Cardiac Actin Research Articles

Novel Insights into Post-Myocardial Infarction Cardiac Remodeling through Algorithmic Detection of Cell-Type Composition Shifts.

Recent advances in single cell sequencing have led to an increased focus on the role of cell-type composition in phenotypic presentation and disease progression. Cell-type composition research in the heart is challenging due to large, frequently multinucleated cardiomyocytes that preclude most single cell approaches from obtaining accurate measurements of cell composition. Our in silico studies reveal that ignoring cell type composition when calculating differentially expressed genes (DEGs) can have significant consequences. For example, a relatively small change in cell abundance of only 10% can result in over 25% of DEGs being false positives. We have implemented an algorithmic approach that uses snRNAseq datasets as a reference to accurately calculate cell type compositions from bulk RNAseq datasets through robust data cleaning, gene selection, and multi-sample cross-subject and cross-cell-type deconvolution. We applied our approach to cardiomyocyte-specific α1A adrenergic receptor (CM-α1A-AR) knockout mice. 8-12 week-old mice (either WT or CM-α1A-KO) were subjected to permanent left coronary artery (LCA) ligation or sham surgery (n=4 per group). Transcriptomes from the infarct border zones were collected 3 days later and analyzed using our algorithm to determine cell-type abundances, corrected differential expression calculations using DESeq2, and validated these findings using RNAscope. Uncorrected DEGs for the CM-α1A-KO X LCA interaction term featured many cell-type specific genes such as Timp4 (fibroblasts) and Aplnr (cardiomyocytes) and overall GO enrichment for terms pertaining to cardiomyocyte differentiation (P=3.1E-4). Using our algorithm, we observe a striking loss of cardiomyocytes and gain in fibroblasts in the α1A-KO + LCA mice that was not recapitulated in WT + LCA animals, although we did observe a similar increase in macrophage abundance in both conditions. This recapitulates prior results that showed a much more severe heart failure phenotype in CM-α1A-KO + LCA mice. Following correction for cell-type, our DEGs now highlight a novel set of genes enriched for GO terms such as cardiac contraction (P=3.7E-5) and actin filament organization (P=6.3E-5). Our algorithm identifies and corrects for cell-type abundance in bulk RNAseq datasets opening new avenues for research on novel genes and pathways as well as an improved understanding of the role of cardiac cell types in cardiovascular disease.

Empagliflozin mitigates ponatinib-induced cardiotoxicity by restoring the connexin 43-autophagy pathway

BackgroundEmpagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AimsTo assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. Methods and ResultsMale C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20 %, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. ConclusionEMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.

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.

Whole-Transcriptome RNA Sequencing Uncovers the Global Expression Changes and RNA Regulatory Networks in Duck Embryonic Myogenesis.

Duck meat is pivotal in providing high-quality protein for human nutrition, underscoring the importance of studying duck myogenesis. The regulatory mechanisms governing duck myogenesis involve both coding and non-coding RNAs, yet their specific expression patterns and molecular mechanisms remain elusive. To address this knowledge gap, we performed expression profiling analyses of mRNAs, lncRNAs, circRNAs, and miRNAs involved in duck myogenesis using whole-transcriptome RNA-seq. Our analysis identified 1733 differentially expressed (DE)-mRNAs, 1116 DE-lncRNAs, 54 DE-circRNAs, and 174 DE-miRNAs when comparing myoblasts and myotubes. A GO analysis highlighted the enrichment of DE molecules in the extracellular region, protein binding, and exocyst. A KEGG analysis pinpointed pathways related to ferroptosis, PPAR signaling, nitrogen metabolism, cell cycle, cardiac muscle contraction, glycerolipid metabolism, and actin cytoskeleton. A total of 51 trans-acting lncRNAs, including ENSAPLT00020002101 and ENSAPLT00020012069, were predicted to participate in regulating myoblast proliferation and differentiation. Based on the ceRNAs, we constructed lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA ceRNA networks involving five miRNAs (miR-129-5p, miR-133a-5p, miR-22-3p, miR-27b-3p, and let-7b-5p) that are relevant to myogenesis. Furthermore, the GO and KEGG analyses of the DE-mRNAs within the ceRNA network underscored the significant enrichment of the glycerolipid metabolism pathway. We identified five different DE-mRNAs, specifically ENSAPLG00020001677, ENSAPLG00020002183, ENSAPLG00020005019, ENSAPLG00020010497, and ENSAPLG00020017682, as potential target genes that are crucial for myogenesis in the context of glycerolipid metabolism. These five mRNAs are integral to ceRNA networks, with miR-107_R-2 and miR-1260 emerging as key regulators. In summary, this study provides a valuable resource elucidating the intricate interplay of mRNA-lncRNA-circRNA-miRNA in duck myogenesis, shedding light on the molecular mechanisms that govern this critical biological process.

