Data on the role of cardiac α-actin (ACTC1) gene mutations on SRF-signaling.

We recently reported a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). We also found that the G247D ACTC1 mutation negatively regulated serum response (SRF)-signaling thereby contributing to the late-onset DCM observed in human patients carrying this mutation ("A cardiac α-actin (ACTC1) p. Gly247Asp mutation inhibits SRF-signaling in vitro in neonatal rat cardiomyocytes" [1]). There are some ACTC1 mutations known to date, majority of which, though, have not been investigated for their functional consequence. We thus aimed at determining the functional impact of various ACTC1 gene mutations on SRF-signaling using SM22-response element driven firefly luciferase activity assays in C2C12 cells.

A cardiac α-actin (ACTC1) p. Gly247Asp mutation inhibits SRF-signaling in vitro in neonatal rat cardiomyocytes.

We recently identified a novel, heterozygous, and non-synonymous ACTC1 mutation (p.Gly247Asp or G247D) in a large, multi-generational family, causing atrial-septal defect followed by late-onset dilated cardiomyopathy (DCM). Molecular dynamics studies revealed possible actin polymerization defects as G247D mutation resides at the juncture of side-chain interaction, which was indeed confirmed by in vitro actin polymerization assays. Since polymerization/de-polymerization is important for the activation of Rho-GTPase-mediated serum response factor (SRF)-signaling, we studied the effect of G247D mutation using luciferase assay. Overexpression of native human ACTC1 in neonatal rat cardiomyocytes (NRVCMs) strongly activated SRF-signaling both in C2C12 cells and NRVCMs, whereas, G247D mutation abolished this activation. Mechanistically, we found reduced GTP-bound Rho-GTPase and increased nuclear localization of globular actin in NRVCMs overexpressing mutant ACTC1 possibly causing inhibition of SRF-signaling activation. In conclusion, our data suggests that human G247D ACTC1 mutation negatively regulates SRF-signaling likely contributing to the late-onset DCM observed in mutation carrier patients.

Myeloid leukemia factor-1 is a novel modulator of neonatal rat cardiomyocyte proliferation.

The present study focuses on the identification of the gene expression profile of neonatal rat cardiomyocytes (NRVCMs) after dynamic mechanical stretch through microarrays of RNA isolated from cells stretched for 2, 6 or 24h. In this analysis, myeloid leukemia factor-1 (MLF1) was found to be significantly downregulated during the course of stretch. We found that MLF1 is highly expressed in the heart, however, its cardiac function is unknown yet. In line with microarray data, MLF1 was profoundly downregulated in in vivo mouse models of cardiomyopathy, and also significantly reduced in the hearts of human patients with dilated cardiomyopathy. Our data indicates that the overexpression of MLF1 in NRVCMs inhibited cell proliferation while augmenting apoptosis. Conversely, knockdown of MLF1 protected NRVCMs from apoptosis and promoted cell proliferation. Moreover, we found that knockdown of MLF1 protected NRVCMs from hypoxia-induced cell death. The observed accelerated apoptosis is attributed to the activation of caspase-3/-7/PARP-dependent apoptotic signaling and upregulation of p53. Most interestingly, MLF1 knockdown significantly upregulated the expression of D cyclins suggesting its possible role in cyclin-dependent cell proliferation. Taken together, we, for the first time, identified an important role for MLF1 in NRVCM proliferation.

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

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 ß-catenin/GSK-3ß 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.

Mice with cardiac-restricted overexpression of Myozap are sensitized to biomechanical stress and develop a protein-aggregate-associated cardiomyopathy.

The intercalated disc (ID) is a major component of the cell-cell contact structures of cardiomyocytes and has been recognized as a hot spot for cardiomyopathy. We have previously identified Myozap as a novel cardiac-enriched ID protein, which interacts with several other ID proteins and is involved in RhoA/SRF signaling in vitro. To now study its potential role in vivo we generated a mouse model with cardiac overexpression of Myozap. Transgenic (Tg) mice developed cardiomyopathy with hypertrophy and LV dilation. Consistently, these mice displayed upregulation of the hypertrophy-associated and SRF-dependent gene expression. Pressure overload (transverse aortic constriction, TAC) caused exaggerated cardiac hypertrophy, further loss of contractility and LV dilation. Similarly, a physiological stimulus (voluntary running) also led to significant LV dysfunction. On the ultrastructural level, Myozap-Tg mouse hearts exhibited massive protein aggregates composed of Myozap, desmoplakin and other ID proteins. This aggregate-associated pathology closely resembled the alterations observed in desmin-related cardiomyopathy. Interestingly, desmin was not detectable in the aggregates, yet was largely displaced from the ID. Molecular analyses revealed induction of autophagy and dysregulation of the unfolded protein response (UPR), associated with apoptosis. Taken together, cardiac overexpression of Myozap leads to cardiomyopathy, mediated, at least in part by induction of Rho-dependent SRF signaling in vivo. Surprisingly, this phenotype was also accompanied by protein aggregates in cardiomyocytes, UPR alteration, accelerated autophagy and apoptosis. Thus, this mouse model may also offer additional insight into the pathogenesis of protein-aggregate-associated cardiomyopathies and represents a new candidate gene itself.