G protein-coupled receptor kinase 5 (GRK5) contributes to impaired cardiac function and immune cell recruitment in post-ischemic heart failure

Figure 1. Cardiomyocyte-specific GRK5 overexpression impairs cardiac function after myocardial infarction. Representative Western blot (A) and quantification (B) showing GRK5 protein levels in cardiomyocytes isolated from WT mice at 4-days post-Sham operation or post–myocardial infarction (MI). GAPDH used as loading control (n = 6–7). Overall design of the 8-week study in NLC and TgGRK5 mice subjected to Sham-operation or MI (C). Ejection fracti) (D) on (EFand left ventricular internal diameter in diastole (LVIDd) (E) as measured by echocardiography at 0- (baseline), 4- and 8-weeks post-surgery in Sham and MI groups (n = 11–38). Quantification of LV +dP/dt Max (F), −dP/dt Min (G) and heart rate (HR) (H) performed at baseline and with increasing doses of Isoproterenol. Different color asterisks have been used to show significant differences between groups (n = 7–18). Representative images of hearts from MI groups (8 weeks post-MI) (I). Measures of heart weight to body weight ratio (HW/BW) at 8 weeks post-surgery (J) (n = 15–27). Quantification of RT-PCR data showing fold change of NLC-Sham for NPPA (K), NPPB (L) and MYH7 (M) in LV samples at 8-weeks post-surgery (n = 10–14). *P < 0.05, **P…

Figure 2. Effect of GRK5 expression on transcriptome in cardiomyocyte after myocardial infarction. Transcriptome evaluation in cardiomyocytes isolated at 4-days post-myocardial infarction (MI) from TgGRK5 mice compared to NLC mice (5 mice/group). Heat map illustrating top genes upregulated (A) and top genes downregulated (B) in TgGRK5-MI (compared to NCL-MI) that pass FDR < 10% threshold. Volcano plot showing upregulated and downregulated genes in TgGRK5-MI compared to NLC-MI (C). Highlighted significantly affected genes. Pathways enriched among genes significantly affected by GRK5-overexpression in cardiomyocytes (D). Quantification of RT-PCR data showing fold change of NLC-MI for Bex1, Cxcl11, Cxcl13 and IL-15 in cardiomyocytes isolated at 4 days post-MI (E) (n = 5–7). *P < 0.05, **P < 0.01. Mann–Whitney test was used among groups.

Figure 3. Transgenic cardiomyocyte GRK5 overexpression impacts cardiac remodeling and survival in ischemic heart failure. Representative TTC staining of hearts at 24 h post-myocardial infarction (MI) of NLC and TgGRK5 groups (A) as well as quantification of infarct size (% of LV) (B) (n = 5–7). TUNEL quantification of images of left ventricular (LV) tissue from NLC and TgGRK5 mice at remote area (RA), border zone (BZ), and infarct area at 24 h post-MI (C) (n = 6–7). Representative TUNEL staining (red) of LV tissue from NLC and TgGRK5 mice at the BZ counterstained with Troponin T (green) to visualize cardiomyocytes, at 4-days post-MI; DAPI was used as a nuclear marker (blue) (D). Scale bar in white (500 μm). TUNEL quantification of images of LV tissue from NLC and TgGRK5 mice at the RA, BZ, and infarct area, at 4-days post-MI (E) (n = 6–12). Representative images (F) and quantitative data (G) showing the percentage of fibrosis via Picrosirius red staining in LV tissue from NLC and TgGRK5 at 8-weeks post-Sham operation or MI (n = 5–8). Scale bar in black (500 μm). Quantification of RT-PCR data showing fold change of NLC-Sham for Col-1 (H), Col-3 (I), CTGF (J) and MMP-2 (K) in LV samples…

Figure 4. TgGRK5 showed increased early immune cell recruitment to the heart post-myocardial infarction. Representative images and quantification of CD45 (A), MPO (B), CD68 (C) and tryptase (D) stainings for remote area (RA) and border zone (BZ) of hearts from NLC and TgGRK5 mice at 4-days post-myocardial infarction (MI) (n = 6–11). Scale bar in white or black (100 μm). Arrowheads indicate positive staining. Insets show higher magnification at ×250. Flow-cytometric quantification of immune cells isolated from NLC and TgGRK5 hearts at 4 days post-MI (E–R) (n = 7 per group). AV= Annexin V. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey’s post hoc test, t-test or Mann–Whitney test were used between groups.

