RESUMO
Atrial fibrillation (AF) is a common cardiac arrhythmia and a major risk factor for stroke, heart failure, and premature death. The pathogenesis of AF remains poorly understood, which contributes to the current lack of highly effective treatments. To understand the genetic variation and biology underlying AF, we undertook a genome-wide association study (GWAS) of 6,337 AF individuals and 61,607 AF-free individuals from Norway, including replication in an additional 30,679 AF individuals and 278,895 AF-free individuals. Through genotyping and dense imputation mapping from whole-genome sequencing, we tested almost nine million genetic variants across the genome and identified seven risk loci, including two novel loci. One novel locus (lead single-nucleotide variant [SNV] rs12614435; p = 6.76 × 10-18) comprised intronic and several highly correlated missense variants situated in the I-, A-, and M-bands of titin, which is the largest protein in humans and responsible for the passive elasticity of heart and skeletal muscle. The other novel locus (lead SNV rs56202902; p = 1.54 × 10-11) covered a large, gene-dense chromosome 1 region that has previously been linked to cardiac conduction. Pathway and functional enrichment analyses suggested that many AF-associated genetic variants act through a mechanism of impaired muscle cell differentiation and tissue formation during fetal heart development.
Assuntos
Fibrilação Atrial/genética , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Coração/embriologia , Sequências Reguladoras de Ácido Nucleico/genética , Humanos , Padrões de Herança/genética , Herança Multifatorial/genética , Especificidade de Órgãos/genética , Mapeamento Físico do Cromossomo , Locos de Características Quantitativas/genética , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 (DLG1) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current (Ito) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease.NEW & NOTEWORTHY The gene encoding SAP97 (DLG1) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1-encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.
Assuntos
Arritmias Cardíacas/metabolismo , Proteína 1 Homóloga a Discs-Large/metabolismo , Coração/fisiopatologia , Miocárdio/metabolismo , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Proteína 1 Homóloga a Discs-Large/genética , Humanos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismoRESUMO
RATIONALE: In cardiomyocytes, NaV1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. OBJECTIVE: The objective of this study was to test whether NaV1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. METHODS AND RESULTS: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1Δ314-315 or Kir2.1R44A/R46A with wild-type (WT) NaV1.5WT in heterologous cells reduced inward sodium current compared with NaV1.5WT alone or coexpressed with Kir2.1WT. In cell surface biotinylation experiments, expression of Kir2.1Δ314-315 reduced NaV1.5 channel surface expression. Glycosylation analysis suggested that NaV1.5WT and Kir2.1WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of NaV1.5WT, and vice versa, whereas coexpression with Kir2.1Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with NaV1.5WT and Kir2.1WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1WT, NaV1.5WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that NaV1.5WT channels interact with AP1 through the NaV1.5Y1810 residue, suggesting that, like for Kir2.1WT, AP1 can mark NaV1.5 channels for incorporation into clathrin-coated vesicles at the trans-Golgi. Silencing the AP1 Ï-adaptin subunit in human induced pluripotent stem cell-derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. CONCLUSIONS: The NaV1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of NaV1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sarcolema/metabolismo , Potenciais de Ação , Animais , Corantes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-Dawley , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
There is accumulating evidence during sepsis that cardiomyocyte (CM) homeostasis is compromised, resulting in cardiac dysfunction. An important role for complement in these outcomes is now demonstrated. Addition of C5a to electrically paced CMs caused prolonged elevations of intracellular Ca(2+) concentrations during diastole, together with the appearance of spontaneous Ca(2+) transients. In polymicrobial sepsis in mice, we found that three key homeostasis-regulating proteins in CMs were reduced: Na(+)/K(+)-ATPase, which is vital for effective action potentials in CMs, and two intracellular Ca(2+) concentration regulatory proteins, that is, sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and the Na(+)/Ca(2+) exchanger. Sepsis caused reduced mRNA levels and reductions in protein concentrations in CMs for all three proteins. The absence of either C5a receptor mitigated sepsis-induced reductions in the three regulatory proteins. Absence of either C5a receptor (C5aR1 or C5aR2) diminished development of defective systolic and diastolic echocardiographic/Doppler parameters developing in the heart (cardiac output, left ventricular stroke volume, isovolumic relaxation, E' septal annulus, E/E' septal annulus, left ventricular diastolic volume). We also found in CMs from septic mice the presence of defective current densities for Ik1, l-type calcium channel, and Na(+)/Ca(2+) exchanger. These defects were accentuated in the copresence of C5a. These data suggest complement-related mechanisms responsible for development of cardiac dysfunction during sepsis.
