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1.
Nat Commun ; 10(1): 5754, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848331

RESUMO

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.


Assuntos
Insuficiência Cardíaca/etiologia , Músculo Esquelético/patologia , Distrofias Musculares/complicações , Miócitos Cardíacos/patologia , Transferases/genética , Adulto , Fatores Etários , Animais , Animais Recém-Nascidos , Sistemas CRISPR-Cas/genética , Células Cultivadas , Modelos Animais de Doenças , Distroglicanas/metabolismo , Feminino , Técnicas de Inativação de Genes , Glicosilação , Células HEK293 , Insuficiência Cardíaca/patologia , Ventrículos do Coração/citologia , Ventrículos do Coração/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Contração Miocárdica/genética , Miócitos Cardíacos/citologia , Cultura Primária de Células , Sarcolema/patologia , Transferases/metabolismo
2.
BMC Med Genet ; 20(1): 178, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718582

RESUMO

BACKGROUND: Oxytocin receptor (OXTR) gene variants have been shown to affect the prevalence of preterm birth, mode of delivery and oxytocin (OXT) requirements for labor induction and augmentation. We hypothesized that this might be associated with different myometrium responses to oxytocin. Our aim was to investigate the influence of a selection of eight OXTR gene single nucleotide variants on oxytocin-induced stimulation of human myometrium contractility in vitro. METHODS: Human myometrium biopsies were collected during elective cesarean sections at term, if patients had given informed consent. Myometrial strips were submerged under tension in an organ bath and allowed to contract; the remaining material was stored at - 80 °C for further determination of relevant genetics and mRNA level. The area under the curve (AUC) of all contractions taking place in the absence of OXT and of those occurring upon OXT addition (for 30 min each) was measured. OXT stimulation, defined as the ratio between AUC measurements after OXT addition and those in the absence of OXT was calculated for each strip. TaqMan™ Assays were used to detect the allele distribution of the eight OXTR variants and to determine the relative amounts of OXTR-mRNA in the samples. For each variant, oxytocin stimulation of contractility was compared between samples homozygous for the reference allele (reference group) and samples with at least one variant allele (variant group) by linear regression. RESULTS: Sixty samples were included in the present study. For rs1042778, rs11706648, rs4686301, rs53576, rs237895, and rs237902, OXT stimulation was similar in the reference and in the variant groups. However, the values of OXT stimulation differed significantly between the reference and the variant groups for rs4686302 (3.1 vs. 4.1 times; p = 0.022) and rs237888 (3.2 vs. 5.5 times; p = 0.001). No significant differences between the levels of OXTR-mRNA in the various reference and corresponding variant groups were detected. CONCLUSIONS: Patients with variant alleles of rs237888 and/or rs4686302 may be more sensitive to oxytocin stimulation, explaining why these sequence variants have been associated with lower cesarean section prevalence and premature birth, respectively.


Assuntos
Contração Miocárdica/genética , Polimorfismo de Nucleotídeo Único , Receptores de Ocitocina/genética , Adulto , Alelos , Biópsia , Feminino , Humanos , Técnicas In Vitro , Miométrio/metabolismo , Miométrio/patologia
3.
Nat Commun ; 10(1): 2685, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213605

RESUMO

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human ß-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human ß-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif (IHM) lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, which could lead to hypercontractility at the molecular level.


Assuntos
Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Contração Miocárdica/genética , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Animais , Miosinas Cardíacas/genética , Linhagem Celular , Movimento Celular/genética , Coração/fisiopatologia , Humanos , Camundongos , Mutação , Mioblastos , Cadeias Pesadas de Miosina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Yakugaku Zasshi ; 139(5): 793-805, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31061349

RESUMO

Angiotensin II (Ang II) is an intrinsic peptide having strong vasopressor effects, and thus, it plays an important role in the physiological regulation of blood pressure. The vasopressor effects of Ang II include direct contraction of myocardium and vascular smooth muscles (SMs) along with aldosterone-mediated sodium retention. In addition, indirect vascular contractions induced by noradrenaline (NA), the release of which is mediated through Ang II receptor type 1 (AT1) existing at the sympathetic nerve terminals (SNTs), also contribute to the vasopressor effects of Ang II. Stimulation of NA release from SNTs by Ang II also occurs in the myocardium leading to an increase in heart rate and cardiac contraction. Furthermore, Ang II enhances the contractions of non-vascular SMs, such as vas deferens, through induction of NA release from the SNTs. We have found that Ang II attenuated vagus nerve stimulation-induced bradycardia in a losartan-sensitive manner. This suggests that Ang II attenuates vagus nerve stimulation-induced bradycardia by inhibiting acetylcholine (ACh) release from the parasympathetic nerve terminals (PNTs) through activation of the AT1 receptor. Ang II was also reported to attenuate the release of ACh from the PNTs in SMs, such as stomach and airway, thus suppressing their contractile functions. There are, however, conflicting reports of the effects of Ang II on parasympathetic nerve-mediated contractile regulation of SMs. In this review, we have highlighted the relevant research articles including our experimental reports on the regulation of sympathetic and parasympathetic nerve-mediated excitation and contraction by Ang II along with the future prospects.


