RESUMO
BACKGROUND: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. METHODS: We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). RESULTS: Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. CONCLUSIONS: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.
Assuntos
Insuficiência Cardíaca Sistólica , Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Contração Miocárdica/fisiologia , RNA Mensageiro/genética , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismoRESUMO
Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.
Assuntos
Cardiomiopatias , Proteínas de Membrana/fisiologia , Miocárdio/metabolismo , Adulto , Idoso , Animais , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação , Miócitos Cardíacos , RatosRESUMO
Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.
Assuntos
Expressão Gênica/fisiologia , Mecanotransdução Celular/fisiologia , Miócitos Cardíacos/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Animais , Cardiomegalia/metabolismo , Insuficiência Cardíaca/metabolismo , Proteínas com Domínio LIM , Camundongos Transgênicos , Proteínas Musculares , Peptídeo Natriurético Encefálico/genética , Peptídeos Natriuréticos/genética , Peptídeos Natriuréticos/metabolismo , Ratos , Ativação Transcricional/genética , Ativação Transcricional/fisiologiaRESUMO
Ambulatory blood pressure monitoring (ABPM) is used for the evaluation of out-of-office blood pressure (BP), however, knowledge concerning the detailed behavior of nocturnal blood pressure (BP) and pulse rate (PR) is limited.A total of 190 participants (64 ± 15 years, 46.3% males) underwent ABPM for diagnosis of hypertension or evaluation of hypertensive therapy. BP and PR were measured automatically by the oscillometric method. From the hourly average ABPM values, the nocturnal time courses (0 AM to 6 AM) of SBP and PR were determined and compared to each other.In general, SBP fell to the lowest level at around midnight and started to increase progressively towards dawn while PR stayed unchanged until 7 AM. Age and gender affected the time course of SBP, most distinctly in the female patients aged ≥ 60 years. The time course of the increase of SBP was very similar in the patients, with BP dipping and non-dipping. The cardiothoracic ratio (CTR) slightly and renal dysfunction modestly facilitated the increase of nocturnal SBP. The nocturnal increase in SBP was not accompanied by an increase of PR in any group or subgroup. The pathophysiology and clinical significance of the early and exclusive increase in nocturnal BP need to be investigated.Average ABPM values in these hypertensive patients showed that BP starts to increase toward dawn without an increase in PR and that this discrepant behavior between BP and RP was most distinct in females 60 or older. The mechanism and clinical significance of such a discordant variation in BP and PR need to be elucidated.
Assuntos
Pressão Sanguínea , Ritmo Circadiano , Frequência Cardíaca , Hipertensão/fisiopatologia , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.
Assuntos
Senescência Celular/genética , Dano ao DNA/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/patologiaRESUMO
BACKGROUND: Fabry disease is an X-linked disease caused by mutations in α-galactosidase A (GLA); these mutations result in the accumulation of its substrates, mainly globotriaosylceramide (Gb3). The accumulation of glycosphingolipids induces pathogenic changes in various organs, including the heart, and Fabry cardiomyopathy is the most frequent cause of death in patients with Fabry disease. Existing therapies to treat Fabry disease have limited efficacy, and new approaches to improve the prognosis of patients with Fabry cardiomyopathy are required. METHODS AND RESULTS: We generated induced pluripotent stem cell (iPSC) lines from a female patient and her son. Each iPSC clone from the female patient showed either deficient or normal GLA activity, which could be used as a Fabry disease model or its isogenic control, respectively. Erosion of the inactivated X chromosome developed heterogeneously among clones, and mono-allelic expression of the GLA gene was maintained for a substantial period in a subset of iPSC clones. Gb3 accumulation was observed in iPSC-derived cardiomyocytes (iPS-CMs) from GLA activity-deficient iPSCs by mass-spectrometry and immunofluorescent staining. The expression of ANP was increased, but the cell surface area was decreased in iPS-CMs from the Fabry model, suggesting that cardiomyopathic change is ongoing at the molecular level in Fabry iPS-CMs. We also established an algorithm for selecting proper Gb3 staining that could be used for high-content analysis-based drug screening. CONCLUSIONS: We generated a Fabry cardiomyopathy model and a drug screening system by using iPS-CMs from a female Fabry patient. Drug screening using our system may help discover new drugs that would improve the prognosis of patients with Fabry cardiomyopathy.