Assuntos
Coinfecção/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Sepse/imunologia , Sepse/fisiopatologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/imunologia , Coinfecção/microbiologia , Coinfecção/fisiopatologia , Complemento C5a/imunologia , Citoplasma/química , Citoplasma/metabolismo , Coração/fisiopatologia , Camundongos , Miócitos Cardíacos/microbiologia , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sepse/complicaçõesRESUMO
BACKGROUND: In catecholaminergic polymorphic ventricular tachycardia (CPVT), cardiac Purkinje cells (PCs) appear more susceptible to Ca(2+) dysfunction than ventricular myocytes (VMs). The underlying mechanisms remain unknown. Using a CPVT mouse (RyR2(R4496C+/Cx40eGFP)), we tested whether PC intracellular Ca(2+) ([Ca(2+)]i) dysregulation results from a constitutive [Na(+)]i surplus relative to VMs. METHODS AND RESULTS: Simultaneous optical mapping of voltage and [Ca(2+)]i in CPVT hearts showed that spontaneous Ca(2+) release preceded pacing-induced triggered activity at subendocardial PCs. On simultaneous current-clamp and Ca(2+) imaging, early and delayed afterdepolarizations trailed spontaneous Ca(2+) release and were more frequent in CPVT PCs than CPVT VMs. As a result of increased activity of mutant ryanodine receptor type 2 channels, sarcoplasmic reticulum Ca(2+) load, measured by caffeine-induced Ca(2+) transients, was lower in CPVT VMs and PCs than respective controls, and sarcoplasmic reticulum fractional release was greater in both CPVT PCs and VMs than respective controls. [Na(+)]i was higher in both control and CPVT PCs than VMs, whereas the density of the Na(+)/Ca(2+) exchanger current was not different between PCs and VMs. Computer simulations using a PC model predicted that the elevated [Na(+)]i of PCs promoted delayed afterdepolarizations, which were always preceded by spontaneous Ca(2+) release events from hyperactive ryanodine receptor type 2 channels. Increasing [Na(+)]i monotonically increased delayed afterdepolarization frequency. Confocal imaging experiments showed that postpacing Ca(2+) spark frequency was highest in intact CPVT PCs, but such differences were reversed on saponin-induced membrane permeabilization, indicating that differences in [Na(+)]i played a central role. CONCLUSIONS: In CPVT mice, the constitutive [Na(+)]i excess of PCs promotes triggered activity and arrhythmogenesis at lower levels of stress than VMs.
Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/fisiologia , Sódio/metabolismo , Taquicardia Ventricular/metabolismo , Animais , Sinalização do Cálcio , Humanos , Camundongos , Células de PurkinjeRESUMO
Maternal hyperglycemia is a risk factor for fetal cardiac anomalies. This study aimed to assess the effect of high glucose on human induced pluripotent stem cell-derived cardiomyocyte self-assembly into 3D microtissues and their calcium handling. Stem cells were differentiated to beating cardiomyocytes using established protocols. On the final day of the differentiation process, cells were treated with control media, 12 mM glucose, or 12 mM mannitol (an osmolality control). Once beating, the cardiac cells were dissociated with trypsin, collected, mixed with collagen, and plated into custom-made silicone micro molds in order to generate 3D cardiac microtissues. A time-lapse microscope took pictures every 4 h to quantify the kinetics of cellular self-assembly of 3D cardiac tissues. Fiber widths were recorded at 4-h intervals and plotted over time to assess cardiomyocyte 3D fiber self-assembly. Microtissue calcium flux was recorded with optical mapping by pacing microtissues at 0.5 and 1.0 Hz. Exposure to high glucose impaired the ability of cardiomyocytes to self-assemble into compact microtissues, but not their ability to spontaneously contract. Glucose-exposed cardiomyocytes took longer to self-assemble and finished as thicker fibers. When cardiac microtissues were paced at 0.5 and 1.0 Hz, those exposed to high glucose had altered calcium handling with shorter calcium transient durations, but larger amplitudes of the calcium transient when compared to controls. Additional studies are needed to elucidate a potential mechanism for these findings. This model provides a novel method to assess the effects of exposures on the cardiomyocytes' intrinsic abilities for organogenesis in 3D.
