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1.
Arthritis Res Ther ; 22(1): 5, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31915059

RESUMO

OBJECTIVE: To describe successful therapeutic strategies in statin-induced anti-HMGCR myopathy. METHODS: Retrospective data from a cohort of 55 patients with statin-induced anti-HMGCR myopathy, sequentially stratified by the presence of proximal weakness, early remission, and corticosteroid and IVIG use at treatment induction, were analyzed for optimal successful induction and maintenance of remission strategies. RESULTS: A total of 14 patients achieved remission with a corticosteroid-free induction strategy (25%). In 41 patients treated with corticosteroids, only 4 patients (10%) failed an initial triple steroid/IVIG/steroid-sparing immunosuppressant (SSI) induction strategy. Delay in treatment initiation was independently associated with lower odds of successful maintenance with immunosuppressant monotherapy (OR 0.92, 95% CI 0.85 to 0.97, P = 0.015). While 22 patients (40%) presented with normal strength, only 9 had normal strength at initiation of treatment. CONCLUSION: While corticosteroid-free treatment of anti-HMGCR myopathy is now a safe option in selected cases, initial triple steroid/IVIG/SSI was very efficacious in induction. Delays in treatment initiation and, as a corollary, delays in achieving remission decrease the odds of achieving successful maintenance with an SSI alone. Avoiding such delays, most notably in patients with normal strength, may reset the natural history of anti-HMGCR myopathy from a refractory entity to a treatable disease.

2.
J Mol Cell Cardiol ; 135: 1-9, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31362018

RESUMO

BACKGROUND: K channel interacting protein 2 (KChIP2), initially cloned as Kv4 channel modulator, is a multi-tasking protein. In addition to modulating several cardiac ion channels at the plasma membrane, it can also modulate microRNA transcription inside nuclei, and interact with presenilins to modulate Ca release through RyR2 in the cytoplasm. However, the mechanism regulating its subcellular distribution is not clear. OBJECTIVE: We tested whether palmitoylation drives KChIP2 trafficking and distribution in cells, and whether the distribution pattern of KChIP2 in cardiac myocytes is sensitive to cellular milieu. METHOD: We conducted imaging and biochemical experiments on palmitoylatable and unpalmitoylatable KChIP2 variants expressed in COS-7 cells and in cardiomyocytes, and on native KChIP2 in myocytes. RESULTS: In COS-7 cells, palmitoylatable KChIP2 clustered to plasma membrane, while unpalmitoylatable KChIP2 exhibited higher cytoplasmic mobility and faster nuclear entry. The same differences in distribution and mobility were observed when these KChIP2 variants were expressed in cardiac myocytes, indicating that the palmitoylation-dependent distribution and trafficking are intrinsic properties of KChIP2. Importantly, acute stress in a rat model of cardiac arrest/resuscitation induced changes in native KChIP2 resembling those of KChIP2 depalmitoylation, promoting KChIP2 nuclear entry. CONCLUSION: The palmitoylation status of KChIP2 determines its subcellular distribution in cardiac myocytes. Stress promotes nuclear entry of KChIP2, diverting it from ion channel modulation at the plasma membrane to other functions in the nuclear compartment.

3.
Int J Cardiol ; 273: 168-176, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30279005

RESUMO

Diminished cardiac contractile function is a characteristic feature of dilated cardiomyopathy (DCM) and many other heart failure (HF) causing etiologies. We tested the hypothesis that targeting the sarcomere to increase cardiac contractility can effectively prevent the DCM phenotype in muscle-LIM protein knockout (MLP-/-) mice. The ablation of cardiac myosin binding protein C (MYBPC3-/-) protected the MLP-/- mice from developing the DCM phenotype. We examined the in vivo cardiac function and morphology of the resultant mouse model lacking both MLP and MYBPC3 (DKO) by echocardiography and pressure-volume catheterization and found a significant reduction in hypertrophy, as evidenced by normalized wall thickness and chamber dimensions, and improved systolic function, as evidenced by enhanced ejection fraction (~26% increase compared MLP-/- mice) and rate of pressure development (DKO 7851.0 ±â€¯504.8 vs. MLP-/- 4496.4 ±â€¯196.8 mmHg/s). To investigate the molecular basis for the improved DKO phenotype we performed mechanical experiments in skinned myocardium isolated from WT and the individual KO mice. Skinned myocardium isolated from DKO mice displayed increased Ca2+ sensitivity of force generation, and significantly accelerated rate of cross-bridge detachment (+63% compared to MLP-/-) and rate of XB recruitment (+58% compared to MLP-/-) at submaximal Ca2+ activations. The in vivo and in vitro functional enhancement of DKO mice demonstrates that enhancing the sarcomeric contractility can be cardioprotective in HF characterized by reduced cardiac output, such as in cases of DCM.


