Early Onset Hypertrophic Cardiomyopathy Research Articles

The W792R HCM missense mutation in the C6 domain of cardiac myosin binding protein-C increases contractility in neonatal mouse myocardium

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model. We crossed heterozygous W792RWR mice to produce homozygous mutant W792RRR, heterozygous W792RWR, and control W792RWW mice. W792RRR mice present with cardiac hypertrophy, myofibrillar disarray and fibrosis by postnatal day 10 (PND10), and do not survive past PND21. Full-length cMyBP-C is present at similar levels in W792RWW, W792RWR and W792RRR mice and is properly incorporated into the sarcomere. Heterozygous W792RWR mice displayed normal heart morphology and contractility. Permeabilized myocardium from PND10 W792RRR mice showed increased Ca2+ sensitivity, accelerated cross-bridge cycling kinetics, decreased cooperativity in the activation of force, and increased expression of hypertrophy-related genes. In silico modeling suggests that the W792R mutation destabilizes the fold of the C6 domain and increases torsion in the C5-C7 region, possibly impacting regulatory interactions of cMyBP-C with myosin and actin. Based on the data presented here, we propose a model in which mutant W792R cMyBP-C preferentially forms Ca2+ sensitizing interactions with actin, rather than inhibitory interactions with myosin. The W792R-cMyBP-C mouse model provides mechanistic insights into the pathology of this mutation and may provide a mechanism by which other central domain missense mutations in cMyBP-C may alter contractility, leading to HCM.

Preclinical evaluation of CRISPR-based therapies for Noonan syndrome caused by deep-intronic LZTR1 variants

Gene variants in LZTR1 are implicated to cause Noonan syndrome associated with a severe and early-onset hypertrophic cardiomyopathy. Mechanistically, LZTR1 deficiency results in accumulation of RAS GTPases and, as a consequence, in RAS-MAPK signaling hyperactivity, thereby causing the Noonan syndrome-associated phenotype. Despite its epidemiological relevance, pharmacological as well as invasive therapies remain limited. Here, personalized CRISPR/Cas9 gene therapies might offer a novel alternative for a curative treatment in this patient cohort. In this study, by utilizing a patient-specific screening platform based on iPSC-derived cardiomyocytes from two Noonan syndrome patients, we evaluated different clinically translatable therapeutic approaches using small Cas9 orthologs targeting a deep-intronic LZTR1 variant to cure the disease-associated molecular pathology. Despite high editing efficiencies in cardiomyocyte cultures transduced with lentivirus or all-in-one AAVs, we observed crucial differences in editing outcomes in proliferative iPSCs versus non-proliferative cardiomyocytes. While editing in iPSCs rescued the phenotype, the same editing approaches did not robustly restore LZTR1 function in cardiomyocytes, indicating critical differences in the activity of DNA double-strand break repair mechanisms between proliferative and non-proliferative cell types and highlighting the importance of cell type-specific screens for testing CRISPR/Cas9 gene therapies.

Abstract 14479: Compound Heterozygosity With Two Novel Mutations in MYBPC3 as a Cause of Severe and Early-Onset Hypertrophic Cardiomyopathy

Case presentation: A 8-year-old male was diagnosed with severe hypertrophic cardiomyopathy (HCM) after a cardiac arrest from which he was successfully defibrillated. Maximal wall thickness was 23 mm (z-score 6.36) on echocardiography. An implantable cardioverter defibrillator (ICD) was implanted and atenolol was started. After 4 ventricular fibrillation-terminating ICD shocks in the following 2 months, further treatment is under consideration. A genetic test identified the likely pathogenic intronic variant (c.1927+337G>T) in the MYBPC3 gene. An additional variant in the same gene (p.Arg817Gly), of uncertain significance but potentially relevant for the phenotype, was also found. His 10-year-old sister was also diagnosed with severe HCM, genetic test identified the two previously referred variants and ICD was also implanted. In the 4-year-old one sister, who is apparently healthy, only the likely pathogenic intronic variant (c.1927+337G>T) was found. Regarding their parents, both with no evidence of cardiomyopathy, c.1927+337G>T variant was identified in the father, while p.Arg817Gly was present in the mother. Discussion: This clinical case is noteworthy as an example of compound heterozygosity in which each parent donates one alternate allele, being these alleles located at different loci within the same gen, which emphasizes the importance of family genetic testing. The variant of uncertain significance p.Arg817Gly appears to be a disease modifier, causing the phenotype to be expressed early and severely in the presence of the probably pathogenic intronic variant c.1927+337G>T in MYBPC3. This deep intronic variant has not been described previously but was found in thirteen index cases referred for HCM and a history of familial sudden death at an early age, being overrepresented in the cohort of HCM in the north of Spain. Being a carrier of both these two novel variants appears to cause an extremely aggressive childhood-onset phenotype.

