Myozap Deficiency Promotes Adverse Cardiac Remodeling via Differential Regulation of Mitogen-activated Protein Kinase/Serum-response Factor and β-Catenin/GSK-3β Protein Signaling

Abstract

The intercalated disc (ID) is a "hot spot" for heart disease, as several ID proteins have been found mutated in cardiomyopathy. Myozap is a recent addition to the list of ID proteins and has been implicated in serum-response factor signaling. To elucidate the cardiac consequences of targeted deletion of myozap in vivo, we generated myozap-null mutant (Mzp(-/-)) mice. Although Mzp(-/-) mice did not exhibit a baseline phenotype, increased biomechanical stress due to pressure overload led to accelerated cardiac hypertrophy, accompanied by "super"-induction of fetal genes, including natriuretic peptides A and B (Nppa/Nppb). Moreover, Mzp(-/-) mice manifested a severe reduction of contractile function, signs of heart failure, and increased mortality. Expression of other ID proteins like N-cadherin, desmoplakin, connexin-43, and ZO-1 was significantly perturbed upon pressure overload, underscored by disorganization of the IDs in Mzp(-/-) mice. Exploration of the molecular causes of enhanced cardiac hypertrophy revealed significant activation of β-catenin/GSK-3β signaling, whereas MAPK and MKL1/serum-response factor pathways were inhibited. In summary, myozap is required for proper adaptation to increased biomechanical stress. In broader terms, our data imply an essential function of the ID in cardiac remodeling beyond a mere structural role and emphasize the need for a better understanding of this molecular structure in the context of heart disease.

Keywords: animal model; cadherin; cardiac development; cardiac hypertrophy; cardiac muscle; cardiomyocyte; cardiomyopathy; cardiovascular disease; heart; heart failure.

FIGURE 1.

Knockdown of myozap in NRVCMs and basal characterization of Mzp^−/− mice. A, primary NRVCMs were infected with adenovirus encoding synthetic microRNA to knock down myozap in the absence or presence of PE and immunostained against α-actinin. B, cell surface area was measured using Keyence's HybridCellCount software module in fluorescence intensity single-extraction mode. Statistical significance was determined using Shapiro-Wilk (for equal distribution) and Kruskal-Wallis (one-way ANOVA) tests. n > 500. Expression of fetal genes Nppa (C) and Nppb (D) was determined by quantitative real time PCR. Data represented are mean of two independent experiments performed in triplicate. E, strategic diagram showing the targeting vector and myozap genomic locus. Homologous recombination replaces exon 1 encoding the translation initiation site with a neomycin-lacZ reporter cassette. Deletion of myozap was confirmed by genotyping (F), qRT-PCR (G), immunoblotting (H), and immunofluorescence microscopy (I). J, electron micrographs showing the structural architecture of cardiac muscle. As to the intercalated discs, fasciae adherents (FA) are shown at higher magnification (see boxes). There are no ultrastructural differences between wild-type (WT) and myozap^−/− (Mzp^−/−) mice. GJ, gap junction; M, M-line; Mi, mitochondria; Z, Z-line. Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; ***, p < 0.001.

FIGURE 2.

Mild hypertrophy and cardiomyopathy upon aging and isoproterenol treatment in Mzp^−/− mice. Comparative images of 1-year-old myozap-KO (Mzp^−/−) and WT mouse hearts (A) and H&E-stained whole heart sections (B). Comparative phenotypic parameters from 1-year-old Mzp^−/− and WT mice, heart weight/body weight (HW/BW, C), lung weight/body weight (Lung W/BW, D), diastolic left ventricular internal dimension (LVIDd, E), fractional shortening (FS, F), and the expression of fetal genes Nppa (G) and Nppb (H) show no severe abnormalities in Mzp^−/− mice. n = 6. Similarly, comparative phenotypic parameters from isoproterenol (Iso)/PBS-treated Mzp^−/− and WT mice, heart weight/body weight (HW/BW, I), lung weight/body weight (Lung W/BW, J), diastolic left ventricular internal dimension (LVIDd, K), fractional shortening (FS, L), and the expression of myozap (M) fetal genes Nppa (N) and Nppb (O) show no severe abnormalities in Mzp^−/− mice. n = 8. Statistical significance was determined using two-tailed Student's t test. Error bars show mean ± S.E. *, p < 0.05; ***, p < 0.001. Cont, control.

FIGURE 3.

