Cardiac Alpha-actin Research Articles

Class I and II Histone Deacetylase Inhibitors as Therapeutic Modulators of Dilated Cardiac Tissue-Derived Mesenchymal Stem/Stromal Cells.

The prevalence of dilated cardiomyopathy (DCM) is increasing globally, highlighting the need for innovative therapeutic approaches to prevent its onset. In this study, we examined the energetic and epigenetic distinctions between dilated and non-dilated human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and assessed the effects of class I and II HDAC inhibitors (HDACi) on these cells and their cardiomyogenic differentiation. Cells were isolated from myocardium biopsies using explant outgrowth methods. Mitochondrial and histone deacetylase activities, ATP levels, cardiac transcription factors, and structural proteins were assessed using flow cytometry, PCR, chemiluminescence, Western blotting, and immunohistochemistry. The data suggest that the tested HDAC inhibitors improved acetylation and enhanced the energetic status of both types of cells, with significant effects observed in dilated myocardium-derived hmMSCs. Additionally, the HDAC inhibitors activated the cardiac transcription factors Nkx2-5, HOPX, GATA4, and Mef2C, and upregulated structural proteins such as cardiac troponin T and alpha cardiac actin at both the protein and gene levels. In conclusion, our findings suggest that HDACi may serve as potential modulators of the energetic status and cardiomyogenic differentiation of human heart hmMSCs. This avenue of exploration could broaden the search for novel therapeutic interventions for dilated cardiomyopathy, ultimately leading to improvements in heart function.

Alpha-cardiac Actin Serum Expression Levels Detect Acute Cellular Rejection in Heart Transplant Patients.

Given the central role of sarcomeric dysfunction in cardiomyocyte biology and sarcomere alterations described in endomyocardial biopsies of transplant patients with rejection, we hypothesized that the serum expression levels of genes encoding sarcomeric proteins were altered in acute cellular rejection (ACR). The aim of this study is to identify altered sarcomere-related molecules in serum and to evaluate their diagnostic accuracy for detecting rejection episodes. Serum samples from transplant recipients undergoing routine endomyocardial biopsies were included in an RNA sequencing analysis (n = 40). Protein concentrations of alpha-cardiac actin were determined using a specific enzyme-linked immunoassay (n = 80). We identified 17 sarcomeric genes differentially expressed in patients with clinically relevant rejection (grade ≥2R ACR). A receiver operating characteristic curve was done to assess their accuracy for ACR detection and found that 6 relevant actins, myosins, and other sarcomere-related genes showed great diagnostic capacity with an area under the curve (AUC) > 0.800. Specifically, the gene encoding alpha-cardiac actin ( ACTC1 ) showed the best results (AUC = 1.000, P < 0.0001). We determine ACTC1 protein levels in a larger patient cohort, corroborating its overexpression and obtaining a significant diagnostic capacity for clinically relevant rejection (AUC = 0.702, P < 0.05). Sarcomeric alterations are reflected in peripheral blood of patients with allograft rejection. Because of their precision to detect ACR, we propose sarcomere ACTC1 serum expression levels as potential candidate for to be included in the development of molecular panel testing for noninvasive ACR detection.

