Cardiac G-Protein–Coupled Receptor Kinase 2 Ablation Induces a Novel Ca2+ Handling Phenotype Resistant to Adverse Alterations and Remodeling After Myocardial Infarction

Background— G-protein–coupled receptor kinase 2 (GRK2) is a primary regulator of &bgr;-adrenergic signaling in the heart. G-protein–coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study. Methods and Results— Myocyte contractility, Ca2+ handling and excitation-contraction coupling were studied in isolated cardiomyocytes from wild-type and GRK2 knockout (GRK2KO) mice without (sham) or with myocardial infarction (MI). In cardiac myocytes isolated from unstressed wild-type and GRK2KO hearts, myocyte contractions and Ca2+ transients were similar, but GRK2KO myocytes had lower sarcoplasmic reticulum (SR) Ca2+ content because of increased sodium-Ca2+ exchanger activity and inhibited SR Ca2+ ATPase by local protein kinase A–mediated activation of phosphodiesterase 4 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. After &bgr;-adrenergic stimulation, GRK2KO myocytes revealed significant increases in contractility and Ca2+ transients, which were not mediated through cardiac L-type Ca2+ channels 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 because of better-maintained L-type Ca2+ channel current density and no increase in sodium-Ca2+ exchanger in GRK2KO myocytes. An L-type Ca2+ channel blocker, verapamil, reversed some beneficial effects of GRK2KO. Conclusions— These data argue for novel differential regulation of L-type Ca2+ channel currents and SR load by GRK2. G-protein–coupled receptor kinase 2 ablation represents a novel beneficial Ca2+ handling phenotype resisting adverse remodeling after MI.

open probability of the ryanodine receptor, causing SR Ca 2ϩ leak. 2,3 Alterations of the L-type Ca 2ϩ channel (LTCC), the trigger for the Ca 2ϩ -induced SR Ca 2ϩ release, also contribute to the pathophysiological changes in Ca 2ϩ homeostasis in failing myocytes. 4 In addition to these changes causing dysfunctional contractile performance, the rise of diastolic Ca 2ϩ may increase the risk of arrhythmias and induce pathological cardiac remodeling. 5

Editorial see p 2054 Clinical Perspective on p 2118
The increased activity of the sympathetic nervous system associated with HF is a compensation to normalize cardiac function by enhancing Ca 2ϩ cycling and maximize contractile force through the ␤-adrenergic signaling pathway. In acute HF, these changes can improve systemic perfusion whereas in chronic HF the augmentation in catecholamines is associated with mortality 6 and results in a downregulation of ␤-adrenergic receptors (␤ARs) promoted by upregulated G protein-coupled receptor kinase 2 (GRK2). G protein-coupled receptor kinase 2 is the primary GRK in the heart and a prototype regulator of ␤AR signaling. 7 We have previously identified GRK2 as a culprit in the progression of HF, and GRK2 inhibition (by expression of its c-terminal domain, called ␤ARKct) or gene silencing has rescued disparate models of HF. 8 -10 Our recent study indicates that the benefits of ␤ARKct could be related to enhanced myocyte contractility by increasing LTCC currents and its responsiveness to ␤-adrenergic agonists. 11 However, the exact underlying cellular mechanisms for these beneficial effects in HF after ␤ARKct expression or GRK2 ablation are not clearly defined. It is especially important to define these mechanisms because in light of the recent success of ␤AR blocker therapy in clinical HF management, the results with ␤ARKct and GRK2 silencing appear paradoxical, as the major function of GRK2 in cardiac myocytes is to dampen ␤AR signaling in a manner similar to that of ␤AR blockade. We have found that ␤ARKct expression can cause a molecular remodeling of the cardiac ␤AR system with receptor upregulation and improved ␤AR signaling, and a recent study with chronic mediated ␤ARKct expression in a rat HF model showed that myocardial ␤AR changes are probably down-stream of neurohormonal lowering including reduction in sympathetic nervous system activity. 12 In this regard, the role of GRK2 inhibition must mechanistically go beyond resensitizing ␤ARs 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 Ca 2ϩ 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 cardiacspecific GRK2KO mice 10,13 to study myocyte ECC coupling and Ca 2ϩ 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 Ca 2ϩ handling phenotype: Myocyte contractility and Ca 2ϩ handling are normal even though the SR Ca 2ϩ content is reduced because there is an increase in LTCC activities and resulting increases in LTCC currents (I Ca,L ) with a compensatory increase in NCX activity. This Ca 2ϩ handling phenotype brought about by GRK2 ablation is resistant to adverse Ca 2ϩ handling remodeling after MI and leads to better cardiac function in GRK2KO mice post MI.

