Germline deletion of Rgs2 and/or Rgs5 in male mice does not exacerbate left ventricular remodeling induced by subchronic isoproterenol infusion

Figures

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  FIGURE 1. Loss of RGS2 or RGS5 leads to left ventricular dilation and reduced left ventricular (LV) function. (a) Schematic illustration of the timeline for non‐invasive cardiac structure and function assessment by echocardiography (echo), systemic infusion of saline or isoproterenol (ISO) in conscious mice, organ harvest, and measurement of heart weight and tibia length after euthanasia. (b) Representative parasternal long‐axis M‐mode LV echocardiograms at mid‐ventricular level, with representative measurements of left ventricular internal diameter at diastole (LVID; d) and systole (LVID; s). Mice (n = 8–14 per group) received subchronic (3 days) infusion of saline or isoproterenol (ISO, 30 mg/kg/day) via osmotic mini‐pumps (schema in a). Echocardiography was performed under anesthesia (isoflurane) 72 h after mini‐pump implantation. Measurement of chamber size at end‐diastole and end‐systole, internal diameter (c and d, respectively), and volume (g and h) reveals LV dilation due to the loss of either RGS2 or RGS5. ISO infusion induced an increase in end‐diastole chamber size in WT mice. No significant differences in septum (e and f) or posterior wall (i and j) thickness due to treatment…

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  FIGURE 2. Deletion of Rgs2 and 5 induces differential expression of other R4 members in the ventricular myocardium. To examine any potential compensatory mechanisms that occur due to the loss of RGS2 and/or RGS5, we performed quantitative PCR analysis to determine mRNA expression of different members of the R4 protein family that have previously been identified as having cardiac expression. (a) Expression of Rgs1 is significantly decreased compared to WT due to the loss of either RGS2 or RGS5 but not both. Regardless of genotype, ISO infusion increased the expression of Rgs1. (b) Expression of Rgs2 is not influenced by the loss of Rgs5 or by ISO infusion. (c) Expression of Rgs3 was not influenced by the loss of RGS2 and/or RGS5, regardless of saline or ISO treatment. (d) Expression of Rgs4 was significantly increased due to the loss of RGS5, and this difference was unchanged by ISO infusion. (e) Expression of Rgs5 was significantly increased due to loss of RGS2 and was unaffected by ISO infusion, regardless of genotype. Symbols in the graphs represent individual samples from separate animals (n = 8 for each genotype and treatment). mRNA expression is expressed as a fold change from WT SAL…

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  FIGURE 3. Dual loss of RGS2 and RGS5 increases susceptibility to subchronic ISO infusion‐induced eccentric cardiac hypertrophy. (a, b) Gravimetric analysis of changes due to saline and ISO infusion in heart and lung weight, respectively. Heart samples were harvested shortly after echocardiography at the end of ISO infusion on day 3. (c) Representative images of trichrome‐stained midventricular tissue sections used to determine cross‐sectional cardiomyocyte area. The scale bar in the low magnification image denotes 50 μm, whereas the one in the high magnification denotes 18.5 μm. (d) Summary graph of quantified cross‐sectional area of midventricular cardiomyocytes in saline (SAL)‐ and isoproterenol (ISO)‐treated ventricular tissue sections. (e) Quantitative PCR analysis of changes in Nppa mRNA expression in SAL‐ and ISO‐treated ventricular tissue from WT (closed circles) and Rgs2/5 dbKO (open squares) mice. (f) Representative Western blot for α‐actinin and CaMKII, with summary plots in (g and h), respectively, of SAL‐ and ISO‐treated ventricular tissue from WT (closed circles) and Rgs2/5 dbKO (open squares) mice. Symbols in the graphs represent individual samples from separate animals (n =…

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  FIGURE 4. Phosphorylation of AKT but not ERK is increased due to the dual loss of RGS2 and RGS5. (a) Representative Western blot for pAKT, AKT, pERK, and ERK expression in heart base tissue harvested from WT and Rgs2/5 dbKO mice. (b, d) Quantification of p‐AKT/AKT and p‐ERK/ERK expression, respectively, demonstrating increased phosphorylation of AKT but not ERK in Rgs2/5 dbKO saline‐treated mice. (c, e) Quantification of total AKT or total ERK expression relative to β‐Actin, respectively. Symbols in the graphs represent individual samples from separate animals (n = 8 for WT and Rgs2/5 dbKO for p‐AKT/AKT, and n = 4 for WT and Rgs2/5 dbKO for p‐ERK/ERK). Data in the summarized plots are mean ± standard deviation. A two‐way ANOVA mixed model, with Šidák post hoc analysis was applied for multiple comparisons for statistical significance. Stated numbers in the graphs are p‐values for individual comparisons. SAL, saline; ISO, isoproterenol.