Assuntos
Angiotensina II/fisiologia , Vias Autônomas/fisiologia , Músculo Liso/fisiologia , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sistema Nervoso Simpático/fisiologia , Transmissão Sináptica/genética , Acetilcolina/metabolismo , Angiotensina II/biossíntese , Animais , Frequência Cardíaca/genética , Humanos , Norepinefrina/metabolismo , Ratos , Sistema Nervoso Simpático/metabolismo
5.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1627-1641, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30946956

RESUMO

Lipopolysaccharide (LPS), an essential component of outer membrane of the Gram-negative bacteria, plays a pivotal role in myocardial anomalies in sepsis. Recent evidence depicted an essential role for mitochondrial aldehyde dehydrogenase (ALDH2) in cardiac homeostasis. This study examined the effect of ALDH2 on endotoxemia-induced cardiac anomalies. Echocardiographic, cardiac contractile and intracellular Ca2+ properties were examined. Our results indicated that LPS impaired cardiac contractile function (reduced fractional shortening, LV end systolic diameter, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration, oxidation of SERCA, and intracellular Ca2+ mishandling), associated with ER stress, inflammation, O2- production, increased autophagy, CAMKKß, phosphorylated AMPK and suppressed phosphorylation of mTOR, the effects of which were significantly attenuated or negated by ALDH2. LPS promoted early endosomal formation (as evidenced by RAB4 and RAB5a), apoptosis and necrosis (MTT and LDH) while decreasing late endosomal formation (RAB7 and RAB 9), the effects were reversed by ALDH2. In vitro study revealed that LPS-induced SERCA oxidation, autophagy and cardiac dysfunction were abrogated by ALDH2 activator Alda-1, the ER chaperone TUDCA, the autophagy inhibitor 3-MA, or the AMPK inhibitor Compound C. The beneficial effect of Alda-1 against LPS was nullified by AMPK activator AICAR or rapamycin. CAMKKß inhibition failed to rescue LPS-induced ER stress. Tunicamycin-induced cardiomyocyte dysfunction was ameliorated by Alda-1 and autophagy inhibition, the effect of which was abolished by rapamycin. These data suggested that ALDH2 protected against LPS-induced cardiac anomalies via suppression of ER stress, autophagy in a CAMKKß/AMPK/mTOR-dependent manner.


Assuntos
Aldeído-Desidrogenase Mitocondrial/genética , Cálcio/metabolismo , Cardiomiopatias/genética , Lipopolissacarídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Aldeído-Desidrogenase Mitocondrial/antagonistas & inibidores , Aldeído-Desidrogenase Mitocondrial/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Sinalização do Cálcio , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/enzimologia , Cardiomiopatias/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Cultura Primária de Células , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
6.
PLoS One ; 14(4): e0214669, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30934005

RESUMO

The sarcolemmal membrane associated proteins (SLMAPs) belong to the super family of tail anchored membrane proteins which serve diverse roles in biology including cell growth, protein trafficking and ion channel regulation. Mutations in human SLMAP have been linked to Brugada syndrome with putative deficits in trafficking of the sodium channel (Nav1.5) to the cell membrane resulting in aberrant electrical activity and heart function. Three main SLMAP isoforms (SLMAP1 (35 kDa), SLMAP2 (45 kDa), and SLMAP3 (91 kDa)) are expressed in myocardium but their precise role remains to be defined. Here we generated transgenic (Tg) mice with cardiac-specific expression of the SLMAP3 isoform during postnatal development which present with a significant decrease (20%) in fractional shortening and (11%) in cardiac output at 5 weeks of age. There was a lack of any notable cardiac remodeling (hypertrophy, fibrosis or fetal gene activation) in Tg hearts but the electrocardiogram indicated a significant increase (14%) in the PR interval and a decrease (43%) in the R amplitude. Western blot analysis indicated a selective and significant decrease (55%) in protein levels of Nav1.5 while 45% drop in its transcript levels were detectable by qRT-PCR. Significant decreases in the protein and transcript levels of the calcium transport system of the sarcoplasmic reticulum (SERCA2a/PLN) were also evident in Tg hearts. These data reveal a novel role for SLMAP3 in the selective regulation of important ion transport proteins at the level of gene expression and suggest that it may be a unique target in cardiovascular function and disease.


