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The genetic reference population of recombinant inbred BXD mice has been derived from crosses between C57BL/6J and DBA/2J strains. The DBA/2J parent exhibits cardiomyopathy phenotypes, while C57BL/6J has normal heart. BXD mice are sequenced for studying genetic interactions in cardiomyopathies.The study aimed to assess cardiomyopathy traits in BXDs and investigate the quantitative genetic architecture of those traits.Echocardiography, blood pressure, and cardiomyocyte size parameters obtained from 44 strains of BXD family (N5/sex) at 4-5 months of age were associated with heart transcriptomes and expression quantitative trait loci (eQTL) mapping was performed.More than 2-fold variance in ejection fraction (EF%), fractional shortening (FS%), left ventricular volumes (LVVol), internal dimensions (LVID), mass (LVM), and posterior wall (LVPW) thickness was found among BXDs. In male BXDs, eQTL mapping identified Ndrg4 on chromosome 8 QTL to be positively correlated with LVVol and LVID and negatively associated with cardiomyocyte diameter. In female BXDs, significant eQTLs were found on chromosomes 7 and 3 to be associated with LVPW and EF% and FS%, respectively, and Josd2, Dap3 and Tmp3 were predicted as strong candidate genes.Our study found variable cardiovascular traits among BXD strains and identified multiple associated QTLs, suggesting an influence of genetic background on expression of echocardiographic and cardiomyocyte diameter traits. Increased LVVol and reduced EF% and FS% represented dilated cardiomyopathy, whereas increased LV mass and wall thickness indicated hypertrophic cardiomyopathy traits. The BXD family is ideal for identifying candidate genes, causal and modifier, that influence cardiovascular phenotypes.

The transmembrane protein 43 (TMEM43/LUMA) p.S358L mutation causes arrhythmogenic cardiomyopathy named as ARVC5, a fully penetrant disease with high risk of ventricular arrhythmias, sudden death, and heart failure. Male gender and vigorous exercise independently predicted deleterious outcome. Our systems genetics analysis revealed the importance of Tmem43 for cardiac and metabolic pathways associated with elevated lipid absorption from small intestine. This study sought to delineate gender-specific cardiac, intestinal, and metabolic phenotypes in vivo and investigate underlying pathophysiological mechanisms of S358L mutation. Serial echocardiography, surface electrocardiography (ECG), treadmill running, and body EchoMRI have been used in knock-in heterozygous (Tmem43WT/S358L), homozygous (Tmem43S358L), and wildtype (Tmem43WT) littermate mice. Electron microscopy, histology, immunohistochemistry, transcriptome, and protein analysis have been performed in cardiac and intestinal tissues. Systolic dysfunction was apparent in 3-mo-old Tmem43S358L and 6-mo-old Tmem43WT/S358L mutants. Both mutant lines displayed intolerance to acute stress at 6 mo of age, arrhythmias, fibro-fatty infiltration, and subcellular abnormalities in the myocardium. Microarray analysis found significantly differentially expressed genes between left ventricular (LV) and right ventricular (RV) myocardium. Mutants displayed diminished PPARG activities and significantly reduced TMEM43 and β-catenin expression in the heart, whereas junctional plakoglobin (JUP) translocated into nuclei of mutant cardiomyocytes. Conversely, elongated villi, fatty infiltration, and overexpression of gut epithelial proliferation markers, β-catenin and Ki-67, were evident in small intestine of mutants. We defined Tmem43 S358L-induced pathological effects on cardiac and intestinal homeostasis via distinctly disturbed WNT-β-catenin and PPARG signaling thereby contributing to ARVC5 pathophysiology. Results suggest that cardiometabolic assessment in mutation carriers may be important for predictive and personalized care.NEW & NOTEWORTHY This manuscript describes the findings of our investigation of cardiac, small intestine, and metabolic features of Tmem43-S358L mouse model. By investigating interorgan pathologies, we uncovered multiple mechanisms of the S358L-induced disease, and these unique mechanisms likely appear to contribute to the disease pathogenesis. We hope our findings are important and novel and open new avenues in the hunting for additional diagnostic and therapeutic targets in subjects carrying TMEM43 mutation.

Introduction: Heart rhythm disorders or cardiac arrhythmias can cause sudden death and heart failure. Risk factors for cardiac arrhythmias vary including genetics, age, and other environmental and lifestyle factors. To sought how genetic background affects an expression of cardiac rhythm phenotypes, we assessed electrocardiography (ECG) traits in BXD family of mice derived from crosses between DBA/2J (D2) and C57BL/6J (B6) strains and explored the quantitative genetic architecture of those traits. Methods: ECG tracings were recorded in 44 BXD male (M) and female (F) strains (N >5 mice/sex) at 4-5 months of age anesthetized with 2% isoflurane. Heart rate (HR), ECG parameters, and frequency of arrhythmias in percentile (‰) were associated with blood pressure, echocardiography, and heart transcriptome values followed by quantitative trait loci (QTLs) mapping. Results: Parental D2 strains had the lowest HR and significantly prolonged QT intervals (62.82 ± 9.42 ms in D2F and 53.42 ± 1.73 ms in D2M) compared to sex matched B6 parental strains. Significantly widened QRS complexes were seen in BXD65F (14.27±2.97 ms) compared to 11.67±1.03 ms in B6F controls. In males, BXD83M had the widest QRS (13.48±1.74 ms) vs 10.89±0.41 ms seen in B6M controls. We found a significant association between QRS duration and increased left ventricular internal diameter (LVID) and reduced ejection fraction and fractional shortening. Further, varied cardiac arrhythmias including bradycardia, atrioventricular block (AVB), premature atrial or ventricular complexes (PAC and PVC, respectively), ventricular tachycardia (VT) and sick sinus syndrome (SSS) were seen among BXD strains. PACs were recorded in BXD73F (14.57 ‰), 40M, 101F, 51M, and 101M strains. Strains, BXD79M (14.97 ‰), 83M, 78M, 69F, and 171F, had frequent PVCs compared to B6 controls. AVB II was recorded in BXD48M and BXD66M had SSS. Both PAC and PVC were positively associated with cardiac phenotype burden. Specifically, PVCs were significantly correlated with echocardiographic pulmonary vein peak pressure, LVID and volumes at end-diastole (P = 0.04) among male BXDs. In females, right ventricular internal diameters (RVID) and cardiac output were significantly correlated with PVC and PAC frequencies. Moreover, PVCs were significantly correlated with systolic blood pressure in both male and female mice. QTL mapping identified a significant locus on Chromosome 3 associated with QTC and JT durations, while the same locus was suggestive for association with QT interval, suggesting that Chromosome 3 loci may be associated with repolarization abnormalities such as long QT syndromes. Conclusions: ECG parameters, heart rate, type and frequency of arrhythmias significantly varied between mouse strains of the BXD family suggesting an influence of genetic background on expression of those traits. Abnormal heart rhythms detected in BXD strains mimic cardiac rhythm and conduction disorders in humans. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder of desmosomal dysfunction, and PKP2 (plakophilin-2) has been reported to be the most common disease-causing gene when mutation-positive. In the early concealed phase, the ACM heart is at high risk of sudden cardiac death before cardiac remodeling occurs because of mistargeted ion channels and altered Ca 2+ handling. However, the results of pathogenic PKP2 variants on myocyte contraction in ACM pathogenesis remain unknown. Methods: We studied the outcomes of a human truncating variant of PKP2 on myocyte contraction using a novel knock-in mouse model with insertion of thymidine in exon 5 of Pkp2 , which mimics a familial case of ACM (PKP2-L404fsX5). We used serial echocardiography, electrocardiography, blood pressure measurements, histology, cardiomyocyte contraction, intracellular calcium measurements, and gene and protein expression studies. Results: Serial echocardiography of Pkp2 heterozygous (Pkp2-Het) mice revealed progressive failure of the right ventricle (RV) in animals older than 3 months. By contrast, left ventricular function remained normal. ECGs of 6-month-old anesthetized Pkp2-Het mice showed normal baseline heart rates and QRS complexes. Cardiac responses to β-adrenergic agonist isoproterenol (2 mg/kg) plus caffeine (120 mg/kg) were also normal. However, adrenergic stimulation enhanced the susceptibility of Pkp2-Het hearts to tachyarrhythmia and sudden cardiac death. Histological staining showed no significant fibrosis or adipocyte infiltration in the RVs and left ventricles of 6- and 12-month-old Pkp2-Het hearts. Contractility assessment of isolated myocytes demonstrated progressively reduced Pkp2-Het RV cardiomyocyte function consistent with RV failure measured by echocardiography. However, aging Pkp2-Het and control RV myocytes loaded with intracellular Ca 2+ indicator Fura-2 showed comparable Ca 2+ transients. Western blotting of Pkp2-RV homogenates revealed a 40% decrease in actin, whereas actin immunoprecipitation followed by a 2,4-dinitrophenylhydrazine staining showed doubled oxidation level. This correlated with a 39% increase in troponin-I phosphorylation. In contrast, Pkp2-Het left ventricular myocytes had normal contraction, actin expression and oxidation, and troponin-I phosphorylation. Last, Western blotting of cardiac biopsies revealed that actin expression was 40% decreased in RVs of patients with end-stage ACM. Conclusions: During the early concealed phase of ACM, reduced actin expression drives loss of RV myocyte contraction, contributing to progressive RV dysfunction.

