INSL6 Protective Effects in Heart Failure

Background The insulin/insulin‐like growth factor/relaxin family represents a group of structurally related but functionally diverse proteins. The family member relaxin‐2 has been evaluated in clinical trials for its efficacy in the treatment of acute heart failure. In this study, we assessed the role of insulin‐like peptide 6 (INSL6), another member of this protein family, in murine heart failure models using genetic loss‐of‐function and protein delivery methods. Methods and Results Insl6‐deficient and wild‐type (C57BL/6N) mice were administered angiotensin II or isoproterenol via continuous infusion with an osmotic pump or via intraperitoneal injection once a day, respectively, for 2 weeks. In both models, Insl6‐knockout mice exhibited greater cardiac systolic dysfunction and left ventricular dilatation. Cardiac dysfunction in the Insl6‐knockout mice was associated with more extensive cardiac fibrosis and greater expression of fibrosis‐associated genes. The continuous infusion of chemically synthesized INSL6 significantly attenuated left ventricular systolic dysfunction and cardiac fibrosis induced by isoproterenol infusion. Gene expression profiling suggests liver X receptor/retinoid X receptor signaling is activated in the isoproterenol‐challenged hearts treated with INSL6 protein. Conclusions Endogenous Insl6 protein inhibits cardiac systolic dysfunction and cardiac fibrosis in angiotensin II– and isoproterenol‐induced cardiac stress models. The administration of recombinant INSL6 protein could have utility for the treatment of heart failure and cardiac fibrosis

miR-410 and miR-495 Are Dynamically Regulated in Diverse Cardiomyopathies and Their Inhibition Attenuates Pathological Hypertrophy.

Noncoding RNAs have emerged as important modulators in cardiac development and pathological remodeling. Recently, we demonstrated that regulation of the Gtl2-Dio3 noncoding RNA locus is dependent on the MEF2 transcription factor in cardiac muscle, and that two of its encoded miRNAs, miR-410 and miR-495, induce robust cardiomyocyte proliferation. Given the possibility of manipulating the expression of these miRNAs to repair the damaged heart by stimulating cardiomyocyte proliferation, it is important to determine whether the Gtl2-Dio3 noncoding RNAs are regulated in cardiac disease and whether they function downstream of pathological cardiac stress signaling. Therefore, we examined expression of the above miRNAs processed from the Gtl2-Dio3 locus in various cardiomyopathies. These noncoding RNAs were upregulated in all cardiac disease models examined including myocardial infarction (MI) and chronic angiotensin II (Ang II) stimulation, and in the cardiomyopathies associated with muscular dystrophies. Consistent with these observations, we show that the Gtl2-Dio3 proximal promoter is activated by stress stimuli in cardiomyocytes and requires MEF2 for its induction. Furthermore, inhibiting miR-410 or miR-495 in stressed cardiomyocytes attenuated the hypertrophic response. Thus, the Gtl2-Dio3 noncoding RNA locus is a novel marker of cardiac disease and modulating the activity of its encoded miRNAs may mitigate pathological cardiac remodeling in these diseases

Knockdown of miR-410 and miR-495 results in decreased cardiomyocyte hypertrophy.

<p><i>A</i>, Quantitative RT-PCR expression of miR-410 in NRVMs transfected with antimiR-410. <i>B</i>, Quantitative RT-PCR expression of miR-495 in NRVMs transfected with antimiR-495. <i>C</i>, Representative images of PE-treated NRVMs transfected with antimiR-NC, antimiR-410, and antimiR-495. <i>Top panels</i>, Immunofluorescence images of NRVMs stained with α-actinin (green) and Dapi (blue). <i>Bottom panels</i>, Computer generated image area analysis of cardiomyocytes. <i>D</i>, Relative CM area in NRVMs transfected with antimiR-410 and antimiR-495 compared to antimiR-NC shows significantly reduced cell size. <i>E</i>, Quantitative RT-PCR expression of <i>Nppa</i> and <i>Nppb</i> in PE-treated NRVMs transfected with antimiR-410, anti-miR-495, or both (anti-miR-410 + miR-495) compared to antimiR-NC. Error bars represent mean ± S.E. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

MEF2-dependent regulation of the <i>Gtl2-Dio3</i> proximal promoter and miRNAs in cardiac homeostasis and stress.

<p><i>A</i>, Luciferase analysis of the wild type (WT) and MEF2 site mutant (MUT) <i>Gtl2-Dio3</i> proximal promoter in neonatal cardiomyocytes stimulated with PE or Ang II compared to control (PBS). <i>B</i>, Quantitative RT-PCR expression of miR-410 and miR-495 in control (sh<i>lacZ</i>) and MEF2A depleted (sh<i>Mef2a</i>) NRVMs treated with PE or Ang II, respectively. <i>C</i>, Quantitative RT-PCR expression of miR-410 and miR-495 in adult MEF2A knockout hearts. <i>D</i>, Quantitative RT-PCR expression of miR-410 and miR-495 in NRVMs depleted of MEF2A (sh<i>Mef2a</i>), MEF2D (sh<i>Mef2d</i>), or both (sh<i>Mef2a/d</i>) compared to control (sh<i>lacZ</i>). Error bars represent mean ± S.E. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

<i>Gtl2-Dio3</i> miRNAs are upregulated <i>in vitro</i> in cardiac hypertrophy.

<p><i>A</i>, Relative cardiomyocyte (CM) area in control (PBS), phenylephrine-treated (PE), and angiotensin II-treated (Ang II) NRVMs. <i>B</i>, Quantitative RT-PCR expression of the hypertrophic markers <i>Nppa</i> and <i>Nppb</i> in PE- and Ang II-treated NRVMs. Values are normalized to PBS control (CTL). <i>C</i>, Quantitative RT-PCR expression of miR-410 and miR-495 in PE- and AngII-treated NRVMs, respectively. Error bars represent mean ± S.E. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Follistatin-like 1 promotes cardiac fibroblast activation and protects the heart from rupture

Follistatin-like 1 (Fstl1) is a secreted protein that is acutely induced in heart following myocardial infarction (MI). In this study, we investigated cell type-specific regulation of Fstl1 and its function in a murine model of MI. Fstl1 was robustly expressed in fibroblasts and myofibroblasts in the infarcted area compared to cardiac myocytes. The conditional ablation of Fstl1 in S100a4-expressing fibroblast lineage cells (Fstl1-cfKO mice) led to a reduction in injury-induced Fstl1 expression and increased mortality due to cardiac rupture during the acute phase. Cardiac rupture was associated with a diminished number of myofibroblasts and decreased expression of extracellular matrix proteins. The infarcts of Fstl1-cfKO mice displayed weaker birefringence, indicative of thin and loosely packed collagen. Mechanistically, the migratory and proliferative capabilities of cardiac fibroblasts were attenuated by endogenous Fstl1 ablation. The activation of cardiac fibroblasts by Fstl1 was mediated by ERK1/2 but not Smad2/3 signaling. This study reveals that Fstl1 is essential for the acute repair of the infarcted myocardium and that stimulation of early fibroblast activation is a novel function of Fstl1