Regulator of G Protein Signaling 6 Facilities Cardiac Hypertrophy by Activating Apoptosis Signal–Regulating Kinase 1–P38/c‐JUN N‐Terminal Kinase 1/2 Signaling

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  Figure 1. Regulator of G protein signaling 6 (RGS6) is expressed in heart and is upregulated in response to hypertrophic stimuli. A, Tissue lysates were prepared from the normal donor hearts (n=8) and failing hearts of patients with dilated cardiomyopathy (DCM) (n=8) and were subjected to Western blotting analysis with anti–atrial natriuretic peptide (ANP), β‐myosin heavy chain (MYH7), RGS6, and GAPDH antibodies. B, Aortic banding (AB) procedures were performed on wild‐type mice, and the heart tissue was harvested 4 and 8 weeks after surgery (n=4 per group). ANP, MYH7, RGS6, and GAPDH were detected by Western blotting. C, Neonatal rat cardiomyocytes were treated with 1 μmol/L angiotensin II (Ang II) for 24 to 48 hours. ANP, MYH7, RGS6, and GAPDH were detected by Western blotting. Representative Western blots from the indicated groups and quantification of the results are shown. Protein levels were normalized to GAPDH levels. The graph shows the quantitative data from 4 to 5 independent experiments. Data are presented as means±SDs. **P<0.01 compared with the controls. ## P<0.01 compared with the group 4 weeks after AB or 24 hours after Ang II treatment.

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  Figure 2. Regulator of G protein signaling 6 (RGS6) regulates angiotensin II (Ang II)–induced cardiomyocyte hypertrophy in vitro. A, Neonatal rat cardiomyocyte (NRCM) lysates were prepared from cells infected with adenoviruses expressing short hairpin RNA (AdshRNA), a short hairpin RNA targeting RGS6 (AdshRGS6), green fluorescent protein (AdGFP), or RGS6 (AdRGS6) and subjected to Western blotting with anti‐RGS6 and GAPDH antibodies. Representative Western blots are shown. B, Representative fluorescent images of NRCMs infected with the indicated adenoviruses and treated with 1 μmol/L Ang II for 48 hours are shown. NRCMs were stained with an α‐actinin antibody (green). Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; blue). Cell surface areas were quantified by planimetry after treatments. Fifty cells were measured for each group. C, Atrial natriuretic peptide (ANP) and β‐myosin heavy chain (MYH7) mRNA levels were measured in NRCMs 48 hours after Ang II exposure using quantitative real‐time polymerase chain reaction and normalized to levels of the GAPDH transcript. The graph shows the quantitative data from 4 to 5 independent experiments. Data are presented as means±SDs.…

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  Figure 3. Cardiac‐specific deletion of regulator of G protein signaling 6 (RGS6) attenuates aortic banding (AB)–induced cardiac hypertrophy. A, We established the cardiac‐specific RGS6 knockout (RGS6 CKO) strain. Tissue lysates from the heart, kidney, liver, and spleen were prepared from wild‐type (WT) mice (n=4) and RGS6 CKO mice (n=4) and subjected to Western blotting analysis. Representative Western blots showing RGS6 expression levels in the 2 strains. B, Tissue lysates from the heart in the RGS6‐floxed mice (n=4), α‐myosin heavy chain (αMHC)–MerCreMer transgenic (MCM‐Cre) mice (n=4), and RGS6 CKO mice (n=4) and subjected to Western blotting analysis. Representative Western blots showing RGS6 expression levels in the 3 strains. C through E, Pressure overload–induced cardiac hypertrophy was alleviated in RGS6 CKO mice. The heart weight/body weight (HW/BW), lung weight/body weight (LW/BW), and heart weight/tibia length (HW/TL) ratios were calculated in RGS6‐floxed mice, MCM‐Cre mice, and RGS6 CKO mice 4 weeks after the AB procedure (n=12 mice per group). F, Representative images of hematoxylin and eosin (H&E)–stained heart sections from RGS6‐floxed mice, MCM‐Cre mice, and RGS6 CKO mice…