Methods
Conditional mice bearing floxed GRK2 alleles were described previously. 10,13 G-protein-coupled receptor kinase 2 KO (␣ myosin heavy chain Ϫ Cre-recombinase ϫ GRK2flox/flox) and wild-type (WT) (GRK2flox/flox) mice were maintained on a C57BL6 genetic background. All animal procedures and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Thomas Jefferson University. G-proteincoupled receptor kinase 2 KO and WT mice were 8 to 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. Myocardial infarction was induced by ligating the left anterior descending coronary artery at 2 to 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 after MI or sham operation until the end of the study period (42 days after MI). Verapamiltreated mice received oral supplementation of verapamil (Sigma-Aldrich, St. Louis, MO) as described previously 15 and were evaluated by echocardiography. Cardiac myocytes were isolated cultured from animals. Myocyte Ca 2ϩ transients and contractions, SR Ca 2ϩ load, I Ca,L , and NCX-Activity I NCX were measured. Quantitative real-time polymerase chain reaction and Western blot analysis were performed for gene-expression assessment. Detailed description of experimental procedures is available in the online-only Data Supplement.
Data are expressed as meanϮSEM. An unpaired 2-tailed t test or a 1-way ANOVA and a 2-way ANOVA (linear mixed effects model) were performed with SAS 9.3 for between-group comparisons, followed by a posthoc Bonferroni adjustment. For all tests, a P value Ͻ0.05 was considered significant.

Myocyte GRK2 Ablation Enhances Adrenergic Responsiveness of Cellular Contractility and Ca 2؉ Transients
Our previous study has shown that GRK2 KO mice have cardiac function comparable to control mice at baseline (online-only Data Supplement Table 1), but their cardiac function responds better to ␤-adrenergic stimulation. 10 Here, we determine the cellular mechanisms for this observation. Myocyte contraction and intracellular Ca 2ϩ transients were recorded from WT and GRK2KO myocytes under baseline conditions and after ␤AR stimulation. Myocytes from both lines revealed a similar fractional shortening when paced at both 0.5Hz and 2Hz ( Figure 1A-1C). However, after isoproterenol (ISO), myocytes from GRK2KO mice showed a significantly greater increase in fractional shortening ( Figure  1A-1C and online-only Data Supplement Table II). In a good agreement, as shown in Figure 1D, at baseline, the characteristics and amplitude of the Ca 2ϩ transients (as ⌬Fura-2 340/380 nm 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-1F, online-only Data Supplement Table II). We observed no differences in the Ca 2ϩ transient decay time constants at baseline and after ISO between groups.

GRK2 Silencing in Myocytes Enhances ECC Efficiency
Myocyte contractility and Ca 2ϩ transients are determined by Ca 2ϩ release from the SR. 16 We measured SR Ca 2ϩ content by rapid application of caffeine (caffeine spritz) at baseline or after ISO stimulation. Figure 2A shows representative tracings of cytosolic Ca 2ϩ transients (measured with indo-1 AM) induced by caffeine spritz after 4 field stimulations in WT and GRK2KO cardiac myocytes to measure the SR Ca 2ϩ load. At baseline, SR Ca 2ϩ load was less in GRK2 myocytes than in control myocytes ( Figure 2B); in response to ISO, the SR Ca 2ϩ 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 Ca 2ϩ was observed in GRK2KO cardiac myocytes compared with WT myocytes at both baseline and after ISO ( Figure 2C and online-only Data Supplement Table II).