CNSC-32. ACTIN SUBUNIT STRUCTURAL DIVERSITY IN SHH MEDULLOBLASTOMA: IMPLICATIONS FOR CANCER BIOLOGY AND NEURODEVELOPMENT

Abstract BACKGROUND Altered expression of actin subunits is found in multiple cancers. Alpha-cardiac actin (ACTC1) is expressed in sonic hedgehog medulloblastoma (SHH MB) and regulates both cell survival and migration. We hypothesized that cardiac actin has a unique structural and functional role in SHH MB and may be expressed in post-natal granule cell progenitors (GCPs) which share similar gene expression profiles. METHODS Actin subunit protein expression (ACTC1, ACTB, ACTG1, and ACTA2) was evaluated by mRNA expression profiling within established datasets and across MB cell lines by Western blot (WB). Immunofluorescence microscopy was performed to assess intracellular actin subunit localization. The composition of each subunit in filamentous (F-) actin was evaluated. Furthermore immunoprecipitation of filamentous ACTC1 was performed to assess protein-protein interactions. The ratio of F-actin to globular (G-) actin was determined by WB in SHH MB cells exposed to mitotic inhibitor. Cell viability was determined by colony formation following over-expression of dominant-negative mutant ACTC1 (P116C &amp; G247D). RESULTS ACTC1, ACTG1, and ACTB are the predominant actin subunits in SHH MB. Immunofluorescence microscopy demonstrated co-localization of ACTC1 with ACTG1 and ACTB but not ACTA2. ACTC1 showed a unique cortical localization pattern. Post-natal MATH1 positive mouse GCPs (P3-5) demonstrate similar distribution of ACTC1 in cortical F-actin. WB analysis of ACTC1 pulldown from SHH MB cell F-actin fractions showed co-precipitation with ACTG1 and ACTB but not ACTA2. ACTC1 F-actin to G-actin ratio is maintained in cells that show resistance to mitotic inhibition. Expression of mutant ACTC1 G247D resulted in reduced cell survival. CONCLUSIONS ACTC1 subunit is incorporated into F-actin in SHH MB and GCPs. Filamentous ACTC1 forms a protein complex with ACTG1 and ACTB. Disruption of ACTC1 polymerization results in reduced cell survival. These findings suggest a functional role of ACTC1 in both SHH MB and in GCPs which are important in cerebellar development.

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.

BackgroundAlterations in actin subunit expression have been reported in multiple cancers, but have not been investigated previously in medulloblastoma.MethodsBioinformatic 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.ConclusionsAlpha-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.

Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Improves Energetic Status and Cardiomyogenic Differentiation of Human Dilated Myocardium-Derived Primary Mesenchymal Cells.

Background. In this study the effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) on the energetic status and cardiomyogenic differentiation of human healthy and dilated myocardium-derived mesenchymal stromal cells (hmMSC) have been investigated. Methods. The hmMSC were isolated from the healthy and dilated post-operation heart biopsies by explant outgrowth method. Cell proliferation, HDAC activity, mitochondrial membrane potential, and level of adenosine triphosphate (ATP) were evaluated. The effect of SAHA on mitochondrial parameters has been investigated also by Seahorse XF analyzer and cardiomyogenic differentiation was confirmed by the expression of transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T and alpha cardiac actin at gene and protein levels. Results. Dilated myocardium-derived hmMSC had almost 1.5 folds higher HDAC activity compared to the healthy cells and significantly lower mitochondrial membrane potential and ATP level. HDAC class I and II inhibitor SAHA improved energetic status of mitochondria in dilated myocardium-isolated hmMSC and increased expression of cardiac specific proteins during 14 days of exposure of cells to SAHA. Conclusions. HDAC inhibitor SAHA can be a promising therapeutic for dilated cardiomyopathy (DCM). Dilated hmMSC exposed to SAHA improved energetic status and, subsequently, cardiomyogenic differentiation. Data suggest that human dilated myocardium-derived MSC still have cardio tissue regenerative potential, which might be stimulated by HDAC inhibitors.

Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes

The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.

Proteome and transcriptome profiling of equine myofibrillar myopathy identifies diminished peroxiredoxin 6 and altered cysteine metabolic pathways.

Equine myofibrillar myopathy (MFM) causes exertional muscle pain and is characterized by myofibrillar disarray and ectopic desmin aggregates of unknown origin. To investigate the pathophysiology of MFM, we compared resting and 3 h postexercise transcriptomes of gluteal muscle and the resting skeletal muscle proteome of MFM and control Arabian horses with RNA sequencing and isobaric tags for relative and absolute quantitation analyses. Three hours after exercise, 191 genes were identified as differentially expressed (DE) in MFM vs. control muscle with >1 log2 fold change (FC) in genes involved in sulfur compound/cysteine metabolism such as cystathionine-beta-synthase (CBS, ↓4.51), a cysteine and neutral amino acid membrane transporter (SLC7A10, ↓1.80 MFM), and a cationic transporter (SLC24A1, ↓1.11 MFM). In MFM vs. control at rest, 284 genes were DE with >1 log2 FC in pathways for structure morphogenesis, fiber organization, tissue development, and cell differentiation including > 1 log2 FC in cardiac alpha actin (ACTC1 ↑2.5 MFM), cytoskeletal desmoplakin (DSP ↑2.4 MFM), and basement membrane usherin (USH2A ↓2.9 MFM). Proteome analysis revealed significantly lower antioxidant peroxiredoxin 6 content (PRDX6, ↓4.14 log2 FC MFM), higher fatty acid transport enzyme carnitine palmitoyl transferase (CPT1B, ↑3.49 MFM), and lower sarcomere protein tropomyosin (TPM2, ↓3.24 MFM) in MFM vs. control muscle at rest. We propose that in MFM horses, altered cysteine metabolism and a deficiency of cysteine-containing antioxidants combined with a high capacity to oxidize fatty acids and generate ROS during aerobic exercise causes chronic oxidation and aggregation of key proteins such as desmin.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