Generation of a genetically-modified induced pluripotent stem cell line harboring a Noonan syndrome-associated gene variant MRAS p.G23V

Patients harboring causative gene variants in RAS GTPase MRAS develop Noonan syndrome and early-onset hypertrophic cardiomyopathy. Here, we describe the generation of a human iPSC line harboring the Noonan syndrome-associated MRAS p.G23V variant by using CRISPR/Cas9 technology. The established MRASG23V iPSC line allows to study MRAS-specific pathomechanisms and to test novel therapeutic strategies in various disease-relevant cell types and tissues.

CMyBP-C ablation in human engineered cardiac tissue causes progressive Ca2+-handling abnormalities.

Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/- ECTs were haploinsufficient. cMyBP-C-/- cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/- and cMyBP-C-/- ECTs, and force production became severely depressed in cMyBP-C-/- ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C-/- than cMyBP-C+/- ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.

Succesful MEK-inhibition of severe hypertrophic cardiomyopathy in RIT1-related Noonan Syndrome

Infants with Noonan Syndrome and hypertrophic cardiomyopathy have a poor prognosis and a high mortality especially when diagnosed before six months of age. As for the majority of the RASopathies, no medical treatment has been approved for Noonan Syndrome. Meanwhile, several approved agents targeting the same RAS/MAPK signaling pathway are used in cancer treatment. In this case report we describe a child with Noonan Syndrome caused by a pathogenic RIT1 variant, who developed severe early-onset hypertrophic cardiomyopathy and pulmonary valve stenosis. She received off-label treatment with the MEK-inhibitor trametinib which resulted in complete remission of the cardiac hypertrophy and a significant improvement of the pulmonary valve stenosis. Our case emphasizes the potential of existing cancer agents targeting the RAS/MAPK signaling pathway as successful treatment for RASopathy manifestations.

Gender- and age-related differences in distinct phenotypes of hypertrophic cardiomyopathy-associated mutation MYBPC3–E334K

The mutation MYBPC3-E334K is a culprit mutation of hypertrophic cardiomyopathy (HCM). The pathogenicity of MYBPC3-E334K is conflicting in ClinVar because of the limited segregation data and the relatively high frequency in gnomAD (0.03% overall, with 0.3% in East Asians and 0.8% in Japanese). The main aim is to clarify the clinical importance and phenotype-genotype correlations in subjects with or without MYBPC3-E334K alone. The prevalence of MYBPC3-E334K was sequenced in 1017 HCM unrelated probands. The clinical features, morphology phenotypes, and electrical phenotypes were further analyzed according to the phenotype and genotype status in families with single-mutation MYBPC3-E334K. Nine of 1017 (0.88%) unrelated HCM probands were detected harboring MYBPC3-E334K, and three of them harbored a second variant in sarcomere protein gene. Family study and co-segregation analyses indicated that patients with single-mutation MYBPC3-E334K showed autosomal dominant mode of inheritance with incomplete penetrance. The overall disease penetrance was 52.6%, and the disease penetrance was higher in males than in females (100% in men vs 25% in women, p = 0.003). The mean age at diagnosis of males was approximately 25years younger than females (36.57 ± 18.65 vs 62.33 ± 12.10, p = 0.062). The variant MYBPC3-E334K was classified as a likely pathogenic variant, and a second sarcomere variant did not reveal obvious cumulative effects. The patients harboring single-mutation MYBPC3-E334K had incomplete penetrance, and males demonstrated higher penetrance and early onset HCM than females. A second sarcomere variant did not reveal obvious cumulative effects.

A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy.

ABSTRACTDuchenne muscular dystrophy (DMD) is a life-threatening neuromuscular disease caused by the lack of dystrophin, resulting in progressive muscle wasting and locomotor dysfunctions. By adulthood, almost all patients also develop cardiomyopathy, which is the primary cause of death in DMD. Although there has been extensive effort in creating animal models to study treatment strategies for DMD, most fail to recapitulate the complete skeletal and cardiac disease manifestations that are presented in affected patients. Here, we generated a mouse model mirroring a patient deletion mutation of exons 52-54 (Dmd Δ52-54). The Dmd Δ52-54 mutation led to the absence of dystrophin, resulting in progressive muscle deterioration with weakened muscle strength. Moreover, Dmd Δ52-54 mice present with early-onset hypertrophic cardiomyopathy, which is absent in current pre-clinical dystrophin-deficient mouse models. Therefore, Dmd Δ52-54 presents itself as an excellent pre-clinical model to evaluate the impact on skeletal and cardiac muscles for both mutation-dependent and -independent approaches.