Assuntos
Coração/fisiologia , Proteínas de Membrana/fisiologia , Miocárdio/metabolismo , Animais , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(14): 6969-6974, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886088

RESUMO

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Morte Súbita do Lactente/genética , Troponina I , Cálcio/química , Cálcio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Recém-Nascido , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Sarcômeros/genética , Sarcômeros/metabolismo , Sarcômeros/patologia , Morte Súbita do Lactente/patologia , Troponina I/química , Troponina I/genética , Troponina I/metabolismo
8.
PLoS One ; 14(2): e0213081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30818334

RESUMO

c-Jun dimerization protein (JDP2) and Activating Transcription Factor 3 (ATF3) are closely related basic leucine zipper proteins. Transgenic mice with cardiac expression of either JDP2 or ATF3 showed maladaptive remodeling and cardiac dysfunction. Surprisingly, JDP2 knockout (KO) did not protect the heart following transverse aortic constriction (TAC). Instead, the JDP2 KO mice performed worse than their wild type (WT) counterparts. To test whether the maladaptive cardiac remodeling observed in the JDP2 KO mice is due to ATF3, ATF3 was removed in the context of JDP2 deficiency, referred as double KO mice (dKO). Mice were challenged by TAC, and followed by detailed physiological, pathological and molecular analyses. dKO mice displayed no apparent differences from WT mice under unstressed condition, except a moderate better performance in dKO male mice. Importantly, following TAC the dKO hearts showed low fibrosis levels, reduced inflammatory and hypertrophic gene expression and a significantly preserved cardiac function as compared with their WT counterparts in both genders. Consistent with these data, removing ATF3 resumed p38 activation in the JDP2 KO mice which correlates with the beneficial cardiac function. Collectively, mice with JDP2 and ATF3 double deficiency had reduced maladaptive cardiac remodeling and lower hypertrophy following TAC. As such, the worsening of the cardiac outcome found in the JDP2 KO mice is due to the elevated ATF3 expression. Simultaneous suppression of both ATF3 and JDP2 activity is highly beneficial for cardiac function in health and disease.


Assuntos
Fator 3 Ativador da Transcrição/deficiência , Proteínas Repressoras/deficiência , Remodelação Ventricular/fisiologia , Fator 3 Ativador da Transcrição/genética , Fator 3 Ativador da Transcrição/fisiologia , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Feminino , Fibrose , Coração/fisiopatologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miocárdio/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Remodelação Ventricular/genética
9.
J Am Coll Cardiol ; 73(10): 1173-1184, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30871701

RESUMO

BACKGROUND: The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit. OBJECTIVES: The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF. METHODS: PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis. RESULTS: The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF. CONCLUSIONS: A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF.


Assuntos
Insuficiência Cardíaca , Inibidores da Fosfodiesterase 3/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/fisiopatologia , Humanos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miócitos Cardíacos/metabolismo , Testes Farmacogenômicos , Polimorfismo Genético , Transdução de Sinais
10.
Nat Commun ; 10(1): 117, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30631059

RESUMO

In vitro models incorporating the complexity and function of adult human tissues are highly desired for translational research. Whilst vital slices of human myocardium approach these demands, their rapid degeneration in tissue culture precludes long-term experimentation. Here, we report preservation of structure and performance of human myocardium under conditions of physiological preload, compliance, and continuous excitation. In biomimetic culture, tissue slices prepared from explanted failing human hearts attain a stable state of contractility that can be monitored for up to 4 months or 2000000 beats in vitro. Cultured myocardium undergoes particular alterations in biomechanics, structure, and mRNA expression. The suitability of the model for drug safety evaluation is exemplified by repeated assessment of refractory period that permits sensitive analysis of repolarization impairment induced by the multimodal hERG-inhibitor pentamidine. Biomimetic tissue culture will provide new opportunities to study drug targets, gene functions, and cellular plasticity in adult human myocardium.


