Mouse Model Of Dilated Cardiomyopathy Research Articles

Epidermal Growth Factor-Like Repeats and Discoidin I-Like Domains 3 Deficiency Attenuates Dilated Cardiomyopathy by Inhibiting Ubiquitin Specific Peptidase 10 Dependent Smad4 Deubiquitination.

Dilated cardiomyopathy (DCM) is the leading cause of heart failure with a poor prognosis. Recent studies suggest that endothelial to mesenchymal transition (EndMT) may be involved in the pathogenesis and cardiac remodeling during DCM development. EDIL3 (epidermal growth factor-like repeats and discoidin I-like domains 3) is an extracellular matrix glycoprotein that has been reported to promote EndMT in various diseases. However, the roles of EDIL3 in DCM still remain unclear. A mouse model of DCM and human umbilical vein endothelial cells were used to explore the roles and mechanisms of EDIL3 in DCM. The results indicated that EndMT and EDIL3 were activated in DCM mice. EDIL3 deficiency attenuated cardiac dysfunction and remodeling in DCM mice. EDIL3 knockdown alleviated EndMT by inhibiting USP10 (ubiquitin specific peptidase 10) dependent Smad4 deubiquitination invivo and invitro. Recombinant human EDIL3 promoted EndMT via reinforcing deubiquitination of Smad4 in human umbilical vein endothelial cells treated with IL-1β (interleukin 1β) and TGF-β (transforming growth factor beta). Inhibiting USP10 abolished EndMT exacerbated by EDIL3. In addition, recombinant EDIL3 also aggravates doxorubicin-induced EndMT by promoting Smad4 deubiquitination in HUVECs. Taken together, these results indicate that EDIL3 deficiency attenuated EndMT by inhibiting USP10 dependent Smad4 deubiquitination in DCM mice.

Cas13b-mediated RNA targeted therapy alleviates genetic dilated cardiomyopathy in mice

BackgroundRecent advances in gene editing technology have opened up new avenues for in vivo gene therapy, which holds great promise as a potential treatment method for dilated cardiomyopathy (DCM). The CRISPR-Cas13 system has been shown to be an effective tool for knocking down RNA expression in mammalian cells. PspCas13b, a type VI-B effector that can be packed into adeno-associated viruses and improve RNA knockdown efficiency, is a potential treatment for diseases characterized by abnormal gene expression.ResultsUsing PspCas13b, we were able to efficiently and specifically knockdown the mutant transcripts in the AC16 cell line carrying the heterozygous human TNNT2R141W (hTNNT2R141W) mutation. We used adeno-associated virus vector serotype 9 to deliver PspCas13b with specific single guide RNA into the hTNNT2R141W transgenic DCM mouse model, effectively knocking down hTNNT2R141W transcript expression. PspCas13b-mediated knockdown significantly increased myofilament sensitivity to Ca2+, improved cardiac function, and reduced myocardial fibrosis in hTNNT2R141W DCM mice.ConclusionsThese findings suggest that targeting genes through Cas13b is a promising approach for in vivo gene therapy for genetic diseases caused by aberrant gene expression. Our study provides further evidence of Cas13b’s application in genetic disease therapy and paves the way for future applicability of genetic therapies for cardiomyopathy.

Torsional and strain dysfunction precede overt heart failure in a mouse model of dilated cardiomyopathy pathogenesis.