Assuntos
Cardiomiopatias/genética , Avaliação Pré-Clínica de Medicamentos , Doença de Fabry/genética , alfa-Galactosidase/genética , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/fisiopatologia , Doença de Fabry/tratamento farmacológico , Doença de Fabry/fisiopatologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Pacientes , Triexosilceramidas/genética , Inativação do Cromossomo X/genéticaRESUMO
Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentration in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Programas de Rastreamento/métodos , Síndrome de Noonan/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Adulto , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/patologia , Pré-Escolar , Humanos , Masculino , Mutação , Miocárdio/patologia , Miócitos Cardíacos/patologia , Síndrome de Noonan/diagnóstico , Síndrome de Noonan/tratamento farmacológico , Síndrome de Noonan/patologia , Fenótipo , Prevalência , Canais de Potencial de Receptor Transitório/uso terapêuticoRESUMO
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene which encodes dystrophin protein. Dystrophin defect affects cardiac muscle as well as skeletal muscle. Cardiac dysfunction is observed in all patients with DMD over 18 years of age, but there is no curative treatment for DMD cardiomyopathy. To establish novel experimental platforms which reproduce the cardiac phenotype of DMD patients, here we established iPS cell lines from T lymphocytes donated from two DMD patients, with a protocol using Sendai virus vectors. We successfully conducted the differentiation of the DMD patient-specific iPS cells into beating cardiomyocytes. DMD patient-specific iPS cells and iPS cell-derived cardiomyocytes would be a useful in vitro experimental system with which to investigate DMD cardiomyopathy.
Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/citologia , Adolescente , Adulto , Diferenciação Celular , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Miócitos Cardíacos/metabolismo , RNA/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: We assessed radiographic kidney enhancement following an emergency coronary procedure as a predictor of contrast-induced nephropathy (CIN) and poor long-term outcome. METHODS AND RESULTS: We enrolled 126 consecutive patients who underwent an emergency coronary procedure and abdominal X-ray within 24h. We defined kidney enhancement as positive when the density of the kidneys was equal to or higher than that of the lumbar vertebrae. Of the 126 patients, 11 showed kidney enhancement and 115 did not. There were no significant differences in the baseline characteristics of patients with and without kidney enhancement. The incidence of CIN was significantly higher in patients with than in those without kidney enhancement (91% vs. 6%, P<0.01). During a mean follow-up of 21±16 months, 5 of 11 patients with kidney enhancement had poor outcomes, such as renal replacement therapy or death, whereas poor outcomes were observed in only 12 of 115 patients without kidney enhancement. Kaplan-Meier analysis revealed a significant difference in the probability of a poor outcome between patients with and those without kidney enhancement (46% vs. 10%, P<0.01). CONCLUSIONS: Radiographic kidney enhancement following a percutaneous coronary procedure predicts the occurrence of CIN and poor clinical outcome.
Assuntos
Meios de Contraste/efeitos adversos , Nefropatias , Rim/diagnóstico por imagem , Intervenção Coronária Percutânea/efeitos adversos , Complicações Pós-Operatórias , Sistema de Registros , Idoso , Idoso de 80 Anos ou mais , Meios de Contraste/administração & dosagem , Feminino , Seguimentos , Humanos , Nefropatias/induzido quimicamente , Nefropatias/diagnóstico por imagem , Nefropatias/mortalidade , Nefropatias/terapia , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/induzido quimicamente , Complicações Pós-Operatórias/diagnóstico por imagem , Complicações Pós-Operatórias/mortalidade , Complicações Pós-Operatórias/terapia , Radiografia , Terapia de Substituição Renal/métodos , Estudos RetrospectivosRESUMO
The MYH7 R453 variant has been identified in inherited hypertrophic cardiomyopathy (HCM) and is associated with sudden death and a poor prognosis. The detailed clinical course of HCM with the MYH7 R453 variant, from a preserved to a reduced left ventricular ejection fraction, has not been reported. We identified the MYH7 R453C and R453H variants in three patients who progressively developed advanced heart failure requiring circulatory support and summarized the clinical course and echocardiographic parameters of these patients over the years. Because of the rapid disease progression, we consider genetic screening for patients with HCM imperative for future prognosis stratification.
