Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients’ death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Expression of cell type incongruent alpha-cardiac actin 1 subunit in medulloblastoma reveals a novel mechanism for cancer cell survival and control of migration

Background Alterations in actin subunit expression have been reported in multiple cancers, but have not been investigated previously in medulloblastoma. Methods Bioinformatic analysis of multiple medulloblastoma tumor databases was performed to profile ACTC1 mRNA levels. Western blot was used to verify protein expression in established medulloblastoma cell lines. Immunofluorescence microscopy was performed to assess ACTC1 localization. Stable cell lines with ACTC1 overexpression were generated and shRNA knockdown of ACTC1 was accomplished. We used PARP1 cleavage by Western blot as a marker of apoptosis and cell survival was determined by FACS viability assay and colony formation. Cell migration with overexpression or knockdown of ACTC1 was determined by the scratch assay. Stress fiber length distribution was assessed by fluorescence microscopy. Results : ACTC1 mRNA expression is highest in SHH and WNT medulloblastoma among all subgroups. ACTC1 protein was confirmed by Western blot in SHH subgroup and Group 3 subgroup cell lines with the lowest expression in Group 3 cells. Microscopy demonstrated ACTC1 co-localization with F-actin. Overexpression of ACTC1 in Group 3 cells abolished the apoptotic response to Aurora kinase B inhibition. Knockdown of ACTC1 in SHH cells and in Myc overexpressing SHH cells induced apoptosis impaired colony formation, and inhibited migration. Changes in stress fiber length distribution in medulloblastoma cells are induced by alterations in ACTC1 abundance. Conclusions Alpha-cardiac actin (ACTC1) is expressed in SHH medulloblastoma. Expression of this protein in medulloblastoma modifies stress fiber composition and functions in promoting resistance to apoptosis induced by mitotic inhibition, enhancing cell survival, and controlling migration.

5-Azacytidine: Effects on the Expression of alpha-Cardiac Actin in Pericytes from Human Adipose Tissue

DNA methylation patterns are closely related to the chromatin structure, and its remodeling is considered an important mechanism in the control of gene transcription during cell differentiation. In rodent, several studies have related the possibility that multipotent mesenchymal stromal cells (MSCs) undergo cardiomyogenesis. However, it has not been completely elucidated if human adult stem cell exhibits true differentiation potential for a cardiac lineage. In this study, the action of the DNA methylation inhibitor 5-azacytidine (5-aza) was examined in human adipose tissue pericytes (hATPCs: 3G5+) regarding their possible capacity to induce myocytes in vitro. Real-Time PCR revealed that cells treated with 5-aza presented time-dependent decrease in the mRNA expression of α-cardiac actin (α-CA). At 24 h, this diminution was statistically significant; however, there was not a correlation with the highest level of DNA demethylation at the same period using Methylation-Sensitive High Resolution Melting-PCR (MS-HRM-PCR). An evident increase in the α-CA protein expression was observed by Western blotting in hATPCs treated with 5-aza at 24 h. The mRNA expression of α-SMA (α-smooth actin) also showed a time-dependent decrease after the treatment, however, it was not significant. The ultrastructural analysis showed similar structures such as like-cell junctions, caveolae, and actin myofilaments, which aligned in parallel. These phenotypic alterations were found only after the treatment; however, the hTAPCs after 5-aza treatment were not able to form thick myofilaments and consequently sarcomeres. These results indicated that a terminal cardiac differentiation of hTAPCs was not achieved and that the cardiomyogenesis failure could be related to the non-muscle origin of the adipose tissue.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilative Cardiomyopathy (p.R312H and p.E361G)

