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

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

Dystrophin Deficiency Causes Progressive Depletion of Cardiovascular Progenitor Cells in the Heart

Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.     

Cardiomyopathies: An Overview

Background: Cardiomyopathies are a heterogeneous group of pathologies characterized by structural and functional alterations of the heart. Aims: The purpose of this narrative review is to focus on the most important cardiomyopathies and their epidemiology, diagnosis, and management. Methods: Clinical trials were identified by Pubmed until 30 March 2021. The search keywords were “cardiomyopathies, sudden cardiac arrest, dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy, arrhythmogenic cardiomyopathy (ARCV), takotsubo syndrome”. Results: Hypertrophic cardiomyopathy (HCM) is the most common primary cardiomyopathy, with a prevalence of 1:500 persons. Dilated cardiomyopathy (DCM) has a prevalence of 1:2500 and is the leading indication for heart transplantation. Restrictive cardiomyopathy (RCM) is the least common of the major cardiomyopathies, representing 2% to 5% of cases. Arrhythmogenic cardiomyopathy (ARCV) is a pathology characterized by the substitution of the myocardium by fibrofatty tissue. Takotsubo cardiomyopathy is defined as an abrupt onset of left ventricular dysfunction in response to severe emotional or physiologic stress. Conclusion: In particular, it has been reported that HCM is the most important cause of sudden death on the athletic field in the United States. It is needless to say how important it is to know which changes in the heart due to physical activity are normal, and when they are pathological.     

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease

Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na⁺ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.     

Clinical Considerations for a Family with Dilated Cardiomyopathy,Sudden Cardiac Death,and a Novel TTN Frameshift Mutation

Dilated cardiomyopathy (DCM) is the leading indication for heart transplantation. TTN gene truncating mutations account for about 25% of familial DCM cases and for 18% of sporadic DCM cases. The clinical relevance of specific variants in TTN has been difficult to determine because of the sheer size of the protein for which TTN encodes, as well as existing extensive genetic variation. Clinicians should communicate novel clinically-relevant variants and genotype–phenotype associations, so that animal studies evaluating the molecular mechanisms are always conducted with a focus on clinical significance. In the present study, we report for the first time the novel truncating heterozygous variant {"type":"entrez-nucleotide","attrs":{"text":"NM_001256850.1","term_id":"378925624","term_text":"NM_001256850.1"}}NM_001256850.1:c.72777_72783del (p.Phe24259Leufs*51) in the TTN gene and its association with DCM in a family with sudden death. This variant occurs in the A-band region of the sarcomere, in a known mutational hotspot of the gene. Truncating titin variants that occur in this region are the most common cause of DCM and have been rarely reported in asymptomatic individuals, differently from other pathogenic TTN gene variants. Further studies are warranted to better understand this particular clinically-relevant variant.     

Synthesis of N‐Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc‐chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate‐based compounds as inhibitors of HDAC. Nine novel osthole‐based N‐hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9 d , 9 e , 9 g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nM ) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC₅₀=24.5 nM ), a potent inhibitor clinically used for the treatment of cutaneous T‐cell lymphoma (CTCL). While compounds 9 d and 9 e showed SAHA‐like activity towards HDAC1 and HDAC6, compound 9 g was more selective for HDAC1. Compound 9 d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α‐tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9 d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class‐specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N‐hydroxycinnamide‐derived HDAC inhibitors.     

A Low-Sodium Diet Boosts Ang (1–7) Production and NO-cGMP Bioavailability to Reduce Edema and Enhance Survival in Experimental Heart Failure

Sodium restriction is often recommended in heart failure (HF) to block symptomatic edema, despite limited evidence for benefit. However, a low-sodium diet (LSD) activates the classical renin-angiotensin-aldosterone system (RAAS), which may adversely affect HF progression and mortality in patients with dilated cardiomyopathy (DCM). We performed a randomized, blinded pre-clinical trial to compare the effects of a normal (human-equivalent) sodium diet and a LSD on HF progression in a normotensive model of DCM in mice that has translational relevance to human HF. The LSD reduced HF progression by suppressing the development of pleural effusions (p < 0.01), blocking pathological increases in systemic extracellular water (p < 0.001) and prolonging median survival (15%, p < 0.01). The LSD activated the classical RAAS by increasing plasma renin activity, angiotensin II and aldosterone levels. However, the LSD also significantly up-elevated the counter-regulatory RAAS by boosting plasma angiotensin converting enzyme 2 (ACE2) and angiotensin (1–7) levels, promoting nitric oxide bioavailability and stimulating 3′-5′-cyclic guanosine monophosphate (cGMP) production. Plasma HF biomarkers associated with poor outcomes, such as B-type natriuretic peptide and neprilysin were decreased by a LSD. Cardiac systolic function, blood pressure and renal function were not affected. Although a LSD activates the classical RAAS system, we conclude that the LSD delayed HF progression and mortality in experimental DCM, in part through protective stimulation of the counter-regulatory RAAS to increase plasma ACE2 and angiotensin (1–7) levels, nitric oxide bioavailability and cGMP production.     