The ACTC M305L Hypertrophic Cardiomyopathy Mutation Results in Hypercontractility and Impaired Relaxation of Drosophila Muscles

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle characterized by ventricular wall thickening, hyperdynamic contractile properties, and impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. The alpha-cardiac actin ACTC M305L HCM mutation is located near the nucleotide-binding site and residues predicted to help confine tropomyosin to an inhibitory position along thin filaments. We generated several Drosophila models of the disorder to evaluate the effects of the lesion from the organ through molecular level. In all muscles tested, phenotypic disturbances correlated with mutant protein load. For example, relative to wildtype actin, when highly overexpressed in the fly heart, the variant significantly reduced cardiac output, prolonged systole, and restricted diastolic volumes. Preliminary force measurements from jump muscle fibers corroborated a dose-dependent, M305L actin-induced pathological effect. Moreover, we observed greater incremental decreases in flight ability with increasing amounts of mutant versus wildtype actin expression in indirect flight muscles (IFM). Flight was abolished when the mutant was highly overexpressed and destructive myosin-induced hypercontraction was evident, suggesting disrupted contractile regulation and poorly inhibited actomyosin associations. However, regulated in vitro motility parameters, including Vmax, nH, and pCa50, of thin filaments reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin-tropomyosin, were indistinguishable. Likewise, no overt differences in computationally-derived electrostatic interaction energy landscapes between M305L and wildtype actin-tropomyosin filaments were observed, suggesting unperturbed tropomyosin-positioning in the absence of troponin. Nonetheless, over sparsely populated beds of myosin, a significantly higher percentage of troponin-free M305L versus wildtype actin-tropomyosin filaments were motile, implying tropomyosin is less effective at blocking actomyosin associations on mutant filaments. Mispositioning of tropomyosin may contribute to the supranormal contraction and compromised relaxation observed in our in vivo models and, potentially, in human patients.

The Actc A295S Hypertrophic Cardiomyopathy Mutation Promotes Thin Filament Disinhibition and Enhances Contractile Activity

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease of the heart. HCM mutations in sarcomeric genes often engender hypercontractile myocardium that displays increased Ca2+-sensitivity of force, enhanced cross-bridge cycling, and incomplete relaxation, which precede abnormal wall thickening. The alpha-cardiac actin (ACTC) Ala295Ser substitution causes a highly penetrant disease with diverse phenotypes. To help resolve the mechanistic basis of the disorder, we generated several Drosophila models that limit genetic diversity and pathological complexity. We created lines with inducible wildtype UAS-Act57BWTand mutant UAS-Act57BA295Scardiac actin transgenes to examine the effects of A295S actin on the fly heart. High-speed video microscopy and motion analysis of beating hearts revealed A295S actin expression significantly reduced diastolic volumes and prolonged systolic intervals, consistent with elevated contractile properties at rest and during contraction. In situ treatment of Act57BA295S hearts with blebbistatin, under low intracellular Ca2+ conditions, caused significantly larger increases in myocyte length vs. Act57BWTcontrols. These data suggest A295S actin induces excessive thin filament disinhibition, myosin binding, and resting tension. We also expressed indirect flight muscle (IFM)-targeted Act88FWT or Act88FA295S transgenes in an IFM actin-null background. Confocal microscopy resolved hypercontracted Act88FA295S fibers. Reconstituted Act88FA295S thin filaments, using bovine cardiac troponin-tropomyosin, displayed significantly enhanced Ca2+-sensitivity of activation vs. control filaments in regulated in vitro motility experiments. Finally, in silico modelling revealed the A295S mutation influences the orientation of nearby actin residues that mediate critical electrostatic interactions with tropomyosin. Such mutation-induced propagated effects likely lower the energy required to relieve tropomyosin-mediated inhibition and promote force production. Overall, enhanced contractile activity from the molecular through the cellular level is consistent with the early hyperdynamic contractile properties frequently associated with HCM and could trigger ACTC A295S-associated pathology. This work was supported by R37HL36153 and 1R01HL124091.