Figure 5. TgGRK5 mice showed increased late immune cell infiltration in the heart and chronic cardiac inflammation post-myocardial infarction. Representative images and quantification of CD45 (A), MPO (B), CD68 (C) and CD3 (D) stainings for remote area (RA) and infarct area of hearts from NLC and TgGRK5 mice at 8 weeks post-myocardial infarction (MI) (n = 8–10). Scale bar in white or black (100 μm). Arrowheads indicate positive staining. Insets show higher magnification at ×250. Flow-cytometric quantification of immune cells isolated from NLC and TgGRK5 hearts at 4 weeks post-MI (E–K) (n = 8 per group). Quantification of RT-PCR data showing fold change of NLC-Sham for IL-6, IL-1β, VCAM1, CCL2, (L–O) in left ventricular samples collected at 8-weeks post-surgery (n = 8–16). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. One-way ANOVA with Tukey’s post hoc test or t-test were used between groups.

Figure 6. Cardiomyocyte-specific GRK2 overexpression did not alter immune cell recruitment to the heart post-myocardial infarction. Ejection fraction (EF) (A) and left ventricular internal diameter in diastole (LVIDd) (B) as measured by echocardiography at 0 (baseline), 4- and 8-weeks post-Sham operation or myocardial infarction (MI) in NLC and TgGRK2 groups (n = 6–14). Measures of heart weight to body weight ratio (HW/BW) at 8 weeks post-surgery (C) (n = 6–13). Kaplan–Meier survival curves of Sham and MI groups (D) (n = 8–43). Log-rank (Mantel–Cox) test has been used between groups. Representative images and quantification of CD45 (E), MPO (F), CD68 (G), tryptase (H) and CD3 (I) stainings for remote area (RA), border zone (BZ), and infarct area of hearts from NLC and TgGRK2 mice at 4-days post-MI. Scale bar in white or black (100 μm). Arrowheads indicate positive staining. Insets show higher magnification at ×250 (n = 5 per group). Quantification of RT-PCR data showing fold change of NLC-MI for Bex1, Cxcl11, Cxcl13 and IL-15 (J) in LV samples collected at 4 days post-MI (n = 7 per group). Quantification of RT-PCR data showing fold change of NLC-MI for TNFα, IL-6, IL-1β, VCAM1, ICAM1,…

Figure 7. Cardiomyocyte-GRK5 deletion improved contractility, diminished early immune cell recruitment to the heart and increased survival rate post-myocardial infarction. Ejection fraction (EF) (A) and left ventricular internal diameter in diastole (LVIDd) (B) as measured by echocardiography at 0 (baseline), 4- and 8-weeks post-Sham operation or myocardial infarction (MI) in WT and GRK5cKO groups (n = 8–23). Measures of heart weight to body weight ratio (HW/BW) at 8 weeks post-surgery (C) (n = 20–24). Representative images and quantification of CD45 (D), MPO (E) and CD68 (F) stainings for remote area (RA) and border zone (BZ) of hearts from WT and GRK5cKO mice at 4-days post-MI. Scale bar in white or black (100 μm). Arrowheads indicate positive staining. Insets show higher magnification at ×250 (n = 6–13). Flow-cytometric quantification of immune cells isolated from WT and GRK5cKO hearts at 4 days post-MI (G–L) (n = 8 per group). Kaplan–Meier survival curves of Sham and MI groups (M) (n = 11–37). Log-rank (Mantel–Cox) test has been used between groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.0001 vs. WT-Sham and GRK5cKO-Sham. Two- or one-way ANOVA with Tukey’s post hoc…