Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function.
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ABSTRACT:
SUBMITTER: Schmitt JP
PROVIDER: S-EPMC1599993 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the ?-cardiac myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human ?-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human ?-cardiac myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.
| S-EPMC5302870 | biostudies-literature
Project description:ObjectiveTo identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism.BackgroundDCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes.MethodsWe performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model.ResultsA novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure.ConclusionsUsing WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM.
| S-EPMC5166708 | biostudies-literature
Project description:Cardiomyopathies caused by double gene mutations are rare but conferred a remarkably increased risk of end-stage progression, arrhythmias, and poor outcome. Compound genetic mutations leading to complex phenotype in the setting of cardiomyopathies represent an important challenge in clinical practice, and genetic tests allow risk stratification and personalized clinical management of patients. We report a case of a 50-year-old woman with congestive heart failure characterized by dilated cardiomyopathy, diffuse coronary disease, complete atrioventricular block, and missense mutations in cardiac myosin-binding protein C (MYBPC3) and myopalladin (MYPN). We discuss the plausible role of genetic profile in phenotype determination.
| S-EPMC7358828 | biostudies-literature
Project description:We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.
| S-EPMC6873177 | biostudies-literature
Project description:We sought to identify a novel gene for dilated cardiomyopathy (DCM).DCM is a heritable, genetically heterogeneous disorder that remains idiopathic in the majority of patients. Familial cases provide an opportunity to discover unsuspected molecular bases of DCM, enabling pre-clinical risk detection.Two large families with autosomal-dominant DCM were studied. Genome-wide linkage analysis was used to identify a disease locus, followed by fine mapping and positional candidate gene sequencing. Mutation scanning was then performed in 278 unrelated subjects with idiopathic DCM, prospectively identified at the Mayo Clinic.Overlapping loci for DCM were independently mapped to chromosome 10q25-q26. Deoxyribonucleic acid sequencing of affected individuals in each family revealed distinct heterozygous missense mutations in exon 9 of RBM20, encoding ribonucleic acid (RNA) binding motif protein 20. Comprehensive coding sequence analyses identified missense mutations clustered within this same exon in 6 additional DCM families. Mutations segregated with DCM (peak composite logarithm of the odds score >11.49), were absent in 480 control samples, and altered residues within a highly conserved arginine/serine (RS)-rich region. Expression of RBM20 messenger RNA was confirmed in human heart tissue.Our findings establish RBM20 as a DCM gene and reveal a mutation hotspot in the RS domain. RBM20 is preferentially expressed in the heart and encodes motifs prototypical of spliceosome proteins that regulate alternative pre-messenger RNA splicing, thus implicating a functionally distinct gene in human cardiomyopathy. RBM20 mutations are associated with young age at diagnosis, end-stage heart failure, and high mortality.
| S-EPMC2782634 | biostudies-literature
Project description:Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.
| S-EPMC5992122 | biostudies-literature
Project description:BackgroundDilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. Although more than 40 genes have been reported to cause DCM, the role of genetic testing in clinical practice is not well defined. Mutations in the troponin T (TNNT2) gene represent an important subset of known disease-causing mutations associated with DCM. Therefore, the aim of the present study was to determine the genetic variations in TNNT2 and the associations of those variations with DCM in Chinese patients.MethodsAn approximately 4 kb fragment of the TNNT2 gene was isolated from 103 DCM patients and 192 healthy controls and was analyzed by DNA sequence analysis for genetic variations.ResultsA total of 6 TNNT2 mutations were identified in 99 patients, including a G321T missense mutation (Leu84Phe) and 5 novel intronic mutations. Alleles of two novel SNPs (c.192 + 353 C>A, OR = 0.095, 95% CI: 0.013-0.714, P = 0.022; c.192 + 463 G>A, OR = 0.090, 95% CI: 0.012-0.675, P = 0.019) and SNP rs3729843 (OR = 1.889, 95% CI: 1.252-2.852; P = 0.002) were significantly correlated with DCM.ConclusionsThese results suggest that the missense mutation (Leu84Phe) and two novel SNPs (c.192 + 353 C>A, c.192 + 463 G>A) in TNNT2 gene might be associated with DCM in the Chinese population.