Most prominent functional abnormalities seen in the failing human heart are impaired contraction and slowed rates of relaxation of cardiac cells in the face of increased neurohormonal activation, sustained inflammation, mechanical and volume overload, and progressive maladaptive remodeling of the myocardium. Mechanical circulatory support devices (MCS) improve cardiac function and outcomes of patients with end-stage heart failure, allowing to bridge to heart transplantation and permitting the removal of MCS device as a bridge to recovery, in some patients with the sufficient recovery of heart function. Numerous reports have demonstrated favorable myocardial recovery and reverse remodeling after prolonged ventricular unloading by MCS. Ventricular unloading by MCS leads to a decreased concentration of peripheral natriuretic peptides in plasma, reduction in cardiac cytokines, kinases, collagens, and proteins involved in hypertrophy, fibrosis, programmed cell death, and necrosis in the heart. This chapter will summarize and review the effects and underlying mechanisms of myocardial remodeling during prolonged MCS in patients with end-stage heart failure. The mechanisms of myocardial recovery are multifactorial and remain to be further explored on cellular, organ, and systems levels.

Background Understanding the overall variant burden in pediatric patients with left ventricular noncompaction (LVNC) has clinical implications. Whole exome sequencing (WES) allows detection of coding variants in both candidate cardiomyopathy genes and those included on commercial panels. Other lines of evidence, including in silico analysis, are necessary to reduce the overwhelming number of variants to those most likely having a phenotypic impact. Methods Five families, including five pediatric probands with LVNC, 5 other affected, and 10 unaffected family members, had WES performed, followed by bioinformatics filtering and Sanger sequencing. Review of the HGMD, variant classification by ACMG guidelines, and clinical information were used to further refine complex genotypes. Results One nonsense and eleven missense variants were identified. In Family 1, affected siblings carried digenic heterozygous variants: E1350K-MYH7 and A276V-ANKRD1. The proband also carried heterozygous W143X-NRG1. Four affected members of Family 2 carried K184Q-MYH7 while unaffected members did not. In Family 3, homozygous A161T-MYH7 and heterozygous P4935T-OBSCN variants were identified in the proband with the latter being absent in his unaffected brother. In Family 4, proband's father and half-sibling have mild hypertrabeculation and carry T3796I-PLEC. The proband, carrying T3796I-PLEC and V2878A-OBSCN, demonstrated higher trabeculation burden. The proband in Family 5 carried four variants, R3247W-PLEC, C92Y-ERG, T1233M-NCOR2, and E54K-HIST1H4B. Application of ACMG criteria and clinical data revealed that W143X-NRG1, P4935T-OBSCN, and V2878A-OBSCN likely have no phenotypic role. Conclusions We report nine variants, including novel T3796I-PLEC and biallelic A161T-MYH7, likely contributing to phenotypes ranging from asymptomatic hypertrabeculation to severe LVNC with heart failure.

Broad cellular functions and diseases including muscular dystrophy, arrhythmogenic right ventricular cardiomyopathy (ARVC5) and cancer are associated with transmembrane protein43 (TMEM43/ LUMA). The study aimed to investigate biological roles of TMEM43 through genetic regulation, gene pathways and gene networks, candidate interacting genes, and up- or downstream regulators. Cardiac transcriptomes from 40 strains of recombinant inbred BXD mice and two parental strains representing murine genetic reference population (GRP) were applied for genetic correlation, functional enrichment, and coexpression network analysis using systems genetics approach. The results were validated in a newly created knock-in Tmem43-S358L mutation mouse model (Tmem43S358L) that displayed signs of cardiac dysfunction, resembling ARVC5 phenotype seen in humans. We found high Tmem43 levels among BXDs with broad variability in expression. Expression of Tmem43 highly negatively correlated with heart mass and heart rate among BXDs, whereas levels of Tmem43 highly positively correlated with plasma high-density lipoproteins (HDL). Through finding differentially expressed genes (DEGs) between Tmem43S358L mutant and wild-type (Tmem43WT) lines, 18 pathways (out of 42 found in BXDs GRP) that are involved in ARVC, hypertrophic cardiomyopathy, dilated cardiomyopathy, nonalcoholic fatty liver disease, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease were verified. We further constructed Tmem43-mediated gene network, in which Ctnna1, Adcy6, Gnas, Ndufs6, and Uqcrc2 were significantly altered in Tmem43S358L mice versus Tmem43WT controls. Our study defined the importance of Tmem43 for cardiac- and metabolism-related pathways, suggesting that cardiovascular disease-relevant risk factors may also increase risk of metabolic and neurodegenerative diseases via TMEM43-mediated pathways.