Loss of Myocyte GRK2 Enhances I Ca,L by a Local Protein Kinase A-Dependent Mechanism but Blunts Its ␤AR Responsiveness
To explore the underlying cellular mechanisms for the reduced SR Ca 2ϩ content with enhanced EC coupling efficiency in GRK2 KO myocytes, we measured the I Ca,L in GRK2KO and control myocytes because I Ca,L serves as both the trigger of Ca 2ϩ 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 with WT cardiac myocytes ( Figure 3A and 3B and online-only Data Supplement Table II). When stimulated with a saturating dose of ISO (10 Ϫ6 M), peak I Ca,L was increased to the same level in cardiac myocytes from both mouse lines ( Figure  3A and 3B), suggesting that the LTCC density was similar in both groups. 4 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 3D). These results imply that the LTCC in GRK2KO myocytes could be in such a high activity state that it loses responsiveness to ␤-adrenergic stimulation.
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, the increase of channel activities at single-channel levels, or both. Charge movement was used to quantify the number 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 of the LTCC induced by various depolarizing voltages, a result which indicates that there was no significant alteration of LTCC density on the membrane and that the increased whole-cell I Ca,L could be due to the increased single-channel activities. Immunoprecipitation of ␣1c, the pore-forming subunit of the LTCC, from the same amount of proteins in GRK2KO and control hearts showed no difference in ␣1c expression (Figure 3N). These data suggest that the LTCC in GRK2KO myocytes must have higher than normal activity, a result supported by single-channel recording of LTCC activities ( Figure 3J-3M). The availability ( Figure 3K) and the open probabilities ( Figure 3L and 3M) were significantly increased in GRK2KO myocytes, a result that 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 ␤ARs carry constitutive activation to activate PKA locally to phosphorylate the LTCC. 18 Here, we tested whether high LTCC activity was mediated by a PKA-dependent mechanism. H89, a PKA-specific inhibitor, was used, and it normalized the current density and voltagedependent activation of the LTCC in KO myocytes but had no significant effect on LTCCs in WT myocytes ( Figure  3F-3I). 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-3M). The phosphorylation of ␣1c at Ser1928, a PKA site, was shown to be greater in GRK2KO hearts than in control hearts ( Figure  3N and 3O).

Loss of GRK2 in Myocytes Increases Na ؉ /Ca 2؉ -Exchanger Expression
Because there is a decreased SR Ca 2ϩ content with increased I Ca,L in GRK2KO myocytes, there should be increased Ca 2ϩ efflux out of GRK2KO myocytes. In ventricular myocytes, the major route of Ca 2ϩ efflux is through the NCX. One way to measure NCX activity is to examine the decay rate of caffeine-induced Ca 2ϩ transients that can be fit by a single exponential decay equation. 19 The tau value was significantly smaller in the GRK2KO 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 with WT myocytes, a result that was confirmed by direct measurement of NCX current ( Figure 4B) and NCX protein expression ( Figure 4C and 4D).

Loss of GRK2 From Myocytes Decreases Basal PLB Phosphorylation but Increases Its Responsiveness to ␤AR Stimulation
The loading of the SR with Ca 2ϩ depends on the competition between the extrusion of Ca 2ϩ out of the cell (mainly through NCX) and the resequestration of Ca 2ϩ into SR by SERCA, which is regulated by PLB. 16 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-4H). 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 4H). When myocytes were stimulated with the ␤AR 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 4J). However, a significant leftward shift of the dose-response curve was found in GRK2KO myocytes compared with WT cells (Figure 4J), demonstrating higher ␤AR sensitivity in cells isolated from unstressed GRK2KO mice.
To further clarify the mechanism responsible for the hypophosphorylation of PLB, we examined whether phosphodiesterase 4 (PDE4), which is activated by PKA, could play a mechanistic role. A PDE4-specific inhibitor, rolipram (10 mg/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 ( Figure 4K and 4L), 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 Contractility of Myocytes
Our previous studies have shown that cardiac specific loss of GRK2 ameliorates the development of HF after MI. 10 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 (online-only Data Supplement Table I). Because 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 was determined. The basal fractional shortening and Ca 2ϩ transient amplitudes in myocytes from GRK2KO mice after MI (GRK2KO MI) were almost normal compared with GRK2KO myocytes from mice not subject to MI and greater than those of WT cardiac myocytes after MI (WT MI) at both pacing frequencies of 0.5Hz and 2Hz ( Figure 5A, B, D and E and online-only Data Supplement Table II). Furthermore, WT MI myocytes had a blunted functional response to ISO. In contrast, cardiac myocytes from GRK2KO mice after MI displayed significantly improved ␤-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 Ca 2ϩ -handling remodeling after MI and provide a potential cellular mechanism for the benefits of GRK2 lowering or inhibition in the failing heart. nϭ5 animals/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 to 5 hearts were analyzed for each group; 1-way ANOVA for L. WT indicates wild type; KO, knockout; ISO, isoproterenol; I NCX , NaϩϪCa 2ϩ exchange current; NCX1, NaϩϪCa 2ϩ exchange 1; SERCA, sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATpase; PLB, phospholamban; PLBt, total Phospholamban; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; and N.S., not significant.