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  Figure 4. Cardiac‐specific regulator of G protein signaling 6 (RGS6) overexpression exacerbates aortic banding (AB)–induced cardiac hypertrophy. A and B, We established RGS6 transgenic (TG) mice. Tissue lysates were prepared from the hearts of non‐TG control (NTG) mice (CAG‐CAT‐RGS6/myosin heavy chain–Cre littermates without tamoxifen administration) and from the RGS6 TG mice 4 weeks after the AB procedure and were subjected to Western blotting analysis. Representative Western blots showing RGS6 expression levels in the 2 strains are depicted. C through E, Pressure overload–induced cardiac hypertrophy was exacerbated in RGS6 TG mice. The heart weight/body weight (HW/BW), lung weight/body weight (LW/BW), and heart weight/tibia length (HW/TL) ratios in NTG mice and RGS6 TG mice were calculated 4 weeks after the AB procedure (n=12 mice per group). F, Representative images of hematoxylin and eosin (H&E)–stained heart sections from NTG mice and RGS6 TG mice are shown. The cross‐sectional areas of the indicated groups were quantified by planimetry (n=5 mice per group). G through I, left ventricular (LV) end‐diastolic dimension (LVEDd), LV end‐systolic dimension (LVESd), and fractional shortening…

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  Figure 5. Regulator of G protein signaling 6 (RGS6) modulates apoptosis signal–regulating kinase 1 (ASK1)‐P38‐c‐JUN N‐terminal kinase 1/2 (JNK1/2) signaling in hypertrophic cardiomyocytes and hearts. A and B, Tissue lysates were prepared from the hearts of RGS6‐floxed mice (n=4), cardiac‐specific RGS6 knockout (RGS6 CKO) mice (n=4), nontransgenic control (NTG) mice (CAG‐CAT‐RGS6/myosin heavy chain–Cre littermates without tamoxifen administration) (n=4), and RGS6 transgenic (TG) mice (n=4) 4 weeks after the aortic banding (AB) procedure and subjected to Western blotting analysis. Representative Western blots showing the levels of the phosphorylated and total mitogen‐activated protein kinase kinase 1/2 (MEK1/2), extracellular signal‐regulated kinase 1/2 (ERK1/2), JNK1/2, and P38 proteins in the indicated strains are depicted. Bar graphs present the quantitative data for the levels of the phosphorylated and total JNK1/2 and P38 proteins in the indicated strains. **P<0.01 compared with RGS6‐floxed mice subjected to the AB procedure or NTG mice subjected to the AB procedure. C and D, Neonatal rat cardiomyocyte (NRCM) lysates were prepared from cells infected with adenoviruses expressing short…

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  Figure 6. RGS6 mediates Ang II‐induced cardiac hypertrophy by directly binding to ASK1. A‐B. The interaction between RGS6 and ASK1 in HEK293T cells transfected with FLAG‐RGS6 and HA‐ASK1 (or FLAG‐ASK1 and HA‐RGS6) was examined by co‐IP and Western blotting analysis. C. The interaction between mutant RGS6N401V and ASK1 in HEK293T cells transfected with FLAG‐ RGS6N401V (or FLAG‐ RGS6) and HA‐ASK1 was examined by co‐IP and Western blotting analysis. D‐E. Representative Western blots of the mapping analyses showing the binding domains responsible for the RGS6‐ASK1 interaction. F. Representative fluorescent images of NRCMs infected with the indicated adenoviruses and treated with 1 μg/mL Ang II for 48 h are shown (n=50 cells per group). NRCMs were stained with an α‐actinin antibody (green). Nuclei were stained with DAPI (blue). Cell surface areas were quantified by planimetry after treatments. G. Levels of the ANP and MYH7 mRNAs in NRCMs treated with Ang II for 48 h were measured by qRT‐PCR and normalized to levels of the GAPDH transcript. The graph shows quantitative data from four‐five independent experiments. H. An illustration of potential mechanisms in cardiac hypertrophy is depicted. The…