Cardiomyocyte-specific LARP6 overexpression prevents angiotensin II-induced myocardial dysfunction and interstitial fibrosis

Abstract

La ribonucleoprotein 6, translational regulator (LARP6), a multifunctional mRNA-binding protein with well-described profibrotic effects, increases type I collagen mRNA half-life, translation, and deposition in noncardiac tissues. In the heart, LARP6 is expressed in cardiomyocytes, not primarily involved in fibrosis, where its role is unknown. To investigate the role of cardiomyocyte-derived LARP6 on cardiac function and remodeling, we generated a cardiomyocyte-specific LARP6 overexpressing transgenic mouse model (LARP6-Tg). Baseline longitudinal studies up to 10 mo of age revealed that constitutive overexpression of LARP6 had no significant effect on cardiac function or morphology despite inducing mild interstitial fibrosis versus wild-type (WT) littermates. Subsequently, we hypothesized that cardiomyocyte-specific LARP6-Tg mice would exhibit exacerbated cardiac remodeling and dysfunction in response to hypertensive stress via angiotensin II (Ang II) infusion. Ang II (1000 ng/kg/min for 21 days) induced hypertension and cardiac hypertrophy in WT and LARP6-Tg mice of both sexes. Unexpectedly, Ang II-induced cardiac dysfunction was prevented in LARP6-Tg mice. Cardiac gene expression profiling predicted increased fibrosis and cardiomyocyte death in Ang II-treated WT mice and inhibition of cardiomyocyte death in Ang II-treated LARP6-Tg mice versus saline-treated controls. Surprisingly, Ang II-induced interstitial fibrosis was reduced in LARP6-Tg mice and associated with attenuation of cardiomyocyte cell death and reduced fibroblast activation. These data support a mild profibrotic action of cardiomyocyte-specific LARP6 overexpression in unstressed mice and, paradoxically, that LARP6 overexpression is sufficient to prevent Ang II-induced cardiac interstitial fibrosis and dysfunction. Sustained induction of LARP6 has therapeutic potential in hypertensive heart disease.NEW & NOTEWORTHY LARP6 is a novel multifunctional RNA-binding protein whose role in the heart is poorly understood. Transgenic overexpression of LARP6 in cardiomyocytes caused mild cardiac fibrosis under basal conditions with no impact on cardiac function but, unexpectedly, blunted angiotensin-II-induced cardiac fibrosis and dysfunction. This protective effect of LARP6 overexpression was associated with significant shifts in the cardiac transcriptome alongside blunted fibroblast activation and cardiomyocyte apoptosis under hypertension conditions, highlighting LARP6 as a novel therapeutic target.

Keywords: La ribonucleoprotein 6; cardiac hypertrophy; cardiac remodeling; hypertension; sequencing.

Figure 1.. Transgenic cardiomyocyte-specific LARP6 overexpression (LARP6-Tg) increases LARP6 gene and protein expression in the heart and is associated with baseline pro-fibrotic gene changes and fibrotic remodeling.

LARP6 mRNA expression in the heart, liver, and kidney of WT and Tg mice (A). Cardiac LARP6 protein expression, localized to cardiomyocytes via troponin staining, in WT and LARP6-Tg mice (B). Cardiac gene expression of collagen Iα1, collagen Iα2, and collagen IIIα1 at 40 weeks of age in male LARP6-Tg mice (C). Top gene network and enriched biological processes among genes differentially expressed between WT and LARP6-Tg mice (D). Upregulated genes, red; orange connecting lines indicated predicted activation of associated processes. Picrosirius red staining for cardiac collagen in male LARP6-Tg mice at 40-weeks of age compared to WT littermates (E). Biological replicates are as follows; (A) 4 Male WT and 4 Male Tg; (B) 6 Male WT Saline, 5 Female WT Saline, 6 Male Tg Saline, 6 Female Tg Saline; (C) 4 Male WT and 4 Male Tg; (D) 11 WT Saline and 12 Tg Saline; (E) 5 Male WT and 7 Male Tg. Statistical analyses for panels are as follows; (A) Kruskal-Wallis test for multiple comparisons with Dunn correction for multiple comparisons, (B) unpaired two-tailed t test, (C) unpaired one-tailed t test, (E) Mann-Whitney two-tailed t test. Scale bars are 400 μm for panel (B) and 200 μm for panel (E).

Figure 2.. Cardiomyocyte-specific constitutive LARP6 overexpression (LARP6-Tg) did not affect the pressor response or cardiac hypertrophy induced by Ang II infusion.