Assuntos
Glucose/farmacologia , Hiperglicemia/complicações , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Cálcio/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Citometria de Fluxo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Imagens com Corantes Sensíveis à VoltagemRESUMO
AIMS: Mutations of cardiac sarcomere genes have been identified to cause HCM, but the molecular mechanisms that lead to cardiomyocyte hypertrophy and risk for sudden death are uncertain. The aim of this study was to examine HCM disease mechanisms at play during cardiac differentiation of human HCM specific pluripotent stem cells. METHODS AND RESULTS: We generated a human embryonic stem cell (hESC) line carrying a naturally occurring mutation of MYPBC3 (c.2905 +1 G >A) to study HCM pathogenesis during cardiac differentiation. HCM-specific hESC-derived cardiomyocytes (hESC-CMs) displayed hallmark aspects of HCM including sarcomere disarray, hypertrophy and impaired calcium impulse propagation. HCM hESC-CMs presented a transient haploinsufficiency of cMyBP-C during cardiomyocyte differentiation, but by day 30 post-differentiation cMyBP-C levels were similar to control hESC-CMs. Gene transfer of full-length MYBPC3 during differentiation prevented hypertrophy, sarcomere disarray and improved calcium impulse propagation in HCM hESC-CMs. CONCLUSION(S): These findings point to the critical role of MYBPC3 during sarcomere assembly in cardiac myocyte differentiation and suggest developmental influences of MYBPC3 truncating mutations on the mature hypertrophic phenotype.
Assuntos
Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Células-Tronco Embrionárias/citologia , Mutação , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/patologia , Análise Mutacional de DNA , Expressão Gênica , Humanos , Cariótipo , Organogênese , Fenótipo , Sarcômeros/metabolismo , Transcrição Gênica , Transdução GenéticaRESUMO
The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.
Assuntos
Cardiomiopatias/etiologia , Modelos Animais de Doenças , Histonas/efeitos adversos , Mitocôndrias/patologia , Sepse/complicações , Animais , Cálcio/metabolismo , Cardiomiopatias/sangue , Cardiomiopatias/diagnóstico , Proteínas de Transporte/fisiologia , Caspase 1/fisiologia , Células Cultivadas , Histonas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Sepse/sangue , Sepse/patologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologiaRESUMO
Cardiac optical mapping has proven to be a powerful technology for studying cardiovascular function and disease. The development and scientific impact of this methodology are well-documented. Because of its relevance in cardiac research, this imaging technology advances at a rapid pace. Here, we review technological and scientific developments during the past several years and look toward the future. First, we explore key components of a modern optical mapping set-up, focusing on: (1) new camera technologies; (2) powerful light-emitting-diodes (from ultraviolet to red) for illumination; (3) improved optical filter technology; (4) new synthetic and optogenetic fluorescent probes; (5) optical mapping with motion and contraction; (6) new multiparametric optical mapping techniques; and (7) photon scattering effects in thick tissue preparations. We then look at recent optical mapping studies in single cells, cardiomyocyte monolayers, atria, and whole hearts. Finally, we briefly look into the possible future roles of optical mapping in the development of regenerative cardiac research, cardiac cell therapies, and molecular genetic advances.