Skeletal and Cardiac Muscle Disorders Caused by Mutations in Genes Encoding Intermediate Filament Proteins

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery–Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.     

Association of Nicotinamide Phosphoribosyltransferase (NAMPT) Gene Polymorphisms and of Serum NAMPT Levels with Dilated Cardiomyopathy in a Chinese Population

Nicotinamide phosphoribosyltransferase (NAMPT) has crucial roles for myocardial development, cardiomyocyte energy metabolism and cell death/survival by regulating NAD⁺-dependent sirtuin-1 (SIRT1) deacetylase. This study aimed to determine if the single nucleotide polymorphisms (SNPs) of the NAMPT gene may affect the susceptibility and prognosis for patients with dilated cardiomyopathy (DCM) and to describe the association of serum NAMPT levels with clinical features of DCM. Three SNPs (rs61330082, rs2505568, and rs9034) were analyzed by the polymerase chain reaction-restriction fragment length polymorphism method in a case-control study of 394 DCM patients and 395 controls from China. Serum NAMPT levels were measured by enzyme-linked immunosorbent assay kits. The homozygote for the minor allele at rs2505568 and rs9034 could not be detected in this study. Rs9034 T allele and CT genotype were associated with increased DCM risk (OR: 1.63, 95% CI = 1.16–2.27, p = 0.005 and OR: 1.72, 95% CI = 1.20–2.50, p = 0.0027, respectively). Nominally significant decreased DCM risk was found to be associated with the A allele and AT genotype of rs2505568 (OR: 0.48, 95% CI = 0.35–0.67, p < 0.0001 and OR: 0.44, 95% CI = 0.31–0.62, p < 0.0001, respectively), but it should be interpreted with caution because of Hardy-Weinberg disequilibrium in the control group. Of five haplotypes constructed, TAC (rs61330082-rs2505568-rs9034) was a protective haplotype to DCM (OR: 0.22, 95% CI = 0.13–0.39, p = 1.84 × 10⁻⁸). The Cox multivariate survival analysis indicated that the rs9034 CT genotype (hazard ratio (HR): 0.59, 95% CI = 0.37–0.96, p = 0.03) was an independently multivariate predictor for longer overall survival in DCM patients. Serum NAMPT levels were significantly higher in the DCM group than controls (p < 0.0001) and gradually increased with the increase of New York Heart Association grade in DCM patients. However, there was a lack of association of the three SNPs with serum NAMPT levels. Spearman correlation test revealed that the NAMPT level was positively associated with brain natriuretic peptide (r = 0.56, p = 0.001), left ventricular end-diastolic diameter (r = 0.293, p = 0.011) and left ventricular end-diastolic volume (r = 0.294, p = 0.011). Our study suggested that NAMPT may play an important role in the development of DCM.     

Energy Metabolites as Biomarkers in Ischemic and Dilated Cardiomyopathy

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10⁻⁶). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.     

Let-7 in Cardiovascular Diseases,Heart Development and Cardiovascular Differentiation from Stem Cells

The let-7 family is the second microRNA found in C. elegans. Recent researches have found it is highly expressed in the cardiovascular system. Studies have revealed the aberrant expression of let-7 members in cardiovascular diseases, such as heart hypertrophy, cardiac fibrosis, dilated cardiomyopathy (DCM), myocardial infarction (MI), arrhythmia, angiogenesis, atherosclerosis, and hypertension. Let-7 also participates in cardiovascular differentiation of embryonic stem cells. TLR4, LOX-1, Bcl-xl and AGO1 are by now the identified target genes of let-7. The circulating let-7b is suspected to be the biomarker of acute MI and let-7i, the biomarker of DCM. Further studies are necessary for identifying the gene targets and signaling pathways of let-7 in cardiovascular diseases. Let-7 might be a potential therapeutic target for cardiovascular diseases. This review focuses on the research progresses regarding the roles of let-7 in cardiovascular development and diseases.     

Nitro-Oleic Acid (NO2-OA) Improves Systolic Function in Dilated Cardiomyopathy by Attenuating Myocardial Fibrosis

Nitro-oleic acid (NO₂-OA), a nitric oxide (NO)- and nitrite (NO₂⁻)-derived electrophilic fatty acid metabolite, displays anti-inflammatory and anti-fibrotic signaling actions and therapeutic benefit in murine models of ischemia-reperfusion, atrial fibrillation, and pulmonary hypertension. Muscle LIM protein-deficient mice (Mlp^−/−) develop dilated cardiomyopathy (DCM), characterized by impaired left ventricular function and increased ventricular fibrosis at the age of 8 weeks. This study investigated the effects of NO₂-OA on cardiac function in Mlp^−/− mice both in vivo and in vitro. Mlp^−/− mice were treated with NO₂-OA or vehicle for 4 weeks via subcutaneous osmotic minipumps. Wildtype (WT) littermates treated with vehicle served as controls. Mlp^−/− mice exhibited enhanced TGFβ signalling, fibrosis and severely reduced left ventricular systolic function. NO₂-OA treatment attenuated interstitial myocardial fibrosis and substantially improved left ventricular systolic function in Mlp^−/− mice. In vitro studies of TGFβ-stimulated primary cardiac fibroblasts further revealed that the anti-fibrotic effects of NO₂-OA rely on its capability to attenuate fibroblast to myofibroblast transdifferentiation by inhibiting phosphorylation of TGFβ downstream targets. In conclusion, we demonstrate a substantial therapeutic benefit of NO₂-OA in a murine model of DCM, mediated by interfering with endogenously activated TGFβ signaling.     