Biomechanical stress-induced severe cardiomyopathy in myozap-deficient mice. A, 8-week-old wild-type (WT) and myozap-KO (Mzp^−/−) mice underwent TAC or SHAM operations. Four weeks post-operations (n = 9 (WT-SHAM), 15 (WT-TAC), 9 (KO-SHAM), 9 (KO-TAC)), phenotypic abnormalities were analyzed by measuring cross-sectional areas of the heart (A), heart weight/body weight (HW/BW, B), left ventricular weight/body weight (LVW/BW, C), diastolic left ventricular internal dimension (LVIDd, D), systolic left ventricular internal dimension (LVIDs, E), fractional shortening (FS, F), posterior wall thickness at diastole (LVPWd, G) and systole (LVPWs, H), lung weight/body weight (Lung W/BW, I), and mortality rate observed during post-operative 4-week housing (J). Mzp^−/− mice displayed severe cardiac pathology, disturbed contractility, signs of heart failure and increased mortality under the influence of TAC. n = 17 (SHAM) or 24 (TAC). Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Biomechanical stress accelerated cellular hypertrophy in myozap-deficient mice. Heart slices obtained from SHAM or TAC-operated wild-type (WT) or myozap-KO (Mzp^−/−) mice were stained with FITC-conjugated lectin (A), and cell surface area of the cardiomyocytes was measured (B) showing accelerated hypertrophy in Mzp^−/− mice compared with the WT littermates. This was further confirmed by super induction of fetal genes Nppa/Nppb determined by quantitative real time PCR (C), marked up-regulation of β-myosin heavy chain (Myh7, D–F), and reduced expression of alpha-myosin heavy chain (Myh6, G–I), or Serca2a (J–L), both at transcript and protein levels. M, heart sections obtained from SHAM or TAC operated wild-type (WT) or Mzp^−/− mice were stained with Sirius-red/Fast-green (upper panel), and the representative fibrotic area is highlighted in the lower panel. N, fibrotic lesions stained in red were measured using Keyence's HybridCellCount software module in bright field single-extraction mode, which confirms the increased fibrosis in Mzp^−/− mice in comparison with WT littermates. O, increased expression of fibrotic markers collagen III (Col-III) and plasminogen activator inhibitor I (PAI-I) further supports the increased fibrosis in Mzp^−/− mice. n = 6. Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Biomechanical stress causes junctional remodeling in mice lacking myozap. Electron micrographs showing the ID structure of wild-type (A, panels I and II) and myozap-KO (B, panels I and II) mice after TAC indicating disorganized IDs. Light microscopy images of wild-type (A, panel III) and myozap-KO (B, panel III) mice after TAC exhibiting cardiomyocyte degeneration in myozap-KO mice. Expression of intercalated disc proteins that directly or indirectly interact with myozap was determined by Western blotting and showed strong up-regulation of desmoplakin and desmin (C–E), although N-cadherin, connexin-43 (F–H), and ZO-1 (I and J) were strongly reduced after the induction of biomechanical stress due to TAC in Mzp^−/− mice. Expression of Na_v1.5 was unaltered in mice or rat cardiomyocytes (K–N), whereas connexin-43 was again down-regulated after myozap knockdown in stressed or unstressed cardiomyocytes (M and O). n = 6. Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Overview of connexin-43 and actin cytoskeleton in myozap-deficient mice or rat cardiomyocytes. Expression and localization of connexin-43 and filamentous actin and sarcomere were studied by immunoimaging with respective antibodies and phalloidin (for filamentous actin) in mouse heart cryosections (n = 5, A and B), neonatal rat cardiomyocytes (C), or neonatal mouse cardiomyocytes isolated from wild-type or Mzp^−/− mice (D). Images clearly indicate visible differences between both genotypes after TAC with respect to the localization of connexin-43, altered sarcomere, and actin cytoskeleton. Experiments for cultured NRVCMs or NMVCMs were repeated three times in triplicate.

FIGURE 7.

Myozap deficiency inhibits cardiac SRF and MAPK signaling due to pressure overload. A, bar graph showing reduced SRF-RE-driven luciferase activity upon myozap knockdown in cardiomyocytes. Expression levels of selected SRF target gene skeletal muscle α-actin (Acta1), cardiac muscle α-actin (Actc1), c-Fos, and myosin light chain 2 (Myl2) were detected in SHAM/TAC-operated (B), or 1-year-old mice (C). Proteins extracted from post-operative hearts were immunoblotted for ERK1/2 and phospho-ERK1/2 (p-ERK1/2; D), MAPK p38, and phospho-p38 (p-p38; F), and MKL1 (H). Respective densitometric analysis indicates the inhibition of ERK1/2 (E), p38 (G), as well as down-regulation of MKL1 (I) after TAC operations exclusively in Mzp^−/− mice. n = 6. Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 8.

Cardiac hypertrophy in myozap-deletion mutant mice due to biomechanical stress is attributed to β-catenin/GSK-3β signaling. Proteins extracted from post-operative hearts were immunoblotted for Akt and phospho-Akt (p-Akt; A and B), GSK3α/3β and phospho-GSK3β (p-GSK3β; C and D), Rcan1–4 at protein (E and F) and transcript level (G), and β-catenin and phospho-β-catenin (p-β-catenin) (H and I). Respective densitometric analysis revealed no significant difference in Akt activation (B); nevertheless, expression of p-GSK3β (D), Rcan1–4 (F), and β-catenin (I) was significantly increased in Mzp^−/− mice after the induction of biomechanical stress (n = 6). These findings were recapitulated in rat cardiomyocytes after myozap knockdown under mechanical stress with inhibition or MAPKs (J–L), increased phosphorylation of GSK-3β (M and N), and up-regulation of Rcan1–4 (O and P). Effect of myozap expression was also studied on NFAT reporter-mediated firefly luciferase activity by either its overexpression or knockdown in NRVCM or in NMVCM isolated from wild-type or myozap-null mice. Overexpression of myozap had no effect (Q), whereas reduction or the absence of myozap significantly accelerated the luciferase activity in NRVCMs (R), or NMVCMs (S), respectively. Experiments for cultured NRVCMs or NMVCMs were repeated three times in triplicate (hexaplicate for luciferase assay). Statistical significance was calculated by two-way ANOVA. Error bars show mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 9.

Simplified pictorial representation of effects of myozap deficiency on cardiac signaling. Myozap deficiency on the one hand inhibits SRF signaling via inhibition of MAPK ERK1/2, RhoA, and MKL1. On the other hand, it activates prohypertrophic calcineurin-NFAT via inhibition of MAPK p38 and interconnected GSK-3β/β-catenin-driven signaling cascade in response to biomechanical stress. Cn, calcineurin; β-catn, β-catenin.