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. 2008 Aug 15;103(4):413-22.
doi: 10.1161/CIRCRESAHA.107.168336. Epub 2008 Jul 17.

G protein-coupled receptor kinase 2 ablation in cardiac myocytes before or after myocardial infarction prevents heart failure

Philip W Raake  1 , Leif E Vinge, Erhe Gao, Matthieu Boucher, Giuseppe Rengo, Xiongwen Chen, Brent R DeGeorge Jr, Scot Matkovich, Steven R Houser, Patrick Most, Andrea D Eckhart, Gerald W Dorn 2nd, Walter J Koch
Affiliations

G protein-coupled receptor kinase 2 ablation in cardiac myocytes before or after myocardial infarction prevents heart failure

Philip W Raake et al. Circ Res. .

Abstract

Myocardial G protein-coupled receptor kinase (GRK)2 is a critical regulator of cardiac beta-adrenergic receptor (betaAR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated betaAR with a GRK2-derived peptide that binds G(beta)gamma (betaARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and betaARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (alphaMHC-Cre x GRK2 fl/fl) or only after administration of tamoxifen (alphaMHC-MerCreMer x GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved betaAR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring betaAR signaling by other means to improve outcomes in heart failure.

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Figures

Figure 1
Figure 1
Survival of HF is improved after post-MI induced loss of GRK2 in cardiac myocytes. A, Experimental protocol showing the study-design of the rescue-study using Tmx-inducible GRK2KO mice (αMHC-MerCreMer × GRK2 fl/fl, GRK2 fl/fl and αMHC-MerCreMer mice). B, Time-defined controlled loss of GRK2 in cardiac myocytes from Tmx-inducible αMHC-MerCreMer × GRK2 fl/fl and αMHC-MerCreMer mice. Impact of Tmx-treatment on cardiac myocyte GRK2 protein levels determined via Western Blotting. Representative Western Blot of GRK2 and CSQ (as a loading control) and quantification of the results, n=6 animals/group, *P<0.05 αMHC-MerCreMer × GRK2 fl/fl vs. αMHC-MerCreMer, unpaired two-tailed t-test. C, Survival of Tmx treated sham and infarcted αMHC-MerCreMer and αMHC-MerCreMer×GRK2 fl/fl mice throughout the study period. n=8–11 animals/group for Sham mice, n=30–34 animals/group for infarcted mice, survival was analyzed by the Kaplan-Meier method and between-group differences in survival were tested by the log-rank test.
Figure 2
Figure 2
Induced loss of GRK2 rescues adverse LV remodeling and cardiac function post-MI. A, Echocardiographic assessment of EDD pre-MI, 10d post-MI (prior to Tmx treatment), 42d post-MI (28d post-Tmx treatment) and 120d post-MI. B, Echocardiographic assessment of FS pre-MI, 10d post-MI (prior to Tmx treatment), 42d post-MI (28d post-Tmx treatment) and 120d post-MI. n=6–8 animals/group for sham-operated mice, n=12–16 animals/group for MI mice, *P<0.05 vs. MI αMHC-MerCreMer and MI GRK2 fl/fl. Hemodynamic measurements of LV contractility (as measured by LV dP/dtmax) (C), and relaxation (as measured by LV dP/dtmin) (D), and LV end diastolic pressure (LVEDP) (E), 120 d post-MI (106 d post-Tmx). n=5 animals/group for sham-operated mice, n=9–11 animals/group for MI mice, §P<0.05 MI αMHC-MerCreMer × GRK2 fl/fl vs. MI αMHC-MerCreMer under respective condition, #P<0.05 MI αMHC-MerCreMer vs. Sham groups under respective condition, ‡P<0.05 MI αMHC-MerCreMer vs. MI αMHC-MerCreMer × GRK2 fl/fl and Sham groups, two-way ANOVA for A, B, C, and D, one-way ANOVA for E.
Figure 3
Figure 3
GRK2 abolishment in the post-MI failing heart alters gene expression. Quantitative RT-PCR analysis on cardiac myocytes isolated from the LV 5 weeks post MI (3 weeks after the last dose of Tmx). A, ANP; B, BNP; C, β-MHC; and D, GRK2. All values normalized to 18S levels. n=5–6 animals/group for Sham mice, n=7–11 animals/group for MI mice, *P<0.05 MI αMHC-MerCreMer vs. MI αMHC-MerCreMer × GRK2 fl/fl and Sham groups, #P<0.05 MI αMHC-MerCreMer vs. Sham groups, one-way ANOVA.
Figure 4
Figure 4
