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PURPOSE OF REVIEW: Precision genetic medicine is evolving at a rapid pace and bears significant implications for clinical cardiology. Herein, we discuss the latest advancements and emerging strategies in gene therapy for cardiomyopathy and heart failure. RECENT FINDINGS: Elucidating the genetic architecture of heart failure has paved the way for precision therapies in cardiovascular medicine. Recent preclinical studies and early-phase clinical trials have demonstrated encouraging results that support the development of gene therapies for heart failure arising from a variety of etiologies. In addition to the discovery of new therapeutic targets, innovative delivery platforms are being leveraged to improve the safety and efficacy of cardiac gene therapies. Precision genetic therapy represents a potentially safe and effective approach for improving outcomes in patients with heart failure. It holds promise for radically transforming the treatment paradigm for heart failure by directly targeting the underlying etiology. As this new generation of cardiovascular medicines progress to the clinic, it is especially important to carefully evaluate the benefits and risks for patients.
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BACKGROUND: The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. METHODS AND RESULTS: We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca2+-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca2+-sensitivity, incorporation of R278C increased Ca2+-sensitivity at low and intermediate dose, while it decreased Ca2+-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. CONCLUSIONS: Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca2+-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca2+-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.
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Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Mutação/genética , Miócitos Cardíacos/patologia , Miofibrilas/patologia , Troponina T/genética , Adolescente , Adulto , Idoso , Cálcio/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Familial cardiomyopathy is an inherited disease that affects the structure and function of heart muscle and has an extreme range of phenotypes. Among the millions of affected individuals, patients with hypertrophic (HCM), dilated (DCM), or left ventricular non-compaction (LVNC) cardiomyopathy can experience morphologic changes of the heart which lead to sudden death in the most detrimental cases. TNNC1, the gene that codes for cardiac troponin C (cTnC), is a sarcomere gene associated with cardiomyopathies in which probands exhibit young age of presentation and high death, transplant or ventricular fibrillation events relative to TNNT2 and TNNI3 probands. Using GnomAD, ClinVar, UniProt and PhosphoSitePlus databases and published literature, an extensive list to date of identified genetic variants in TNNC1 and post-translational modifications (PTMs) in cTnC was compiled. Additionally, a recent cryo-EM structure of the cardiac thin filament regulatory unit was used to localize each functionally studied amino acid variant and each PTM (acetylation, glycation, s-nitrosylation, phosphorylation) in the structure of cTnC. TNNC1 has a large number of variants (> 100) relative to other genes of the same transcript size. Surprisingly, the mapped variant amino acids and PTMs are distributed throughout the cTnC structure. While many cardiomyopathy-associated variants are localized in α-helical regions of cTnC, this was not statistically significant χ2 (p = 0.72). Exploring the variants in TNNC1 and PTMs of cTnC in the contexts of cardiomyopathy association, physiological modulation and potential non-canonical roles provides insights into the normal function of cTnC along with the many facets of TNNC1 as a cardiomyopathic gene.
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Miocárdio , Troponina C , Humanos , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional/genética , Troponina C/genética , Troponina C/metabolismo , Troponina I , Troponina T/genéticaRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with early childhood onset characterized by profound loss of muscle strength and associated cardiomyopathy. DMD affects is most often caused by deletions involving single or multiple exons that disrupt the open reading frame of the DMD gene. Mutations causing loss or premature truncation of dystrophin result in dystrophin protein deficiency, which renders the plasma membrane of skeletal myofibers and cardiomyocytes weakened. AIM OF REVIEW: Genetic correction is in use to treat DMD, since several drugs have been already approved which partially restore dystrophin production through the use of antisense oligonucleotides. There are multiple ongoing clinical trials to evaluate the efficacy of treating DMD with micro-dystrophins delivered by adeno-associated viruses. Future approaches entail gene editing to target the single copy of the DMD gene on the X-chromosome. The primary, near-term goal is restoration of skeletal muscle dystrophin, and for some of these treatments, the efficacy in the heart is not fully known. Here, we discuss the anticipated cardiac outcomes of dystrophin-targeted therapies, and how this information informs genomic medicine for cardiomyopathies, especially in childhood. KEY SCIENTIFIC CONCEPTS OF REVIEW: Many genetic treatment strategies are being implemented to treat DMD. Since most preclinical testing has focused on skeletal muscle, there is a gap in knowledge about the expected effects of these approaches on cardiac genetic correction and cardiomyopathy progression in DMD. Additional study is needed.
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Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.
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Troponina C/metabolismo , Troponina T/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Feminino , Humanos , Masculino , Mutagênese Sítio-Dirigida , Contração Miocárdica , Miocárdio/metabolismo , Miofibrilas/fisiologia , Ressonância Magnética Nuclear Biomolecular , Linhagem , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Troponina C/química , Troponina T/química , Troponina T/genéticaRESUMO
Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.
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Cálcio/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Miofibrilas/metabolismo , Troponina C/metabolismo , Algoritmos , Animais , Sinalização do Cálcio , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Contração Isométrica , Cinética , Masculino , Camundongos , Modelos Moleculares , Modelos Teóricos , Mutação , Miocárdio/química , Miofibrilas/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Troponina C/química , Troponina C/genéticaRESUMO
The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k-4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.