GRK2KO MI Myocytes Have Preserved SR Ca 2؉ Loading and I Ca,L
A decrease in the SR Ca 2ϩ load is a contributing factor for depressed myocyte contractility, a hallmark of HF. Although GRK2KO mice without MI having a lower SR Ca 2ϩ load than WT mice without MI, the SR Ca 2ϩ load in GRK2KO MI myocytes was not decreased as in WT MI myocytes ( Figure  6A and B and online-only Data Supplement Table II). Fractional Ca 2ϩ release from the SR was higher in GRK2KO MI myocytes as well, explaining the preserved myocyte contractility ( Figure 6C). NaϩϪCa 2ϩ exchanger activity, as indirectly assessed by the decay constant tau of the caffeineinduced Ca 2ϩ transient was normalized in post-MI GRK2KO myocytes, despite significant increases in NCX activity in infarcted WT mice consistent with severe HF ( Figure 6D). Improvements in intracellular Ca 2ϩ transients and SR fractional Ca 2ϩ 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 with pre-MI values (see Figure 3A and 3B), peak I Ca,L in post-MI myocytes were significantly greater with the loss of GRK2 ( Figure 6E and online-only Data Supplement Table II). When stimulated with ISO (10 Ϫ6 M), I Ca,L in GRK2KO post-MI myocytes was only insignificantly increased (12.9Ϯ6.9%, nϭ4), but I Ca,L in post-MI WT myocytes was significantly increased by 84.3Ϯ18.1% (nϭ7) ( Figure 6F). However, after ISO, I Ca,L amplitudes in myocytes were not different between the 2 mouse lines ( Figure 6G and 6H), indicating similar LTCC density. The marked enhancement in basal I Ca,L in the GRK2KO MI myocytes might contribute to the normalization of intracellular Ca 2ϩ 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 6H).

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 with WT mice post-MI (GRK2KO MI 211Ϯ19pF versus WT MI 311Ϯ35pF versus WT Sham 178Ϯ19pF versus KO Sham 188Ϯ15pF; PϽ0.05 between GRK2KO MI and WT MI).

Beneficial Effects of Myocyte GRK2 Silencing Is Suppressed by the LTCC Blocker Verapamil
To determine if the beneficial Ca 2ϩ 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 whereas the LTCC blocker had minimal effects on post-MI WT mice ( Figure 7A). Furthermore, cardiac brain natriuretic peptide messenger RNA expression as a molecular marker of HF was significantly lower in post-MI GRK2KO mice compared with 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 Ca 2ϩ 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 .

Discussion
G-protein-coupled receptor kinase 2 is an important molecule in the heart. It is not only a primary regulator of adrenergic signaling but also claims an important role in the development of HF. 8 -10 It is upregulated 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 silencing 10 or inhibition by ␤ARKct 20 is able to improve cardiac function during HF progression after MI. However, the specific role of GRK2 in the regulation of normal and failing Ca 2ϩ cycling has never been studied. In HF, myocyte Ca 2ϩ cycling is deranged, and abnormalities include altered cardiac LTCC density and properties and reduced SR Ca 2ϩ content due to decreased SERCA and increased NCX activities. These changes result in reduced intracellular Ca 2ϩ transients and depressed myocyte contractility. 1,4 Our current study has revealed that GRK2 can influence myocyte Ca 2ϩ homeostasis and that its absence in cardiac myocytes 10,13 causes a novel Ca 2ϩ handling phenotype that is resistant to cardiac function deterioration after MI. The benefits rendered by GRK2 silencing are associated with the differential regulation of sarcolemmal and SR Ca 2ϩ handling by the ␤-adrenergic system.