Cardiac troponin T is necessary for normal development in the embryonic chick heart.

The heart is the first functioning organ to develop during embryogenesis. The formation of the heart is a tightly regulated and complex process, and alterations to its development can result in congenital heart defects. Mutations in sarcomeric proteins, such as alpha myosin heavy chain and cardiac alpha actin, have now been associated with congenital heart defects in humans, often with atrial septal defects. However, cardiac troponin T (cTNT encoded by gene TNNT2) has not. Using gene‐specific antisense oligonucleotides, we have investigated the role of cTNT in chick cardiogenesis. TNNT2 is expressed throughout heart development and in the postnatal heart. TNNT2‐morpholino treatment resulted in abnormal atrial septal growth and a reduction in the number of trabeculae in the developing primitive ventricular chamber. External analysis revealed the development of diverticula from the ventricular myocardial wall which showed no evidence of fibrosis and still retained a myocardial phenotype. Sarcomeric assembly appeared normal in these treated hearts. In humans, congenital ventricular diverticulum is a rare condition, which has not yet been genetically associated. However, abnormal haemodynamics is known to cause structural defects in the heart. Further, structural defects, including atrial septal defects and congenital diverticula, have previously been associated with conduction anomalies. Therefore, to provide mechanistic insights into the effect that cTNT knockdown has on the developing heart, quantitative PCR was performed to determine the expression of the shear stress responsive gene NOS3 and the conduction gene TBX3. Both genes were differentially expressed compared to controls. Therefore, a reduction in cTNT in the developing heart results in abnormal atrial septal formation and aberrant ventricular morphogenesis. We hypothesize that alterations to the haemodynamics, indicated by differential NOS3 expression, causes these abnormalities in growth in cTNT knockdown hearts. In addition, the muscular diverticula reported here suggest a novel role for mutations of structural sarcomeric proteins in the pathogenesis of congenital cardiac diverticula. From these studies, we suggest TNNT2 is a gene worthy of screening for those with a congenital heart defect, particularly atrial septal defects and ventricular diverticula.

Cardiomyocyte differentiation induced in cardiac progenitor cells by cardiac fibroblast-conditioned medium

Our previous study showed that after being treated with 5-azacytidine, Nkx2.5(+) human cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiomyocytes. Although 5-azacytidine is a classical agent that induces myogenic differentiation in various types of cells, the drug is toxic and unspecific for myogenic differentiation. To investigate the possibility of inducing CPCs to differentiate into cardiomyocytes by a specific and non-toxic method, CPCs of passage 15 and mesenchymal stem cells (MSCs) were treated with cardiac ventricular fibroblast-conditioned medium (CVF-conditioned medium). Following this treatment, the Nkx2.5(+) CPCs underwent cardiomyogenic differentiation. Phase-contrast microscopy showed that the morphology of the treated CPCs gradually changed. Ultrastructural observation confirmed that the cells contained typical sarcomeres. The expression of cardiomyocyte-associated genes, such as alpha-cardiac actin, cardiac troponin T, and beta-myosin heavy chain (MHC), was increased in the CPCs that had undergone cardiomyogenic differentiation compared with untreated cells. In contrast, the MSCs did not exhibit changes in morphology or molecular expression after being treated with CVF-conditioned medium. The results indicated that Nkx2.5(+) CPCs treated with CVF-conditioned medium were capable of differentiating into a cardiac phenotype, whereas treated MSCs did not appear to undergo cardiomyogenic differentiation. Subsequently, following the addition of Dkk1 and the blocking of Wnt signaling pathway, CVF-conditioned medium-induced morphological changes and expression of cardiomyocyte-associated genes of Nkx2.5(+) CPCs were inhibited, which indicates that CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs is associated with Wnt signaling pathway. In addition, we also found that the activation of Wnt signaling pathway was accompanied by higher expression of GATA-4 and the blocking of the pathway inhibited the expression of GATA-4 in CVF-conditioned medium-incubated Nkx2.5(+) CPCs. This finding suggests that Wnt signaling pathway may alter GATA-4 expression and activate the cardiogenic program in the regulation of differentiation. In conclusion, Nkx2.5(+) CPCs have enormous potential for cardiomyogenic differentiation and the CVF-conditioned medium specifically induces CPCs to differentiate into a cardiac phenotype. Wnt signaling pathway is involved in CVF-conditioned medium-induced cardiomyogenic differentiation of Nkx2.5(+) CPCs.

