EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R=−0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N=40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P<0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardiu

EH-myomesin splice isoform is a novel marker for dilated cardiomyopathy

The M-band is the prominent cytoskeletal structure that cross-links the myosin and titin filaments in the middle of the sarcomere. To investigate M-band alterations in heart disease, we analyzed the expression of its main components, proteins of the myomesin family, in mouse and human cardiomyopathy. Cardiac function was assessed by echocardiography and compared to the expression pattern of myomesins evaluated with RT-PCR, Western blot, and immunofluorescent analysis. Disease progression in transgenic mouse models for dilated cardiomyopathy (DCM) was accompanied by specific M-band alterations. The dominant splice isoform in the embryonic heart, EH-myomesin, was strongly up-regulated in the failing heart and correlated with a decrease in cardiac function (R = -0.86). In addition, we have analyzed the expressions of myomesins in human myocardial biopsies (N = 40) obtained from DCM patients, DCM patients supported by a left ventricular assist device (LVAD), hypertrophic cardiomyopathy (HCM) patients and controls. Quantitative RT-PCR revealed that the EH-myomesin isoform was up-regulated 41-fold (P < 0.001) in the DCM patients compared to control patients. In DCM hearts supported by a LVAD and HCM hearts, the EH-myomesin expression was comparable to controls. Immunofluorescent analyses indicate that EH-myomesin was enhanced in a cell-specific manner, leading to a higher heterogeneity of the myocytes' cytoskeleton through the myocardial wall. We suggest that the up-regulation of EH-myomesin denotes an adaptive remodeling of the sarcomere cytoskeleton in the dilated heart and might serve as a marker for DCM in mouse and human myocardium

Eccentric cardiac hypertrophy in <i>cJun^Δmu</i> mice upon TAC.

<p>(a) H/BW ratios increase of <i>Jun^Δmu</i> and <i>Jun^f/f</i> mice before and after TAC. Data are presented as values ± SEM. (****) p<0.0001; n = 11–12 per group. (b) Histological analyses. H&E staining shows TAC-induced concentric growth of the heart in <i>Jun^f/f</i> mice and the heart dilation in <i>Jun^Δmu</i> mice. (c) Relative expression of indicated hypertrophic markers in hearts isolated from sham or TAC operated <i>Jun^f/f</i> and <i>Jun^Δmu</i> mice assessed by quantitative RT-PCR. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01; n = 5 per group.</p

Echocardiographic analyses in <i>Jun^Δmu</i> mice after TAC.

<p>All values are shown as mean ± SEM. n = 5–6 per group. p<0.05 is indicated as: # WT TAC vs WT sham; † KO TAC vs KO sham; § KO TAC vs WT TAC. HR, Heart rate; LVPWd, Left ventricular posterior wall in diastole; LVPWs, Left ventricular posterior wall in systole; LVIDd, Left ventricular internal diameter in diastole; LVIDs, Left ventricular internal diameter in systole; FS, Fractional Shortening; EF, Ejection Fraction.</p

<i>Jun^Δmu</i> mice display impaired myocardial remodeling.

<p>(a) Histological analyses. EvG staining reveals mild spontaneous fibrosis in hearts of <i>Jun^Δmu</i> mice, being markedly enhanced after TAC. (b) Relative expression of indicated fibrotic markers assessed by quantitative RT-PCR in hearts isolated from sham or TAC operated <i>Jun^Δmu</i> and <i>Jun^f/f</i> mice. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01, (***) p<0.001; n = 5 per group. (c) TUNEL staining of heart cross sections. TAC does not induce apoptosis in hearts of <i>Jun^f/f</i> mice. <i>Jun^Δmu</i> mice show slightly more apoptotic cardiomyocytes already at baseline, while the apoptotic rate markedly increased in hearts of <i>Jun^Δmu</i> mice upon TAC. (d) Quantification of TUNEL positive nuclei does in sham or TAC operated animals of indicated genotypes. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01.</p

Upregulation of extracellular matrix proteins and downregulation expression of sarcomeric associated proteins in hearts of <i>Jun^Δmu</i> mice.

<p>(a) Expression of indicated genes in hearts of sham or TAC operated mice of indicated genotypes. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001; n = 5 per group. (b) Western blot analyses of Periostin abundancein hearts from sham and TAC operated <i>Jun^Δmu</i> and <i>Jun^f/f</i> mice. Tubulin was used as a loading control for the extracts. (c) Immunoflorescence staining for Periostin in cross sections of hearts from sham and TAC operated animals of indicated genotypes.</p

Generation of <i>Jun^Δmu</i> mice.

<p>(a) Southern blot analysis of genomic DNA from total heart, skeletal muscle and kidney extracts. Deleted band (Δ band) occurs only in samples from MCK-cre positive heart and skeletal muscle. (b) PCR analysis of genomic DNA. PCR in samples from <i>Jun^+/+</i> (+/+), <i>Jun^f/f</i> (f/f) and <i>Jun^Δmu</i> (f/f Cre) mice yielded a 297 bp band corresponding to the wild type allele, a 344 bp band for the floxed allele and a 450 bp for the Δ allele. (c) Quantitative RT-PCR. <i>Jun</i> mRNA levels are down-regulated in total heart extracts from <i>Jun^Δmu</i> mice. (d) Western blot analysis of c-Jun protein levels in total heart extracts of indicated genotypes. (e) Immunofluorescence of isolated mouse neonatal cardiomycoytes. Nuclear localization of c-Jun can be observed in plated neonatal <i>Jun^f/f</i> cardiomyocytes, but not <i>Jun^Δmu</i> cardiomyocytes.</p

Altered sarcomeric structure of isolated neonatal <i>Jun^Δmu</i> cardiomyocytes.

<p>(a) Immunofluorescent staining. Phalloidin staining shows defects in structural organization of <i>Jun^Δmu</i> cardiomyocytes as compared to <i>Jun^f/f</i> cardiomyocytes. (b) Immunofluorescent staining. alpha actinin (αActinin) and titin staining show disruption of Z-line and M-band struture in <i>Jun^Δmu</i> cardiomyocytes as compared to <i>Jun^f/f</i> cardiomyocytes. (c) Relative percent of cardimyocytes presenting different cytoskeleton organization: blue bars – fully organize cytoskeleton, red – well organized, yellow – moderately and green – poorly organized cytoskeleton.</p