A Novel Ca 2؉ -Handling Phenotype Induced by GRK2 Silencing
Although GRK2 plays an important role in regulating the ␤-adrenergic system, its loss does not affect basal cardiac 10 and cardiomyocyte function. Myocyte loss of GRK2 did not change characteristics of basal intracellular Ca 2ϩ transients and myocyte contractions. However, detailed characterization of myocyte Ca 2ϩ handling has shown many differences in EC coupling between GRK2KO and WT myocytes: (1) The SR Ca 2ϩ content is reduced in GRK2 KO myocytes, but Ca 2ϩ transients and contraction in GRK2KO myocytes are normal because of an increased fractional Ca 2ϩ release from the SR; (2) increased I Ca,L ensures normal Ca 2ϩ transients and cardiac myocyte contractility; (3) decreased SR Ca 2ϩ content in the face of increased I Ca,L is due to increased Ca 2ϩ efflux through the NCX and the inhibition of SERCA by hypophosphorylated PLB; and (4) increased I Ca,L is possibly due to local increase in PKA activity. Most of these aspects of Ca 2ϩ handling in GRK2KO myocytes, except the greater than normal I Ca,L and enhanced ␤-adrenergic regulation, have some similarity with those observed in failing myocytes. 21 These findings could imply that, even in failing myocytes, some of the Ca 2ϩ handling aspects could be more of adaptive mechanisms.

Differential Regulation of Sarcolemmal and SR Ca 2؉ Handling by the ␤-Adrenergic System
Our data clearly show that there is a differential regulation of the LTCC on the sarcolemma and the PLB on the SR by the ␤-adrenergic system in GRK2KO myocytes: At baseline, the LTCC is already in high-activity mode probably because of the high-phosphorylation state of the channel, but the LTCC loses its responses to ␤-adrenergic stimulation; in contrast, the PLB is in a low-phosphorylation state (hypophosphorylation), but it has enhanced responsiveness to ␤-adrenergic receptor (␤-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 ␤-ARs. In normal cardiac physiology, GRK2 mediates the desensitization of ␤-ARs. 20 The loss of GRK2 prevents desensitization of ␤-ARs and thus likely promotes the accumulation of activated ␤-ARs and causes constitutive activity of ␤-ARs in GRK2KO myocytes even after the isolation. The increase in LTCC activity induced by constitutive ␤ 1 -ARs has been shown in cardiac ␤ 1 -AR-overexpression mice. 18 The high LTCC activity in GRK2KO myocytes blunts the responsiveness of the channel to ␤-adrenergic stimulation. Similar situations have been reported in myocytes with high basal LTCC activities. 17,22 Recently, we have shown that overexpression of ␤ARKct, an inhibitor of GRK2, in adult rat myocytes increases basal I Ca,L , as we have seen with GRK2 silencing. However, ␤ARKct overexpression also enhanced the responses of I Ca,L to ISO, 11 which is in contrast to our findings with GRK2KO. These results suggest that potentially different mechanisms are involved in our current study and the ␤ARKct study, 11 with the net effect (increased LTCC) being comparable. Primarily, ␤ARKct reduces the inhibitory effect of the ␤␥ subunits of activated heterotrimeric-G proteins (G␤␥) on the LTCC whereas GRK2KO leads to a local increase in PKA, thereby activating the LTCC. The role of G ␤␥ in this setting was not specifically addressed, however: If G ␤␥ 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 ␤ARKct in GRK2KO myocytes. . nϭ6 to 8/sham group; nϭ15 to 25/MI group without verapamil; nϭ10 to 13/MI group with verapamil; 1-way ANOVA. C, Cardiac myocyte brain natriuretic protein messenger RNA levels 42 days post-MI or sham operation; nϭ5 to 6/group for sham; nϭ8 to 9/group for MI; 1-way ANOVA and unpaired 2-tailed t test. FS indicates fractional shortening; WT, wild type; GRK2KO, G-protein-coupled receptor kinase 2 knockout; MI, myocardial infarction; EDD, end-diastolic diameter; BNP, brain natriuretic protein; and mRNA, messenger RNA. This experiment will finally address the role of G ␤␥ in this setting, but such a goal 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 versus ␤ARKct expression in cultured rat ventricular myocytes 11 in vitro for 24 hours) could also account for different mechanisms in mediating increased basal I Ca,L and different degrees of responsiveness to ISO stimulation.
In contrast to the enhanced LTCC phosphorylation in GRK2KO myocytes, the phosphorylation state of PLB on the SR is lower than normal and the responsiveness of PLB to ␤-adrenergic agonists is enhanced. The underlying mechanism is related to locally activated PDE4 by activated subsarcolemmal PKA because 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. 23 Our results mechanistically explain the differential regulation of sarcolemmal versus SR Ca 2ϩ handling associated with the cardiac myocyte lowering of GRK2, which ultimately can improve cardiac function in HF models.