Detailed assessments of whole heart mechanics are crucial for understanding the consequences of sarcomere perturbations that lead to cardiomyopathy in mice. Echocardiography offers an accessible and cost-effective method of obtaining metrics of cardiac function, but the most routine imaging and analysis protocols might not identify subtle mechanical deficiencies. This study aims to use advanced echocardiography imaging and analysis techniques to identify previously unappreciated mechanical deficiencies in a mouse model of dilated cardiomyopathy (DCM) before the onset of overt systolic heart failure (HF). Mice lacking muscle LIM protein expression (MLP-/-) were used to model DCM-linked HF pathogenesis. Left ventricular (LV) function of MLP-/- and wild-type (WT) controls were studied at 3, 6, and 10 wk of age using conventional and four-dimensional (4-D) echocardiography, followed by speckle-tracking analysis to assess torsional and strain mechanics. Mice were also studied with RNA-seq. Although 3-wk-old MLP-/- mice showed normal LV ejection fraction (LVEF), these mice displayed abnormal torsional and strain mechanics alongside reduced β-adrenergic reserve. Transcriptome analysis showed that these defects preceded most molecular markers of HF. However, these markers became upregulated as MLP-/- mice aged and developed overt systolic dysfunction. These findings indicate that subtle deficiencies in LV mechanics, undetected by LVEF and conventional molecular markers, may act as pathogenic stimuli in DCM-linked HF. Using these analyses in future studies will further help connect in vitro measurements of the sarcomere function to whole heart function.NEW & NOTEWORTHY A detailed study of how perturbations to sarcomere proteins impact whole heart mechanics in mouse models is a major yet challenging step in furthering our understanding of cardiovascular pathophysiology. This study uses advanced echocardiographic imaging and analysis techniques to reveal previously unappreciated subclinical whole heart mechanical defects in a mouse model of cardiomyopathy. In doing so, it offers an accessible set of measurements for future studies to use when connecting sarcomere and whole heart function.

Abstract EC.04: The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and k tr , a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa 50 : 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa 50 ) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased k tr , possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.

M6A regulator-mediated RNA methylation modification remodels immune microenvironment in dilated cardiomyopathy.

The latest evidence suggested that the onset of dilated cardiomyopathy (DCM)is closely associated with immune microenvironment disturbance. Since N6 -methyladenosine (m6A) RNA methylation impacts on immunocyte function and antitumor immunity, it is predictable that m6A RNA methylation may result in immune microenvironment disorder. Here, we attempted to verify this hypothesis. We used single-sample gene set enrichment analysis (ssGSEA)to investigate the infiltration abundance of immunocytes, single-cell RNA-Seq to identify key m6A regulator, and a doxorubicin (Dox)-induced DCM mouse model to confirm our findings. ssGSEA revealed a higher infiltration abundance of CD8+ T lymphocytes, NK cells, monocytes, and B+ lymphocytes in DCM myocardium tissue. Single-cell RNA-Seq indicated a critical role of IGFBP2 in DCM. Cross-checking analysis hinted an interaction between IGFBP2 and NSUN5, ALYREF, RRP8, and ALKBH3. Mechanically, IGFBP2-mediated RNA methylation deteriorated the immune microenvironment and thus increased the risk of DCM by enhancing CD8+ T lymphocyte, NK cell, monocyte, B+ lymphocyte infiltration and activating check-point, MHC-I, and T cell co-stimulation signaling pathways. In the DCM mouse model, echocardiography indicated a significant reduction in ejection fraction (EF)and fractional shortening (FS)and an increase in left ventricular internal dimensions at systole (LVIDs)and diastole (LVIDd).MASSON staining indicated an increased fibrosis in myocardium tissue. qPCR and immunofluorescence staining indicated a significant increase in mRNA and protein levels of IGFBP2. The present study indicated that IGFBP2-mediated RNA methylation remodeled the immune microenvironment and increased the risk of DCM. IGFBP2 may serve as potential therapeutic target for DCM.

Mitophagy Regulation by Kangxian Yixin Granule in a Mouse Model of Dilated Cardiomyopathy

Abstract Objective Kangxian Yixin granule (KXYXG) has been found to be effective in the clinical treatment of dilated cardiomyopathy (DCM). We aim to explore the effect of KXYXG and the underlying mechanism in a mouse model of DCM. Methods Thirty specific pathogen-free (SPF) male cTnTR141W mice with DCM were randomly divided into the model group, KXYXG (20.4 g/kg/d) group and coenzyme Q10 (CoQ10) (1.5 mg/kg/d) group; 10 SPF male C57BL/6J mice were included to form the normal group. The mice in KXYXG group and CoQ10 group were administered by oral gavage for 8 weeks. M-echocardiography was used to evaluate the cardiac function in mice, and hematoxylin and eosin staining and transmission electron microscopy were performed to observe morphological characters. The colocalization and expression levels of mitophagy-related proteins were observed using immunofluorescence and western blot. Results Compared with the normal group, the model group showed ventricular remodeling, cardiac insufficiency, disordered arrangement of cardiomyocytes, as well as disordered mitochondria and irregular and diffuse swelling. Furthermore, the model group had lower mitophagy protein colocalization and autophagy flux. Furthermore, PINK1 and Parkin expression levels decreased in the mice with DCM (p < 0.05). KXYXG could decrease the left ventricular end-diastolic and end-systolic diameters and mitochondrial injury, rescue cardiac dysfunction and remodeling, and protect against myocardial ultrastructure changes in the mice with DCM. KXYXG also increased the colocalization levels of mitophagy-related proteins and PINK1 and Parkin expression levels compared with those in the model group (p < 0.05). Conclusion KXYXG can protect against heart injury by possibly activating the PINK1/Parkin pathway and mitophagy in mice with DCM.