Assuntos
Cardiomiopatia Dilatada/genética , Proteínas de Transporte/genética , Modelos Animais de Doenças , Proteínas com Domínio LIM/genética , Proteínas Musculares/genética , Sarcômeros/genética , Sístole/fisiologia , Animais , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/metabolismo , Proteínas de Transporte/metabolismo , Feminino , Proteínas com Domínio LIM/deficiência , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/deficiência , Miócitos Cardíacos/fisiologia , Sarcômeros/metabolismo
4.
Elife ; 72018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30106376

RESUMO

Computational modeling indicates that cardiac conduction may involve ephaptic coupling - intercellular communication involving electrochemical signaling across narrow extracellular clefts between cardiomyocytes. We hypothesized that ß1(SCN1B) -mediated adhesion scaffolds trans-activating NaV1.5 (SCN5A) channels within narrow (<30 nm) perinexal clefts adjacent to gap junctions (GJs), facilitating ephaptic coupling. Super-resolution imaging indicated preferential ß1 localization at the perinexus, where it co-locates with NaV1.5. Smart patch clamp (SPC) indicated greater sodium current density (INa) at perinexi, relative to non-junctional sites. A novel, rationally designed peptide, ßadp1, potently and selectively inhibited ß1-mediated adhesion, in electric cell-substrate impedance sensing studies. ßadp1 significantly widened perinexi in guinea pig ventricles, and selectively reduced perinexal INa, but not whole cell INa, in myocyte monolayers. In optical mapping studies, ßadp1 precipitated arrhythmogenic conduction slowing. In summary, ß1-mediated adhesion at the perinexus facilitates action potential propagation between cardiomyocytes, and may represent a novel target for anti-arrhythmic therapies.


Assuntos
Arritmias Cardíacas/tratamento farmacológico , Comunicação Celular/genética , Junções Comunicantes/ultraestrutura , Miócitos Cardíacos/fisiologia , Potenciais de Ação , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Adesão Celular/genética , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Biologia Computacional , Impedância Elétrica , Junções Comunicantes/fisiologia , Cobaias , Humanos , Camundongos , Modelos Cardiovasculares , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Patch-Clamp , Peptídeos/química , Sódio/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genética
5.
Am J Physiol Heart Circ Physiol ; 315(5): H1250-H1257, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30118344

RESUMO

Mutations in voltage-gated Na+ channels have been linked to several channelopathies leading to a wide variety of diseases including cardiac arrhythmias, epilepsy, and myotonia. We have previously demonstrated that voltage-gated Na+ channel (Nav)1.5 trafficking-deficient mutant channels could lead to a dominant negative effect by impairing trafficking of the wild-type (WT) channel. We also reported that voltage-gated Na+ channels associate as dimers with coupled gating properties. Here, we hypothesized that the dominant negative effect of mutant Na+ channels could also occur through coupled gating. This was tested using cell surface biotinylation and single channel recordings to measure the gating probability and coupled gating of the dimers. As previously reported, coexpression of Nav1.5-L325R with WT channels led to a dominant negative effect, as reflected by a 75% reduction in current density. Surprisingly, cell surface biotinylation showed that Nav1.5-L325R mutant is capable of trafficking, with 40% of Nav1.5-L325R reaching the cell surface when expressed alone. Importantly, even though a dominant negative effect on the Na+ current is observed when WT and Nav1.5-L325R are expressed together, the total Nav channel cell surface expression was not significantly altered compared with WT channels alone. Thus, the trafficking deficiency could not explain the 75% decrease in inward Na+ current. Interestingly, single channel recordings showed that Nav1.5-L325R exerted a dominant negative effect on the WT channel at the gating level. Both coupled gating and gating probability of WT:L325R dimers were drastically impaired. We conclude that dominant negative suppression exerted by Nav1.5 mutants can also be caused by impairing the WT gating probability, a mechanism resulting from the dimerization and coupled gating of voltage-gated Na+ channel α-subunits. NEW & NOTEWORTHY The presence of dominant negative mutations in the Na+ channel gene leading to Brugada syndrome was supported by our recent findings that Na+ channel α-subunits form dimers. Up until now, the dominant negative effect was thought to be caused by the interaction of the wild-type Na+ channel with trafficking-deficient mutant channels. However, the present study demonstrates that coupled gating of voltage-gated Na+ channels can also be responsible for the dominant negative effect leading to arrhythmias.