Assuntos
Sinalização do Cálcio , Sistema de Condução Cardíaco/metabolismo , Cardiopatias/metabolismo , Miocárdio/metabolismo , Imagens com Corantes Sensíveis à Voltagem , Potenciais de Ação , Animais , Desenho de Equipamento , Corantes Fluorescentes/química , Sistema de Condução Cardíaco/fisiopatologia , Cardiopatias/diagnóstico , Cardiopatias/fisiopatologia , Humanos , Processamento de Imagem Assistida por Computador , Miócitos Cardíacos/metabolismo , Fatores de Tempo , Imagens com Corantes Sensíveis à Voltagem/instrumentação , Imagens com Corantes Sensíveis à Voltagem/métodos , Imagens com Corantes Sensíveis à Voltagem/tendênciasRESUMO
RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a powerful in vitro tool to investigate disease mechanisms and to perform patient-specific drug screening. To date, electrophysiological analysis of iPSC-CMs has been limited to single-cell recordings or low-resolution microelectrode array mapping of small cardiomyocyte aggregates. New methods of generating and optically mapping impulse propagation of large human iPSC-CM cardiac monolayers are needed. OBJECTIVE: Our first aim was to develop an imaging platform with versatility for multiparameter electrophysiological mapping of cardiac preparations, including human iPSC-CM monolayers. Our second aim was to create large electrically coupled human iPSC-CM monolayers for simultaneous action potential and calcium wave propagation measurements. METHODS AND RESULTS: A fluorescence imaging platform based on electronically controlled light-emitting diode illumination, a multiband emission filter, and single camera sensor was developed and utilized to monitor simultaneously action potential and intracellular calcium wave propagation in cardiac preparations. Multiple, large-diameter (≥1 cm), electrically coupled human cardiac monolayers were then generated that propagated action potentials and calcium waves at velocities similar to those commonly observed in rodent cardiac monolayers. CONCLUSIONS: The multiparametric imaging system presented here offers a scalable enabling technology to measure simultaneously action potential and intracellular calcium wave amplitude and dynamics of cardiac monolayers. The advent of large-scale production of human iPSC-CMs makes it possible to now generate sufficient numbers of uniform cardiac monolayers that can be utilized for the study of arrhythmia mechanisms and offers advantages over commonly used rodent models.
Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Engenharia Genética/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Separação Celular/métodos , Células Cultivadas , HumanosRESUMO
RATIONALE: Cardiomyocytes (CMs) differentiated from human pluripotent stem cells (PSCs) are increasingly being used for cardiovascular research, including disease modeling, and hold promise for clinical applications. Current cardiac differentiation protocols exhibit variable success across different PSC lines and are primarily based on the application of growth factors. However, extracellular matrix is also fundamentally involved in cardiac development from the earliest morphogenetic events, such as gastrulation. OBJECTIVE: We sought to develop a more effective protocol for cardiac differentiation of human PSCs by using extracellular matrix in combination with growth factors known to promote cardiogenesis. METHODS AND RESULTS: PSCs were cultured as monolayers on Matrigel, an extracellular matrix preparation, and subsequently overlayed with Matrigel. The matrix sandwich promoted an epithelial-to-mesenchymal transition as in gastrulation with the generation of N-cadherin-positive mesenchymal cells. Combining the matrix sandwich with sequential application of growth factors (Activin A, bone morphogenetic protein 4, and basic fibroblast growth factor) generated CMs with high purity (up to 98%) and yield (up to 11 CMs/input PSC) from multiple PSC lines. The resulting CMs progressively matured over 30 days in culture based on myofilament expression pattern and mitotic activity. Action potentials typical of embryonic nodal, atrial, and ventricular CMs were observed, and monolayers of electrically coupled CMs modeled cardiac tissue and basic arrhythmia mechanisms. CONCLUSIONS: Dynamic extracellular matrix application promoted epithelial-mesenchymal transition of human PSCs and complemented growth factor signaling to enable robust cardiac differentiation.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Colágeno , Matriz Extracelular/fisiologia , Laminina , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , Proteoglicanas , Ativinas/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Combinação de Medicamentos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The R403Q mutation of human cardiac ß-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human ß-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human ß-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at â¼40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after ß-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.