Assuntos
Cardiomiopatia Hipertrófica , Insuficiência Cardíaca , Humanos , Mutação/genética , Volume Sistólico , Função Ventricular Esquerda , Cardiomiopatia Hipertrófica/complicações , Cardiomiopatia Hipertrófica/genética , Insuficiência Cardíaca/genética , Progressão da Doença , Cadeias Pesadas de Miosina/genética , Miosinas Cardíacas/genéticaRESUMO
Study investigators encountered a female Becker muscular dystrophy (BMD) carrier with advanced heart failure (HF) and identified a stop-gain variant in procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3) as a potential second-hit variant. Isogenic induced pluripotent stem cells (iPSCs) with dominant expression of WT-DMD, Δ45-48-DMD, or Δ45-48-DMD with corrected PLOD3 variant were established. Microforce testing using 3-dimensional self-organized tissue rings (SOTRs) generated from iPSC-derived cardiomyocytes (iPSC-CMs) demonstrated that correction of the heterozygous PLOD3 variant did not improve the reduced force, but it significantly recovered the reduced stiffness in Δ45-48-DMD SOTRs. Correction of the PLOD3 variant restored collagen synthesis in iPSC-CMs. Our findings revealed the pathogenesis underlying advanced HF in a female BMD carrier.
RESUMO
BACKGROUND: Restrictive cardiomyopathy in children is rare and outcomes are very poor. However, little information is available concerning genotype-outcome correlations. METHODS: We analyzed the clinical characteristics and genetic testing, including whole exome sequencing, of 28 pediatric restrictive cardiomyopathy patients who were diagnosed from 1998 to 2021 at Osaka University Hospital in Japan. RESULTS: The median age at diagnosis (interquartile range) was 6 (2.25-8.5) years. Eighteen patients received heart transplantations and 5 patients were on the waiting list. One patient died while waiting for transplantation. Pathologic or likely-pathogenic variants were identified in 14 of the 28 (50%) patients, including heterozygous TNNI3 missense variants in 8 patients. TNNT2, MYL2, and FLNC missense variants were also identified. No significant differences in clinical manifestations and hemodynamic parameters between positive and negative pathogenic variants were detected. However, 2- and 5-year survival rates were significantly lower in patients with pathogenic variants (50% and 22%) compared with survival in patients without pathogenic variants (62% and 54%; P=0.0496, log-rank test). No significant differences were detected in the ratio of patients diagnosed at nationwide school heart disease screening program between positive and negative pathogenic variants. Patients diagnosed by school screening showed better transplant-free survival compared with patients diagnosed by heart failure symptoms (P=0.0027 in log-rank test). CONCLUSIONS: In this study, 50% of pediatric restrictive cardiomyopathy patients had pathogenic or likely-pathogenic gene variants, and TNNI3 missense variants were the most frequent. Patients with pathogenic variants showed significantly lower transplant-free survival compared with patients without pathogenic variants.
Assuntos
Cardiomiopatia Restritiva , Cardiopatias , Humanos , Criança , Cardiomiopatia Restritiva/diagnóstico , Cardiomiopatia Restritiva/genética , Testes Genéticos , Genótipo , Heterozigoto , Mutação de Sentido Incorreto , Cardiopatias/genéticaRESUMO
Cardiac sarcoidosis (CS) is the scarring of heart muscles by autoimmunity, leading to heart abnormalities and patients with sarcoidosis with cardiac involvements have poor prognoses. Due to the small number of patients, it is difficult to stratify all patients of CS by human leukocyte antigen (HLA) analysis. We focused on the structure of antigen-recognizing pockets in heterodimeric HLA-class II, in addition to DNA sequences, and extracted high-affinity combinations of antigenic epitopes from candidate autoantigen proteins and HLA. Four HLA heterodimer-haplotypes (DQA1*05:03/05:05/05:06/05:08-DQB1*03:01) were identified in 10 of 68 cases. Nine of the 10 patients had low left ventricular ejection fraction (< 50%). Fourteen amino-acid sequences constituting four HLA anchor pockets encoded by the HLA haplotypes were all common, suggesting DQA1*05:0X-DQB1*03:01 exhibit one group of heterodimeric haplotypes. The heterodimeric haplotypes recognized eight epitopes from different proteins. Assuming that autoimmune mechanisms might be activated by molecular mimicry, we searched for bacterial species having peptide sequences homologous to the eight epitopes. Within the peptide epitopes form the SLC25A4 and DSG2, high-homology sequences were found in Cutibacterium acnes and Mycobacterium tuberculosis, respectively. In this study, we detected the risk heterodimeric haplotypes of ventricular dysfunction in CS by searching for high-affinity HLA-class II and antigenic epitopes from candidate cardiac proteins.
