Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling

Figures

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  Fig. 1. Validation of differentially regulated miRNAs in HCM mouse models by Taqman qPCR analysis. Total RNA was isolated, reverse transcribed and qPCR was performed with specific Taqman miRNA assays (Applied Biosystems). Relative quantity of each miRNA was calculated using the 2-ΔΔCT method (snoRNA202 used as a normalization control). miRNA expression levels were quantified for the following mice models: (A) HCM_CsA vs. WT_CsA mice (n = 3–4); (B) HCM_Aged vs. WT_Aged mice (n = 3); (C) Mybpc3 homozygous mutant (MyBPCt/tvs. WT mice (n = 3). Data are represented as geometric mean of relative quantity ±SD, Statistics: 2way ANOVA with Sidak's multiple comparisons test. Adjusted p value **** p < 0.0001, ***p < 0.001, ** p = 0.0085, *p < 0.05.

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  Fig. 2. miR-199a-3p mimic expression regulates expression of ECM. (A) Adult murine cardiac fibroblasts were transfected with control or miR-199a-3p mimics and conditioned media was collected and processed for mass-spectrometric analysis. Volcano plots depicting proteomics analysis in basal unstimulated conditions (left graph) and in TGFβ1 stimulated conditions (right graph). Blue dots indicate significantly down-regulated proteins and red dots indicate significantly up-regulated proteins in miR-199a-3p mimic expressing cells as compared to control-mimic expressing cells (B) Validation of proteomics data was conducted by corresponding gene expression levels through quantitative PCR. Relative quantity of each gene was calculated using the 2-ΔΔCT method (Tbp used as a normalization control). Data are represented as geometric mean ± SD (n = 4 experiments). Statistics: Paired 2-tailed t-test with multiple comparisons-FDR, p value **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3. Effects of in vivo miR-199a-3p inhibition on echocardiography and gravimetric parameters of HCM mice. HCM mice were treated with either LNA-control (n = 9, blue-filled circles or LNA-antimiR-199a-3p (n = 7, red-filled circles) (12.5 mg/kg monthly). (A) diastolic left ventricular posterior wall thickness (LVPW;d) (B) diastolic left ventricular internal dimension (LVID;d) and (C) fractional shortening (FS) % at baseline and end-point of the study. Data are expressed as mean ± SEM. Statistics: 2way ANOVA with Sidak's multiple comparisons test. P value: *p = 0.0457, **p = 0.0082, ****p < 0.0001. (D) Isovolumetric relaxation time (IVRT) at end-point. (E) Gravimetric analysis shows left atrial /tibia length ratio (LA/TL). Data are expressed as mean ± SEM, Statistics: Unpaired two-tailed t-test. P value p = ns (not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 4. Effects of in vivo miR-199a-3p inhibition on cardiomyocyte response in HCM mice. (A) Micrographs depict 5 μm paraffin-embedded heart sections from LNA-control and LNA-antimiR-199a-3p treated mice, stained with fluorescent-labelled wheat-germ agglutinin (WGA; green) and DAPI (blue) to demarcate cardiomyocyte borders and identify nuclei, respectively. (B) Graph shows quantification of cardiomyocyte cross-sectional area (CSA, um2) as measured by using NIH Image J image analysis software. Statistics: Unpaired two-tailed t-test. There is no statistical difference (ns) between LNA-control (n = 9, blue filled circles) or LNA-antimiR-199a-3p (n = 7, red filled circles). mRNA levels of cardiac stress markers (C) Myh6, Myh7 and (D) Anp were quantified by real time-quantitative PCR in left ventricular heart tissue obtained from aged untreated WT (n = 7, black-filled circles), age-matched HCM (n = 9, empty circles), and aged HCM mice treated with LNA-control (n = 9, blue filled circles) or LNA-antimiR-199a-3p (n = 7, red filled circles). Relative quantity of each gene was calculated using the 2-DDCT method (Tbp was used as a normalization control). Data are expressed as geometric mean ± SD.…

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  Fig. 5. Effects of in vivo miR-199a-3p inhibition on cardiac remodeling in HCM mice. (A) Micrographs depict 5 μm paraffin-embedded heart sections from LNA-control and LNA-antimiR-199a-3p treated mice stained with Masson's Trichrome (MT). Blue staining depicts collagen deposition areas. Scale bar = 0.5 mm (B) Graph shows quantification of fibrosis in heart sections using NIH Image J Software in LNA-control (blue-filled circles) and LNA-antimiR-199a-3p (red-filled circles) treated mice hearts. Data are expressed as mean ± SEM. Statistics: Unpaired 2-tailed t-test with Welch's correction, *p-value <0.05. mRNA levels of collagens (C) Col1a1 and (D) Col3a1 were quantified by real time-quantitative PCR in left ventricular heart tissue obtained from LNA-control (n = 9, blue filled circles) or LNA-antimiR-199a-3p treated HCM mice (n = 7, red filled circles). Relative quantity of each gene was calculated using the 2-DDCT method (Tbp was used as a normalization control). Data are expressed as geometric mean ± SD. Statistics: Unpaired 2-tailed t-test with Welch's correction. P value * p < 0.05 (E) Representative immunoblots show Periostin and Gapdh protein expression (left) and densitometric…

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  Fig. 6. Identification of direct targets of miR-199a-3p in cultured adult mouse cardiac fibroblasts. mRNA levels of computationally predicted miR-199a-3p targets were quantified by real time-quantitative PCR in cardiac fibroblasts, cultured in serum-free media or 10 % FBS/DMEM, transfected with either control mimic (cntr) or miR-199a-3p mimic (199a-3p). (A) Cd151, (B) Itga3, (C) Rgs4, (D) Adamtsl3, (E) Cited2, (F) Rb1 (G) Kdm6a (H) Pnrc1 (I) Dnmt3a (J) Mkrn1 (K) mTor (L) Egr1, (M) Sbno2, (N) Nid2, (O) Itga8 (P) Dnmt1. Relative quantity of each gene was calculated using the 2-DDCT method (Hprt was used as a normalization control, n = 4 per group). Data are presented geometric mean ± SD. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Fig. 7. miR-199a-3p mimic inhibits AKT phosphorylation in cardiac fibroblasts. Cardiac fibroblasts were transfected with control mimic (cntr) or miR-199a-3p mimic (199a-3p) and cultured with or without TGFβ1 (10 ng/ml) for 48 h. (A) Representative immunoblots show phospho-AKT (Ser473) (upper panels) and total AKT (lower panels) (B) Densitometric analysis of phospho-Akt (Ser473) normalized to total Akt (n = 8 per group). Data are presented as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test, ** p < 0.01, **** p < 0.0001. (C) Representative immunoblots show phospho-AKT(Thr308) (upper panel) and total AKT (lower panel) (D) Densitometric analysis of phospho-AKT (Thr308) normalized to total AKT (n = 5–6 per group). Data is represented as mean ± SEM. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test, * p < 0.05, ** p < 0.01 (E) mRNA expression levels of miR-199a-3p targets Pik3cβ, Gna12 and Phlpp2. Relative quantity of each gene was calculated using the 2-DDCT method (Hprt was used as a normalization control, n = 4 per group). Data are presented as geometric mean ± SD. Statistical analysis was…