Identification of Novel Targets for Treatment of Dilated Cardiomyopathy Based on the Ferroptosis and Immune Heterogeneity.

This study aimed to investigate the role of ferroptosis in dilated cardiomyopathy (DCM) and to identify new targets for treatment and diagnosis of DCM. GSE116250 and GSE145154 were downloaded from the Gene Expression Omnibus database. Unsupervised consensus clustering of DCM patients was used to confirm the impact of ferroptosis. Ferroptosis-related hub genes were identified by WGCNA and single cell sequencing analyses. Finally, we established a DCM mouse model via injection of Doxorubicin to verify the expression level of OTUD1 and colocalization between cell markers and OTUD1 in DCM mouse heart. A total of 13 ferroptosis-related differentially expressed genes (DEGs) were identified. The DCM patients were divided into two clusters according to the expression of 13 DEGs. The DCM patients in different clusters showed discrepancies in immune infiltration. Four hub genes were further identified by WGCNA analysis. Single cell data analysis revealed that OTUD1 may regulate B cells and DC cells and then participate in immune infiltration discrepancy. The upregulation of OTUD1 and the colocalization of OTUD1 with CD19 (B cell maker) and CD11c (DCs markers) markers were confirmed in DCM mouse hearts. Ferroptosis and the immune microenvironment are closely associated with DCM, and OTUD1 may play an important role through B cells and DCs.

A Mouse Model of Dilated Cardiomyopathy Produced by Isoproterenol Acute Exposure Followed by 5-Fluorouracil Administration.

Appropriate dilated cardiomyopathy (DCM) animal models are highly desirable considering the pathophysiological and clinical heterogeneity of DCM. Genetically modified mice are the most widely and intensively utilized research animals for DCM. However, to translate discoveries from basic science into new and personalized medical applications, research in non-genetically based DCM models remains a key issue. Here, we characterized a mouse model of non-ischemic DCM induced by a stepwise pharmacologic regime of Isoproterenol (ISO) high dose bolus followed by a low dose systemic injection of the chemotherapy agent, 5-Fluorouracil (5-FU). C57BL/6J mice were injected with ISO and, 3 days after, were randomly assigned to saline or 5-FU. Echocardiography and a strain analysis show that ISO + 5FU in mice induces progressive left ventricular (LV) dilation and reduced systolic function, along with diastolic dysfunction and a persistent global cardiac contractility depression through 56 days. While mice treated with ISO alone recover anatomically and functionally, ISO + 5-FU causes persistent cardiomyocyte death, ensuing in cardiomyocyte hypertrophy through 56 days. ISO + 5-FU-dependent damage was accompanied by significant myocardial disarray and fibrosis along with exaggerated oxidative stress, tissue inflammation and premature cell senescence accumulation. In conclusions, a combination of ISO + 5FU produces anatomical, histological and functional cardiac alterations typical of DCM, representing a widely available, affordable, and reproducible mouse model of this cardiomyopathy.