Assuntos
Sarcoidose , Disfunção Ventricular Esquerda , Humanos , Haplótipos , Volume Sistólico , Cadeias alfa de HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Função Ventricular Esquerda , Antígenos HLA-DQ/genética , Antígenos de Histocompatibilidade Classe I/genética , Sarcoidose/genética , Epitopos , Disfunção Ventricular Esquerda/genética , Peptídeos/genética , Cadeias HLA-DRB1/genética , Frequência do Gene , Alelos , Predisposição Genética para DoençaRESUMO
Phospholamban p.Arg14del is reported to cause hereditary cardiomyopathy with malignant ventricular tachycardia (VT) and advanced heart failure. However, the clinical courses of Japanese cardiomyopathy patients with phospholamban p.Arg14del remain uncharacterized. We identified five patients with this variant. All patients were diagnosed with dilated cardiomyopathy (DCM), developed end-stage heart failure and experienced VT requiring implantable cardioverter defibrillator discharge. Four patients survived after implantation of a left ventricular assist device (LVAD), while one patient who refused LVAD implantation died of heart failure. Based on the severe course of the disease, we propose genetic screening for phospholamban p.Arg14del in DCM patients.
Assuntos
Proteínas de Ligação ao Cálcio , Cardiomiopatia Dilatada , Insuficiência Cardíaca , Taquicardia Ventricular , Arritmias Cardíacas/complicações , Proteínas de Ligação ao Cálcio/genética , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Desfibriladores Implantáveis , Insuficiência Cardíaca/complicações , Humanos , Japão , Taquicardia Ventricular/etiologiaRESUMO
BACKGROUND: The Δ160E mutation in TNNT2, which encodes troponin T, is a rare pathogenic variant identified in patients with hypertrophic cardiomyopathy and is associated with poor prognosis. Thus, a convenient human model recapitulating the pathological phenotype caused by TNNT2 Δ160E is required for therapeutic development. METHODS: We identified a heterozygous in-frame deletion mutation (c.478_480del, p.Δ160E) in TNNT2 in a patient with familial hypertrophic cardiomyopathy showing progressive left ventricular systolic dysfunction, leading to advanced heart failure. To investigate the pathological phenotype caused by Δ160E, we generated a set of isogenic induced pluripotent stem cells carrying the heterozygous Δ160E, homozygously corrected or homozygously introduced Δ160E using genome editing and differentiated them into cardiomyocytes (Hetero-Δ160E-, wild type-, and Homo-Δ160E-induced pluripotent stem cells [iPSC]-derived cardiomyocytes [iPSC-CMs]). RESULTS: Hetero-Δ160E-iPSC-CMs exhibited prolonged calcium decay, relaxation impairment, and hypertrophy compared to wild type-iPSC-CMs. Notably, these phenotypes were further exacerbated in Homo-Δ160E-iPSC-CMs. Overexpression of R-GECO-fused Δ160E mutant troponin T prolonged decay time and time to peak of the myofilament-localized calcium transient in iPSC-CMs, indicating that sarcomeric calcium retention with Δ160E may affect intracellular calcium concentration. High-content imaging analysis detected remarkable nuclear translocation of NFATc1, especially in Homo-Δ160E-iPSC-CMs, indicating that the Δ160E mutation promotes hypertrophic signaling pathway in a dose-dependent manner. Increased phosphorylation of CaMKIIδ (calcium/calmodulin-dependent protein kinase IIδ) and phospholamban at Thr17 was observed in Homo- and Hetero-Δ160E-iPSC-CMs. Epigallocatechin-3-gallate, a calcium desensitizing compound, shortened prolonged calcium decay and relaxation duration in Δ160E-iPSC-CMs. CONCLUSIONS: Isogenic iPSC-CMs recapitulate the prolonged calcium decay, relaxation impairment, and subsequent calcium-regulated signaling pathways caused by the TNNT2 Δ160E mutation and can serve as a human model for therapeutic development to prevent hypertrophic cardiomyopathy pathology.