Visual Abstract, Highlights • AM is an important cause of cardiac dysfunction following some viral infections and has been previously linked with genetic host susceptibilities. • Sequencing data for genes related to cardiomyocyte structure and function was compared between patients with AM and sex- and ancestry-matched heart-healthy control subjects. • Putatively DVs among these genes were found in ∼16% of AM cases, contributing to viral susceptibility. • Implicated genes are ones classically associated with cardiomyopathy or neuromuscular disorders with cardiac involvement, suggesting shared mechanistic pathways. • Phenotypes, including outcomes, were similar in subjects who have AM with and without DV, therefore genetic testing will be necessary to uncover this subtype and inform risk in relatives., Summary Impairments in certain cardiac genes confer risk for myocarditis in children. To determine the extent of this association, we performed genomic sequencing in predominantly adult patients with acute myocarditis and matched control subjects. Putatively deleterious variants in a broad set of cardiac genes were found in 19 of 117 acute myocarditis cases vs 34 of 468 control subjects (P = 0.003). Thirteen genes classically associated with cardiomyopathy or neuromuscular disorders with cardiac involvement were implicated, including >1 associated damaging variant in DYSF, DSP, and TTN. Phenotypes of subjects who have acute myocarditis with or without deleterious variants were similar, indicating that genetic testing is necessary to differentiate them.

Cardiomyopathy or disease of the heart muscle involves abnormal enlargement and a thickened, stiff, or spongy-like appearance of the myocardium. As a result, the function of the myocardium is weakened and does not sufficiently pump blood throughout the body nor maintain a normal pumping rhythm, leading to heart failure. The main types of cardiomyopathies include dilated hypertrophic, restrictive, arrhythmogenic, and noncompaction cardiomyopathy. Abnormal trabeculations of the myocardium in the left ventricle are classified as left ventricular noncompaction cardiomyopathy (LVNC). Myocardial noncompaction most frequently is observed at the apex of the left ventricle and can be associated with chamber dilation or muscle hypertrophy, systolic or diastolic dysfunction, or both, or various forms of congenital heart disease. Animal models are incredibly important for uncovering the etiology and pathogenesis involved in this disease. This chapter will describe the clinical and pathological features of LVNC in humans and present the animal models that have been used for the study of the genetic basis and pathogenesis of this disease.

Studies showed that the gastrointestinal (GI) tract is one of the most important pathways for SARS-CoV-2 infection and coronavirus disease 2019 (COVID-19). As SARS-CoV-2 cellular entry depends on the ACE2 receptor and TMPRSS2 priming of the spike protein, it is important to understand the molecular mechanisms through which these two proteins and their cognate transcripts interact and influence the pathogenesis of COVID-19. In this study, we quantified the expression, associations, genetic modulators, and molecular pathways for Tmprss2 and Ace2 mRNA expressions in GI tissues using a systems genetics approach and the expanded family of highly diverse BXD mouse strains. The results showed that both Tmprss2 and Ace2 are highly expressed in GI tissues with significant covariation. We identified a significant expression quantitative trait locus on chromosome 7 that controls the expression of both Tmprss2 and Ace2. Dhx32 was found to be the strongest candidate in this interval. Co-expression network analysis demonstrated that both Tmprss2 and Ace2 were located at the same module that is significantly associated with other GI-related traits. Protein–protein interaction analysis indicated that hub genes in this module are linked to circadian rhythms. Collectively, our data suggested that genes with circadian rhythms of expression may have an impact on COVID-19 disease, with implications related to the timing and treatment of COVID-19.

Restrictive cardiomyopathy (RCM), a potentially devastating heart muscle disorder, is characterized by diastolic dysfunction due to abnormal muscle relaxation and myocardial stiffness resulting in restrictive filling of the ventricles. Diastolic dysfunction is often accompanied by left atrial or bi-atrial enlargement and normal ventricular size and systolic function. RCM is the rarest form of cardiomyopathy, accounting for 2-5% of pediatric cardiomyopathy cases, however, survival rates have been reported to be 82%, 80%, and 68% at 1-, 2-, and 5-years after diagnosis, respectively. RCM can be idiopathic, familial, or secondary to a systemic disorder, such as amyloidosis, sarcoidosis, and hereditary hemochromatosis. Approximately 30% of cases are familial RCM, and the genes that have been linked to RCM are

The sarcomeres represent the essential contractile units of the cardiac myocyte and are bordered by two Z-lines (disks) that are made by various proteins. The cardiac Z-disk is recognized as one of the nodal points in cardiomyocyte structural organization, mechano-sensation and signal transduction. Rapid progress in molecular and cellular biology has significantly improved the knowledge about pathogenic mechanisms and signaling pathways involved in the development of inherited cardiomyopathies. Genetic insult resulting in expression of mutated proteins that maintain the structure of the heart can perturb cardiac function. The primary mutation in the cardiac contractile apparatus or other subcellular complexes can lead to cardiac pathology on a tissue level, resulting in organ and organism level pathophysiology. The “final common pathway” hypothesis interpreting the genetic basis and molecular mechanisms involved in the development of cardiomyopathies suggests that mutations in cardiac genes encoding proteins with similar structure, function, or location and operating in the same pathway, are responsible for a particular phenotype of cardiomyopathy with unique morpho-histological remodeling of the heart. This chapter will describe genetic abnormalities of cardiac Z-disk and related “final common pathways” that are triggered by a Z-disk genetic insult leading to heart muscle diseases. In addition, animal models carrying mutations in Z-disk proteins will be described.

The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain are known to induce cardiomyopathy. The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. We ablated exon3 encoding the first NR of Nebl and created global Nebl(ex3-/ex3-) knockout mice. Cardiac function, structure, and transcriptome were assessed before and after a 4-wk treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Nebl(ex3-/ex3-) mice demonstrated diastolic dysfunction with preserved systolic function at 6 mo of age. After treadmill running, 4-mo-old Nebl(ex3-/ex3-) mice developed concentric cardiac hypertrophy and left ventricular dilation compared with running Nebl(+/+) and sedentary Nebl(ex3-/ex3-) mice. Disturbance of sarcomeric Z-disks and thin filaments architecture and disruption of intercalated disks and mitochondria were found in exercised Nebl(ex3-/ex3-) mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, β-catenin, MLP, α-actinin2, and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-h poststrain and redistributed along with F-actin at 72-h poststrain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. We conclude that Nebl's NR ablation causes disturbed sarcomere, Z-disks, and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to biomechanical stress. NEW & NOTEWORTHY We demonstrate that ablation of first nebulin-repeats of sarcomeric nebulette (Nebl) causes diastolic dysfunction in Nebl(ex3-/ex3-) mice. Exercise-induced development of diastolic dysfunction, cardiac hypertrophy and ventricular dilation in knockouts. This was associated with sarcomere disturbance, intercalated disks disruption, and mitochondrial distortion upon stress and altered expression of genes involved in Nebl-based stress network. We demonstrate that G202R and A592 mutations alter actin and desmin expression causing disorganization of desmin filaments upon cyclic strain.