Empagliflozin restores autophagy and ameliorates ponatinib-induced cardiomyocyte death

Abstract Background The tyrosine kinase inhibitor ponatinib has proven cardiovascular toxicity, for which no counteractive treatment is available at the moment. Sodium-glucose cotransporter type 2 (SGLT-2) inhibitors are approved drugs treat patients with chronic heart failure of different aetiologies. They play direct anticancer activity in SGLT2-expressing cancer cells, independent of glucose metabolism and through inhibition of oxidative phosphorylation. Ongoing studies are now testing empagliflozin (EMPA) in the prevention of left ventricular dysfunction in patients receiving high cumulative doses of anthracyclines. Aims To determine the effect of EMPA on cardiomyocytes in an in vitro model of PON-induced cardiac toxicity, and to assess underlying cardioprotective mechanisms. Methods and Results We exposed cardiomyocytes to PON (1.7 nM) or vehicle (DMSO) in the presence or absence of EMPA (100 and 500 nM) for 24 hours. Compared with vehicle, incubations with PON significantly reduced cell viability at 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, decreased autophagic cells at immunofluorescence assay (1±0.5 vs 6±1 fluorescence intensity/total area, p=0.0006, Figure 1), and increased cell senescence (15±5 vs 4±2 β-gal positivity/tissue area, p<0.02), effects all reverted by EMPA (p<0.01). We observed no significant effects of PON or EMPA on formation of reactive oxygen species. Compared with vehicle, PON significantly reduced serum response factor (SRF)/serum response elements (SRE) binding activity (25±9 vs 218±15 SRE complex abundance, arbitrary units, p<0.001), in parallel with a reduction in cardiac actin expression (0.32±0.06 vs 0.98±0,12 cardiac actin/GAPDH ratio, arbitrary units, p=0.001), which was reversed by EMPA (p=0.001). Conclusion EMPA attenuates PON cardiotoxicity by reducing senescence and enhancing autophagy, apparently independent of antioxidant mechanisms. This study suggests the design of clinical studies aimed at assessing the effects of EMPA on PON-induced cardiotoxicity.Legend to Figure 1.CON, control (vehicle, DMSO); PON, ponatinib; EMPA 100, empagliflozin 100 nM; EMPA 500, empagliflozin 500 nM

Abstract P2113: Zdhhc7 Uniquely Influences Cardiac Fibroblast Activity Amongst Golgi-localized S-acyltransferases

Cardiac fibroblasts (CF’s), the heart’s most critical regulators of the extra-cellular matrix, can undergo a continuum of activation states when stimulated that influence their proliferation, migration, collagen production, and cell-to-cell communication in response to injury. The signaling pathways that underlie this transformation though, are poorly understood. Previous studies have shown zDHHC3, an S-acyltransferase enzyme that mediates the reversible lipid modification, palmitoylation, to be important in cardiac remodeling in cardiomyocytes due to its ability to regulate intracellular signaling, though the role of zDHHC enzymes and palmitoylation in CF signaling are unknown. To investigate this, we deleted zDHHC3 or the closely related enzyme, zDHHC7, in cardiac fibroblasts to screen for changes in fibroblast activity and myocardial fibrosis. Loss of zDHHC7, but not zDHHC3, resulted in increased expression of the activated cardiac fibroblast marker α-smooth muscle actin in response to TGFb stimulation and a trend towards increased mRNA levels of fibrotic genes. More notably, zDHHC7 deletion significantly blunted migratory ability of adult cardiac fibroblasts in scratch assays. These data suggest zDHHC7 plays a critical role in CF signaling, and future experiments include analyzing the impact of zDHHC3/7 deletion alone or in combination on cardiac fibrosis and identifying key substrates and pathways being modified by these enzymes in CFs.