Circulating cardiac MicroRNAs safeguard against dilated cardiomyopathy

Cardiac-resident or -enriched microRNAs (miRNAs) could be released into the bloodstream becoming circulating cardiac miRNAs, which are increasingly recognized as non-invasive and accessible biomarkers of multiple heart diseases. However, dilated cardiomyopathy (DCM)-associated circulating miRNAs (DACMs) and their roles in DCM pathogenesis remain largely unexplored. Two human cohorts, consisting of healthy individuals and DCM patients, were enrolled for serum miRNA sequencing (10vs. 10) and quantitative polymerase chain reaction validation (46vs. 54), respectively. Rigorous screening strategy was enacted to define DACMs and their potentials for diagnosis. DCM mouse model, different sources of cardiomyocytes, adeno-associated virus 9 (AAV9), gene knockout, RNAscope miRNA in situ hybridization, mRFP-GFP-LC3B reporter, echocardiography and transmission electron microscopy were adopted for mechanistic explorations. Serum miRNA sequencing revealed a unique expression pattern for DCM circulating miRNAs. DACMs miR-26a-5p, miR-30c-5p, miR-126-5p and miR-126-3p were found to be depleted in DCM circulation as well as heart tissues. Their expressions in circulation and heart tissues were proven to be correlated significantly, and a combination of these miRNAs was suggested potential values for DCM diagnosis. FOXO3, a predicted common target, was experimentally demonstrated to be co-repressed within cardiomyocytes by these DACMs except miR-26a-5p. Delivery of a combination of miR-30c-5p, miR-126-5p and miR-126-3p into the murine myocardium via AAV9 carrying an expression cassette driven by cTnT promoter, or cardiac-specific knockout of FOXO3 (Myh6-CreERT2 , FOXO3 flox+/+ ) dramatically attenuated cardiac apoptosis and autophagy involved in DCM progression. Moreover, competitively disrupting the interplay between DACMs and FOXO3 mRNA by specifically introducing their interacting regions into murine myocardium crippled the cardioprotection of DACMs against DCM. Circulating cardiac miRNA-FOXO3 axis plays a pivotal role in safeguarding against myocardial apoptosis and excessive autophagy in DCM development, which may provide serological cues for DCM non-invasive diagnosis and shed light on DCM pathogenesis and therapeutic targets.

Identification of Signature Genes of Dilated Cardiomyopathy Using Integrated Bioinformatics Analysis.

Dilated cardiomyopathy (DCM) is characterized by left ventricular or biventricular enlargement with systolic dysfunction. To date, the underlying molecular mechanisms of dilated cardiomyopathy pathogenesis have not been fully elucidated, although some insights have been presented. In this study, we combined public database resources and a doxorubicin-induced DCM mouse model to explore the significant genes of DCM in full depth. We first retrieved six DCM-related microarray datasets from the GEO database using several keywords. Then we used the "LIMMA" (linear model for microarray data) R package to filter each microarray for differentially expressed genes (DEGs). Robust rank aggregation (RRA), an extremely robust rank aggregation method based on sequential statistics, was then used to integrate the results of the six microarray datasets to filter out the reliable differential genes. To further improve the reliability of our results, we established a doxorubicin-induced DCM model in C57BL/6N mice, using the "DESeq2" software package to identify DEGs in the sequencing data. We cross-validated the results of RRA analysis with those of animal experiments by taking intersections and identified three key differential genes (including BEX1, RGCC and VSIG4) associated with DCM as well as many important biological processes (extracellular matrix organisation, extracellular structural organisation, sulphur compound binding, and extracellular matrix structural components) and a signalling pathway (HIF-1 signalling pathway). In addition, we confirmed the significant effect of these three genes in DCM using binary logistic regression analysis. These findings will help us to better understand the pathogenesis of DCM and may be key targets for future clinical management.

Exploring the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy.

