Regression from pathological hypertrophy in mice is sexually dimorphic and stimulus specific

Abstract

Pathological cardiac hypertrophy is associated with increased morbidity and mortality. Understanding the mechanisms whereby pathological cardiac growth can be reversed could be of therapeutic value. Here, we show that pathways leading to regression of pathological cardiac hypertrophy are strongly dependent on the hypertrophic trigger and are significantly modified by sex. Two pathological stimuli causing hypertrophy via distinct pathways were administered to male and female mice: angiotensin II (ANG II) or isoproterenol (Iso). Stimuli were removed after 7 days of treatment, and left ventricles (LVs) were studied at 1, 4, and 7 days. ANG II-treated females did not show regression after stimulus removal. Iso-treated males showed rapid LV hypertrophy regression. Somewhat surprisingly, RNAseq analysis at day 1 after removal of triggers revealed only 45 differentially regulated genes in common among all the groups, demonstrating distinct responses. Ingenuity pathway analysis predicted strong downregulation of the TGFβ1 pathway in all groups except for ANG II-treated females. Consistently, we found significant downregulation of Smad signaling after stimulus removal including in ANG II-treated females. In addition, the ERK1/2 pathway was significantly reduced in the groups showing regression. Finally, protein degradation pathways were significantly activated only in Iso-treated males 1 day after stimulus removal. Our data indicate that TGFβ1 downregulation may play a role in the regression of pathological cardiac hypertrophy via downregulation of the ERK1/2 pathway and activation of autophagy and proteasome activity in Iso-treated males. This work highlights that the reversal of pathological hypertrophy does not use universal signaling pathways and that sex potently modifies this process.NEW & NOTEWORTHY Pathological cardiac hypertrophy is a major risk factor for mortality and is thought to be largely irreversible in many individuals. Although cardiac hypertrophy itself has been studied extensively, very little is understood about its regression. It is important that we have a better understanding of mechanisms leading to regression, why this process is not reversible in some individuals and that sex differences need to be considered when contemplating therapies.

Keywords: cardiac hypertrophy; heart; regression from hypertrophy.

Figure 1.

Pathological cardiac hypertrophy and regression depend on the hypertrophic trigger and are modulated by sex. A: experimental set-up. Mice were treated with vehicle control, ANG II, or Iso for 7 days, administered through an osmotic pump. Pumps were removed and regression was studied at various time points. B: LV/TL in males and females compared with vehicle control group. n = 4–8/group. Means ± SE. One-way ANOVA post hoc-uncorrected Fisher’s LSD. *P < 0.05, ***P < 0.001, ****P < 0.0001 significance. C: plots of gene expression measured by RNA sequencing comparing postremoval day 1 (P1) and hypertrophy in males and females treated with ANG II (top) and Iso (bottom). D: Venn diagram showing common differentially expressed genes among all groups. E: biological functions identified using ingenuity pathway analysis (IPA) on the 45 differentially expressed genes in common to all groups. ANG II, angiotensin II; Iso, isoproterenol; LV/TL, left ventricle weight/tibia length; P, post hypertrophy day.

Figure 2.

Fibrotic signaling involved upon removal of hypertrophic trigger. A: heatmap showing the top canonical pathways identified by ingenuity pathway analysis (IPA) that are enriched at post-removal day 1 (P1) in male and female mice treated for 7 days with ANG II or Iso. Complete list in Supplemental Fig. S3. B: TGFβ1 in male Iso is the most significant upstream regulator predicted (P = 4.99e-61) compared with any other regulator in all groups. IPA predicts the inhibition of TGFβ1 regulator for male Iso (z score = −6.085). C: selected differentially expressed genes regulated by TGFβ1 in male Iso at P1 and normalized by hypertrophy. D: hydroxyproline content measured in all groups. Seven days of treatment with hypertrophic trigger did not induce an increase of collagen deposition (vehicle vs. hypertrophy). Significant increase of collagen deposition was observed after removal of hypertrophic stimuli when compared with hypertrophy. Means ± SE. One-way ANOVA post hoc-uncorrected Fisher’s LSD. *P < 0.05, **P < 0.01, ***P < 0.001 significance. n = 4–8/group. ANG II, angiotensin II; Iso, isoproterenol.

Figure 3.

TGFβ1 signaling. A: scheme showing the TGFβ1 signaling pathways via SMAD, AKT, ERK, and p38. B: Western blot of SMAD2/3 phosphorylation. C: Western blot of Akt phosphorylation. There was little regulation of Akt in response to ANG II or Iso in males or females. Protein quantifications were normalized to vinculin. D: Western blot of ERK1/2 was activated in male mice with ANG II or Iso, which decreased after stimulus removal. ERK1/2 was not activated in female mice with ANG II or Iso, but was decreased after the removal of Iso. E: Western blot of p38 phosphorylation. Protein quantifications were normalized to vinculin. Means ± SE. One-way ANOVA post hoc-uncorrected Fisher’s LSD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 significance; n = 4–8/group. Hypertrophy group compared with vehicle control; P1, P4, and P7 compared with hypertrophy. ANG II, angiotensin II; Iso, isoproterenol.

Figure 4.

Protein degradation pathways. A: measurements of proteasome. Proteasome increased in males in response to ANG II and Iso; then increased further in response to Iso removal but decreased after ANG II removal. Proteasome activity was unchanged in female mice. B: autophagy activity did not significantly change in male and female ANG II. Autophagy increased after the removal of Iso in male mice. In female mice, autophagy activity decreased with Iso; then increased after Iso removal. Means ± SE. One-way ANOVA post hoc-uncorrected Fisher’s LSD. *P < 0.05, **P < 0.01, ***P < 0.001 significance; n = 4–8/group. Hypertrophy group compared with vehicle control; P1, P4, and P7 compared with hypertrophy. ANG II, angiotensin II; Iso, isoproterenol.

Figure 5.

Summary table of significant results. Arrows indicate P < 0.05. Red arrow indicates upregulation and green arrow downregulation. ANG II, angiotensin II; Iso, isoproterenol; LV/TL, left ventricle weight/tibia length.