RESUMO
Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca2+-handling proteins in atria, including the sarcoplasmic reticulum Ca2+-ATPase (SERCA), a major component of beat-to-beat Ca2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca2+ transient amplitude and SERCA-mediated Ca2+ uptake. Adrenergic stimulation further increases the Ca2+ transient amplitude but does not affect Ca2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca2+-induced Ca2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF.
RESUMO
A common genetic risk factor for bipolar disorder is CACNA1C, a gene that is also critical for cardiac rhythm. The impact of CACNA1C mutations on bipolar patient cardiac rhythm is unknown. Here, we report the cardiac electrophysiological implications of a bipolar disorder-associated genetic risk factor in CACNA1C using patient induced pluripotent stem cell-derived cardiomyocytes. Results indicate that the CACNA1C bipolar disorder-related mutation causes cardiac electrical impulse conduction slowing mediated by impaired intercellular coupling via connexin 43 gap junctions. In vitro gene therapy to restore connexin 43 expression increased cardiac electrical impulse conduction velocity and protected against thioridazine-induced QT prolongation. Patients positive for bipolar disorder CACNA1C genetic risk factors may have elevated proarrhythmic risk for adverse events in response to psychiatric medications that slow conduction or prolong the QT interval. This in vitro diagnostic tool enables cardiac testing specific to patients with psychiatric disorders to determine their sensitivity to off-target effects of psychiatric medications.
Bipolar disorder (BD) is associated with genetic risk factors that present as mutations in specific genes. One gene commonly associated with BD is the calcium channel gene CACNA1C, found in the brain and the heart. The impact of CACNA1C mutation on cardiac function in patients with BD is unclear. Here, we created a BD CACNA1C mutant patient "heart in a dish" using patient-specific stem cells. Gene editing was also used to correct the mutation to create an isogenic control cell line. We found that the BD calcium gene mutation caused slow electrical impulse propagation, reduced the function of the calcium channel, and was associated with low intercellular communication channels called connexin. Using connexin gene therapy in vitro, the the cardiac dysfunction could be corrected and cured. This new approach offers patient-specific hearts-in-a-dish that can be used to ensure that medications will not cause heart racing or arrhythmias.
RESUMO
Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca(2+) sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca(2+) transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.
Assuntos
Contração Muscular , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Troponina I/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Animais , Western Blotting , Cálcio/metabolismo , Meios de Cultura Livres de Soro , Endotelina-1/farmacologia , Feminino , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Miofibrilas/efeitos dos fármacos , Miofibrilas/fisiologia , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Sarcômeros/genética , Sarcômeros/metabolismo , Serina/genética , Serina/metabolismo , Fatores de Tempo , Troponina I/genéticaRESUMO
Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Humanos , Cardiotoxicidade , Cálcio/metabolismo , Diferenciação Celular , Células CultivadasRESUMO
hiPSC-CMs are being considered by the Food and Drug Administration and other regulatory agencies for in vitro cardiotoxicity screening to provide human-relevant safety data. Widespread adoption of hiPSC-CMs in regulatory and academic science is limited by the immature, fetal-like phenotype of the cells. Here, to advance the maturation state of hiPSC-CMs, we developed and validated a human perinatal stem cell-derived extracellular matrix coating applied to high-throughput cell culture plates. We also present and validate a cardiac optical mapping device designed for high-throughput functional assessment of mature hiPSC-CM action potentials using voltage-sensitive dye and calcium transients using calcium-sensitive dyes or genetically encoded calcium indicators (GECI, GCaMP6). We utilize the optical mapping device to provide new biological insight into mature chamber-specific hiPSC-CMs, responsiveness to cardioactive drugs, the effect of GCaMP6 genetic variants on electrophysiological function, and the effect of daily ß-receptor stimulation on hiPSC-CM monolayer function and SERCA2a expression.