Background: A high inflammatory and cytokine burden that induces vascular inflammation, myocarditis, cardiac arrhythmias, and myocardial injury is associated with a lethal outcome in COVID-19. The SARS-CoV-2 virus utilizes the ACE2 receptor for cell entry in a similar way to SARS-CoV. This study investigates the regulation, gene network, and associated pathways of ACE2 that may be involved in inflammatory and cardiovascular complications of COVID-19.Methods: Cardiovascular traits were determined in the one of the largest mouse genetic reference populations: BXD recombinant inbred strains using blood pressure, electrocardiography, and echocardiography measurements. Expression quantitative trait locus (eQTL) mapping, genetic correlation, and functional enrichment analysis were used to identify Ace2 regulation, gene pathway, and co-expression networks.Results: A wide range of variation was found in expression of Ace2 among the BXD strains. Levels of Ace2 expression are negatively correlated with cardiovascular traits, including systolic and diastolic blood pressure and P wave duration and amplitude. Ace2 co-expressed genes are significantly involved in cardiac- and inflammatory-related pathways. The eQTL mapping revealed that Cyld is a candidate upstream regulator for Ace2. Moreover, the protein–protein interaction (PPI) network analysis inferred several potential key regulators (Cul3, Atf2, Vcp, Jun, Ppp1cc, Npm1, Mapk8, Set, Dlg1, Mapk14, and Hspa1b) for Ace2 co-expressed genes in the heart.Conclusions:Ace2 is associated with blood pressure, atrial morphology, and sinoatrial conduction in BXD mice. Ace2 co-varies with Atf2, Cyld, Jun, Mapk8, and Mapk14 and is enriched in the RAS, TGFβ, TNFα, and p38α signaling pathways, involved in inflammation and cardiac damage. We suggest that all these novel Ace2-associated genes and pathways may be targeted for preventive, diagnostic, and therapeutic purposes in cardiovascular damage in patients with systemic inflammation, including COVID-19 patients.

Objectives: This study sought to delineate clinical phenotype and investigate pathophysiological mechanisms of arrhythmogenic right ventricular cardiomyopathy (ARVC5) in vivo.Background: Mutation p.S358L in TMEM43/LUMA causes ARVC5, a fully penetrant gender- and exercise-dependent disease. Exact pathophysiological mechanisms of ARVC5 remain incomplete and controversial. Methods: Knock-in Tmem43S358L mice were generated using conventional targeting. Assessment of cardiac and intestinal phenotypes, transcriptome and protein analysis were performed. Results: Onset of systolic dysfunction and rhythm asymmetry was apparent in 3-month-old homozygote mutants. Heterozygote 6-month-old mutants displayed systolic dysfunction as well. Biventricular myocardial fibro-fatty infiltration and subcellular abnormalities were found in both mutants, while acute stress caused ventricular tachycardia and death in homozygotes. Microarray revealed that WNT-b-catenin and PPARG signaling-associated genes were differentially expressed between LV and RV. Tmem43S358L mutants displayed significantly reduced PPARG activities and downregulation of PPARG-downstream metabolic genes in the heart. Expression of b-catenin were significantly reduced, while JUP was translocated into nuclei of mutant cardiomyocytes. Conversely in small intestine, b-catenin and PPARG-associated genes responsible for cholesterol, bile acid, and lipid transport were upregulated in Tmem43S358L mutants resulting in elevated lipids absorption, hyperlipidemia, and reciprocal fecal lipids reduction. These abnormalities were associated with elongated villi, fatty infiltrations, and gut epithelial proliferation markers in small intestine. Conclusions: We defined novel distinct effects of the TMEM43-S358L mutation on small intestine homeostasis in addition to cardiac damage. Tmem43-S358L perturbs PPARG and WNT-b-catenin signaling in the heart as well as in intestine, elevating lipids absorption from gut lumen whereby triggering ACM pathophysiology.

Nemaline myopathy (NM) is a common form of congenital nondystrophic skeletal muscle disease characterized by muscular weakness of proximal dominance, hypotonia, and respiratory insufficiency but typically not cardiac dysfunction. Wide variation in severity has been reported. Intranuclear rod myopathy is a subtype of NM in which rod-like bodies are seen in the nucleus, and it often manifests as a severe phenotype. Although ten mutant genes are currently known to be associated with NM, only ACTA1 is associated with intranuclear rod myopathy. In addition, the genetic cause remains unclear in approximately 25%-30% of individuals with NM. We performed whole-exome sequencing on individuals with histologically confirmed but genetically unsolved NM. Our study included individuals with milder, later-onset NM and identified biallelic loss-of-function mutations in myopalladin (MYPN) in four families. Encoded MYPN is a sarcomeric protein exclusively localized in striated muscle in humans. Individuals with identified MYPN mutations in all four of these families have relatively mild, childhood- to adult-onset NM with slowly progressive muscle weakness. Walking difficulties were recognized around their forties. Decreased respiratory function, cardiac involvement, and intranuclear rods in biopsied muscle were observed in two individuals. MYPN was localized at the Z-line in control skeletal muscles but was absent from affected individuals. Homozygous knockin mice with a nonsense mutation in Mypn showed Z-streaming and nemaline-like bodies adjacent to a disorganized Z-line on electron microscopy, recapitulating the disease. Our results suggest that MYPN screening should be considered in individuals with mild NM, especially when cardiac problems or intranuclear rods are present.

Background: Arrhythmogenic cardiomyopathies (ACMs) are heritable diseases characterized by arrhythmias, related sudden death, fibro-fatty infiltration and progressive heart failure. Although the pathogenic variant p.S358L in TMEM43/ LUMA causes fully penetrant ACM (ARVC5) in patients, little in vivo evidence has been reported and the roles of Tmem43 in normal cardiac function and disease remain obscure and controversial. This study explored Tmem43-associated genetic modifiers using forward, reverse, and systems genetics analyses in vivo . Methods: Microarray of cardiac and jejunal tissues were performed in a newly created Tmem43 S358L knock-in mouse and in forty BXD strains, used as a murine genetic reference population (GRP). Levels of plasma and fecal lipids and Pparγ activities were defined in heart and jejunum of Tmem43 animals. Immunohistochemistry was performed in the myocardium of patients with non-TMEM43 origin ACM. Results: BXD strain cardiac tissues demonstrated high levels of Tmem43 expression that was significantly negatively correlated with heart mass and heart rate, while positively correlated with plasma HDL and LDL levels. Expression of Tmem43 was also significantly (r>0.5, pPpargc1a ( Pcg1a ) in the heart and intestine of BXDs and Tmem43 mutants. In the Tmem43 S358L mouse heart, Tmem43 and Ppargc1a were downregulated resulting in reduced Pparγ activities. Conversely, Tmem43 and Ppars- regulated genes, including Mogat2, responsible for cholesterol, bile acid, and lipid absorption and re-esterification, were upregulated in the jejunum, resulting in elevated lipid absorption in the gut lumen and hyperlipidemia in Tmem43 S358L mutants. Expression of TMEM43 was disrupted in cardiomyocyte intercalated disks in ACM patients’ myocardium in contrast to normal controls. Conclusions: Tmem43 is an essential gene for cardiac and small intestine signaling and function. The S358L-Tmem43 pathogenic variant is significantly associated with downregulation of Tmem43 , Ppagc1a and Ppar γ in the myocardium and upregulation of Pparγ and Pparα signaling in small intestine in vivo . Testing plasma lipid levels and TMEM43 expression in ACM patients may provide critical information for personalized predictive care.