Cardiac protein expression of LARP6 in WT and LARP6-Tg mice after saline or Ang II infusion (A). Systolic blood pressure in male (B) and female (C) WT and LARP6-Tg mice after saline or Ang II infusion. Cardiac hypertrophy, assessed by heart weight-to-body weight (HW/BW) ratio, in male (D) and female (E) WT and LARP6-Tg mice. Cardiomyocyte cross-sectional area (CSA) in male (F) and female (G) WT and LARP6-Tg mice infused with saline or Ang II. Blue data points represent a males, red points indicate females. Biological replicates are as follows; (A) 3 Male WT Saline, 2 Male WT Ang II, 3 Male Tg Saline, 3 Male Tg Ang II, 2 Female WT Saline, 4 Female WT Ang II, 3 Female Tg Saline, 4 Female Tg Ang II; (B) 6 Male WT Saline, 6 Male WT Ang II, 6 Male Tg Saline, 7 Male Tg Ang II; (C) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II; (D) 6 Male WT Saline, 6 Male WT Ang II, 6 Male Tg Saline, 7 Male Tg Ang II; (E) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II; (F) 6 Male WT Saline, 6 Male WT Ang II, 6 Male Tg Saline, 6 Male Tg Ang II; (G) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II. Statistical analysis for panels of this figure utilized a two-way ANOVA with a Tukey post hoc. Scale bars are 200 μm.

Figure 3.. Cardiomyocyte-specific constitutive LARP6 overexpression (LARP6-Tg) prevented Ang II infusion-induced systolic dysfunction.

Baseline ejection fraction (i.e., pre-saline or Ang II infusion) between WT and LARP6-Tg animals of either sex (A and B). Cardiac ejection fraction following 21 days of saline or Ang II infusion in WT and LARP6-Tg animals of either sex (C and D). Diastolic function, assessed by E/E’, following 21 days of saline or Ang II infusion in WT and LARP6-Tg animals of either sex (E and F). Blue data points represent a males, a red point indicates females. Biological replicates are as follows; (A) 6 Male WT Saline, 6 Male WT Ang II, 6 Male Tg Saline, 7 Male Tg Ang II; (B) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II; (C) 11 Male WT Saline, 11 Male WT Ang II, 12 Male Tg Saline, 11 Male Tg Ang II; (D) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II; (E) 11 Male WT Saline, 11 Male WT Ang II, 12 Male Tg Saline, 11 Male Tg Ang II; (F) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II. Statistical analysis for panels of this figure utilized a two-way ANOVA with a Tukey post hoc.

Figure 4.. Ang II induced interstitial fibrosis and fibroblast activation in WT micethat was blunted in LARP6-Tg mice.

Interstitial fibrosis in male (A) and female (B) WT and LARP6 Tg mice treated with saline of Ang II. Cardiac periostin staining in male (C) and female (D) WT and LARP6-Tg mice following saline or Ang II infusion. Blue data points represent a males, a red point indicates female. Biological replicates are as follows; (A) 6 Male WT Saline, 6 Male WT Ang II, 6 Male Tg Saline, 7 Male Tg Ang II; (B) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II; (C) 6 Male WT Saline, 5 Male WT Ang II, 6 Male Tg Saline, 7 Male Tg Ang II; (D) 5 Female WT Saline, 7 Female WT Ang II, 6 Female Tg Saline, 7 Female Tg Ang II. Statistical analysis for panels (A-C) of this figure utilized a two-way ANOVA with a Tukey post hoc. A Kruskal-Wallis test was performed on panel (D) due to the non-parametric distribution of the data. Scale bars for all panels in this figure are 200 μm.

Figure 5.. LARP6-Tg mice exhibit anti-apoptotic cardiac gene signatures and reduced cardiomyocyte cell death in response to Ang II infusion.

Biological processes enriched in differentially expressed cardiac genes between (A) WT Ang II and WT Saline mice and (B) LARP6-Tg Ang II and WT Ang II mice. TUNEL positive nuclei within cardiomyocytes (red arrows) in male and female mice of both genotypes at baseline and after Ang II infusion (C). Yellow arrows indicate TUNEL positive interstitial cells. Plasma cardiac troponin measured after 21 day Ang II or saline infusion in male and female mice of both genotypes. Dashed line represents the assay absorbance threshold for troponin release indicative of myocardial damage (0.247) (D). Western blotting (E) for pro-apoptotic BAX expression in WT and LARP6-Tg mice treated with saline or Ang II. Biological replicates are as follows; (A) 11 WT Saline and 13 WT Ang II, (B) 13 WT Ang II and 14 Tg Ang II, (C) 6/5 M/F WT Saline, 6/7 M/F WT Ang II, 6/6 M/F Tg Saline, 7/6 M/F Tg Ang II, (D) 5/5 M/F WT Saline, 5/5 M/F WT Ang II, 5/5 Tg Saline, 5/5 Tg Ang II; (E) 3 Male WT Saline, 2 Male WT Ang II, 3 Male Tg Saline, 3 Male Tg Ang II, 2 Female WT Saline, 4 Female WT Ang II, 3 Female Tg Saline, 4 Female Tg Ang II. Statistical analysis for panels of this figure utilized a two-way ANOVA with a Tukey post hoc. Scale bar=100 μm.