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

Abstract

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.

Fig. 1

Sarcomere cytoskeleton and M-band protein components. a Scheme of the sarcomere depicting the main components of the sarcomeric cytoskeleton (M-band, Z-disk and titin). b Myomesin (white), M-protein (gray) and myomesin-3 (dark gray) are composed of immunoglobulin-like domains (ellipses) and fibronectin type 3 domains (rectangles). The alternatively spliced EH-domain (EH) and the N-terminal domains are intrinsically unstructured

Fig. 2

Echocardiography of DCM models. a Left ventricular diastolic volume measurements confirming the DCM phenotype in MLP-KO (black) and more severe in β-catenin c∆ex3 animals (gray) compared to controls (white). b Ejection fraction of control (white), β-catenin c∆ex3 (gray) and MLP-KO (black) mice demonstrating the continuous decrease in heart function during progression of the disease. Error bars represent standard deviation, N ≥ 6 for each subgroup. Asterisks mean significant differences compared to control groups. c M-mode echocardiographic tracings in control (left), β-catenin c∆ex3 mice (center) and MLP-KO (right) at the age of 4 months. Left ventricular dimensions are indicated by white lines

Fig. 3

Expression of myomesin proteins during development of dilated cardiomyopathy. a Immunoblot analysis of mouse heart protein extracts of control, β-catenin c∆ex3 mice and MLP-KO mice (age 2 months). EH-myomesin (EH) is accumulated in several animals suffering from DCM with variability between individual mice. A slight down-regulation of M-protein is detectable in the β-catenin c∆ex3 mice, which is more pronounced in the MLP-KO mice at the age of 2 months (M-pr). Myomesin-3 up-regulation is detectable only in the MLP-KO mice (Myo3). β-Catenin (β-cat) is accumulated in both mouse models, with the β-catenin c∆ex3 mice showing expression of the truncated form (lower bands) in addition to the normal protein (upper bands). b Quantification of EH-myomesin levels during disease development shows an accumulation already at 2 weeks in the MLP-KO mice (black columns). In the β-catenin c∆ex3 model (gray columns) a significant up-regulation of this isoform is detectable at the age of 8 weeks. At 4 months, both DCM models show an accumulation of EH-myomesin. Expression levels are normalized on sarcomeric actin levels and compared to control mice. Error bars represent standard deviation, N ≥ 6 for each subgroup. Asterisks mean significant differences compared to control groups. P values compared to control were as follows: 2 weeks (β-catenin c∆ex3, P = 0.163; MLP KO, P = 0.007), 5 weeks (β-catenin c∆ex3, P = 0.089; MLP KO, P = 0.001), 2 months (β-catenin c∆ex3, P = 0.016; MLP KO, P = 0.067), 4 months (β-catenin c∆ex3, P = 0.016; MLP KO, P = 0.041). w weeks, m months

Fig. 4

Comparison of EH-myomesin expression with heart parameters. a Left ventricular (LV) volume in systole measured by echocardiography at the age of 5 weeks correlates with EH-myomesin expression in MLP-KO (oblique squares, N = 6) and β-catenin c∆ex3 mice (triangles, N = 6). The β-catenin c∆ex3 transgenic animals show a relatively wide distribution in volume, whereas the MLP-KO mice have a significant dilation including up-regulation of EH-myomesin expression already at this age. The strong correlation between LV volume and EH-myomesin accumulation is reflected by a correlation coefficient of R = 0.82. Control mice are shown as squares (N = 6). b At the age of 5 weeks, the controls (squares) and some of the β-catenin c∆ex3 animals (triangles) have a normal ejection fraction (about 60%) including normal levels of EH-myomesin. Animals with a clearly reduced ejection fraction have accumulated this protein significantly. The strong negative correlation is reflected by a correlation coefficient R = −0.86. c Table showing the correlation coefficients for the relations between EH-myomesin protein levels and LV systolic volume (LV Volume, s), LV systolic internal diameter (LVID, s) or % ejection fraction (EF%). The strongest correlations were found at the age of 5 weeks. 5w 5 weeks, 2m 2 months, 4m 4 months