Introduction: Pediatric and adult dilated cardiomyopathy (DCM) represent distinct pathological entities, however, the basis of this phenomena remains obscure. The pathogenic variant p.K69R in MLP/ CSRP3 induced DCM in adult Mlp K69R knock-in mice. A noncompacted ventricular wall and dilated ventricles were seen in embryonic and neonatal Mlp K69R hearts, reminiscent of that seen in infant variant carriers. Hypothesis: Mlp- K69R associated genetic modifiers govern DCM phenotypes in neonatal and adult heart. The study was to find genetic modifiers associated with divergent neonatal and adult phenotypes induced by Mlp- K69R. Methods: RNA extracted from neonatal and adult Mlp K69R and Mlp WT mouse myocardium was applied to Affymetrix microarray and QPCR analysis. Data were normalized, subjected to differentially expression genes and gene set enrichment analysis. Results: We found 1024 genes in adult and 252 genes in neonatal heart that were significantly altered in Mlp K69R vs WT littermates. 23 of these genes were mutually altered in adult and neonatal mutants. In adult mutants, 15 sarcomeric genes were significantly over-expressed compared to WT littermates. The mutation altered expression of genes involved in sarcolemmal and desmosomes unity. Calcium handling, integrin-associated networks, and heart failure-associated genes were upregulated in mutants vs WT littermates. In neonates, genes involved in cardiac and vascular development ( Rhox2c, Tbx3, Mir23a, Tbc1d7 ), cell adhesion and migration ( Tff2 ), and apoptosis ( Mir297a-2 ) were significantly altered. Two cardio-protective miRNAs, Mir98 and Mir451a , were highly reciprocally altered in both, adult and neonatal, mutant vs WT hearts. Their expression was increased in mutant adult hearts, but decreased in neonatal mutant hearts, suggesting novel candidates associated with K69R-MLP-induced DCM and noncompaction phenotypes. Conclusions: Differential transcriptomes in neonatal and adult hearts suggest that pathogenic K69R-MLP variant induces DCM and noncompaction phenotypes via distinct underlying genetic modifiers. Contrary to mutant adult mice, mutant newborn hearts show significant decrease in Mir98 and Mir451a, which may predispose them to developing myocardial noncompaction.

Background Left ventricular non-compaction (LVNC) is a heritable cardiomyopathy characterized by hypertrabeculation, inter-trabecular recesses and thin compact myocardium, but the genetic basis and mechanisms remain unclear. This study identified novel LVNC-associated mutations inNOTCH-dependent genes and investigated their mutational effects.Methods and Results:High-resolution melting screening was performed in 230 individuals with LVNC, followed by whole exome and Sanger sequencing of available family members. Dimerization of bone morphogenetic protein 10 (BMP10) and its binding to BMP receptors (BMPRs) were evaluated. Cellular differentiation, proliferation and tolerance to mechanical stretch were assessed in H9C2 cardiomyoblasts, expressing wild-type (WT) or mutant BMP10 delivered by adenoviral vectors. Rare variants, p.W143*-NRG1and p.V407I-BMP10, were identified in 2 unrelated probands and their affected family members. Although dimerization of mutant V407I-BMP10 was preserved like WT-BMP10, V407I-BMP10 pulled BMPR1a and BMPR2 receptors more weakly compared with WT-BMP10. On comparative gene expression and siRNA analysis, expressed BMPR1a and BMPR2 receptors were responsive to BMP10 treatment in H9C2 cardiomyoblasts. Expression of V407I-BMP10 resulted in a significantly lower rate of proliferation in H9C2 cells compared with WT-BMP10. Cyclic stretch resulted in destruction and death of V407I-BMP10 cells. Conclusions The W143*-NRG1and V470I-BMP10variants are associated with LVNC. Impaired BMPR-binding ability, perturbed proliferation and differentiation processes and intolerance to stretch in V407I-BMP10 mutant cardiomyoblasts may underlie myocardial non-compaction.

Introduction: Causal and modifier genes associated with left ventricular noncompaction (LVNC) often occurring in conjunction with other familial cardiomyopathies remain elusive. Hypothesis: The LVNC-associated di- and multigenic abnormalities can be identified by whole exome sequencing (WES). Methods: Five families with a history of LVNC, including five affected probands, three affected family members, and twelve unaffected relatives, were studied. Genomic DNA was extracted from whole blood samples followed by WES and Sanger sequencing to confirm possibly pathogenic variants predicted by in-silico analysis. Phenotype-genotype correlation and quantitative co-segregation studies are performed. Results: We identified nine missense possibly pathogenic variants, a 2-bp frameshift insertion, and a 9-bp in-frame insertion in the five families. Two affected siblings in Family 1 were found carrying digenic heterozygous variants: c.4048G>A (p.E1350K) in MYH7 and c.827C>T (p.A276V) in ANKRD1. Unaffected parents were carriers for each of the two variants. Three affected members, father and two daughters, of Family 2 carried c.550A>C (p.K184Q) variant in MYH7 in contrast to two unaffected members, mother and another daughter. In Family 3, multigenic heterozygosity (c.673G>T (p.D225Y) in CACNA2D1 ; c.440T>A (p.V147E) in COQ4 and c.3700C>A (p.H1234N) in MYH7) was identified in the proband. These variants were found in none of three unaffected relatives. The proband of Family 4 was positive for heterozygous variants: c.2684_2685insAG (p.A897Kfs*3) in DSC2 , c.8633T>C (p.V2878A) in OBSCN , and c.11717C >T (p.T3906I) in PLEC. The T3906I PLEC variant was identified in his unaffected half-sibling and his father, but not in his mother. In Family 5, c. 2591A>T (p.D864V) in HDAC9 , c.9616C>T (p.R3206W) in PLEC and c.954_955insT (p.L319Sfs*74) in MYH14 were identified in the proband. None of those variants were identified in his unaffected sibling. Conclusions: We report several potential pathogenic LVNC-associated variants in novel genes (ANKRD1, DSC2, OBSCN , PLEC, HDAC9, MYH14, COQ4, CACNA2D1) and known genes ( MYH7 and MYH7B). The diverse profile of inheritance (digenic and multigenic heterogeneity) that may cause and modify the heterogeneous LVNC phenotypes.

Phosphorylation and methylation of desmoplakin are required for proper junction assembly and adhesion strengthening, and inhibition of these modifications might contribute to skin and heart diseases., Intermediate filament (IF) attachment to intercellular junctions is required for skin and heart integrity, but how the strength and dynamics of this attachment are modulated during normal and pathological remodeling is poorly understood. We show that glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT-1) cooperate to orchestrate a series of posttranslational modifications on the IF-anchoring protein desmoplakin (DP) that play an essential role in coordinating cytoskeletal dynamics and cellular adhesion. Front-end electron transfer dissociation mass spectrometry analyses of DP revealed six novel serine phosphorylation sites dependent on GSK3 signaling and four novel arginine methylation sites including R2834, the mutation of which has been associated with arrhythmogenic cardiomyopathy (AC). Inhibition of GSK3 or PRMT-1 or overexpression of the AC-associated mutant R2834H enhanced DP–IF associations and delayed junction assembly. R2834H blocked the GSK3 phosphorylation cascade and reduced DP–GSK3 interactions in cultured keratinocytes and in the hearts of transgenic R2834H DP mice. Interference with this regulatory machinery may contribute to skin and heart diseases.