The sarco/endoplasmic Ca2+-ATPase (SERCA2a) and its natural inhibitor phospholamban (PLN) play a pivotal role in cardiac excitation-contraction coupling. A heterozygous deletion of arginine 14 of the PLN protein (R14del) is associated with a dilated cardiomyopathy (DCM). It has been previously shown that overexpression of PLN-R14del leads to a substantial decrease in SERCA2a affinity for Ca2+, thus proposing that a “superinhibitory” effect of mutant PLN on SERCA2a may lead to the DCM phenotype. This theory is not consistently supported by recent experimental evidences, thus suggesting alternative pathogenetic mechanisms. To shed light on the debated pathophysiological mechanisms of PLN-R14del DCM exploiting a novel heterozygous transgenic model (Mut) that recapitulates the clinical human phenotype. SERCA2a activity and intracellular Ca2+ dynamics have been measured in cardiomyocytes (CMs) isolated from 8-12 weeks old Mut mice and WT littermates. To compare mutation effect with pharmacological SERCA2a modulation, CMs were perfused with the pure SERCA2a activator PST-3093. To test the involvement of metabolic alterations, oxidative phosphorylation and glycolysis have been investigated. In myocardial homogenates, the KdCa of SERCA2a ATPase activity was lower in Mut. In Mut CMs, the Ca2+ τdecay was shorter and diastolic Ca2+ was lower compared to WT cells. When applied to WT CMs, PST-3093 decreased τdecay to approach the value found in Mut. When applied to Mut, PST-3093 had no effect on CaT parameters. In Mut cells O2 consumption and glycolytic acidification were reduced. These results indicate enhancement of SR Ca2+ uptake rate in Mut, which is compatible with diminished PLN inhibitory function. Future work will be directed to establish if the metabolic abnormalities may be related to altered Ca2+ dynamics, or be an independent mechanism for PLN-R14del DCM.

PS-B08-3: DILATED CARDIOMYOPATHY MOUSE MODEL AGGRAVATES ACUTE KIDNEY INJURY

Objective: The relation between cardiac and renal diseases is well known, and the concept of the cardio-renal syndrome has attracted much attention in recent years. However, the molecular mechanism of the cardio-renal syndrome is still unclear. In this study, we used αMHC-dominant negative Neuron-Restrictive Silencer Factor (NRSF) transgenic mice (Tg mice), a mouse model of dilated cardiomyopathy with gradually worsening ejection fraction, to elucidate the pathogenesis of acute kidney injury in the setting of chronic heart failure. Design and Methods: Sixteen-week-old Tg and wild-type mice were subjected to unilateral nephrectomy and 45-minute ischemia reperfusion injury (IRI) to the remaining kidney, and then the kidneys and hearts were retrieved 72 hours later. The control group were subjected to unilateral nephrectomy without IRI. Results: Heart weights were significantly heavier in Tg mice in both control and IRI-treated groups. In the control group, serum creatinine and blood urea nitrogen were unchanged between wild-type and Tg mice. However, serum creatinine and blood urea nitrogen were significantly elevated in Tg mice compared to wild-type mice in the IRI-treated group, and only IRI-treated Tg mice died within 72 hours after IRI (6/14). Furthermore, mRNA expression of Ccl2 and Lcn2 (NGAL) in the whole kidney was significantly elevated in IRI-treated wild-type mice, and was more upregulated in Tg mice. Periodic acid-Schiff staining showed that tubular injuries, such as dilation of tubules, cast formation and tubular atrophy, were exacerbated in IRI-treated Tg mice. In microarray analysis of IRI-treated kidneys from wild-type and Tg mice, several candidate genes were upregulated in Tg mice. Conclusions: In dilated cardiomyopathy model mouse, inflammation and histological changes were aggravated after IRI, suggesting that chronic heart failure augments renal impairment in acute kidney injury.

Abstract 13146: Cardioprotective Effects of Mtss1 Reduction in Dilated Cardiomyopathy

Introduction: MTSS1 is an I-BAR protein that interacts with the actin cytoskeleton and the cell membrane and is speculated to play a role in cell motility, tumor metastasis, and cardiogenesis. Enhancer variants that reduce the expression of MTSS1 in the human left ventricle have been associated with cardioprotective traits in genome-wide association studies. However, the effect of MTSS1 downregulation on the development of heart disease has not been studied in experimental models. Here we tested the hypothesis that reduction of MTSS1 protects against dilated cardiomyopathy (DCM) in vitro and in vivo. Methods: We used CRISPR-Cas9 to knock out MTSS1 in induced pluripotent stem cells (iPSCs) harboring pathogenic variants from patients with severe DCM. DCM iPSC lines with and without MTSS1 were then differentiated into cardiomyocytes and phenotyped in vitro. A DCM mouse model containing a TPM1 D230N transgene was crossed with a mouse model heterozygous for Mtss1 knockout. Progeny from this F1 cross were assessed for differences in cardiac structure and function via echocardiography. Results: MTSS1 deletion restored the beta-adrenergic response in DCM iPSC cardiomyocytes. In parallel, Mtss1 deletion partially rescued DCM-associated phenotypes including left ventricular dilation and reduced ejection fraction in mice. Conclusions: Our initial results suggest a causal link between MTSS1 reduction and cardioprotection in DCM. Inhibiting cardiac MTSS1 may hold therapeutic potential for DCM, though the cellular mechanisms through which MTSS1 affects cardiac phenotypes remain under investigation.