Infarct-related mortality is reduced with loss of GRK2 in cardiac myocytes prior to MI. A, Experimental protocol showing the study-design of the prevention-study using conditional GRK2KO mice (αMHC-Cre × GRK2 fl/fl and GRK2 fl/fl mice). B, Conventional conditional loss of GRK2 in cardiac myocytes in αMHC-Cre × GRK2 fl/fl and GRK2 fl/fl mice. Western blot analysis for GRK2 protein and actin (as a loading control) in isolated cardiac myocytes. C, Survival of male GRK2 fl/fl and αMHC-MHC × GRK2 fl/fl mice throughout the study period. n=8 animals/group for Sham mice, n=49–58 animals/group for infarcted mice, survival was analyzed by the Kaplan-Meier method and between-group differences in survival were tested by the log-rank test.
Figure 5
Figure 5
Constitutive loss of GRK2 in cardiac myocytes prior to MI reduces the extent of cardiac dysfunction. Echocardiographic assessment 28d post-MI of EDD (A) and FS (B), n=8–10 animals/group for Sham mice, n=21–30 animals/group for MI mice, *P<0.01 MI vs. Sham for respective group, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl, one-way ANOVA. Hemodynamic measurements of LV contractility (as measured by LV dP/dtmax) (C), and relaxation (as measured by LV dP/dtmin) (D) at baseline and in response to increasing intraperitoneal injected doses of isoproterenol. E, left ventricular enddiastolic pressure (LVEDP) n=5 animals/group for sham-operated mice, n=9–10 animals/group for MI mice; †P<0.05 Iso 0.5µg vs. basal for each group, *P<0.05 MI GRK2 fl/fl vs. Sham GRK2 fl/fl for each condition, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl for each condition, two-way ANOVA for C, and D, one-way ANOVA for E. F, Heart-to-body weight ratio at day 28 post MI. *P<0.01 MI vs. Sham, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl, one-way ANOVA.
Figure 5
Figure 5
Constitutive loss of GRK2 in cardiac myocytes prior to MI reduces the extent of cardiac dysfunction. Echocardiographic assessment 28d post-MI of EDD (A) and FS (B), n=8–10 animals/group for Sham mice, n=21–30 animals/group for MI mice, *P<0.01 MI vs. Sham for respective group, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl, one-way ANOVA. Hemodynamic measurements of LV contractility (as measured by LV dP/dtmax) (C), and relaxation (as measured by LV dP/dtmin) (D) at baseline and in response to increasing intraperitoneal injected doses of isoproterenol. E, left ventricular enddiastolic pressure (LVEDP) n=5 animals/group for sham-operated mice, n=9–10 animals/group for MI mice; †P<0.05 Iso 0.5µg vs. basal for each group, *P<0.05 MI GRK2 fl/fl vs. Sham GRK2 fl/fl for each condition, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl for each condition, two-way ANOVA for C, and D, one-way ANOVA for E. F, Heart-to-body weight ratio at day 28 post MI. *P<0.01 MI vs. Sham, #P<0.05 MI αMHC-Cre × GRK2 fl/fl vs. MI GRK2 fl/fl, one-way ANOVA.
Figure 6
Figure 6
Loss of GRK2 in cardiac myocytes alters gene expression in the failing heart. Quantitative RT-PCR analysis 28 days post MI in the remote non-infarcted region of the LV. A, ANP; B, BNP; C, β-MHC; D, Coll-1; E, MMP-9; F, GRK2; and G, GRK5. All values normalized to 28S levels. n=6–8 animals/group, *P<0.05 MI GRK2 fl/fl vs. MI αMHC-Cre × GRK2 fl/fl and Sham GRK2 fl/fl and Sham αMHC-Cre × GRK2 fl/fl, #P<0.05 MI GRK2 fl/fl vs. Sham GRK2 fl/fl, one-way ANOVA.
Figure 6
Figure 6
Loss of GRK2 in cardiac myocytes alters gene expression in the failing heart. Quantitative RT-PCR analysis 28 days post MI in the remote non-infarcted region of the LV. A, ANP; B, BNP; C, β-MHC; D, Coll-1; E, MMP-9; F, GRK2; and G, GRK5. All values normalized to 28S levels. n=6–8 animals/group, *P<0.05 MI GRK2 fl/fl vs. MI αMHC-Cre × GRK2 fl/fl and Sham GRK2 fl/fl and Sham αMHC-Cre × GRK2 fl/fl, #P<0.05 MI GRK2 fl/fl vs. Sham GRK2 fl/fl, one-way ANOVA.
Figure 7
Figure 7
Loss of GRK2 improves post-MI βAR responsiveness of single cardiac myocytes. A, Representative tracings of single myocyte contractility under baseline conditions and stimulation with isoproterenol. B, Quantitative data of FS. C, Maximum rate of cardiac myocyte contractility and relengthening. Cardiac myocytes were measured at baseline conditions and after stimulation with isoproterenol. n=3 animals/group for Sham mice, n= 6 animals/group for MI mice, *P<0.05 MI αMHC-Cre/GRK2(fl/fl) vs. MI GRK2(fl/fl) for respective condition, two-way ANOVA.
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