Assuntos
Coração/fisiologia , Miocárdio/metabolismo , Preservação Biológica/métodos , Técnicas de Cultura de Tecidos/métodos , Adulto , Fenômenos Biomecânicos , Estimulação Elétrica , Expressão Gênica , Humanos , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Fatores de Tempo
11.
Circulation ; 139(16): 1913-1936, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30646747

RESUMO

BACKGROUND: Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. METHODS: Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well. RESULTS: FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction. CONCLUSIONS: We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Adulto , Idoso , Animais , Sinalização do Cálcio , Linhagem Celular , Cardiomiopatias Diabéticas/patologia , Retículo Endoplasmático/ultraestrutura , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Contração Miocárdica/genética , Ratos
12.
Biochim Biophys Acta Mol Basis Dis ; 1865(3): 661-677, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30611859

RESUMO

Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substitution of tyrosine to serine at residue 237 in humans (residue 235 in mice) has been linked to HCM with strong disease association. Although cMyBPC truncations, deletions and insertions, and frame shift mutations have been studied, relatively little is known about the functional consequences of missense mutations in cMyBPC. In this study, we characterized the functional and structural effects of the HCM-causing Y235S mutation by performing mechanical experiments and molecular dynamics simulations (MDS). cMyBPC null mouse myocardium was virally transfected with wild-type (WT) or Y235S cMyBPC (KOY235S). We found that Y235S cMyBPC was properly expressed and incorporated into the cardiac sarcomere, suggesting that the mechanism of disease of the Y235S mutation is not haploinsufficiency or poison peptides. Mechanical experiments in detergent-skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT hearts, evidenced by accelerated cross-bridge kinetics and increased Ca2+ sensitivity of force generation. In addition, MDS revealed that the Y235S mutation causes alterations in important intramolecular interactions, surface conformations, and electrostatic potential of the C1 domain of cMyBPC. Our combined in vitro and in silico data suggest that the Y235S mutation directly disrupts internal and surface properties of the C1 domain of cMyBPC, which potentially alters its ligand-binding interactions. These molecular changes may underlie the mechanism for hypercontractile cross-bridge behavior, which ultimately results in the development of cardiac hypertrophy and in vivo cardiac dysfunction.


Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Mutação de Sentido Incorreto , Contração Miocárdica/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Cardiomiopatia Hipertrófica/metabolismo , Proteínas de Transporte/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Proteínas Mutantes/fisiologia , Mutação de Sentido Incorreto/fisiologia , Miocárdio/metabolismo , Domínios Proteicos/genética , Sarcômeros/genética , Sarcômeros/metabolismo , Serina/genética , Tirosina/genética
13.
Int Heart J ; 60(1): 220-225, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30464138

RESUMO

A 70-year-old man with dyspnea was admitted to our department and received standard therapy for recurrent heart failure. He was diagnosed with polycystic kidney disease (PKD) in his thirties and received hemodialysis for 4 years before undergoing renal transplantation at age 45. Although his left ventricular ejection fraction (LVEF) was preserved in his 50s, LVEF decreased progressively from 61% to 24%, while left ventricular diastolic dimension (LVDd) increased from 54 mm to 65 mm between 63 and 69 years of age. Right ventricular endomyocardial biopsy demonstrated myocardial disarray and interstitial fibrosis. Genetic analysis identified a heterozygous frameshift mutation in PKD1, which encodes polycystin-1, a major causative gene of PKD. We detected PKD1 protein expression in myocardial tissue by immunostaining. Recent epidemiological studies and animal models have clarified the pathological correlation between ventricular contractile dysfunction and PKD1 function. Here, we present a case of old-age onset progressive cardiac contractile dysfunction with a PKD1 gene mutation.


Assuntos
Mutação da Fase de Leitura/genética , Cardiopatias/fisiopatologia , Miocárdio/metabolismo , Doenças Renais Policísticas/complicações , Doenças Renais Policísticas/genética , Idoso , Ecocardiografia , Fibrose/patologia , Cardiopatias/etiologia , Cardiopatias/genética , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Humanos , Transplante de Rim , Masculino , Contração Miocárdica/genética , Miocárdio/patologia , Doenças Renais Policísticas/terapia , Volume Sistólico/fisiologia , Canais de Cátion TRPP
14.
FASEB J ; 33(3): 3152-3166, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30365366