Zebrafish as a model for cardiac disease; Cryo-EM structure of native cardiac thin filaments from Danio Rerio

Actin, tropomyosin and troponin, the proteins that comprise the contractile apparatus of the cardiac thin filament, are highly conserved across species. We have used cryo-EM to study the three-dimensional structure of the zebrafish cardiac thin and actin filaments. With 70% of human genes having an obvious zebrafish orthologue, and conservation of 85% of disease-causing genes, zebrafish are a good animal model for the study of human disease. Our structure of the zebrafish thin filament reveals the molecular interactions between the constituent proteins, showing that the fundamental organisation of the complex is the same as that reported in the human reconstituted thin filament. A reconstruction of zebrafish cardiac F-actin demonstrates no deviations from human cardiac actin over an extended length of 14 actin subunits. Modelling zebrafish homology models into our maps enabled us to compare, in detail, the similarity with human models. The structural similarities of troponin-T in particular, a region known to contain a hypertrophic cardiomyopathy ‘hotspot’, confirm the suitability of zebrafish to study these disease-causing mutations.

Cardiac myosin's mechanics and load dependent kinetics are not altered by the thin-filament proteins.

The force necessary to power human heart contraction is generated by beta cardiac myosin pulling on regulated thin filaments. Myosin's force generation is a tightly regulated process, and its disruption causes diseases including genetic cardiomyopathies. Deciphering the details of force generation at the molecular scale is critical for our understanding of both cardiac physiology and disease. While excellent studies have elucidated the mechanics and kinetics underlying the interactions between cardiac myosin and actin at the single molecule scale, the majority of these studies have been conducted in the absence of regulatory proteins. In the case of several other myosin isoforms, regulatory proteins alter the mechanics and load-dependent kinetics of the myosin working stroke, and it is not clear what role, if any they play in tuning the cardiac myosin working stroke and load-dependent kinetics. Here, we used a single molecule optical trapping assay to measure myosin's mechanics in the presence and absence of reconstituted thin-filaments (RTFs). We observed that regulatory proteins do not affect the myosin working stroke or its substeps. Interestingly, at low ATP, we observed that regulatory proteins slow the rate of actomyosin dissociation, and we showed using stopped flow kinetics that this is due to tuning of the rate of ATP-induced dissociation. The difference in the dissociation rate disappears at physiologically relevant ATP concentrations. We also used an isometric force clamp to investigate the load dependence of the myosin working stroke at physiologically relevant ATP concentrations, and this was also unchanged with RTFs. Taken together, these results show that cardiac myosin's mechanics are not altered by regulatory proteins at physiologically relevant ATP concentrations, and they provide important insights into the molecular mechanisms underlying heart contraction.

Empagliflozin inhibits excessive autophagy through the AMPK/GSK3β signalling pathway in diabetic cardiomyopathy.

