Fig 1.
Cardiac-specific deletion of Med1 causes dilated cardiomyopathy.
(A) Relative Med1 mRNA expression in Med1fl/fl (WT) and csMed1-/- mouse (KO) heart, liver, kidney and skeletal muscle. (B & C) Immunohistochemical localization of Med1 in 25-day old Med1 fl/fl (B, arrow), and csMed1-/- (C) heart. (D) Nppb (BNP) mRNA level in 15-day old KO mouse heart is not increased as compared to 25-day old KO mouse. (E) Representative photographs of intact 15- day to-29 day-old csMed1-/- mice and their corresponding Med1fl/fl littermate. In 29-day old csMed1-/- mice the heart was flaccid and flabby. (F) Heart sections of 15- day (upper pair), 25-day (middle pair) and 29- day old (lower pair) Med1 KO heart stained with H&E reveal thinning of ventricular walls and dilation of chambers. (G) Survival curve. 41 mice for each group of Med1fl/fl and csMed1-/- were used for the generation of survival curve. (H) Heart weight / body weight ratio. I, dry heart/wet heart ratio. Results, as indicated, are expressed as the mean ±SD. *p<0.05, **p<0.01, NS: non-significant.
Fig 2.
Cardiac fibrosis, increased cardiomyocyte apoptosis and mitochondrial damage in csMed1-/- mouse heart.
(A) Morphological changes in csMed1-/- mouse heart as assessed by H&E, Masson’s trichrome (MT), TUNEL and DAPI staining. MERGE indicates DAPI and TUNEL overlap. (B) TUNEL positive cells in myocardium and (C) mitochondrial DNA content were determined by measuring the levels of the mitochondrial encoded gene cytochrome oxidase 1 (CO1) and the nuclear encoded 18S gene in Med1fl/fl and csMed1-/-mouse heart. (D) qPCR data represent collagen mRNA transcript in Med1fl/fl and csMed1-/-mouse heart. (E&F) Represent respectively the electron micrographs of 29-day old Med1fl/f and csMed1-/- mouse hearts. Red arrows in F indicate lipid droplets and electron dense degenerative bodies and black arrow points to irregularities in Z band pattern. Results are expressed as the mean ±SD. *p<0.05, **p<0.01.
Fig 3.
(A&B) Representative profiles of M-mode echocardiographic analyses of 29-day old Med1fl/fl (A) and csMed1-/- (B) mice. (C, D, & E) Depict left ventricular dimension, fractional shortening and ejection fraction respectively. (F) Relative mRNA levels of ANP (Nppa) in Med1fl/fl and csMed1-/- mouse hearts. Results are expressed as the mean ±SD. *p<0.05, **p<0.01.
Table 1.
Gene expression changes in csMed1-/- relative to Med1fl/fl mouse heart.
Table 2.
Up-regulated genes in csMed1-/- relative to Med1fl/fl mouse heart.
Table 3.
Genes down-regulated in csMed1-/- heart as assessed by RNA-Seq KEGG pathway.
Table 4.
Genes up-regulated in csMed1-/- heart as assessed by RNA-Seq KEGG pathway.
Fig 4.
Changes in selected proteins involved in fatty acid oxidation in heart.
(A) Homogenates of heart from two 29-day old Med1fl/fl and csMed1-/- mice were immunoblotted with antibodies against the proteins shown. (B) Densitometry of Western blot to analyze the relative protein expression. The protein expression of each gene was normalized to GAPDH. Results are expressed as the mean ±SD. *p<0.05, **p<0.01, NS: non-significant. 2-Enoyl-CoA hydratase (MH); Mitochondrial trifunctional protein (MTP); Pyruvate carboxylase (PCS); 3-ketoacyl-CoA thiolase (PTL); Long chain, medium chain, and short chain acyl-CoA dehydrogenases (LCAD, MCAD, and SCAD, respectively); Enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE).
Fig 5.
Tamoxifen-inducible cardiac-specific disruption of Med1 (TmcsMed1-/-) in adult mice causes dilated cardiomyopathy.
(A) Relative Med1 mRNA expression in Med1fl/fl and TmcsMed1-/- mouse heart, liver and kidney. (B) Western blot analysis of Med1 in TmMed1fl/fl and TmcsMed1-/- hearts. Crude nuclear extracts from the heart tissues of appropriate mice were prepared as described (see Materials and Methods). They were then Western immunoblotted and probed with an anti-Med1 antibody (Abcam ab64965) and the protein bands were quantified using ImageJ software. The data were normalized to β-actin bands. Note that in the experiments shown in (A) to (H), mice were killed 14 days after first tamoxifen injection. (C) Representative photographs of adult hearts after tamoxifen-inducible heart-specific Cre mediated Med1 deletion. It is evident that TmcsMed-/- mouse heart is flaccid and flabby. Lower panel in (C) shows cross sections of TmcsMed1-/- and the littermate control hearts stained with H&E. (D and E) Representative profiles of M-mode echocardiographic analyses of TmcsMed1-/- and littermate mice. (F) and (G) represent ejection fraction and fractional shortening respectively. Data were derived from (D) and (E). (H), relative mRNA levels of BNP (Nppb) in Med1 fl/fl and TmcsMed1-/- mouse hearts. (I), survival curve. 13 mice for each group of Med1fl/fl and TmcsMed1-/- were used for the generation of survival curve. The mice survival time was between 13 and 28 days, and the day of initial injection of Tamoxifen was counted as day 1. Results are expressed as the mean ±SD. *p<0.05, **p<0.01.
Fig 6.
Cardiac fibrosis in TmcsMed1-/- mouse heart.
TmcsMed1-/- mice given daily injections of tamoxifen for 5 days were killed 14 days after the first tamoxifen injection. (A&B) Masson’s trichrome staining was used to assess myocardial fibrosis. Inset in panel B shows intensely stained (blue color) interstitial fibrous strands. (C,D &E) Quantification of mRNA levels for Tgfβ2, Ctgf and Col9a2 mRNA levels in TmcsMed1-/- (KO) and TmMed1fl/fl (WT) mouse hearts. mRNA levels were quantified by qPCR assays and the results were expressed as the mean ±SD. **p<0.01.
Fig 7.
Analysis of expression of selected genes in Med1fl/fl and TmcsMed1-/- mouse heart.
(A) qPCR data of RNA samples for key genes linked to heart function. (B) Western blot analysis of key proteins. Protein homogenates extracted from hearts of tamoxifen-inducible Med1fl/fl (14 days after initial injection) and TmcsMed1-/- mice were immunoblotted. The protein bands were quantified using a densitometer. Percent change for TmcsMed1-/- heart as compared to littermate controls are: MTP (Complex II), 28% reduction; MTP (Mitochondrial trifunctional protein), 67% reduction; PCS (Pyruvate carboxylase), 61% reduction; MCAD (Medium chain acyl-CoA dehydrogenases), 42% reduction; L-PBE (Enoyl-CoA hydratase/L- hydroxyacyl-CoA dehydrogenase), 44% reduction; Tgfβ (transforming growth factor beta), 119% increase; CTGF (connective tissue growth factor), 68% increase; SERCA2 (ATPase, Ca++ transporting, cardiac muscle, slow twitch 2), 29% reduction). The protein expression was normalized to GAPDH.