RESUMO

Myosin light chain 2 ( MYL2) gene encodes the myosin regulatory light chain (RLC) simultaneously in heart ventricles and in slow-twitch skeletal muscle. Using transgenic mice with cardiac-specific expression of the human R58Q-RLC mutant, we sought to determine whether the hypertrophic cardiomyopathy phenotype observed in papillary muscles (PMs) of R58Q mice is also manifested in slow-twitch soleus (SOL) muscles. Skinned SOL muscles and ventricular PMs of R58Q animals exhibited lower contractile force that was not observed in the fast-twitch extensor digitorum longus muscles of R58Q vs. wild-type-RLC mice, but mutant animals did not display gross muscle weakness in vivo. Consistent with SOL muscle abnormalities in R58Q vs. wild-type mice, myosin ATPase staining revealed a decreased proportion of fiber type I/type II only in SOL muscles but not in the extensor digitorum longus muscles. The similarities between SOL muscles and PMs of R58Q mice were further supported by quantitative proteomics. Differential regulation of proteins involved in energy metabolism, cell-cell interactions, and protein-protein signaling was concurrently observed in the hearts and SOL muscles of R58Q mice. In summary, even though R58Q expression was restricted to the heart of mice, functional similarities were clearly observed between the hearts and slow-twitch skeletal muscle, suggesting that MYL2 mutated models of hypertrophic cardiomyopathy may be useful research tools to study the molecular, structural, and energetic mechanisms of cardioskeletal myopathy associated with myosin RLC.-Kazmierczak, K., Liang, J., Yuan, C.-C., Yadav, S., Sitbon, Y. H., Walz, K., Ma, W., Irving, T. C., Cheah, J. X., Gomes, A. V., Szczesna-Cordary, D. Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain.


Assuntos
Miosinas Cardíacas/genética , Miosinas Cardíacas/fisiologia , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/fisiopatologia , Fibras Musculares de Contração Lenta/fisiologia , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/fisiologia , Substituição de Aminoácidos , Animais , Cardiomiopatia Hipertrófica/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Contração Muscular/genética , Contração Muscular/fisiologia , Fibras Musculares de Contração Lenta/patologia , Mutação de Sentido Incorreto , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miocárdio/patologia , Músculos Papilares/patologia , Músculos Papilares/fisiopatologia , Proteômica
15.
Biosens Bioelectron ; 124-125: 129-135, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30366257

RESUMO

Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs' drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca2+ was studied. The differential evaluation explained the observed effects despite variability of biological samples. The ß-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.


Assuntos
Técnicas Biossensoriais , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Distrofia Muscular de Duchenne/fisiopatologia , Miócitos Cardíacos/citologia , Diferenciação Celular/genética , Distrofina/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Isoproterenol/farmacologia , Microeletrodos , Microscopia de Força Atômica , Contração Miocárdica/genética , Contração Miocárdica/fisiologia , Verapamil/farmacologia
16.
Circulation ; 139(7): 932-948, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30586714

RESUMO

BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.


Assuntos
Pressão Arterial/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Mutação , Contração Miocárdica/genética , Artéria Pulmonar/fisiopatologia , Função Ventricular Direita/genética , Potenciais de Ação , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Predisposição Genética para Doença , Hipertensão Pulmonar/metabolismo , Hipóxia/complicações , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Fosforilação , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Artéria Pulmonar/metabolismo , Ratos Mutantes , Proteínas Smad/metabolismo
17.
JACC Cardiovasc Imaging ; 12(3): 446-455, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29550307

RESUMO

OBJECTIVES: The aim of this study was to investigate the prognostic value of echocardiographic deformation imaging in arrhythmogenic right ventricular cardiomyopathy (ARVC) to optimize family screening protocols. BACKGROUND: ARVC is characterized by variable disease expressivity among family members, which complicates family screening protocols. Previous reports have shown that echocardiographic deformation imaging detects abnormal right ventricular (RV) deformation in the absence of established disease expression in ARVC. METHODS: First-degree relatives of patients with ARVC were evaluated according to 2010 task force criteria, including RV deformation imaging (n = 128). Relatives fulfilling structural task force criteria were excluded for further analysis. At baseline, deformation patterns of the subtricuspid region were scored as type I (normal deformation), type II (delayed onset, decreased systolic peak, and post-systolic shortening), or type III (systolic stretching and large post-systolic shortening). The final study population comprised relatives who underwent a second evaluation during follow-up. Disease progression was defined as the development of a new 2010 task force criterion during follow-up that was absent at baseline. RESULTS: Sixty-five relatives underwent a second evaluation after a mean follow-up period of 3.7 ± 2.1 years. At baseline, 28 relatives (43%) had normal deformation (type I), and 37 relatives (57%) had abnormal deformation (type II or III) in the subtricuspid region. Disease progression occurred in 4% of the relatives with normal deformation at baseline and in 43% of the relatives with abnormal deformation at baseline (p < 0.001). Positive and negative predictive values of abnormal deformation were, respectively, 43% (95% confidence interval: 27% to 61%) and 96% (95% confidence interval: 82% to 100%). CONCLUSIONS: Normal RV deformation in the subtricuspid region is associated with absence of disease progression during nearly 4-year follow-up in relatives of patients with ARVC. Abnormal RV deformation seems to precede the established signs of ARVC. RV deformation imaging may potentially play an important role in ARVC family screening protocols.