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Cardiomiopatia Hipertrófica/metabolismo , Miofibrilas/metabolismo , Subfragmentos de Miosina/metabolismo , Músculos Papilares/metabolismo , Troponina C/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Cromatografia Líquida , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Immunoblotting , Cinética , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Fosforilação , Espectrometria de Massas em Tandem , Troponina C/genéticaRESUMO
Background Pediatric dilated cardiomyopathy (DCM) is a well-known clinical entity; however, phenotype-genotype correlations are inadequately described. Our objective was to provide genotype associations with life-threatening cardiac outcomes in pediatric DCM probands. Methods and Results We performed a retrospective review of children with DCM at a large pediatric referral center (2007-2016), excluding syndromic, chemotherapy-induced, and congenital heart disease causes. Genetic variants were adjudicated by an expert panel and an independent clinical laboratory. In a cohort of 109 pediatric DCM cases with a mean age at diagnosis of 4.2 years (SD 5.9), life-threatening cardiac outcomes occurred in 47% (42% heart transplant, 5% death). One or more pathogenic/likely pathogenic variants were present in 40/109 (37%), and 36/44 (82%) of pathogenic/likely pathogenic variants occurred in sarcomeric genes. The frequency of pathogenic/likely pathogenic variants was not different in patients with familial cardiomyopathy (15/33 with family history versus 25/76 with no family history, P=0.21). TTN truncating variants occurred in a higher percentage of children diagnosed as teenagers (26% teenagers versus 6% younger children, P=0.01), but life-threatening cardiac outcomes occurred in both infants and teenagers with these TTN variants. DCM with left ventricular noncompaction features occurred in 6/6 patients with MYH7 variants between amino acids 1 and 600. Conclusions Sarcomeric variants were common in pediatric DCM. We demonstrated genotype-specific associations with age of diagnosis and cardiac outcomes. In particular, MYH7 had domain-specific association with DCM with left ventricular noncompaction features. Family history did not predict pathogenic/likely pathogenic variants, reinforcing that genetic testing should be considered in all children with idiopathic DCM.
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Cardiomiopatia Dilatada , Adolescente , Criança , Estudos de Associação Genética , Testes Genéticos , Genótipo , Humanos , Mutação , SarcômerosRESUMO
Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on ß-myosin heavy chain (ß-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and nonischemic failing hearts compared to nondiseased hearts. Molecular dynamics (MD) simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent-exposed SH3 domain surface - known for protein-protein interactions - but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1's structure and dynamics - known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that ß-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between ß-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and failing hearts.
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Cadeias Pesadas de Miosina , Sarcômeros , Difosfato de Adenosina/metabolismo , Humanos , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Processamento de Proteína Pós-Traducional , Sarcômeros/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Mutations in the gene that codes for lamin A/C (LMNA) are a common cause of adult-onset cardiomyopathy and heart failure. In this issue of the JCI, Guénantin and Jebeniani et al. identify impaired cardiomyocyte development and maturation as a prenatal feature in a model of laminopathy. Cardiomyocytes carrying the Lmna point mutation H222P misexpressed genes involved in the epithelial-mesenchymal transition and showed decreased methylation at the fourth lysine of histone H3 (H3K4). Notably, inhibiting lysine-specific demethylase 1 in the LMNA H222P mouse model treated this congenital form of cardiomyopathy and improved survival in utero. These data highlight early epigenomic modifications in lamin A/C-mediated pathology and indicate a unique therapeutic strategy for cardiomyopathy.
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Cardiomiopatias , Epigenômica , Animais , Cardiomiopatias/genética , Epigênese Genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Camundongos , MutaçãoRESUMO
Cardiac TnC (cTnC) is highly conserved among mammals, and genetic variants can result in disease by perturbing Ca2+-regulation of myocardial contraction. Here, we report the molecular basis of a human mutation in cTnC's αD-helix (TNNC1-p.C84Y) that impacts conformational dynamics of the D/E central-linker and sampling of discrete states in the N-domain, favoring the "primed" state associated with Ca2+ binding. We demonstrate cTnC's αD-helix normally functions as a central hub that controls minimally frustrated interactions, maintaining evolutionarily conserved rigidity of the N-domain. αD-helix perturbation remotely alters conformational dynamics of the N-domain, compromising its structural rigidity. Transgenic mice carrying this cTnC mutation exhibit altered dynamics of sarcomere function and hypertrophic cardiomyopathy. Together, our data suggest that disruption of evolutionary conserved molecular frustration networks by a myofilament protein mutation may ultimately compromise contractile performance and trigger hypertrophic cardiomyopathy.
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Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including TNNC1 (encoding cardiac troponin C, cTnC). Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca2+ sensitivity of isometric force. CMPs reconstituted 50-50% with both variants mirrored WT in regard to myofilament Ca2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (k TR) at saturating Ca2+ were similar to WT for all preparations. Modeling of Ca2+-dependence of k TR support the observation from Ca2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.
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Troponin is a heterotrimeric Ca2+-binding protein that has a well-established role in regulating striated muscle contraction. However, mounting evidence points to novel cellular functions of troponin, with profound implications in cancer, cardiomyopathy pathogenesis and skeletal muscle aging. Here, we highlight the non-canonical roles and aberrant expression patterns of troponin beyond the sarcomeric milieu. Utilizing bioinformatics tools and online databases, we also provide pathway, subcellular localization, and protein-protein/DNA interaction analyses that support a role for troponin in multiple subcellular compartments. This emerging knowledge challenges the conventional view of troponin as a sarcomere-specific protein exclusively involved in muscle contraction and may transform the way we think about sarcomeric proteins, particularly in the context of human disease and aging.