Fig. 5

Heterogeneous accumulation of EH-myomesin in cardiomyocytes of mouse DCM models. Cryosections of mouse heart ventricles of control (a, d, g, k, n), β-catenin c∆ex3 (b, e, h, l, o) and MLP KO (c, f, i, m, p) mice at the age of 4 months quadruple-stained with antibodies against M-protein (d–f; green in overlays), EH-myomesin (g–i; blue in overlays), DAPI (k–m; red in overlays) and N-cadherin (n–p). A down-regulation of M-protein is apparent in single cardiomyocytes of the β-catenin c∆ex3 transgenic animals. In contrast, M-protein is relatively homogenously expressed in MLP KO (f) and control mice (d). EH-myomesin is accumulated in both DCM models (h, i; blue in b, c) with certain heterogeneity. In the β-catenin c∆ex3 transgenic animals, the same cardiomyocytes, which show an up-regulation of EH-myomesin (h), have reduced levels of M-protein (e), leading to a more embryonic-like phenotype. A big accumulation of nuclei can be detected in the hearts of β-catenin c∆ex3 mice (l, red in overlay), including fibrosis (regions without M-band staining) and some hypertrophied nuclei. The accumulation of the intercalated disk component N-cadherin (n–p) is visible in both DCM mouse models. Scale bar 100 μm

Fig. 6

Expression of M-band components at the end-stage of DCM. Cryosections of mouse heart ventricles of control (a, d, g, k), β-catenin c∆ex3 (b, e, h, l) and MLP-KO (c, f, i, m) mice at the age of 5 months triple-stained with antibodies against M-protein (d–f; green in overlays), EH-myomesin (g–i; blue in overlays), and β-catenin (k–m; red in overlays). M-protein is homogenously expressed in control mice (d), whereas in β-catenin c∆ex3 (e) and MLP-KO (f) transgenic animals it is significantly down-regulated. This down-regulation is homogenous in the MLP-KO, whereas in the β-catenin c∆ex3 animals it is extremely heterogeneous. EH-myomesin is up-regulated extremely heterogeneously in the β-catenin c∆ex3 transgenic animals. Some single cardiomyocytes show a switch to an embryonic phenotype of M-bands, with higher level of EH-myomesin expression and very low M-protein expression level (asterisk in e, h). β-Catenin is strongly accumulated in both DCM models (l, m; red in b, c). Scale bar 20 μm

Fig. 7

EH-myomesin is re-expressed in human patients suffering from DCM. RT-qPCR analysis of human patients suffering from DCM or HCM using primers specific for EH-myomesin. This isoform is massively up-regulated (41 times) in the hearts of patients suffering from DCM (right column, N = 10) compared to non-dilated control hearts (N = 5) and hearts of patients suffering from HCM (N = 7). DCM patients under treatment with a left ventricular assist device (LVAD, N = 3) show no significant re-expression of the EH-myomesin isoform. Sarcomeric α-actinin was used for normalization. HCM hypertrophic cardiomyopathy, DCM dilated cardiomyopathy, LVAD left ventricular assist device. Error bars represent standard deviation. Asterisk means significant difference compared to the other tested groups

Fig. 8

EH-myomesin is up-regulated in the heart of human DCM patients in cell-specific fashion. Confocal images of cryosections of human hearts of control (a, c, e, g) and a DCM patient (left ventricle; b, d, f, h) triple-stained with antibodies against M-protein (c, d; green in overlays), EH-myomesin (e, f; blue in overlays), and with β-catenin (g, h; red in overlay). M-protein is homogenously expressed in both hearts (c, d), whereas EH-myomesin is up-regulated in the DCM patient in a cell-specific manner. Scale bar 10 μm