The ACTC A295S Mutation Increases Heart and Skeletal Muscle Contractile Activity in Drosophila Models of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by thickened heart walls, cardiac arrhythmias, myocyte disarray and interstitial fibrosis. Over 10 mutations in alpha-cardiac actin (ACTC) are associated with HCM. The ACTC Ala295Ser substitution causes a highly penetrant disease with diverse phenotypes. To investigate the in vivo consequences of the ACTC mutation we generated Drosophila lines with muscle-specific expression of A295S actin. Our unique models limit genetic diversity and pathological complexity to help resolve molecular mechanisms of diseases. For expression in the fly heart we used UAS-Act57BWT and UAS-Act57BA295S actin transgenes in conjunction with the HandGal4 cardiac-specific driver. Confocal microscopy confirmed heart-restricted expression of UAS-Act57BWTGFP and UAS-Act57BA295SGFP and co-polymerization of transgenic and endogenous actin. High-speed video microscopy and motion analysis revealed A295S actin expression significantly reduced diastolic volumes and increased systolic intervals consistent with elevated contractile properties at rest and during contraction. For indirect flight muscle (IFM)-specific actin expression, we used the IFM targeted Act88FWTand Act88FA295S transgenes, in an IFM actin null background. Muscle mechanics on heterozygous IFM fibers revealed that the A295S actin mutation increased power, the frequency of maximum power generation (fmax), and stiffness relative to control fibers. The higher fmax and stiffness are likely causes of an observed increase in wing beat frequency (WBF) of Act88FA295Sheterozygous flies. The WBF increase had a negative impact on flight ability which decreased further as mutant: wildtype actin ratios were increased. Overall, enhanced contractile activity of actin HCM cardiac myocytes and IFM fibers is consistent with the hypercontractile properties frequently associated with HCM mutations.

A cocktail method for promoting cardiomyocyte differentiation from bone marrow-derived mesenchymal stem cells.

A growing body of evidence supports the argument that bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into cardiomyocyte-like cells in an appropriate cellular environment, but the differentiation rate is low. A cocktail method was designed: we investigated the role of 5-azacytidine (5-aza), salvianolic acid B (SalB), and cardiomyocyte lysis medium (CLM) in inducing MSCs to acquire the phenotypical characteristics of cardiomyocytes. The fourth-passage MSCs were treated with 5-aza, SalB, CLM, 5-aza+salB, 5-aza+CLM, SalB+CLM, and 5-aza+SalB+CLM for 2 weeks. Immunofluorescence results showed that cTnT expression in the 5-aza+salB+CLM group was stronger than other groups. Real-time qPCR and Western blotting analyses showed that cTnT, alpha-cardiac actin, mef-2c, Cx43, and GSK-3beta expression increased while beta-catenin expression decreased. The salB+5-aza+CLM group had the most evident effects. SalB combined with 5-aza and CLM improved cardiomyocyte differentiation from MSCs. In the MSCs differentiation process, the Wnt/beta-catenin signaling pathway had been inhibited.