Investigating LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

LMNA-related dilated cardiomyopathy is an inherited heart disease caused by mutations in the LMNA gene encoding for lamin A/C. The disease is characterized by left ventricular enlargement and impaired systolic function associated with conduction defects and ventricular arrhythmias. We hypothesized that LMNA-mutated patients’ induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) display electrophysiological abnormalities, thus constituting a suitable tool for deciphering the arrhythmogenic mechanisms of the disease, and possibly for developing novel therapeutic modalities. iPSC-CMs were generated from two related patients (father and son) carrying the same E342K mutation in the LMNA gene. Compared to control iPSC-CMs, LMNA-mutated iPSC-CMs exhibited the following electrophysiological abnormalities: (1) decreased spontaneous action potential beat rate and decreased pacemaker current (I_f) density; (2) prolonged action potential duration and increased L-type Ca²⁺ current (I_Ca,L) density; (3) delayed afterdepolarizations (DADs), arrhythmias and increased beat rate variability; (4) DADs, arrhythmias and cessation of spontaneous firing in response to β-adrenergic stimulation and rapid pacing. Additionally, compared to healthy control, LMNA-mutated iPSC-CMs displayed nuclear morphological irregularities and gene expression alterations. Notably, KB-R7943, a selective inhibitor of the reverse-mode of the Na⁺/Ca²⁺ exchanger, blocked the DADs in LMNA-mutated iPSC-CMs. Our findings demonstrate cellular electrophysiological mechanisms underlying the arrhythmias in LMNA-related dilated cardiomyopathy.     

Anti-Fibrotic Effects of Class I HDAC Inhibitor,Mocetinostat Is Associated with IL-6/Stat3 Signaling in Ischemic Heart Failure

Background: Recent studies have linked histone deacetylases (HDAC) to remodeling of the heart and cardiac fibrosis in heart failure. However, the molecular mechanisms linking chromatin remodeling events with observed anti-fibrotic effects are unknown. Here, we investigated the molecular players involved in anti-fibrotic effects of HDAC inhibition in congestive heart failure (CHF) myocardium and cardiac fibroblasts in vivo. Methods and Results: MI was created by coronary artery occlusion. Class I HDACs were inhibited in three-week post MI rats by intraperitoneal injection of Mocetinostat (20 mg/kg/day) for duration of three weeks. Cardiac function and heart tissue were analyzed at six week post-MI. CD90⁺ cardiac fibroblasts were isolated from ventricles through enzymatic digestion of heart. In vivo treatment of CHF animals with Mocetinostat reduced CHF-dependent up-regulation of HDAC1 and HDAC2 in CHF myocardium, improved cardiac function and decreased scar size and total collagen amount. Moreover, expression of pro-fibrotic markers, collagen-1, fibronectin and Connective Tissue Growth Factor (CTGF) were reduced in the left ventricle (LV) of Mocetinostat-treated CHF hearts. Cardiac fibroblasts isolated from Mocetinostat-treated CHF ventricles showed a decrease in expression of collagen I and III, fibronectin and Timp1. In addition, Mocetinostat attenuated CHF-induced elevation of IL-6 levels in CHF myocardium and cardiac fibroblasts. In parallel, levels of pSTAT3 were reduced via Mocetinostat in CHF myocardium. Conclusions: Anti-fibrotic effects of Mocetinostat in CHF are associated with the IL-6/STAT3 signaling pathway. In addition, our study demonstrates in vivo regulation of cardiac fibroblasts via HDAC inhibition.     

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca²⁺ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.     

Compound Heterozygous FKTN Variants in a Patient with Dilated Cardiomyopathy Led to an Aberrant α-Dystroglycan Pattern

Fukutin encoded by FKTN is a ribitol 5-phosphate transferase involved in glycosylation of α-dystroglycan. It is known that mutations in FKTN affect the glycosylation of α-dystroglycan, leading to a dystroglycanopathy. Dystroglycanopathies are a group of syndromes with a broad clinical spectrum including dilated cardiomyopathy and muscular dystrophy. In this study, we reported the case of a patient with muscular dystrophy, early onset dilated cardiomyopathy, and elevated creatine kinase levels who was a carrier of the compound heterozygous variants p.Ser299Arg and p.Asn442Ser in FKTN. Our work showed that compound heterozygous mutations in FKTN lead to a loss of fully glycosylated α-dystroglycan and result in cardiomyopathy and end-stage heart failure at a young age.