RESUMO
We have recently shown that antibody-induced blockade of C5a, C5a receptors, or IL-17A greatly reduced the harmful outcomes of sepsis. In the current study, normal cardiomyocytes from young (300 g) male Sprague-Dawley rats responded in vitro to C5a (ED(50)=55 nM) with release of IL-6 and TNFα, peaking between 2 to 8 h. Neutralizing antibodies to mouse C5a or IL-17A (ED(50)=40 µg for each, based on improved survival) reduced spontaneous in vitro release of cardiosuppressive cytokines and chemokines in cardiomyocytes obtained from mice with polymicrobial sepsis. A non-neutralizing C5a antibody had no such effects. Cardiomyocytes from septic mice (C57Bl/6) showed increased mRNA for TNFR1, IL-6 (gp80), and C5aR at 6 h after sepsis. Cardiomyocytes from septic C5aR(-/-) or C5L2(-/-) mice did not show spontaneous in vitro release of cytokines and chemokines. These data suggest that cardiomyocytes from septic mice release suppressive cytokines in a C5a-, C5aR-, and IL-17A-dependent manner, followed by mediator reactivity with receptors on cardiomyocytes, resulting in defective contractility and relaxation. These data may be relevant to a strategy for the treatment of heart dysfunction developing during sepsis.
Assuntos
Complemento C5a/metabolismo , Citocinas/metabolismo , Miócitos Cardíacos/metabolismo , Sepse/metabolismo , Animais , Quimiocina CCL3/sangue , Quimiocina CCL3/metabolismo , Quimiocina CXCL2/sangue , Quimiocina CXCL2/metabolismo , Quimiocinas/sangue , Quimiocinas/metabolismo , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Interleucina-6/sangue , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/genética , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Diastolic dysfunction (DD) underlies heart failure with preserved ejection fraction (HFpEF), a clinical syndrome associated with aging that is becoming more prevalent. Despite extensive clinical studies, no effective treatment exists for HFpEF. Recent findings suggest that oxidative stress contributes to the pathophysiology of DD, but molecular mechanisms underpinning redox-sensitive cardiac remodeling in DD remain obscure. Using transgenic mice with mitochondria-targeted NOX4 overexpression (Nox4TG618) as a model, we demonstrate that NOX4-dependent mitochondrial oxidative stress induces DD in mice as measured by increased E/E', isovolumic relaxation time, Tau Glantz and reduced dP/dtmin while EF is preserved. In Nox4TG618 mice, fragmentation of cardiomyocyte mitochondria, increased DRP1 phosphorylation, decreased expression of MFN2, and a higher percentage of apoptotic cells in the myocardium are associated with lower ATP-driven and maximal mitochondrial oxygen consumption rates, a decrease in respiratory reserve, and a decrease in citrate synthase and Complex I activities. Transgenic mice have an increased concentration of TGFß and osteopontin in LV lysates, as well as MCP-1 in plasma, which correlates with a higher percentage of LV myocardial periostin- and ACTA2-positive cells compared with wild-type mice. Accordingly, the levels of ECM as measured by Picrosirius Red staining as well as interstitial deposition of collagen I are elevated in the myocardium of Nox4TG618 mice. The LV tissue of Nox4TG618 mice also exhibited increased ICaL current, calpain 2 expression, and altered/disrupted Z-disc structure. As it pertains to human pathology, similar changes were found in samples of LV from patients with DD. Finally, treatment with GKT137831, a specific NOX1 and NOX4 inhibitor, or overexpression of mCAT attenuated myocardial fibrosis and prevented DD in the Nox4TG618 mice. Together, our results indicate that mitochondrial oxidative stress contributes to DD by causing mitochondrial dysfunction, impaired mitochondrial dynamics, increased synthesis of pro-inflammatory and pro-fibrotic cytokines, activation of fibroblasts, and the accumulation of extracellular matrix, which leads to interstitial fibrosis and passive stiffness of the myocardium. Further, mitochondrial oxidative stress increases cardiomyocyte Ca2+ influx, which worsens CM relaxation and raises the LV filling pressure in conjunction with structural proteolytic damage.
RESUMO
Background: Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. Methods: To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Results: Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. Conclusions: We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Funding: Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.