The human mutant cardiac α-actins p.A295S or p.R312H (plus p.R312K) and p.E361G correlated with hypertrophic or dilative cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. After purification their biochemical and cell biological properties were analysed and compared to wild type (wt) cardiac actin identically obtained or conventionally isolated from bovine hearts. DNase I inhibition and their polymerization behaviour indicated that all c-α-actins had maintained their native state. Cardiomyopathy type specific differences were observed except for the p.R312K mutant, which behaved like wt c-α-actin. The extent of myosin-S1 ATPase stimulation by the c-actin variants and its Ca2+-sensitivity after decoration with tropomyosin (cTm) and troponin complex (cTn) varied being highest for the HCM p.A295S and lower for both DCM mutants. Similar Ca2+-sensitivity differences were observed by recording the fluorescence increase of pyrene-cTm in the absence or presence of myosin-S1 and/or the actin-binding N-terminal fragment of cardiac myosin binding protein C (N-cMyBP-C). Transfection experiments showed the incorporation of the c-actin variants into existing cytoskeletal elements of non-muscle cells. Wt and p.A295S c-α-actin preferably incorporated into the microfilament system and p.R312H and p.E361G into the submembranous actin network of MDCK cells. Transduction of neonatal rat cardiomyocytes with adenoviral constructs coding for HA-tagged c-α-actins showed their incorporation into thin filaments of nascent sarcomeric structures at their plus ends (Z-lines) except the p.E361G mutant, which preferably incorporated at the minus ends. Our data indicate functional differences of the c-α-actins that may be causative for the different cardiomyopathy phenotypes.

Which actin genes are necessary for zebrafish heart development and function?

Heart failure is the number one cause of mortality in the world, contributed to by cardiovascular disease. Many diseases of the heart muscle are caused by mutations in genes encoding contractile proteins, including cardiac actin mutations. Zebrafish are an advantageous system for modeling cardiac disease since embryos can develop without a functional heart. However, genome duplication in the teleost lineage creates a unique obstacle by increasing the number of genes involved in heart development. Four actin genes are expressed in the zebrafish heart: acta1b; actc1c; and duplicates of actc1a on chromosome 19 and 20. Here, we characterize the actin genes involved in early zebrafish heart development using in situ hybridization and CRISPR targeting to determine which gene is best to model changes seen in human patients with heart disease. The actc1a and acta1b genes are predominant during embryonic heart development, resulting in severe cardiac phenotypes when targeted with CRISPRs. Targeting these two cardiac genes with CRISPRs simultaneously results in a more severe phenotype than their individual counterparts, with the results suggesting compensation for lost actin genes by other actin paralogues. Given the duplication of the actc1a gene, we recommend acta1b as the best gene for targeted cardiac actin research.

An autoantibody profile detects Brugada syndrome and identifies abnormally expressed myocardial proteins

Aims Brugada syndrome (BrS) is characterized by a unique electrocardiogram (ECG) pattern and life-threatening arrhythmias. However, the Type 1 Brugada ECG pattern is often transient, and a genetic cause is only identified in <25% of patients. We sought to identify an additional biomarker for this rare condition. As myocardial inflammation may be present in BrS, we evaluated whether myocardial autoantibodies can be detected in these patients. Methods and results For antibody (Ab) discovery, normal human ventricular myocardial proteins were solubilized and separated by isoelectric focusing (IEF) and molecular weight on two-dimensional (2D) gels and used to discover Abs by plating with sera from patients with BrS and control subjects. Target proteins were identified by mass spectrometry (MS). Brugada syndrome subjects were defined based on a consensus clinical scoring system. We assessed discovery and validation cohorts by 2D gels, western blots, and ELISA. We performed immunohistochemistry on myocardium from BrS subjects (vs. control). All (3/3) 2D gels exposed to sera from BrS patients demonstrated specific Abs to four proteins, confirmed by MS to be α-cardiac actin, α-skeletal actin, keratin, and connexin-43, vs. 0/8 control subjects. All (18/18) BrS subjects from our validation cohorts demonstrated the same Abs, confirmed by western blots, vs. 0/24 additional controls. ELISA optical densities for all Abs were elevated in all BrS subjects compared to controls. In myocardium obtained from BrS subjects, each protein, as well as SCN5A, demonstrated abnormal protein expression in aggregates. Conclusion A biomarker profile of autoantibodies against four cardiac proteins, namely α-cardiac actin, α-skeletal actin, keratin, and connexin-43, can be identified from sera of BrS patients and is highly sensitive and specific, irrespective of genetic cause for BrS. The four involved proteins, along with the SCN5A-encoded Nav1.5 alpha subunit are expressed abnormally in the myocardium of patients with BrS.

Actin Mutations and Their Role in Disease

Actin is a widely expressed protein found in almost all eukaryotic cells. In humans, there are six different genes, which encode specific actin isoforms. Disease-causing mutations have been described for each of these, most of which are missense. Analysis of the position of the resulting mutated residues in the protein reveals mutational hotspots. Many of these occur in regions important for actin polymerization. We briefly discuss the challenges in characterizing the effects of these actin mutations, with a focus on cardiac actin mutations.