Unraveling the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy

Unraveling the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy

NMR-Based Metabolomic Analysis of Cardiac Tissues Clarifies Molecular Mechanisms of CVB3-Induced Viral Myocarditis and Dilated Cardiomyopathy.

Viral myocarditis (VMC), which is defined as inflammation of the myocardium with consequent myocardial injury, may develop chronic disease eventually leading to dilated cardiomyopathy (DCM). Molecular mechanisms underlying the progression from acute VMC (aVMC), to chronic VMC (cVMC) and finally to DCM, are still unclear. Here, we established mouse models of VMC and DCM with Coxsackievirus B3 infection and conducted NMR-based metabolomic analysis of aqueous metabolites extracted from cardiac tissues of three histologically classified groups including aVMC, cVMC and DCM. We showed that these three pathological groups were metabolically distinct from their normal counterparts and identified three impaired metabolic pathways shared by these pathological groups relative to normal controls, including nicotinate and nicotinamide metabolism; alanine, aspartate and glutamate metabolism; and D-glutamine and D-glutamate metabolism. We also identified two extra impaired metabolic pathways in the aVMC group, including glycine, serine and threonine metabolism; and taurine and hypotaurine metabolism Furthermore, we identified potential cardiac biomarkers for metabolically distinguishing these three pathological stages from normal controls. Our results indicate that the metabolomic analysis of cardiac tissues can provide valuable insights into the molecular mechanisms underlying the progression from acute VMC to DCM.

Exploration of dilated cardiomyopathy for biomarkers and immune microenvironment: evidence from RNA-seq

BackgroundThe pathogenic mechanism of dilated cardiomyopathy (DCM) remains to be defined. This study aimed to identify hub genes and immune cells that could serve as potential therapeutic targets for DCM.MethodsWe downloaded four datasets from the Gene Expression Omnibus (GEO) database: GSE141910, GSE3585, GSE42955 and GSE79962. Weighted gene coexpression network analysis (WGCNA) and differential expression analysis were performed to identify gene panels related to DCM. Meanwhile, the CIBERSORT algorithm was used to estimate the immune cells in DCM tissues. Multiple machine learning approaches were used to screen the hub genes and immune cells. Finally, the diagnostic value of the hub genes was assessed by receiver operating characteristic (ROC) analysis. An experimental mouse model of dilated cardiomyopathy was used to validate the bioinformatics results.ResultsFRZB and EXT1 were identified as hub biomarkers, and the ROC curves suggested an excellent diagnostic ability of the above genes for DCM. In addition, naive B cells were upregulated in DCM tissues, while eosinophils, M2 macrophages, and memory CD4 T cells were downregulated in DCM tissues. The increase in two hub genes and naive B cells was validated in animal experiments.ConclusionThese results indicated that FRZB and EXT1 could be used as promising biomarkers, and eosinophils, M2 macrophages, resting memory CD4 T cells and naive B cells may also affect the occurrence of DCM.

