Myocardin ablation in a cardiac-renal rat model

Figures

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  Figure 1. Myocardin (MYOCD) mRNA and protein expression in myocardial tissues of DCM patients with and without renal failure and controls. (a) Quantitative mRNA expression of MYOCD in DCM cardiac tissues (n = 30) with and without renal failure and control group (n = 15) as determined by qRT-PCR using Taqman probe chemistry. (b) Representative western blot for MYOCD and GAPDH proteins in control (C1–C4) and DCM patients (P1–P5) in LV myocardium samples. Semi-Quantitative protein expression of MYOCD in DCM patients and controls as estimated by software ImageJ. Normalization of total RNA and protein was done using GAPDH as an internal control. Data given is Mean ± SEM; *p < 0.05 Control vs DCM; ***p < 0.001 Control vs DCM or DCM with cardio-renal syndrome.

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  Figure 2. MYOCD siRNA treatment on expression of hypertrophic genes and fibrotic genes in H9c2 and cardiac fibroblast respectively. (a) mRNA expression of ANP, Beta myosin heavy chain (β-MHC) and MYOCD after treatment with MYOCD siRNA in Ang II treated H9c2 cells. (b) ANP and MYOCD protein expression after treatment with MYOCD siRNA in Ang II treated H9c2 cells. (c) mRNA expression of MYOCD expression after treatment with MYOCD siRNA in cardiac fibroblast cells. (d) mRNA expression of fibrotic gene (Col1a, Col3a, Col4a, CTGF, TGF β and FGF β) after treatment with MYOCD siRNA in Ang-II treated fibroblast cells. Normalization of total RNA and protein was done by using GAPDH as an internal control. Data given is Mean ± SEM of triplicates; *p < 0.05; **p < 0.01; ***p < 0.001Control vs Ang II and Ang II vs Ang II + MYOCD siRNA.

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  Figure 3. Effect of MYOCD siRNA treatment on expression of hypertrophic genes (ANP and β-MHC) and on cardiomyocyte size in RAL rats (n = 8). (a) mRNA expression of MYOCD, ANP and β-MHC in RAL and MYOCD siRNA treated RAL rats. (b) Representative western blot for MYOCD, ANP and GAPDH proteins in RAL and MYOCD siRNA treated RAL rats (before and after ligation). (c) Haematoxylin and Eosin (H & E) stained photomicrographs (10X) of rat hearts: (I) Control group, (II) RAL group (III) Scrambled treated RAL group IV) MYOCD siRNA treated (before ligation) RAL Group V) MYOCD siRNA treated (after ligation) RAL group. Bar diagram shows mean of cardiomyocyte area (n = 50) as measured by Image J analyser. Normalization of total RNA and protein was done using GAPDH as an internal control. Data given is Mean ± SEM; **p < 0.01; ***p < 0.001 for Control vs RAL or scrambled; ##p < 0.01 for RAL or Scrambled vs RAL treated with MYOCD siRNA before ligation (Pre-ligation); $p < 0.05, $$p < 0.01 $$$p < 0.001 for RAL or Scrambled vs RAL treated with MYOCD siRNA after ligation (Post-Ligation).

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  Figure 4. Effect of MYOCD siRNA treatment on expression of fibrotic genes (Col 1a, Col 3a, Col 4a, TGF β, CTGF and FGF β) in RAL rats (n = 8). (a) mRNA expression of Col 1a, Col 3a, Col 4a, TGF β, CTGF and FGF β in RAL rats. (b) Representative western blot for CTGF, FGF and GAPDH proteins in RAL rats. (c) mRNA expression of Col 1a, Col 3a, Col 4a, TGF β, CTGF and FGF β in MYOCD siRNA treated RAL rats (before and after ligation). (d) Representative western blot for CTGF, FGF and GAPDH proteins in MYOCD siRNA treated RAL rats (before and after ligation). (e) Photomicrographs (10X) of rat heart tissues with Masson’s Trichrome staining which is collagen specific and evaluates fibrosis: (I) control, (II) RAL rats with extensive collagen deposition (III) Scrambled treated RAL group III) MYOCD siRNA (before ligation) treated RAL rats show decreased collagen deposition (IV) MYOCD siRNA (after ligation) treated RAL rats show decreased collagen deposition. Normalization of RNA and protein was done using GAPDH as an internal control. Data given is Mean ± SEM; *p < 0.05; **p < 0.01 for Control vs RAL or scrambled; #p < 0.05; ##p < 0.01 for RAL or scrambled vs RAL treated with MYOCD siRNA before…