CRISPR-Cas9 generated Pompe knock-in murine model exhibits early-onset hypertrophic cardiomyopathy and skeletal muscle weakness

Infantile-onset Pompe Disease (IOPD), caused by mutations in lysosomal acid alpha-glucosidase (Gaa), manifests rapidly progressive fatal cardiac and skeletal myopathy incompletely attenuated by synthetic GAA intravenous infusions. The currently available murine model does not fully simulate human IOPD, displaying skeletal myopathy with late-onset hypertrophic cardiomyopathy. Bearing a Cre-LoxP induced exonic disruption of the murine Gaa gene, this model is also not amenable to genome-editing based therapeutic approaches. We report the early onset of severe hypertrophic cardiomyopathy in a novel murine IOPD model generated utilizing CRISPR-Cas9 homology-directed recombination to harbor the orthologous Gaa mutation c.1826dupA (p.Y609*), which causes human IOPD. We demonstrate the dual sgRNA approach with a single-stranded oligonucleotide donor is highly specific for the Gaac.1826 locus without genomic off-target effects or rearrangements. Cardiac and skeletal muscle were deficient in Gaa mRNA and enzymatic activity and accumulated high levels of glycogen. The mice demonstrated skeletal muscle weakness but did not experience early mortality. Altogether, these results demonstrate that the CRISPR-Cas9 generated Gaac.1826dupA murine model recapitulates hypertrophic cardiomyopathy and skeletal muscle weakness of human IOPD, indicating its utility for evaluation of novel therapeutics.

Identification of LAMP2 mutations in early-onset hypertrophic cardiomyopathy by targeted exome sequencing

Identification of <i>LAMP2</i> mutations in early-onset hypertrophic cardiomyopathy by targeted exome sequencing

Dysregulation of the calcium handling protein, CCDC47, is associated with diabetic cardiomyopathy

BackgroundDiabetes mellitus is associated with an increased risk in diabetic cardiomyopathy (DCM) that is distinctly not attributed to co-morbidities with other vasculature diseases. To date, while dysregulation of calcium handling is a key hallmark in cardiomyopathy, studies have been inconsistent in the types of alterations involved. In this study human cardiomyocytes were exposed to an environmental nutritional perturbation of high glucose, fatty acids, and l-carnitine to model DCM and iTRAQ-coupled LC–MS/MS proteomic analysis was used to capture proteins affected by the perturbation. The proteins captured were then compared to proteins currently annotated in the cardiovascular disease (CVD) gene ontology (GO) database to identify proteins not previously described as being related to CVD. Subsequently, GO analysis for calcium regulating proteins and endoplasmic/sarcoplasmic reticulum (ER/SR) associated proteins was carried out.ResultsHere, we identified CCDC47 (calumin) as a unique calcium regulating protein altered in our in vitro nutritional perturbation model. The cellular and functional role of CCDC47 was then assessed in rat cardiomyocytes. In rat H9C2 myocytes, overexpression of CCDC47 resulted in increase in ionomycin-induced calcium release and reuptake. Of interest, in a diet-induced obese (DIO) rat model of DCM, CCDC47 mRNA expression was increased in the atrium and ventricle of the heart, but CCDC47 protein expression was significantly increased only in the atrium of DIO rats compared to lean control rats. Notably, no changes in ANP, BNP, or β-MHC were observed between DIO rats and lean control rats.ConclusionsTogether, our in vitro and in vivo studies demonstrate that CCDC47 is a unique calcium regulating protein that is associated with early onset hypertrophic cardiomyopathy.

Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation mayfurther increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

Mutations of the Mitochondrial-tRNA Modifier MTO1 Cause Hypertrophic Cardiomyopathy and Lactic Acidosis