Polymorphism in the Alpha Cardiac Muscle Actin 1 Gene Is Associated to Susceptibility to Chronic Inflammatory Cardiomyopathy

AimsChagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America, and may lead to a life-threatening inflammatory dilated, chronic Chagas cardiomyopathy (CCC). One third of T. cruzi-infected individuals progress to CCC while the others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Since mutations in multiple sarcomeric genes, including alpha-cardiac actin (ACTC1) have been involved in hereditary dilated cardiomyopathy, we investigated the involvement of the ACTC1 gene in CCC pathogenesis. Methods and ResultsWe conducted a proteomic and genetic study on a Brazilian study population. The genetic study was done on a main cohort including 118 seropositive asymptomatic subjects and 315 cases and the replication was done on 36 asymptomatic and 102 CCC cases. ACTC1 protein and mRNA levels were lower in myocardial tissue from patients with end-stage CCC than those found in hearts from organ donors. Genotyping a case-control cohort of CCC and ASY subjects for all informative single nucleotide polymorphism (SNP) in the ACTC1 gene identified rs640249 SNP, located at the 5’ region, as associated to CCC. Associations are borderline after correction for multiple testing. Correlation and haplotype analysis led to the identification of a susceptibility haplotype. Functional assays have shown that the rs640249A/C polymorphism affects the binding of transcriptional factors in the promoter regions of the ACTC1 gene. Confirmation of the detected association on a larger independent replication cohort will be useful.ConclusionsGenetic variations at the ACTC1 gene may contribute to progression to chronic Chagas Cardiomyopathy among T. cruzi-infected patients, possibly by modulating transcription factor binding to ACTC1 promoter regions.

Exome Sequencing Identifies a Novel Variant in ACTC1 Associated With Familial Atrial Septal Defect

BackgroundThe genetics of congenital heart disease (CHD) remain incompletely understood. Exome sequencing has been successfully used to identify disease-causing mutations in familial disorders in which candidate gene analyses and linkage mapping have failed. MethodsWe studied a large family characterized by autosomal dominant isolated secundum atrial septal defect (ASD) (MIM No. 612794). Candidate gene resequencing and linkage analysis were uninformative. ResultsWhole-exome sequencing of 2 affected family members identified 44 rare shared variants, including a nonsynonymous mutation (c.532A>T, p.M178L, NM_005159.4) in alpha-cardiac actin (ACTC1). This mutation was absent from 1834 internal controls as well as from the 1000 Genomes and the Exome Sequencing Project (ESP) databases, but predictions regarding its effect on protein function were divergent. However, p.M178L was the only rare mutation segregating with disease in our family. ConclusionsOur results provide further evidence supporting a causative role for ACTC1 mutations in ASD. Massively parallel sequencing of the exome allows for the detection of novel rare variants causing CHD without the limitations of a candidate gene approach. When mutation prediction algorithms are not helpful, studies of familial disease can help distinguish rare pathologic mutations from benign variants. Consideration of the family history can lead to genetic insights into CHD.

The W-Loop of Alpha-Cardiac Actin Is Critical for Heart Function and Endocardial Cushion Morphogenesis in Zebrafish

Mutations in cardiac actin (ACTC) have been associated with different cardiac abnormalities in humans, including dilated cardiomyopathy and septal defects. However, it is still poorly understood how altered ACTC structure affects cardiovascular physiology and results in the development of distinct congenital disorders. A zebrafish mutant (s434 mutation) was identified that displays blood regurgitation in a dilated heart and lacks endocardial cushion (EC) formation. We identified the mutation as a single nucleotide change in the alpha-cardiac actin 1a gene (actc1a), resulting in a Y169S amino acid substitution. This mutation is located at the W-loop of actin, which has been implicated in nucleotide sensing. Consequently, s434 mutants show loss of polymerized cardiac actin. An analogous mutation in yeast actin results in rapid depolymerization of F-actin into fragments that cannot reanneal. This polymerization defect can be partially rescued by phalloidin treatment, which stabilizes F-actin. In addition, actc1a mutants show defects in cardiac contractility and altered blood flow within the heart tube. This leads to downregulation or mislocalization of EC-specific gene expression and results in the absence of EC development. Our study underscores the importance of the W-loop for actin functionality and will help us to understand the structural and physiological consequences of ACTC mutations in human congenital disorders.

