myocardium, indicating that it participates in the progression of ventricular dysfunction and cardiomyopathy^8-10. Our previous studies have shown that GRK2 silencing¹⁰ or inhibition by βARKct²⁰ is able to improve cardiac function during HF progression after MI. However, the specific role of GRK2 in the regulation of normal and failing Ca²⁺ cycling has never been studied. In heart failure, myocyte Ca²⁺ cycling is deranged, and abnormalities include altered cardiac LTCC density and properties and reduced SR Ca²⁺ content due to decreased SERCA2a and increased NCX-activities. These changes result in reduced intracellular Ca²⁺ transients and depressed myocyte contractility^{1, 4}. Our current study has revealed that GRK2 can influence myocyte Ca²⁺ homeostasis and its absence in cardiac myocytes^{10, 13} causes a novel Ca²⁺ handling phenotype that is resistant to cardiac function deterioration after MI. The benefits rendered by GRK2 silencing is associated with the differential regulation of sarcolemmal and SR Ca²⁺ handling by the β-adrenergic system.

1. A novel Ca²⁺ handling phenotype induced by GRK2 silencing

Although GRK2 plays an important role in regulating the β-adrenergic system, its KO does not affect basal cardiac¹⁰ and cardiomyocyte function. Myocyte loss of GRK2 did not change characteristics of basal intracellular Ca²⁺ transients and myocyte contractions. However, detailed characterization of myocyte Ca²⁺ handling have shown many differences in ECC coupling between GRK2KO and WT myocytes: (1) The SR Ca²⁺ content is reduced in GRK2 KO myocytes but Ca²⁺ transient and contraction in GRK2KO myocytes are normal because an increased fractional Ca²⁺ release from the SR; (2) Increased I_Ca,L ensures normal Ca²⁺ transients and cardiac myocyte contractility; (3) Decreased SR Ca²⁺ content in the face of increased I_Ca,L is due to increased Ca²⁺ efflux through the NCX and the inhibition of SERCA by hypophosphorylated PLB; (4) Increased I_Ca,L is possibly due to local increase in PKA activity. Most of these aspects of Ca²⁺ handling in GRK2 KO myocytes, except the greater than normal I_Ca,L and enhanced β-adrenergic regulation, have some similarity to these observed in failing myocytes²¹. These findings could imply that even in failing myocytes, some of the Ca²⁺ handling aspects could be more of adaptation.

2. Differential regulation of sarcolemmal and SR Ca²⁺ handling by the β-adrenergic system

Our data clearly show that there is a differential regulation of the LTCC and the PLB on the SR by the β-adrenergic system in GRK2 KO myocytes: at baseline, the LTCC is already in high activity mode probably due to high phosphorylation state of the channel but the LTCC loses its responses to β -adrenergic stimulation; in contrast, the PLB is in low phosphorylation state (hypophosphorylation) but it has enhanced responsiveness to β-AR stimulation. Our study indicates that the increased LTCC activity could be due to an increase in subsarcolemmal (local) PKA activation brought about by constitutive activity of the β-ARs. In normal cardiac physiology, GRK2 mediates the desensitization of β-ARs²⁰. The loss of GRK2 prevents desensitization of β-ARs and thus likely promotes the accumulation of activated β-ARs and causes constitutive activity of β-ARs in GRK2 KO myocytes even after the isolation. The increase in LTCC activity induced by constitutive β₁-ARs has been shown in cardiac β₁-AR overexpression mice¹⁸. The high LTCC activity in GRK2KO myocytes blunts the responsiveness of the channel to β-adrenergic stimulation. Similar situations have been reported in myocytes with high basal LTCC activities^{17, 22}. Recently, we have shown that overexpression of βARKct, an inhibitor of GRK2, in adult rat myocytes increases basal I_Ca,L as we have seen with GRK2 silencing. However, βARKct overexpression also enhanced the responses of I_Ca,L to ISO¹¹, which contrasts to our findings with GRK2 KO. These results suggest a potentially different mechanism is involved in our current study and the βARKct study¹¹ with the net effect (increased LTCC) being comparable. βARKct mainly reduces the inhibory G_βγ effect on the LTCC while in contrast, GRK2KO leads to a local increase in PKA, thereby activating the LTCC. The role of G_βγ in this setting was not specifically addressed, however, if G_βγ was released with the KO of GRK2 the inhibitory effect on the LTCC must be at least overcome by the PKA-dependent activation of the LTCC. An interesting experiment for future studies will indeed be the expression of βARKct in GRK2KO myocytes. This experiment will finally address the role of G_βγ in this setting but goes far beyond the scope of our current study. The use of different models (GRK2KO in mice in vivo for a relatively long period of time vs. βARKct expression in cultured rat ventricular myocytes¹¹ in vitro for 24 hours) could also account for different mechanisms in mediating increased basal I_Ca,L and different responsiveness to ISO stimulation.