Sodium-glucose cotransporter 2 inhibitors have beneficial effects on heart failure and cardiovascular mortality in diabetic and non-diabetic patients, with unclear mechanisms. Autophagy is a cardioprotective mechanism under acute stress conditions, but excessive autophagy accelerates myocardial cell death leading to autosis. We evaluated the protective role of empagliflozin (EMPA) against cardiac injury in murine diabetic cardiomyopathy. Male mice, rendered diabetics by one single intraperitoneal injection of streptozotocin and treated with EMPA (30 mg/kg/day), had fewer apoptotic cells (4.9 ± 2.1 vs. 1 ± 0.5 TUNEL-positive cells %, P < 0.05), less senescence (10.1 ± 2 vs. 7.9 ± 1.2 β-gal positivity/tissue area, P < 0.05), fibrosis (0.2 ± 0.05 vs. 0.15 ± 0.06, P < 0.05 fibrotic area/tissue area), autophagy (7.9 ± 0.05 vs. 2.3 ± 0.6 fluorescence intensity/total area, P < 0.01), and connexin (Cx)-43 lateralization compared with diabetic mice. Proteomic analysis showed a down-regulation of the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway and upstream activation of sirtuins in the heart of diabetic mice treated with EMPA compared with diabetic mice. Because sirtuin activation leads to the modulation of cardiomyogenic transcription factors, we analysed the DNA binding activity to serum response elements (SRE) of serum response factor (SRF) by electromobility shift assay. Compared with diabetic mice [0.5 ± 0.01 densitometric units (DU)], non-diabetic mice treated with EMPA (2.2 ± 0.01 DU, P < 0.01) and diabetic mice treated with EMPA (2.0 ± 0.1 DU, P < 0.01) significantly increased SRF binding activity to SRE, paralleled by increased cardiac actin expression (4.1 ± 0.1 vs. 2.2 ± 0.01 target protein/β-actin ratio, P < 0.01). EMPA significantly reversed cardiac dysfunction on echocardiography in diabetic mice and inhibited excessive autophagy in high-glucose-treated cardiomyocytes by inhibiting the autophagy inducer glycogen synthase kinase 3 beta (GSK3β), leading to reactivation of cardiomyogenic transcription factors. Taken together, our results describe a novel paradigm in which EMPA inhibits hyperactivation of autophagy through the AMPK/GSK3β signalling pathway in the context of diabetes.

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.

Empagliflozin alleviates diabetic cardiomyopathy via inhibiting excessive autophagy and adverse remodeling in type 1 diabetic mice

Abstract Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with and without diabetes mellitus, but underlying mechanisms remain unclear. Autophagy is an important cardioprotective mechanism under acute stress conditions, but excessive autophagy can accelerate myocardial cell death leading to autosis. In this study we evaluated the role of empagliflozin (EMPA) against cardiac injury in streptozotocin (STZ)-induced diabetic mice, and assessed underlying mechanisms. EMPA (10 mg/kg/die)-treated diabetic male mice had a lower number of TUNEL-positive apoptotic cells (%TdT+ 4.9±2, p&amp;lt;0.05), lower percentage of SA-β-gal positive senescent cardiac area (%SA-β-gal 7.9±1, p&amp;lt;0.05), lower fibrotic area (%picrosirius red 0.15±0.06, p&amp;lt;0.05), lower autophagy (%green fluorescence 2.3±0.6, p&amp;lt;0.01), compared to diabetic mice (%TdT+ 4.9±2; %SA-β-gal 10±2; %picrosirius red fibrotic area 0.24±0.08; %green fluorescence autophagic area 3.98±0.5). Proteomics analysis of cardiac tissue showed downregulation of apoptosis, necrosis and 5' adenosine monophosphate-activated protein kinase (AMPK) pathways, along with upstream activation of vascular endothelial growth factor and sirtuins in the heart of EMPA+STZ treated mice compared to STZ mice. Since activation of sirtuins leads to modulation of cardiomyogenic transcription factors, we analyzed total expression of serum response factor (SRF), myocardin and myocardin-related transcription factor (MRTF), and DNA binding activity of SRF by electromobility shift assay. While EMPA only increased MRTF, EMPA (2.2±0.01, p&amp;lt;0.01) and EMPA+STZ (2.0±0.1, p&amp;lt;0.01) significantly increased SRF/SRE binding activity compared with STZ alone (0.5±0.01), which was paralleled by the increase in cardiac actin expression (4.06±0.1 vs 2.2±0.01, p&amp;lt;0.01). EMPA significantly reversed STZ-induced systolic cardiac dysfunction at echocardiography, and downregulation of pS368-connexin 43 active isoform (Table). In parallel in vitro experiments, EMPA inhibited excessive autophagy in high glucose-treated cardiomyocytes by inhibiting the autophagy inducer GSK3β, leading to reactivation of transcriptional complex for cardiomyocyte contractile genes. Our results indicate that EMPA plays a cardioprotective effect by inhibiting excessive autophagy through the mTOR/AMPK axis in the mouse model of STZ-induced type 1 diabetes. EMPA also promotes cardiac reverse remodeling by inhibiting fibrosis, activates cardiac Cx43 and the promyogenic transcriptional program through the sirtuins-SRF axis (Figure 1), in the context of diabetes. Funding Acknowledgement Type of funding sources: None.

Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H

Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such, ACTA1 mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated ACTA1 R256H/+ human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that ACTA1 R256H/+ hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human ACTA1 mutations.

Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

ObjectiveTo investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation. MethodsNeonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes. ResultsCFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30–100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest. ConclusionThe exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.

A murine model of ischemia–reperfusion: the perfusion with leptin promotes the apoptosis-related relocation of mitochondrial proteins Bax and cytochrome c

BackgroundLeptin exerts both protective and deleterious effects on the heart; the first occurs under hypoxia- or ischemia-associated damage, the second is a pro-hypertrophic factor on cardiomyocytes. Therefore, leptin could represent a link between obesity and cardiovascular diseases. The study aimed to investigate the effect of leptin—the same concentration that is frequently measured in obesity and induces cardiac hypertrophy—on murine hearts following an episode of ischemia–reperfusion; moreover, we evaluated the heart's performance, hypertrophy, and activation of apoptosis. Rat hearts were perfused continuously with or without 3.1 nM leptin for one h before and 1 h after an episode of ischemia. Cardiac performance was evaluated, homogenates and mitochondria were prepared for western blot analysis of cardiac actin, leptin receptor, STAT3, pSTAT3, and apoptosis-related proteins Bax, Bcl-2, cytochrome c, and caspase 3.ResultsLeptin worsened heart recovery after ischemia (p < 0.05 Control vs IR + Lep of Heart Perform, Fig. 2). Although no hypertrophic response was observed, leptin induced the migration of Bax to the mitochondria and the release of cytochrome c into the cytosol (p < 0.05 Control vs IR + Lep, Fig. 5), essential events in the intrinsic/mitochondrial apoptosis.ConclusionsOur results indicate that the presence of leptin for 1 h before and after the ischemic insult reduces heart recovery and amplifies apoptotic signaling through the mitochondrial pathway.

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

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Chronic exposure to ozone induces cardiac antioxidant response and overexpression of either mitochondrial fision protein DRP1 and hipertrophyc-related proteins.

Pollution is considered a risk factor for cardiovascular disease; however, the mechanisms to explain this relationship are not well understood; ozone is one of the most abundant and studied air contaminants. Our study aimed to evaluate the effect of chronic exposition of rats to controlled low doses of ozone on oxidative stress, apoptosis, mitochondrial dynamics, and cardiac hypertrophy. Male Wistar rats were daily exposed to low ozone doses during 7, 15, 30, and 60days, 4h/day. Hearts were dissected, and homogenates were prepared. Oxidative stress was evaluated by TBARS and protein nitrosylation in addition to Superoxide dismutase 1 (SOD1) and Catalase levels; the apoptosis related-proteins caspase 3, caspase 9, Bax, Bcl-2, and the mitochondrial dynamic-associated proteins Fis1, Drp1, OPA1, and Mfn1 were quantified by western blot among the cardiac hypertrophy indicator alpha-actin (cardiac actin). There were no changes in the oxidative stress markers, however SOD1 expression increases. Caspase 3 expression decreased, whereas caspase 9 increased without changes in Bax or Bcl-2. Mitochondrial fission may be favored according to the increased expression of Drp1 but not changes in fusion-related proteins OPA1 and Mfn1. Finally, the molecular marker for cardiac hypertrophy was overexpressed after 30 and 60days of ozone exposition. The chronic exposition to ozone induces a deleterious effect on cardiac mitochondria. Antioxidant defenses also show changes in relation to exposure time, as well as an apparent pro-hypertrophic effect associated with altered mitochondrial dynamics.