Phenotypic screening with deep learning identifies HDAC6 inhibitors as cardioprotective in a BAG3 mouse model of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM. To identify candidate therapeutics, we developed an in vitro DCM model using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) deficient in B-cell lymphoma 2 (BCL2)-associated athanogene 3 (BAG3). With these BAG3-deficient iPSC-CMs, we identified cardioprotective drugs using a phenotypic screen and deep learning. From a library of 5500 bioactive compounds and siRNA validation, we found that inhibiting histone deacetylase 6 (HDAC6) was cardioprotective at the sarcomere level. We translated this finding to a BAG3 cardiomyocyte-knockout (BAG3cKO) mouse model of DCM, showing that inhibiting HDAC6 with two isoform-selective inhibitors (tubastatin A and a novel inhibitor TYA-018) protected heart function. In BAG3cKO and BAG3E455K mice, HDAC6 inhibitors improved left ventricular ejection fraction and reduced left ventricular diameter at diastole and systole. In BAG3cKO mice, TYA-018 protected against sarcomere damage and reduced Nppb expression. Based on integrated transcriptomics and proteomics and mitochondrial function analysis, TYA-018 also enhanced energetics in these mice by increasing expression of targets associated with fatty acid metabolism, protein metabolism, and oxidative phosphorylation. Our results demonstrate the power of combining iPSC-CMs with phenotypic screening and deep learning to accelerate drug discovery, and they support developing novel therapies that address underlying mechanisms associated with heart disease.

NMR-Based Metabolomic Analysis of Sera in Mouse Models of CVB3-Induced Viral Myocarditis and Dilated Cardiomyopathy

Viral myocarditis (VMC) is an inflammatory heart condition which can induce dilated cardiomyopathy (DCM). However, molecular mechanisms underlying the progression of VMC into DCM remain exclusive. Here, we established mouse models of VMC and DCM by infecting male BALB/c mice with Coxsackievirus B3 (CVB3), and performed NMR-based metabonomic analyses of mouse sera. The mouse models covered three pathological stages including: acute VMC (aVMC), chronic VMC (cVMC) and DCM. We recorded 1D 1H-NMR spectra on serum samples and conducted multivariate statistical analysis on the NMR data. We found that metabolic profiles of these three pathological stages were distinct from their normal controls (CON), and identified significant metabolites primarily responsible for the metabolic distinctions. We identified significantly disturbed metabolic pathways in the aVMC, cVMC and DCM stages relative to CON, including: taurine and hypotaurine metabolism; pyruvate metabolism; glycine, serine and threonine metabolism; glycerolipid metabolism. Additionally, we identified potential biomarkers for discriminating a VMC, cVMC and DCM from CON including: taurine, valine and acetate for aVMC; glycerol, valine and leucine for cVMC; citrate, glycine and isoleucine for DCM. This work lays the basis for mechanistically understanding the progression from acute VMC to DCM, and is beneficial to exploitation of potential biomarkers for prognosis and diagnosis of heart diseases.

Comparison of voluntary exercise and renin angiotensin inhibitor administration in a mouse model of dilated cardiomyopathy

Comparison of voluntary exercise and renin angiotensin inhibitor administration in a mouse model of dilated cardiomyopathy

Abstract 11154: Cardioprotective Effects of Mtss1 Reduction Iin Dilated Cardiomyopathy

Introduction: MTSS1 is an I-BAR protein that interacts with the actin cytoskeleton and the cell membrane and is speculated to play a role in cell motility, tumor metastasis, and cardiogenesis. Enhancer variants that reduce the expression of MTSS1 in the human left ventricle have been associated with cardioprotective traits in genome-wide association studies. However, the effect of MTSS1 downregulation on the development of heart disease has not been studied in experimental models. Here we tested the hypothesis that reduction of MTSS1 protects against dilated cardiomyopathy (DCM) in vitro and in vivo . Methods: We used CRISPR-Cas9 to knock out MTSS1 in induced pluripotent stem cells (iPSCs) harboring pathogenic variants from patients with severe DCM. DCM iPSC lines with and without MTSS1 were then differentiated into cardiomyocytes and phenotyped in vitro . A DCM mouse model containing a TPM1 D230N transgene was crossed with a mouse model heterozygous for Mtss1 knockout. Progeny from this F1 cross were assessed for differences in cardiac structure and function via echocardiography. Results: MTSS1 deletion restored the beta-adrenergic response in DCM iPSC cardiomyocytes. In parallel, Mtss1 deletion partially rescued DCM-associated phenotypes including left ventricular dilation and reduced ejection fraction in mice. Conclusions: Our initial results suggest a causal link between MTSS1 reduction and cardioprotection in DCM. Inhibiting cardiac MTSS1 may hold therapeutic potential for DCM, though the cellular mechanisms through which MTSS1 affects cardiac phenotypes remain under investigation.