BackgroundFamilial restrictive cardiomyopathy (FRCM) has a poor prognosis due to diastolic dysfunction and restrictive physiology (RP). Myocardial stiffness, with or without fibrosis, underlie RP, but the mechanism(s) of restrictive remodeling is unclear. Myopalladin (MYPN) is a messenger molecule that links structural and gene regulatory molecules via translocation from the Z-disk and I-bands to the nucleus in cardiomyocytes. Expression of N-terminal MYPN peptide results in severe disruption of the sarcomere.ObjectivesThe aim was to study a nonsense MYPN-Q529X mutation previously identified in the FRCM family in an animal model to explore the molecular and pathogenic mechanisms of FRCM.MethodsFunctional (echocardiography, cardiac magnetic resonance [CMR] imaging, electrocardiography), morphohistological, gene expression, and molecular studies were performed in knock-in heterozygote (MypnWT/Q526X) and homozygote mice harboring the human MYPN-Q529X mutation.ResultsEchocardiographic and CMR imaging signs of diastolic dysfunction with preserved systolic function were identified in 12-week-old MypnWT/Q526X mice. Histology revealed interstitial and perivascular fibrosis without overt hypertrophic remodeling. Truncated MypnQ526X protein was found to translocate to the nucleus. Levels of total and nuclear cardiac ankyrin repeat protein (Carp/Ankrd1) and phosphorylation of mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 (Erk1/2), Erk1/2, Smad2, and Akt were reduced. Up-regulation was evident for muscle LIM protein (Mlp), desmin, and heart failure (natriuretic peptide A [Nppa], Nppb, and myosin heavy chain 6) and fibrosis (transforming growth factor beta 1, alpha–smooth muscle actin, osteopontin, and periostin) markers.ConclusionsHeterozygote MypnWT/Q526X knock-in mice develop RCM due to persistence of mutant MypnQ526X protein in the nucleus. Down-regulation of Carp and up-regulation of Mlp and desmin appear to augment fibrotic restrictive remodeling, and reduced Erk1/2 levels blunt a hypertrophic response in MypnWT/Q526X hearts.

Pediatric dilated cardiomyopathy (DCM) is the most common indication for heart transplantation in children. Despite similar genetic etiologies, medications routinely used in adult heart failure patients do not improve outcomes in the pediatric population. The mechanistic basis for these observations is unknown. We hypothesized that pediatric and adult DCM comprise distinct pathological entities, in that children do not undergo adverse remodeling, the target of adult heart failure therapies. To test this hypothesis, we examined LV specimens obtained from pediatric and adult donor controls and DCM patients. Consistent with the established pathophysiology of adult heart failure, adults with DCM displayed marked cardiomyocyte hypertrophy and myocardial fibrosis compared with donor controls. In contrast, pediatric DCM specimens demonstrated minimal cardiomyocyte hypertrophy and myocardial fibrosis compared with both age-matched controls and adults with DCM. Strikingly, RNA sequencing uncovered divergent gene expression profiles in pediatric and adult patients, including enrichment of transcripts associated with adverse remodeling and innate immune activation in adult DCM specimens. Collectively, these findings reveal that pediatric and adult DCM represent distinct pathological entities, provide a mechanistic basis to explain why children fail to respond to adult heart failure therapies, and suggest the need to develop new approaches for pediatric DCM.

Cardiopulmonary complications are the leading cause of mortality in sickle cell anemia (SCA). Elevated tricuspid regurgitant jet velocity, pulmonary hypertension, diastolic, and autonomic dysfunction have all been described, but a unifying pathophysiology and mechanism explaining the poor prognosis and propensity to sudden death has been elusive. Herein, SCA mice underwent a longitudinal comprehensive cardiac analysis, combining state-of-the-art cardiac imaging with electrocardiography, histopathology, and molecular analysis to determine the basis of cardiac dysfunction. We show that in SCA mice, anemia-induced hyperdynamic physiology was gradually superimposed with restrictive physiology, characterized by progressive left atrial enlargement and diastolic dysfunction with preserved systolic function. This phenomenon was absent in WT mice with experimentally induced chronic anemia of similar degree and duration. Restrictive physiology was associated with microscopic cardiomyocyte loss and secondary fibrosis detectable as increased extracellular volume by cardiac-MRI. Ultrastructural mitochondrial changes were consistent with severe chronic hypoxia/ischemia and sarcomere diastolic-length was shortened. Transcriptome analysis revealed up-regulation of genes involving angiogenesis, extracellular-matrix, circadian-rhythm, oxidative stress, and hypoxia, whereas ion-channel transport and cardiac conduction were down-regulated. Indeed, progressive corrected QT prolongation, arrhythmias, and ischemic changes were noted in SCA mice before sudden death. Sudden cardiac death is common in humans with restrictive cardiomyopathies and long QT syndromes. Our findings may thus provide a unifying cardiac pathophysiology that explains the reported cardiac abnormalities and sudden death seen in humans with SCA.

Cardiomyopathies are a heterogeneous group of disorders of heart muscle that ultimately result in congestive heart failure (CHF), sudden cardiac death, and/or arrhythmias. Contraction of heart muscle is the prime force for efficient pumping of the blood during systole, and relaxation is main force that collects a sufficient volume of blood during diastole, preparing ventricles for next efficient contraction. Efficient contraction and relaxation is maintained by cadre of intracellular and intercellular molecular signaling though paracrine communication. However, persistent unfavorable imbalance of these molecular pathways results in systolic and/or diastolic dysfunction leading to failure of the heart to maintain hemodynamic balance. Rapid progress in genetics as well as in molecular and cellular biology over the past 25 years has greatly improved the understanding of pathogenic signaling pathways in inherited cardiomyopathies. This chapter will focus on intracellular and intercellular molecular signaling pathways that are activated by a genetic insult in cardiomyocytes to maintain tissue and organ level regulation and resultant cardiac remodeling in certain forms of cardiomyopathies. In addition, animal models of different clinical forms of human cardiomyopathies with summaries of triggered key molecules and signaling pathways will be described.

Objectives Four variants (K60N, Q128R, G202R, and A592E) in the nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if these mutations are cardiomyopathy causing. Background Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. Methods We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain. Results We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. Conclusions Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.