Dysfunction of mitochondrial respiration is an increasingly recognized cause of isolated hypertrophic cardiomyopathy. To gain insight into the genetic origin of this condition, we used next-generation exome sequencing to identify mutations in MTO1, which encodes mitochondrial translation optimization 1. Two affected siblings carried a maternal c.1858dup (p.Arg620Lysfs(∗)8) frameshift and a paternal c.1282G>A (p.Ala428Thr) missense mutation. A third unrelated individual was homozygous for the latter change. In both humans and yeast, MTO1 increases the accuracy and efficiency of mtDNA translation by catalyzing the 5-carboxymethylaminomethylation of the wobble uridine base in three mitochondrial tRNAs (mt-tRNAs). Accordingly, mutant muscle and fibroblasts showed variably combined reduction in mtDNA-dependent respiratory chain activities. Reduced respiration in mutant cells was corrected by expressing a wild-type MTO1 cDNA. Conversely, defective respiration of a yeast mto1Δ strain failed to be corrected by an Mto1(Pro622∗) variant, equivalent to human MTO1(Arg620Lysfs∗8), whereas incomplete correction was achieved by an Mto1(Ala431Thr) variant, corresponding to human MTO1(Ala428Thr). The respiratory yeast phenotype was dramatically worsened in stress conditions and in the presence of a paromomycin-resistant (P(R)) mitochondrial rRNA mutation. Lastly, in vivo mtDNA translation was impaired in the mutant yeast strains.

Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy

BackgroundNADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinson's disease, Alzheimer's disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.MethodsA series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.ResultsWe identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.ConclusionsThe mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

Loss of function of Sco1 and its interaction with cytochrome c oxidase

Sco1 and Sco2 are mitochondrial copper-binding proteins involved in the biogenesis of the Cu(A) site in the cytochrome c oxidase (CcO) subunit Cox2 and in the maintenance of cellular copper homeostasis. Human Surf1 is a CcO assembly factor with an important but poorly characterized role in CcO biogenesis. Here, we analyzed the impact on CcO assembly and tissue copper levels of a G132S mutation in the juxtamembrane region of SCO1 metallochaperone associated with early onset hypertrophic cardiomyopathy, encephalopathy, hypotonia, and hepatopathy, assessed the total copper content of various SURF1 and SCO2-deficient tissues, and investigated the possible physical association between CcO and Sco1. The steady-state level of mutant Sco1 was severely decreased in the muscle mitochondria of the SCO1 patient, indicating compromised stability and thus loss of function of the protein. Unlike the wild-type variant, residual mutant Sco1 appeared to migrate exclusively in the monomeric form on blue native gels. Both the activity and content of CcO were reduced in the patient's muscle to approximately 10-20% of control values. SCO1-deficient mitochondria showed accumulation of two Cox2 subcomplexes, suggesting that Sco1 is very likely responsible for a different posttranslational aspect of Cox2 maturation than Sco2. Intriguingly, the various SURF1-deficient samples analyzed showed a tissue-specific copper deficiency similar to that of SCO-deficient samples, suggesting a role for Surf1 in copper homeostasis regulation. Finally, both blue native immunoblot analysis and coimmunoprecipitation revealed that a fraction of Sco1 physically associates with the CcO complex in human muscle mitochondria, suggesting a possible direct relationship between CcO and the regulation of cellular copper homeostasis.

Sarcomere protein gene mutations in hypertrophic cardiomyopathy of the elderly.

Hypertrophic cardiomyopathy, a familial myocardial condition caused by sarcomere protein mutations, is usually recognized by early adulthood. Hypertrophic cardiomyopathy of the elderly has similar clinical features but, notably, a later age of onset and noncontributory family history. Causes of elderly-onset hypertrophic cardiomyopathy are unknown. Eighteen women and 13 men diagnosed with late-onset hypertrophic cardiomyopathy were studied. Initial symptoms occurred at 59.3 (+/-12.3) years, and diagnosis was made at 62.8 (+/-10.8) years. None had family histories of cardiomyopathy. Echocardiography demonstrated maximal left ventricular wall thickness of 19.9+/-3.8 mm, systolic anterior motion of the mitral valve (58%), and, in 11 individuals, left ventricular outflow tract gradients (average, 63+/-42.8 mm). Sarcomere protein gene analyses revealed 8 sequence variants in cardiac myosin binding protein-C (1 nonsense, 1 splice acceptor site, and 3 missense), cardiac troponin I (2 missense), and alpha-cardiac myosin heavy chain (1 missense). Seven variants were not found in over 170 normal chromosomes; 1 variant (cardiac myosin binding protein-C Arg326Gln) also occurred in a healthy adult. Hypertrophic cardiomyopathy of the elderly can be a genetic disorder caused by dominant sarcomere protein mutations. The distribution of mutations in elderly-onset disease is strikingly different (P<0.00001) from that of familial, early onset hypertrophic cardiomyopathy. Whereas defects in beta-cardiac myosin heavy chain, cardiac troponin T, and alpha-tropomyosin account for > 45% of familial hypertrophic cardiomyopathy, none were found here. Rather, mutations in cardiac myosin binding protein-C, troponin I, and alpha-cardiac myosin heavy chain caused elderly-onset hypertrophic cardiomyopathy.