Adverse prognostic value of supernormal left ventricular force noninvasively assessed by resting transthoracic echocardiography in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: None. OnBehalf Stress Echo 2030 study group of the Italian Society of Echocardiography and Cardiovascular Imaging (SIECVI). Background The excessive cross-bridging of cardiac myosin and actin, resulting in increased left ventricular (LV) force development, is one of the biomechanical abormalities inhypertrophic cardiomyopathy (HCM). Purpose To assess the prognostic value of increased LV force development at rest in patients with HCM Methods We enrolled 918 HCM patients (age 48 ± 16 years, 502 males, New York Heart Association I or II, Class III in 48 patients, 6% and with LVOTG &amp;gt;30 mmHg in 211patients, 23%) with ejection fraction (EF) ≥50%, referred for rest transthoracic echocardiography (TTE) in 10 quality-controlled labs from 7 countries (Belgium, Hungary, Italy, Portugal, Serbia, Spain, Brazil). The maximal wall thickness was 21 ± 5 mm. TTE assessment included left ventricular outflow tract gradient (LVOTG, mmHg), EF (%), and LV force (systolic arterial pressure + LVOTG/LV endsystolic volume assessed with 2-D, mmHg/mL). All patients were followed-up. An age- and gender matched control group of 95 healthy subjects was also evaluated. Results. Compared to normals, HCM showed higher values of EF (68 ± 8 vs 65 ± 5%, p &amp;lt; 0.001) and force (7.2 ± 5.5 vs 4.0 ± 1.8 mmHg/mL, p &amp;lt; 0.001). At a median follow-up of 94 months [interquartile range 40-140 months], 95 all-cause deaths occurred. Mortality was significantly increased in the force highest quartile compared to other quartiles (see figure). At multivariable Cox analysis, increased Force (highest quartile &amp;gt;8.5 mmHg/mL hazard ratio= 2.189 95% CI = 1.095-4.377, p = 0.027 and the intermediate quartile: Force 5.7-8.5 mmHg/mL hazard ratio= 2.525 95% CI = 1.2205.228, p = 0.013) were independent predictors of mortality with age (hazard ratio= 1.065 95% CI = 1.047-1.084, p &amp;lt; 0.001) and maximal wall thickness (hazard ratio= 1.094, 95% CI = 1.055-1.135, p &amp;lt; 0.001). At univariate analysis neither LVOTG (hazard ratio= 1.430, 95% CI = 0.920-2.222, p = 0.112) nor quartiles of EF (hazard ratio= 1.497, 95% CI = 0.868-2.582, p = 0.147) were significant. Conclusion HCM patients with preserved baseline LV function and higher values of resting LV force ("too good to be normal") show a worse survival, highlighting the dark prognostic side of an excess of force. The hypercontractile phenotype possibly indicates an increased activity of myosin resulting in increased force production at the sarcomere and cellular levels that propagates at the whole-organ level with unfavorable long-term effects on outcome. Figure Mortality rate based on quartiles of resting LV Force in HCM. Abstract Figure. Mortality rate

14 Telomerase and myocardin co-expressing mesenchymal cells increase survival and induce cardiac and vascular markers in cardiac stromal cells undergoing simulated ischemia/reperfusion

Abstract Aims Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced coexpression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities of MSCs isolated from the adipose tissue (AT-MSCs), may increase CSC survival, favouring their paracrine activities. To investigate the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment toward the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT-MSCs overexpressing TERT and MYOCD (T/M AT-MSCs). Methods Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. Results T/M AT-MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT-MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs. 10 ± 3%, P = 0.02). Pre-treatment with CM (15 ± 2, P = 0.02) or with the EV-enriched fraction (10 ± 1%, P = 0.02) obtained from mock-transduced AT-MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = 0.01) or the EV-enriched fraction (2 ± 1%, P = 0.01) obtained from T/M AT-MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase-3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α-actinin and cardiac actin. Conclusions The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.