Background Mutations in the SCN5A gene, which encodes the cardiac sodium channel, have been implicated in the pathogenesis of Brugada syndrome (BrS). Febrile illnesses have been recognized to unmask and/or trigger the BrS phenotype. However, the pathophysiological mechanism has not been fully elucidated. Objective A novel SCN5A missense mutation, V1340I, was identified in a patient with BrS suffering from frequent episodes of polymorphic ventricular tachycardia (VT) and syncope associated with fever. The biophysical modifications of hNa v 1.5 by V1340I were studied. Methods The effects of the V1340I mutation were studied in the 2 splice variants, SCN5A and SCN5A-Q1077del (delQ), using patch-clamp techniques at various temperatures between 22°C and 40°C. Results At 22°C, V1340I-SCN5A generated markedly diminished sodium currents compared to the wild-type (WT) SCN5A. On the contrary, V1340I-delQ generated almost identical current density compared to the WT-delQ. However, V1340I-delQ significantly attenuated the peak current density compared to the WT-delQ at 32°C, 37°C and 40°C. The voltage dependency of steady-state activation was leftward shifted both in WT-delQ and V1340I-delQ at 40°C. In addition, the V1340I-delQ accelerated the recovery time course from fast inactivation compared to the WT-delQ at 40°C. Immunohistochemical staining showed that both V1340I-SCN5A and V1340I-dQ were expressed in the plasma membrane. Conclusion Our study supports the concept that febrile illness predisposes individuals who carry a loss of function SCN5A mutation, such as V1340I, to fever-induced ventricular arrhythmias in BrS by significantly reducing the sodium currents in the hyperthermic state.

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.

AIM To investigate the regulation of Myopalladin (Mypn) and identify its gene network involved in restrictive cardiomyopathy (RCM). METHODS Gene expression values were measured in the heart of a large family of BXD recombinant inbred (RI) mice derived from C57BL/6J and DBA/2J. The proteomics data were collected from Mypn knock-in and knock-out mice. Expression quantitative trait locus (eQTL) mapping methods and gene enrichment analysis were used to identify Mypn regulation, gene pathway and co-expression networks. RESULTS A wide range of variation was found in expression of Mypn among BXD strains. We identified upstream genetic loci at chromosome 1 and 5 that modulate the expression of Mypn. Candidate genes within these loci include Ncoa2, Vcpip1, Sgk3, and Lgi2. We also identified 15 sarcomeric genes interacting with Mypn and constructed the gene network. Two novel members of this network (Syne1 and Myom1) have been confirmed at the protein level. Several members in this network are already known to relate to cardiomyopathy with some novel genes candidates that could be involved in RCM. CONCLUSION Using systematic genetics approach, we constructed Mypn co-expression networks that define the biological process categories within which similarly regulated genes function. Through this strategy we have found several novel genes that interact with Mypn that may play an important role in the development of RCM.

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is associated with ventricular arrhythmias and sudden cardiac death. We previously demonstrated that cardiac-specific overexpression of human mutant desmoplakin (DSPR2834H) leads to ARVC in mice at 6 months of age. However, the potential role and mechanism(s) of DSP in preclinical ARVC under cardiac stress remains unclear. Objectives: This study is aimed to elucidate the impact of DSP in the development of ARVC under adrenergic stimulation. Methods: Three-month-old non-transgenic (NTg), wild-type DSP (Tg-DSPWT) and Tg-DSPR2834H mice without obvious signs of ARVC, including right and left ventricular dysfunction, arrhythmias, were infused with either vehicle or isoproterenol (30mg/kg/d) for 2 weeks using mini-osmotic pumps. During isoproterenol infusion, electrocardiography (ECG) was monitored daily on conscious mice. Echocardiography and cardiac MRI were performed before and after 2-week of isoproterenol infusion. Myocardial tissues from both RV and LV were subjected to cellular, biochemical and histopathological analysis. Results: Isoproterenol resulted in cardiac hypertrophy to a similar degree amongst all genotypes; however, mortality occurred only in Tg-DSPR2834H mice. Vehicle-treated mice from all genotypes showed largely normal ECGs, whereas isoproterenol led to various types of arrhythmia in Tg-DSPR2834H mice including ventricular tachycardia and QT prolongation. Echocardiography analysis revealed LV dysfunction (decreased factional shortening) in isoproterenol-treated Tg-DSPR2834H mice compared to other treatment groups. Interstitial fibrosis and lipid infiltration was prominent in the Tg-DSPR2834H myocardium. MRI analysis is being performed to understand the RV and LV geometry and function. Conclusion: Our preliminary data confirms the essential role of desmoplakin in response to adrenergic stimulation. Studies investigating the electrophysiological, geometrical and cellular mechanisms of the pro-arrhythmic nature of DSPR2834H dysfunction are ongoing which may ultimately provide critical experimental data on prevention of asymptomatic preclinical ARVC in humans.

The very long-chain acyl-CoA dehydrogenase (VLCAD) enzyme catalyzes the first step of mitochondrial β-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia and cardiomyopathy, which can be exacerbated by fasting and/or cold stress. Global VLCAD knockout mice recapitulate these phenotypes: mice develop cardiomyopathy, and cold exposure leads to rapid hypothermia and death. However, the contribution of different tissues to development of these phenotypes has not been studied. We generated cardiac-specific VLCAD-deficient (cVLCAD−/−) mice by Cre-mediated ablation of the VLCAD in cardiomyocytes. By 6 mo of age, cVLCAD−/− mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Surprisingly, selective VLCAD gene ablation in cardiomyocytes was sufficient to evoke severe cold intolerance in mice who rapidly developed severe hypothermia, bradycardia, and markedly depressed cardiac function in response to fasting and cold exposure (+5°C). We conclude that cardiac-specific VLCAD deficiency is sufficient to induce cold intolerance and cardiomyopathy and is associated with reduced ATP production. These results provide strong evidence that fatty acid oxidation in myocardium is essential for maintaining normal cardiac function under these stress conditions.

Nebulette (NEBL) is a sarcomeric Z-disk protein involved in mechanosensing and force generation via its interaction with actin and tropomyosin-troponin complex. Genetic abnormalities in NEBL lead to dilated cardiomyopathy (DCM) in humans and animal models. The objectives of this study are to determine the earliest preclinical mechanical changes in the myocardium and define underlying molecular mechanisms by which NEBL mutations lead to cardiac dysfunction. We examined cardiac function in 3-month-old non-transgenic (non-Tg) and transgenic (Tg) mice (WT-Tg, G202R-Tg, A592E-Tg) by cardiac magnetic resonance (CMR) imaging. Contractility and calcium transients were measured in isolated cardiomyocytes. A592E-Tg mice exhibited enhanced in vivo twist and untwisting rate compared to control groups. Ex vivo analysis of A592E-Tg cardiomyocytes showed blunted calcium decay response to isoproterenol. CMR imaging of G202R-Tg mice demonstrated reduced torsion compared to non-Tg and WT-Tg, but conserved twist and untwisting rate after correcting for geometric changes. Ex vivo analysis of G202R-Tg cardiomyocytes showed elevated calcium decay at baseline and a conserved contractile response to isoproterenol stress. Protein analysis showed decreased α-actinin and connexin43, and increased cardiac troponin I phosphorylation at baseline in G202R-Tg, providing a molecular mechanism for enhanced ex vivo calcium decay. Ultrastructurally, G202R-Tg cardiomyocytes exhibited increased I-band and sarcomere length, desmosomal separation, and enlarged t-tubules. A592E-Tg cardiomyocytes also showed abnormal ultrastructural changes and desmin downregulation. This study showed distinct effects of NEBL mutations on sarcomere ultrastructure, cellular contractile function, and calcium homeostasis in preclinical DCM in vivo. We suggest that these abnormalities correlate with detectable myocardial wall motion patterns.