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  FIGURE 5. Loss of RGS2 and RGS5 induces increased basal GSK3β phosphorylation. (a) Representative Western blot for p‐GSK3β, GSK3β, p‐P70S6K, and P70S6K expression in heart base tissue harvested from WT and Rgs2/5 dbKO mice. (b, d) quantification of p‐P70S6K/P70S6K and p‐GSK3β/GSK3β expression, respectively, demonstrating increased phosphorylation of GSK3β but not P70S6K in Rgs2/5 dbKO saline‐treated mice. (c, e) Quantification of total GSK3β or total P70S6K expression relative to β‐Actin, respectively. Symbols in the graphs represent individual samples from separate animals (n = 4–5 for each genotype and treatment). Data in the summarized plots are mean ± standard deviation. Two‐way ANOVA mixed model, with Šidák post hoc analysis, was applied for multiple comparisons for statistical significance. Stated numbers in the graphs are p‐values for individual comparisons. SAL, saline; ISO, isoproterenol.

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  FIGURE 6. Loss of RGS2 results in increased sarcomere disorganization. (a–d) Representative confocal images of adult LVCM isolated from WT, Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice, where α‐actinin is green and titin is red. Quantitative assessment of (e) cell length, (f) width, (g) width/length ratio, (h) sarcomere length, and (i) Z‐disc thickness are presented by bar graphs for each genotype. (j) Stacked bar graphs showing the ratio of LVCM displaying normal vs. abnormal sarcomere arrangements. The graphs show increased disorganization in LVCM isolated from Rgs2 KO and Rgs2/5 dbKO relative to WT mice. Symbols in the graphs represent individual samples from separate animals. Data in the summarized plots are mean ± standard deviation. For e–i, n = 8 hearts per group were used, from which at least 100 cells per heart were analyzed, and for J, we quantified sarcomere disorganization based on titin immunolabeling as a percentage of total isolated cells (at least 20 cells per heart) from each mouse. Two‐way ANOVA mixed model, with Šidák post hoc analysis, was applied for multiple comparisons for statistical significance. Stated numbers in the graphs are p‐values for individual comparisons.

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  FIGURE 7. Loss of RGS2 or RGS5 increases immune cell presence in ventricular tissue. Representative CD45 labeling (brown) (a, quantified in b (n = 8 hearts)) and CD68 labeling (c, quantified in d (n = 4–5 hearts)) demonstrates increased immune cell infiltration in saline‐treated Rgs2 and Rgs5 KO ventricular tissue. Symbols in the graphs represent individual samples from separate animals. Data in the summarized plots are mean ± standard deviation. Two‐way ANOVA mixed model, with Šidák post hoc analysis, was applied for multiple comparisons for statistical significance. Stated numbers in the graphs are p‐values for individual comparisons. SAL, saline; ISO, isoproterenol.

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  FIGURE 8. Loss of RGS2 or RGS5 protects against ISO‐induced interstitial and pericardial fibrosis. (b) Representative low (2x)‐magnification photomicrographs of Masson's trichrome stained midventricular cross‐sections from WT and Rgs2/5 db KO mice infused with saline or ISO for 3 days. (g–j) Representative Masson's trichrome staining, where collagen fibers are stained blue, the cytoplasm is stained red, and nuclei are stained black of cardiac tissue harvested from the vehicle or ISO‐infused mice. Interstitial (quantified in c and represented in g), pericardial (d and h), perivascular (e and i), and endocardial (f and j) fibrosis was scored on an arbitrary scale of 1–4, with 1 displaying little to no fibrosis and 4 displaying extensive fibrosis. ISO infusion significantly increased the amount of interstitial fibrosis present in WT and Rgs2/5 dbKO tissue but not tissue from Rgs2 or Rgs5 KO mice. (a) Col3a1 expression increased in both WT and Rgs2/5 dbKO tissue due to ISO infusion. Symbols in the graphs represent individual samples from separate animals. Data in the summarized plots are mean ± standard error. We used nonparametric ANOVA with Kruskal–Wallis test, followed by a two‐stage linear…

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  FIGURE 9. Loss of RGS2 and/or RGS5 does not increase apoptosis in cardiac tissue in either saline or ISO‐infused animals. (a) Representative TUNEL staining, where TUNEL‐positive (TUNEL+) nuclei are stained blue. The tissue sections were counterstained with nuclear fast red, which stains TUNEL‐negative nuclei pink. (b) The total number of TUNEL‐positive and ‐negative nuclei was quantified from 10 fields per section and the total percent of TUNEL‐positive nuclei was calculated. Symbols in the graphs represent individual samples from separate animals. Data in the summarized plots are mean ± standard deviation.