Intracoronary artery transplantation of cardiomyoblast-like cells from human adipose tissue-derived multi-lineage progenitor cells improve left ventricular dysfunction and survival in a swine model of chronic myocardial infarction

Transplantation of human cardiomyoblast-like cells (hCLCs) from human adipose tissue-derived multi-lineage progenitor cells improved left ventricular function and survival of rats with myocardial infarction. Here we examined the effect of intracoronary artery transplantation of human CLCs in a swine model of chronic heart failure. Twenty-four pigs underwent balloon-occlusion of the first diagonal branch followed by reperfusion, with a second balloon-occlusion of the left ascending coronary artery 1week later followed by reperfusion. Four weeks after the second occlusion/reperfusion, 17 of the 18 surviving animals with severe chronic MI (ejection fraction <35% by echocardiography) were immunosuppressed then randomly assigned to receive either intracoronary artery transplantation of hCLCs hADMPCs or placebo lactic Ringer’s solution with heparin. Intracoronary artery transplantation was followed by the distribution of DiI-stained hCLCs into the scarred myocardial milieu. Echocardiography at post-transplant days 4 and 8weeks showed rescue and maintenance of cardiac function in the hCLCs transplanted group, but not in the control animals, indicating myocardial functional recovery by hCLCs intracoronary transplantation. At 8week post-transplantation, 7 of 8 hCLCs transplanted animals were still alive compared with only 1 of the 5 control (p=0.0147). Histological studies at week 12 post-transplantation demonstrated engraftment of the pre DiI-stained hCLCs into the scarred myocardium and their expression of human specific alpha-cardiac actin. Human alpha cardiac actin-positive cells also expressed cardiac nuclear factors; nkx2.5 and GATA-4. Our results suggest that intracoronary artery transplantation of hCLCs is a potentially effective therapeutic strategy for future cardiac tissue regeneration.

Abstract 56: Shifting AAV Mediated Expression Profile to Heart by an Integrated Transcriptional Targeting Strategy

Adeno-associated viral (AAV) vectors are promising platforms for cardiac gene transport, but their broad tropism is a drawback after systematic delivery. To divert gene expression of AAV6 or 9 to heart, an integrated transcriptional targeting strategy was explored. By comparing cardiac GFP expression of scAAV6 and 9-CMV-GFP in C57/B6 or Balb/C mice, a heart-bias in C57/B6 and liver bias in Balb/C was seen. This makes the native liver biased tropism of AAV6 or 9 in Balb/C mice more suitable for the transcriptional targeting study. Luciferase (Luc) gene expression driven by cardiac specific promoters; brain natriuretic peptide (BNP), cardiac ankyrin repeat kinase (CARK), myoglobin (Myo), alpha cardiac actin (a-CARD), Troponin I (Tni), and Troponin T (Tnt), were compared to CMV promoter post retro orbital injection of 2.5x1010 viral particles of AAV6 or 9. In contrast to the previously reported heart biased signal seen in C57/B6 mice, signal was observed mainly in the liver of Balb/C mice. In vitro Luc activity, mRNA abundance and viral genomes (VG) assays were performed in liver and heart samples. Surprisingly, ∼10 folds higher Luc activity and RNA abundance in heart were observed for Tnt, Tni and a-CARD promoters than CMV promoter post AAV9 delivery, while a reduction of 100 fold was observed in liver. Copy number assay revealed six fold higher VG in liver than in heart for all promoters used including CMV. This indicates Tnt, Tni and a-CARD promoters have strong heart biased activity, without a tropism change as determined by VG copy numbers. To further develop the transcriptional targeting strategy three copies of mir122 binding site were inserted into the 3'UTR of AAV9-CMV-Luc and Balb/C mice were injected with 5x1010 viral particles. This resulted in ∼30 fold less Luc activity when compared to AAV9 without mir122 binding sites, with no change in VG copy number. In conclusion, in a mouse strain biased for liver expression after systemic delivery, transcriptional targeting to heart can be accomplished by using strong cardiac biased promoter integrated with mir122 binding sites in the 3’UTR.