Ablation of lncRNA Miat attenuates pathological hypertrophy and heart failure

Abstract

Rationale: The conserved long non-coding RNA (lncRNA) myocardial infarction associate transcript (Miat) was identified for its multiple single-nucleotide polymorphisms that are strongly associated with susceptibility to MI, but its role in cardiovascular biology remains elusive. Here we investigated whether Miat regulates cardiac response to pathological hypertrophic stimuli. Methods: Both an angiotensin II (Ang II) infusion model and a transverse aortic constriction (TAC) model were used in adult WT and Miat-null knockout (Miat-KO) mice to induce pathological cardiac hypertrophy. Heart structure and function were evaluated by echocardiography and histological assessments. Gene expression in the heart was evaluated by RNA sequencing (RNA-seq), quantitative real-time RT-PCR (qRT-PCR), and Western blotting. Primary WT and Miat-KO mouse cardiomyocytes were isolated and used in Ca²⁺ transient and contractility measurements. Results: Continuous Ang II infusion for 4 weeks induced concentric hypertrophy in WT mice, but to a lesser extent in Miat-KO mice. Surgical TAC for 6 weeks resulted in decreased systolic function and heart failure in WT mice but not in Miat-KO mice. In both models, Miat-KO mice displayed reduced heart-weight to tibia-length ratio, cardiomyocyte cross-sectional area, cardiomyocyte apoptosis, and cardiac interstitial fibrosis and a better-preserved capillary density, as compared to WT mice. In addition, Ang II treatment led to significantly reduced mRNA and protein expression of the Ca²⁺ cycling genes Sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a) and ryanodine receptor 2 (RyR2) and a dramatic increase in global RNA splicing events in the left ventricle (LV) of WT mice, and these changes were largely blunted in Miat-KO mice. Consistently, cardiomyocytes isolated from Miat-KO mice demonstrated more efficient Ca²⁺ cycling and greater contractility. Conclusions: Ablation of Miat attenuates pathological hypertrophy and heart failure, in part, by enhancing cardiomyocyte contractility.

Keywords: Miat; RNA splicing; cardiac hypertrophy; cardiomyocytes; heart failure; lncRNA.

Figure 1

Deletion of Miat attenuates Ang II-induced cardiac dysfunction. Miat-KO mice and WT littermates were treated with Ang II (2 mg/kg BW/day) or saline control (CNT) via a subcutaneously-implanted mini-osmotic pump for 28 days, then cardiac functions were evaluated by echocardiography. (A) Representative image of echocardiography. (B) Thicknesses of LV anterior wall (LVAW) and posterior wall (LVPW). (C) LV internal diameter (LVID; upper panels) and volume (LV Vol; lower panels) at diastole (d) and systole (s). (D) LV ejection fraction (EF) and fractional shortening (FS). (E) Ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/E'; left panel) and isovolumic relaxation time (IVRT; right panel). (F) stroke volume (SV; left panel) and cardiac output (CO; right panel). *p < 0.05, **p < 0.01.

Figure 2

Deletion of Miat attenuates Ang II-induced cardiac pathological hypertrophy. At 28 days after continuous Ang II (2 mg/Kg BW/day) or saline treatment, mice were euthanized and examined. (A) Heart weight (HW) versus tibia length (TL) ratio. (B) Representative images of wheat germ agglutinin (WGA) staining (left panel) and quantification of cardiomyocyte cross-section areas (right panel). (C) Representative images of TUNEL and cTnI staining (left panel) and quantification of apoptotic cardiomyocytes (right panel, % of nuclei). Yellow arrows indicate TUNEL⁺ cardiomyocytes. Scale bar = 50µm. (D) Representative images of Sirius Red/Fast Green staining (left panel) and quantification of interstitial fibrosis areas (right panel). (E) Representative images of CD31 staining (left panel) and quantification of capillary density (right panel). HPF, high-powered field. (F) qRT-PCR analyses of the mRNA levels of Bnp and Anf in the heart tissue. *p < 0.05, **p < 0.01.

Figure 3

Deletion of Miat attenuates TAC-induced heart failure. Cardiac functions were evaluated with echocardiography in Miat-KO mice and WT littermates 6 weeks after TAC or Sham surgery. (A) Representative image of echocardiography. (B) Thicknesses of LV anterior wall (LVAW) and posterior wall (LVPW). (C) LV internal diameter (LVID; upper panels) and volume (LV Vol; lower panels) at diastole (d) and systole (s). (D) LV ejection fraction (EF) and fractional shortening (FS). *p < 0.05, **p < 0.01.

Figure 4

Deletion of Miat attenuates TAC-induced cardiac adverse remodeling. At 6 weeks after TAC or Sham surgery, mice were euthanized and examined. (A) Heart-weight (HW) to tibia-length (TL) ratio. (B) Representative images of WGA staining (left panel) and quantification of cardiomyocyte cross-section areas (right panel). (C) Representative images of TUNEL and cTnI staining (left panel) and quantification of apoptotic cardiomyocytes (right panel, % of nuclei). Yellow arrows indicate TUNEL⁺ cardiomyocytes. Scale bar = 50 µm. (D) Representative images of Sirius Red/Fast Green staining (left panel) and quantification of interstitial fibrosis areas (right panel). (E) Representative images of CD31 staining (left panel) and quantification of capillary density (right panel). HPF, high-powered field. (F) qRT-PCR analyses of the mRNA levels of Bnp and Anf in the heart tissue. *p < 0.05, **p < 0.01.

Figure 5

Deletion of Miat blunts Ang II-induced hypertrophic gene program in the myocardium. WT and Miat-KO mice were treated with Ang II (WTA, KOA) or saline control (WTC, KOC) for 7 days, then RNAs were isolated from LV and sequenced. (A) Heatmaps of genes associated with cardiac hypertrophy (left panel) and contractility (right panel). ATP2A2 = SERCA2a. (B & C) validation of gene expression levels by qRT-PCR (B) and Western blotting (C, n = 3-6). *p < 0.05, **p < 0.01.

Figure 6

Deletion of Miat increases cardiomyocyte contractility. Cardiomyocytes were isolated from adult WT and Miat-KO mice. (A) Representative confocal line-scan images of Ca²⁺ transients (upper panels) along with their spatial averages and cell contractility by Fractional shortening (FS) (lower panels) in response to field stimulation (1 Hz). (B) Average calcium amplitude (ΔF/F₀), cell fraction shortening, half-time of decay (T50) and the rise time of Ca²⁺ transient. *p < 0.05, **p < 0.01. n = 41-88.