Assuntos
Displasia Arritmogênica Ventricular Direita/diagnóstico por imagem , Ecocardiografia Doppler , Imagem por Ressonância Magnética , Contração Miocárdica , Função Ventricular Direita , Adolescente , Adulto , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Progressão da Doença , Diagnóstico Precoce , Eletrocardiografia , Feminino , Predisposição Genética para Doença , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Contração Miocárdica/genética , Linhagem , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Sistema de Registros , Função Ventricular Direita/genética , Adulto Jovem
18.
Biomolecules ; 8(4)2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30486323

RESUMO

Genetic model organisms have the potential of removing blind spots from the underlying gene regulatory networks of human diseases. Allowing analyses under experimental conditions they complement the insights gained from observational data. An inevitable requirement for a successful trans-species transfer is an abstract but precise high-level characterization of experimental findings. In this work, we provide a large-scale analysis of seven weak contractility/heart failure genotypes of the model organism zebrafish which all share a weak contractility phenotype. In supervised classification experiments, we screen for discriminative patterns that distinguish between observable phenotypes (homozygous mutant individuals) as well as wild-type (homozygous wild-types) and carriers (heterozygous individuals). As the method of choice we use semantic multi-classifier systems, a knowledge-based approach which constructs hypotheses from a predefined vocabulary of high-level terms (e.g., Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways or Gene Ontology (GO) terms). Evaluating these models leads to a compact description of the underlying processes and guides the screening for new molecular markers of heart failure. Furthermore, we were able to independently corroborate the identified processes in Wistar rats.


Assuntos
Insuficiência Cardíaca/genética , Redes e Vias Metabólicas/genética , Contração Miocárdica/genética , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Ontologia Genética , Genótipo , Insuficiência Cardíaca/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Mutação , Contração Miocárdica/fisiologia , Ratos , Semântica
19.
Science ; 362(6421)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30409805

RESUMO

Junctophilin-2 (JP2) is a structural protein required for normal excitation-contraction (E-C) coupling. After cardiac stress, JP2 is cleaved by the calcium ion-dependent protease calpain, which disrupts the E-C coupling ultrastructural machinery and drives heart failure progression. We found that stress-induced proteolysis of JP2 liberates an N-terminal fragment (JP2NT) that translocates to the nucleus, binds to genomic DNA, and controls expression of a spectrum of genes in cardiomyocytes. Transgenic overexpression of JP2NT in mice modifies the transcriptional profile, resulting in attenuated pathological remodeling in response to cardiac stress. Conversely, loss of nuclear JP2NT function accelerates stress-induced development of hypertrophy and heart failure in mutant mice. These data reveal a self-protective mechanism in failing cardiomyocytes that transduce mechanical information (E-C uncoupling) into salutary transcriptional reprogramming in the stressed heart.


Assuntos
Cardiomegalia/genética , Núcleo Celular/metabolismo , Acoplamento Excitação-Contração/genética , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Remodelação Ventricular/genética , Transporte Ativo do Núcleo Celular , Adaptação Fisiológica/genética , Animais , Calpaína/metabolismo , Cardiomegalia/fisiopatologia , Humanos , Fatores de Transcrição MEF2/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Musculares/química , Proteínas Musculares/genética , Miócitos Cardíacos/metabolismo , Proteólise , Transcrição Genética
20.
PLoS One ; 13(10): e0205719, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30332462

RESUMO

AIMS: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome-decreased ventricular function. Titin (TTN)-a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS). METHODS AND RESULTS: iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to ß-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells. CONCLUSIONS: These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.


Assuntos
Cardiomiopatia Dilatada/genética , Diferenciação Celular/genética , Conectina/genética , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Adulto , Idoso , Cardiomiopatia Dilatada/patologia , Acoplamento Excitação-Contração/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Isoproterenol/farmacologia , Masculino , Mutação , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteoma
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