In contrast to the enhanced LTCC phosphorylation in GRK2 KO myocytes, the phosphorylation state of PLB on the SR is lower than normal and the responsiveness of PLB to β-adrenergic agonists is enhanced. The underlying mechanism is related to locally activated PDE4 by activated subsarcolemmal PKA since rolipram - a selective PDE4 inhibitor - blunted the hypophosphorylation of PLB in GRK2KO hearts. Subsarcolemmal PDEs are generally able to diffuse to the SR and thus, can limit local cAMP production and PKA activation. Our results mechanistically explain the differential regulation of sarcolemmal versus SR Ca²⁺ handling associated with the cardiac myocyte lowering of GRK2, which ultimately can improve cardiac function in HF models.

3. The novel Ca²⁺ handling phenotype induced by the loss of GRK2 resists to adverse remodeling in hearts post-MI

As summarized above, GRK2 KO induces a novel Ca²⁺ handling phenotype which maintains a normal myocyte contractility in a way less dependent on SR Ca²⁺ but more dependent on I_Ca,L. We also show that this type of Ca²⁺ handling in GRK2KO myocytes is more resistant to adverse remodeling induced by MI in that the SR Ca²⁺ content and its regulation by the βAR system, and I_Ca,L density are better preserved and NCX activity is not increased. The cellular processes responsible for better remodeling after MI in GRK2 KO mice are potentially due to a combination of increased I_Ca,L and normalized NCX activity resulting into reduced SR Ca²⁺ content at baseline. We suspect that the post-MI relatively unchanged and small NCX activity predisposes the GRK2KO MI myocyte to maintain an unchanged SR Ca²⁺ loading in contrast to WT mice. Overall, this combination renders cardiac myocytes less susceptible to SR Ca²⁺ overload, which is known to induce myocyte apoptosis and necrosis ^{24, 25}. Importantly, the beneficial effects of GRK2 KO are negated by an LTCC antagonist, verapamil. Elevated SR Ca²⁺ content might also participate in myocyte hypertrophy ²⁶. In this study, GRK2KO myocytes develop less cardiac myocyte hypertrophy post-MI. This could be due to the concomitant decrease of SR Ca²⁺ load induced by the loss of GRK2 expression in the cardiac myocyte. The enhanced β-adrenergic responsiveness in GRK2 KO myocytes may also contribute to the beneficial effects of GRK2 silencing.

These results obtained here explain our previous studies showing that the loss of GRK2 in cardiac myocytes reduces HF associated mortality and enhances global cardiac function post-MI¹⁰. The improvements associated with the loss of GRK2 expression and activity are in a large part attributable to the normalization of intracellular Ca²⁺ cycling and cardiac myocyte function.

Conclusions

In summary, our data provide novel and important insights into the role of GRK2 in normal and failing hearts. Loss of GRK2 in cardiac myocytes enhances ECC efficiency in the presence of a lower than normal SR Ca²⁺ loading condition and better β-adrenergic responsiveness in unstressed hearts. This enhancement of ECC occurs through differential regulation of sarcolemmal versus SR Ca²⁺ handling, with the net result being improved Ca²⁺ transients leading to the amelioration of the HF phenotype. This is seen at the myocytes level and also globally in vivo with improved cardiac function of GRK2KO mice post-MI. Our data revealed for the first time that the beneficial effects seen with a loss of myocyte GRK2 activity after MI were associated with marked amelioration of cardiac myocyte contractility and significant improvements in intracellular Ca²⁺ cycling effected by modulation of I_Ca,L. These effects seen at the myocytes level may also contribute to the beneficial effects seen in various HF models treated with the βARKct peptide as a GRK2 inhibitor ^{8, 9, 12}. Further, GRK2 appears to induce novel regulatory modulation on the LTCC since currents were enhanced with a loss of GRK2. Overall, our current results mechanistically explain the beneficial effects of GRK2 silencing or inhibition after MI in the heart at the cellular and molecular levels and validates GRK2 as a potential target for HF prevention and treatment.

Clinical Perspective.

GRK2 is a molecular culprit in the development and progression of HF. We now provide the molecular basis for potential benefits of therapeutic strategies aiming at GRK2 inhibition. With our current study we demonstrate that loss of GRK2 in cardiac myocytes enhances ECC in unstressed hearts and in failing myocytes. We show that this enhancement of ECC occurs through differential regulation of sarcolemmal versus sarcoplasmic reticulum Ca²⁺ handling, with the net result being improved Ca²⁺ transients without SR Ca²⁺ overload leading to reversal of the HF phenotype. Further, we demonstrate that the beneficial effects seen with GRK2 inhibition in HF were associated with marked amelioration of cardiac myocyte contractility and significant improvements in intracellular Ca²⁺ cycling effected by modulation of the cardiac LTCC. Clear novelty is the compartmentalization of intracellular signaling by GRK2 and thereby differential regulation of Ca²⁺ signaling. Overall, our current results explain the beneficial effects of GRK2 knockdown in cardiac myocytes with intracellular changes in Ca²⁺ cycling being orchestrated to improve overall cardiac-myocyte contractility. Furthermore, our study demonstrates that this interplay between GRK2 and the LTCC might represent an attractive target to correct deranged Ca²⁺ cycling in the failing heart. Future gene therapeutic or pharmacological strategies will thus potentially directly target this interplay and might thus contribute to further improvements in clinical HF treatment.