Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice

Abstract

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.

Fig 1. Cardiac-specific deletion of Med1 causes dilated cardiomyopathy.

(A) Relative Med1 mRNA expression in Med1^fl/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 Med1^fl/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 Med1^fl/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 Med1^fl/fl and csMed1^-/-mouse heart. (D) qPCR data represent collagen mRNA transcript in Med1^fl/fl and csMed1^-/-mouse heart. (E&F) Represent respectively the electron micrographs of 29-day old Med1^fl/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. Echocardiography.

(A&B) Representative profiles of M-mode echocardiographic analyses of 29-day old Med1^fl/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 Med1^fl/fl and csMed1^-/- mouse hearts. Results are expressed as the mean ±SD. *p<0.05, **p<0.01.

Fig 4. Changes in selected proteins involved in fatty acid oxidation in heart.

(A) Homogenates of heart from two 29-day old Med1^fl/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 Med1^fl/fl and TmcsMed1^-/- mouse heart, liver and kidney. (B) Western blot analysis of Med1 in TmMed1^fl/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 Med1^fl/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 TmMed1^fl/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 Med1^fl/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 Med1^fl/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.