Abnormalities in Z-disc proteins cause hypertrophic (HCM), dilated (DCM) and/or restrictive cardiomyopathy (RCM), but disease-causing mechanisms are not fully understood. Myopalladin (MYPN) is a Z-disc protein expressed in striated muscle and functions as a structural, signaling and gene expression regulating molecule in response to muscle stress. MYPN was genetically screened in 900 patients with HCM, DCM and RCM, and disease-causing mechanisms were investigated using comparative immunohistochemical analysis of the patient myocardium and neonatal rat cardiomyocytes expressing mutant MYPN. Cardiac-restricted transgenic (Tg) mice were generated and protein-protein interactions were evaluated. Two nonsense and 13 missense MYPN variants were identified in subjects with DCM, HCM and RCM with the average cardiomyopathy prevalence of 1.66%. Functional studies were performed on two variants (Q529X and Y20C) associated with variable clinical phenotypes. Humans carrying the Y20C-MYPN variant developed HCM or DCM, whereas Q529X-MYPN was found in familial RCM. Disturbed myofibrillogenesis with disruption of α-actinin2, desmin and cardiac ankyrin repeat protein (CARP) was evident in rat cardiomyocytes expressing MYPN(Q529X). Cardiac-restricted MYPN(Y20C) Tg mice developed HCM and disrupted intercalated discs, with disturbed expression of desmin, desmoplakin, connexin43 and vinculin being evident. Failed nuclear translocation and reduced binding of Y20C-MYPN to CARP were demonstrated using in vitro and in vivo systems. MYPN mutations cause various forms of cardiomyopathy via different protein-protein interactions. Q529X-MYPN causes RCM via disturbed myofibrillogenesis, whereas Y20C-MYPN perturbs MYPN nuclear shuttling and leads to abnormal assembly of terminal Z-disc within the cardiac transitional junction and intercalated disc.

Fas receptor and its ligand, Fas-L, are the most well characterized co-stimulatory molecules playing an essential role in the induction of programmed cell death or apoptosis. Increase of circulating Fas-L in serum and in cardiomyocytes are described in different models of cardiac diseases such as myocarditis, pressure overload, AND myocardial infarction/ischemia. In addition, associated activation of inflammatory pathwayshas been observed. However, no studies have yet revealed mechanisms underlying the pathogenecity of FasL in the heart. Transgenic mice (C57/Bl6) with cardiac-specific over-expression of Fas-L were investigated. We characterized cardiac function by echocardiography. Cellular and molecular studies were performed on heart tissue using genomic, histologic, immunological, and protein analysis techniques. Fas-L transgenic mice had a significant rate of mortality (p Our findings suggest that Fas-L induces cardiac failure, via an activation of the ERK1/2 pathway, which is an important player involved in the hypertrophic response in the heart. Targeting the ERK1/2 pathway in cardiac diseases associated with Fas-L expression may therefore be able to prevent progression to cardiomyopathy.

Background— Long-QT syndrome (LQTS) is an inherited disorder associated with sudden cardiac death. The cytoskeletal protein syntrophin-α 1 (SNTA1) is known to interact with the cardiac sodium channel (hNa v 1.5), and we hypothesized that SNTA1 mutations might cause phenotypic LQTS in patients with genotypically normal hNa v 1.5 by secondarily disturbing sodium channel function. Methods and Results— Mutational analysis of SNTA1 was performed on 39 LQTS patients (QTc≥480 ms) with previously negative genetic screening for the known LQTS-causing genes. We identified a novel A257G- SNTA1 missense mutation, which affects a highly conserved residue, in 3 unrelated LQTS probands but not in 400 ethnic-matched control alleles. Only 1 of these probands had a preexisting family history of LQTS and sudden death with an additional intronic variant in KCNQ1. Electrophysiological analysis was performed using HEK-293 cells stably expressing hNa v 1.5 and transiently transfected with either wild-type or mutant SNTA1 and, in neonatal rat cardiomyocytes, transiently transfected with either wild-type or mutant SNTA1. In both HEK-293 cells and neonatal rat cardiomyocytes, increased peak sodium currents were noted along with a 10-mV negative shift of the onset and peak of currents of the current-voltage relationships. In addition, A257G-SNTA1 shifted the steady-state activation ( V h ) leftward by 9.4 mV, whereas the voltage-dependent inactivation kinetics and the late sodium currents were similar to wild-type SNTA1. Conclusion— SNTA1 is a new susceptibility gene for LQTS. A257G- SNTA1 can cause gain-of-function of Na v 1.5 similar to the LQT3.

Background Dilated cardiomyopathy (DCM) and myocarditis occur in many HIV-infected individuals, resulting in symptomatic heart failure in up to 5% of patients. Highly active antiretroviral therapy (HAART) has significantly reduced morbidity and mortality of acquired immunodeficiency syndrome (AIDS), but has resulted in an increase in cardiac and skeletal myopathies. Methods and Results In order to investigate whether the HAART component zidovudine (3′-azido-2′,3′-deoxythymidine; AZT) triggers the Fas-dependent cell-death pathway and cause cytoskeletal disruption in a murine model of DCM, 8-week-old transgenic (expressing Fas ligand in the myocardium: FasL Tg) and non-transgenic (NTg) mice received water ad libitum containing different concentrations of AZT (0, 0.07, 0.2, and 0.7 mg/ml). After 6 weeks, cardiac function was assessed by echocardiography and morphology was assessed by histopathologic and immunohistochemical methods. NTg and untreated FasL Tg mice showed little or no change in cardiac structure or function. In contrast, AZT-treated FasL Tg mice developed cardiac dilation and depressed cardiac function in a dose-dependent manner, with concomitant inflammatory infiltration of both ventricles. These changes were associated with an increased sarcolemmal expression of Fas and FasL, as well as increased activation of caspase 3, translocation of calpain 1 to the sarcolemma and sarcomere, and increased numbers of cells undergoing apoptosis. These were associated with changes in dystrophin and cardiac troponin I localization, as well as loss of sarcolemmal integrity. Conclusions The expression of Fas ligand in the myocardium, as identified in HIV-positive patients, might increase the susceptibility to HAART-induced cardiomyopathy due to activation of apoptotic pathways, resulting in cardiac dilation and dysfunction.

Enterovirus-induced myocardial injury can lead to severe heart failure. To date, little is known about the early innate stress response that contributes to host defense in the heart. Toll-like receptor 3 (TLR3) is important in the initiation of the innate antiviral response. We investigated the involvement of TLR3, which recognizes viral double-stranded RNA, on encephalomyocarditis virus (EMCV) infection. To examine the contribution of TLR3 in protection from EMCV infection, we infected mice deficient in TLR3 with 50 plaque-forming units of EMCV. TLR3-deficient (TLR3−/−) mice were more susceptible to EMCV infection and had a significantly higher viral load in the heart compared with TLR3+/+mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TLR3−/−than in TLR3+/+mice. TLR3−/−mice had impaired proinflammatory cytokine and chemokine expression in the heart following EMCV infection. However, the expression of interferon-β was not impaired in EMCV-infected TLR3−/−mice. EMCV infection leads to a TLR3-dependent innate stress response, which is involved in mediating protection against virus-induced myocardial injury.