12 Sex-related differential susceptibility to ponatinib-induced cardiotoxicity and its relationship to modulation of notch signalling in a muine model

Abstract Aims Ponatinib (PON), a tyrosine kinase inhibitor approved in chronic myeloid leukaemia, has proven cardiovascular toxicity. Although sex is a risk factor for PON-induced cardiotoxicity in humans, little is known about its mechanisms, in general, and sex-related mechanisms in particular. To determine the mechanisms of sex-related PON-induced cardiotoxicity and identify potential rescue strategies in a murine model. Methods Twenty-four-month-old male and female C57B5 mice were treated with 3 mg/kg/day of PON or vehicle via oral gavage for 28 days, with/without siRNA-Notch1 or siRNA-scrambled via tail vein every 3 days. Results PON + scrambled siRNA-treated male mice had a higher number of TUNEL-positive cells, a higher percentage of senescence-associated β-galactosidase positive senescent cardiac areas, as well as a lower reactivity degree for the survival marker Bmi1 than female counterparts. Proteomics analysis of cardiac tissue showed upstream activation of nitric oxide synthase (NOS) type 2, downstream activation of cell death and production of reactive oxygen species in PON + scrambled siRNA- compared to vehicle or PON + Notch1 siRNA-treated male mice. Upstream analysis showed beta-estradiol activation, while downstream analysis showed activation of cell survival and inhibition of cell death in PON + scrambled siRNA compared to vehicle-treated female mice. PON + scrambled siRNA-treated mice also showed a down-regulation of cardiac actin, which was more marked in male; as well as vessel density, which was more marked in female mice. Female hearts showed a greater extent of cardiac fibrosis than male counterparts at baseline, with no significant changes after PON treatment. In contrast, PON + scrambled siRNA-treated mice had less fibrosis than vehicle or PON + Notch1-siRNA-treated mice. Left ventricular systolic dysfunction shown in PON + scrambled siRNA-treated male mice and—to a lesser extent—by female mice was similarly reversed in both PON + Notch1-siRNA-treated male and female mice (Table 1). Conclusions We found a sex-related differential susceptibility and Notch1 modulation in PON-induced cardiotoxicity. This can improve our understanding of sex-related differences and help identify biomarkers in PON cardiotoxicity.

Toxicity of perfluorooctanoic acid on zebrafish early embryonic development determined by single-cell RNA sequencing

The perfluorooctanoic acid (PFOA) poses a high risk for aquatic organisms. Nevertheless, the current toxicity studies rarely report how PFOA affects different cell populations during the embryonic development of fish. Here, the zebrafish embryos at 2–30 hpf were exposed to 1–100 μg/L PFOA. The heartbeat and locomotor behavior were significantly decreased after ≥ 25 μg/L PFOA exposure. The single-cell RNA sequencing showed that PFOA exposure influenced nine cell populations, including heart cells, hatching gland cells, macrophages, lens cells, ionocytes, melanoblasts, optic cup cells, periderm cells, and differentiating neurons cells. Among them, heart cells were the most affected cell population. Functions of cardiac muscle contraction, actin cytoskeleton and oxygen binding were significantly changed in the heart cells, which were involved in the altered expressions of tnni2a.4, acta1a, atp1a1a.2, mylpfa, and so on. Besides, the changes of apoptotic process, innate immune response, and translation in lens cells, hatching gland cells, macrophages and ionocytes should also be of concern. Our study indicates that 2–30 hpf of embryonic development is the sensitivity window for the PFOA exposure. Identification of the target cell population provides clear information of the toxic endpoint of PFOA, which sheds new light on the risk assessment of PFOA on aquatic organisms.