| S-EPMC4109665 | biostudies-literature
Project description:Heart failure (HF) is a worldwide pandemic with an unacceptable high level of morbidity and mortality. Understanding the different pathophysiological mechanisms will contribute to prevention and individualized therapy of HF. We established mouse models for ischemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM) by inducing myocardial infarction (MI) and Coxsackievirus B3 infection, respectively. Isobaric tags for relative and absolute quantitation and liquid chromatography coupled with tandem mass spectrometry technology was used to identify the protein expression profiles in control and failing hearts. A total of 1,638 proteins were identified and compared in this proteomics analysis. Among them, 286 proteins were differently expressed. Gene ontology, KEGG pathway and ingenuity pathway analysis was performed to systematically assess the potential connections of the differentially expressed proteins to biological functions. Compared with control group, the differentially expressed proteins derived from the hearts of ICM and DCM mice were partially similar and mainly modulated in oxidative phosphorylation, metabolism and protein folding pathways. Moreover, difference still existed, the differentially expressed proteins between DCM and ICM hearts were significantly modulated in oxidative phosphorylation, metabolic and AMPK signaling pathways. Confirmatory western bolt analysis demonstrated that SDHB was down-regulated in both ICM and DCM hearts, while UQCRQ, GLUT4 and adiponectin were up-regulated in ICM hearts. Adenosine triphosphate (ATP) concentration significantly decreased in both DCM and ICM hearts. The protein expression of phospho-AMPK? decreased significantly in DCM hearts, but increased in ICM. In summary, oxidative phosphorylation, cardiac metabolism, and protein folding play critical roles in the pathogenesis of HF. The diverse changes in protein expression profiles between failing hearts induced by either MI or CVB3 infection demonstrated the heterogeneity of HF. Understanding the differences in proteome profiles could offer more precise therapeutic options for HF.
| S-EPMC6591471 | biostudies-literature
Project description:BackgroundThe titin gene (TTN) encodes the largest human protein, which plays a central role in sarcomere organization and passive myocyte stiffness. TTN truncating mutations cause dilated cardiomyopathy (DCM); however, the role of TTN missense variants in DCM has been difficult to elucidate because of the presence of background TTN variation.Methods and resultsA cohort of 147 DCM index subjects underwent DNA sequencing for 313 TTN exons covering the N2B and N2BA cardiac isoforms of TTN. Of the 348 missense variants, we identified 44 "severe" rare variants by using a bioinformatic filtering process in 37 probands. Of these, 5 probands were double heterozygotes (additional variant in another DCM gene) and 7 were compound heterozygotes (2 TTN "severe" variants). Segregation analysis allowed the classification of the "severe" variants into 5 "likely" (cosegregating), 5 "unlikely" (noncosegregating), and 34 "possibly" (where family structure precluded segregation analysis) disease-causing variants. Patients with DCM carrying "likely" or "possibly" pathogenic TTN "severe" variants did not show a different outcome compared with "unlikely" and noncarriers of a "severe" TTN variant. However, the "likely" and "possibly" disease-causing variants were overrepresented in the C-zone of the A-band region of the sarcomere.ConclusionsTTN missense variants are common and present a challenge for bioinformatic classification, especially when informative families are not available. Although DCM patients carrying bioinformatically "severe" TTN variants do not appear to have a worse clinical course than noncarriers, the nonrandom distribution of "likely" and "possibly" disease-causing variants suggests a potential biological role for some TTN missense variants.
| S-EPMC4845231 | biostudies-literature
Project description:Dilated cardiomyopathy (DCM) is characterized by impaired cardiac function due to myocardial hypo-contractility and is associated with point mutations in β-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super relaxed" state (SRX), which is further stabilized by a structural state known as the "Interacting Heads Motif" (IHM). Therefore, hypo-contractility of DCM myocardium may result from: 1) reduced function of individual myosin, and/or; 2) decreased myosin availability due to increased IHM/SRX stabilization. To define the molecular impact of an established DCM myosin mutation, E525K, we characterized the biochemical and mechanical activity of wild-type (WT) and E525K human β-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. Single-headed (S1) and a short-tailed, double-headed (2HEP) myosin constructs exhibited low (~10%) IHM/SRX content, actin-activated ATPase activity of ~5s-1 and fast velocities in unloaded motility assays (~2000nm/s). Double-headed, longer-tailed (15HEP, 25HEP) constructs exhibited higher IHM/SRX content (~90%), and reduced actomyosin ATPase (<1s-1) and velocity (~800nm/s). A simple analytical model suggests that reduced velocities may be attributed to IHM/SRXdependent sequestration of myosin heads. Interestingly, the E525K 15HEP and 25HEP mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength. However, at higher ionic strength, the E525K mutation stabilized the IHM/SRX state. Therefore, the E525K-associated DCM human cardiac hypo-contractility may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability.
| S-EPMC10723396 | biostudies-literature