The Novel Ca 2؉ -Handling Phenotype Induced by the Loss of GRK2 Is Resistant to Adverse Remodeling in Hearts After MI
As summarized above, GRK2KO induces a novel Ca 2ϩhandling phenotype that maintains a normal myocyte contractility in a way that is less dependent on SR Ca 2ϩ but more dependent on I Ca,L . We also show that this type of Ca 2ϩ handling in GRK2KO myocytes is more resistant to adverse remodeling induced by MI in that the SR Ca 2ϩ content and its regulation by the ␤AR 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 GRK2KO mice are potentially due to a combination of increased I Ca,L and normalized NCX activity resulting in reduced SR Ca 2ϩ content at baseline. We suspect that the relatively unchanged and small NCX activity after MI predisposes the GRK2KO MI myocyte to maintain an unchanged SR Ca 2ϩ loading, which is in contrast to WT mice. Overall, this combination renders cardiac myocytes less susceptible to SR Ca 2ϩ overload, which is known to induce myocyte apoptosis and necrosis. 24,25 Importantly, the beneficial effects of GRK2KO are negated by an LTCC antagonist, verapamil. Elevated SR Ca 2ϩ 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 Ca 2ϩ load induced by the loss of GRK2 expression in the cardiac myocyte. The enhanced ␤-adrenergic responsiveness in GRK2KO myocytes may also contribute to the beneficial effects of GRK2 silencing.
The 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-MI. 10 The improvements associated with the loss of GRK2 expression and activity are in large part attributable to the normalization of intracellular Ca 2ϩ 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 Ca 2ϩ loading condition and better ␤-adrenergic responsiveness in unstressed hearts. This enhancement of ECC occurs through differential regulation of sarcolemmal versus SR Ca 2ϩ handling, with the net result being improved Ca 2ϩ transients leading to the amelioration of the HF phenotype. This is seen at the myocyte 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 Ca 2ϩ cycling effected by modulation of I Ca,L . These effects seen at the myocyte level may also contribute to the beneficial effects seen in various HF models treated with the ␤ARKct peptide as a GRK2 inhibitor. 8,9,12 Further, GRK2 appears to induce novel regulatory modulation in the LTCC because 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.

Sources of Funding
This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Ra 1668/1-1 and Ra 1668/3-1 to Dr Raake). Drs Chen, Li, and Tang were supported by National Institutes of Health (NIH) grant R01 HL088243 and American Heart Association grant AHA0730347N. Dr Koch is the W.W. Smith Professor of Medicine, and this research was supported by NIH grants R37 HL61690, R01 HL56205, R01 HL085503, and P01 HL075443 (Project 2). Also, Drs. Koch and Gao were supported through P01 HL091799 (Project 1 and Core B). The effort of Dr Dorn II on this project was supported by NIH grant R01 HL87871, and Dr Houser was supported by NIH grant P01 HL091799 (Project 3).