Assuntos
Cardiomiopatias , Cardiomiopatia Hipertrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Troponina T/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatias/patologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismoRESUMO
Dilated cardiomyopathy (DCM) is a major cause of heart failure, characterized by ventricular dilatation and systolic dysfunction. Familial DCM is reportedly caused by mutations in more than 50 genes, requiring precise disease stratification based on genetic information. However, the underlying genetic causes of 60 to 80% of familial DCM cases remain unknown. Here, we identified that homozygous truncating mutations in the gene encoding Bcl-2associated athanogene (BAG) co-chaperone 5 (BAG5) caused inherited DCM in five patients among four unrelated families with complete penetrance. BAG5 acts as a nucleotide exchange factor for heat shock cognate 71 kDa protein (HSC70), promoting adenosine diphosphate release and activating HSC70-mediated protein folding. Bag5 mutant knock-in mice exhibited ventricular dilatation, arrhythmogenicity, and poor prognosis under catecholamine stimulation, recapitulating the human DCM phenotype, and administration of an adeno-associated virus 9 vector carrying the wild-type BAG5 gene could fully ameliorate these DCM phenotypes. Immunocytochemical analysis revealed that BAG5 localized to junctional membrane complexes (JMCs), critical microdomains for calcium handling. Bag5-mutant mouse cardiomyocytes exhibited decreased abundance of functional JMC proteins under catecholamine stimulation, disrupted JMC structure, and calcium handling abnormalities. We also identified heterozygous truncating mutations in three patients with tachycardia-induced cardiomyopathy, a reversible DCM subtype associated with abnormal calcium homeostasis. Our study suggests that loss-of-function mutations in BAG5 can cause DCM, that BAG5 may be a target for genetic testing in cases of DCM, and that gene therapy may potentially be a treatment for this disease.
Assuntos
Cardiomiopatia Dilatada , Transplante de Coração , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Humanos , Camundongos , Mutação/genética , Miócitos Cardíacos/metabolismo , FenótipoRESUMO
BACKGROUND: Atrial fibrillation (AF) is observed in patients with Brugada syndrome (BS), especially those showing coved-type electrocardiogram (ECG) pattern. Using P-wave signal-averaged ECG (P-SAE), we investigated whether increased intraatrial conduction abnormality contributed to AF generation in BS patients. METHODS: Twenty BS patients and 20 age- and gender-matched healthy controls were enrolled. At the P-SAE recording, 12 of the 20 BS patients showed coved-type (C-BS) and eight showed saddleback-type (S-BS). The total duration (Ad) and root mean square voltage for the terminal 20 ms (LP(20) ) of the filtered P wave were measured. P-wave dispersion (P-disp) was defined as the difference between the maximum and minimum, measured from 16 precordial recording sites. RESULTS: BS patients had a significantly longer Ad (128.2 ± 7.6 vs 116.3 ± 8.2 ms, P < 0.0001), lower LP(20) (2.6 ± 0.9 vs 3.4 ± 0.8 µV, P < 0.01), and greater P-disp (15.5 ± 7.0 vs 7.4 ± 3.2 ms, P < 0.0001) than the controls. C-BS patients had significantly longer Ad (131.0 ± 7.2 vs 124.1 ± 6.8 ms, P < 0.05) and lower LP(20) (2.2 ± 0.6 vs 3.2 ± 1.0 µV, P < 0.05) than S-BS patients. All C-BS patients and only three S-BS patients had atrial late potential (100% vs 38%, P < 0.01). CONCLUSION: Intraatrial conduction delay and its heterogeneity may exist in BS patients, especially those showing coved-type ECG patterns. These atrial electrical abnormalities could be a substrate for atrial reentrant tachycardia such as AF.
Assuntos
Síndrome de Brugada/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Adulto , Idoso , Eletrocardiografia , Feminino , Átrios do Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Processamento de Sinais Assistido por ComputadorRESUMO
Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irreversible or was altered after withdrawal of the structural support, the hiPS-CMs were harvested from the three different patterns and reseeded on the non-scaffold (flat) pattern. After culturing for one more week, the improvements in morphological and functional properties diminished, although hiPS-CMs pre-cultured on the aligned pattern retained the molecular features of development, which were even more significant as compared to that observed during the pre-culture stage. Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of the external support, which might persist throughout hiPS-CM development.