Assuntos
Ativação do Canal Iônico/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Sódio/metabolismo , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Células HEK293 , Frequência Cardíaca/genética , Humanos , Cadeias de Markov , Potenciais da Membrana , Modelos Biológicos , Multimerização Proteica , Transporte Proteico , Fatores de Tempo
6.
J Gen Physiol ; 150(9): 1333-1347, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30082431

RESUMO

Slow inactivation in voltage-gated sodium channels (NaVs) directly regulates the excitability of neurons, cardiac myocytes, and skeletal muscles. Although NaV slow inactivation appears to be conserved across phylogenies-from bacteria to humans-the structural basis for this mechanism remains unclear. Here, using site-directed labeling and EPR spectroscopic measurements of membrane-reconstituted prokaryotic NaV homologues, we characterize the conformational dynamics of the selectivity filter region in the conductive and slow-inactivated states to determine the molecular events underlying NaV gating. Our findings reveal profound conformational flexibility of the pore in the slow-inactivated state. We find that the P1 and P2 pore helices undergo opposing movements with respect to the pore axis. These movements result in changes in volume of both the central and intersubunit cavities, which form pathways for lipophilic drugs that modulate slow inactivation. Our findings therefore provide novel insight into the molecular basis for state-dependent effects of lipophilic drugs on channel function.


Assuntos
Canais de Sódio Disparados por Voltagem/metabolismo , Escherichia coli , Células HEK293 , Humanos , Conformação Proteica , Domínios Proteicos , Análise Espectral
7.
Naunyn Schmiedebergs Arch Pharmacol ; 391(10): 1119-1131, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30008082

RESUMO

K2P17.1 (TASK-4, TALK-2) potassium channels are expressed in the heart and represent potential targets for pharmacological management of atrial and ventricular arrhythmias. Reduced K2P17.1 expression was found in atria and ventricles of heart failure (HF) patients. Modulation of K2P17.1 currents by antiarrhythmic compounds has not been comprehensively studied to date. The objective of this study was to investigate acute effects of clinically relevant antiarrhythmic drugs on human K2P17.1 channels to provide a more complete picture of K2P17.1 electropharmacology. Whole-cell patch clamp and two-electrode voltage clamp electrophysiology was employed to study human K2P17.1 channel pharmacology. K2P17.1 channels expressed in Xenopus laevis oocytes were screened for sensitivity to antiarrhythmic drugs, revealing significant activation by propafenone (+ 296%; 100 µM), quinidine (+ 58%; 100 µM), mexiletine (+ 21%; 100 µM), propranolol (+ 139%; 100 µM), and metoprolol (+ 17%; 100 µM) within 60 min. In addition, the currents were inhibited by amiodarone (- 13%; 100 µM), sotalol (- 10%; 100 µM), verapamil (- 21%; 100 µM), and ranolazine (- 8%; 100 µM). K2P17.1 channels were not significantly affected by ajmaline and carvedilol. Concentration-dependent K2P17.1 activation by propafenone was characterized in more detail. The onset of activation was fast, and current-voltage relationships were not modulated by propafenone. K2P17.1 activation was confirmed in mammalian Chinese hamster ovary cells, revealing 7.8-fold current increase by 100 µM propafenone. Human K2P17.1 channels were sensitive to multiple antiarrhythmic drugs. Differential pharmacological regulation of repolarizing K2P17.1 background K+ channels may be employed for personalized antiarrhythmic therapy.


Assuntos
Canais de Potássio de Domínios Poros em Tandem/fisiologia , Idoso , Animais , Antiarrítmicos/farmacologia , Células CHO , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/fisiopatologia , Cricetulus , Feminino , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Humanos , Masculino , Oócitos , Xenopus laevis
8.
Int J Mol Sci ; 19(5)2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29734659

RESUMO

Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) holds a great promise for regenerative medicine and has been studied in several major directions. However, cell-cycle regulation, a fundamental biological process, has not been investigated during iCM-reprogramming. Here, our time-lapse imaging on iCMs, reprogrammed by Gata4, Mef2c, and Tbx5 (GMT) monocistronic retroviruses, revealed that iCM-reprogramming was majorly initiated at late-G1- or S-phase and nearly half of GMT-reprogrammed iCMs divided soon after reprogramming. iCMs exited cell cycle along the process of reprogramming with decreased percentage of 5-ethynyl-20-deoxyuridine (EdU)⁺/α-myosin heavy chain (αMHC)-GFP⁺ cells. S-phase synchronization post-GMT-infection could enhance cell-cycle exit of reprogrammed iCMs and yield more GFPhigh iCMs, which achieved an advanced reprogramming with more expression of cardiac genes than GFPlow cells. However, S-phase synchronization did not enhance the reprogramming with a polycistronic-viral vector, in which cell-cycle exit had been accelerated. In conclusion, post-infection synchronization of S-phase facilitated the early progression of GMT-reprogramming through a mechanism of enhanced cell-cycle exit.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , Miócitos Cardíacos/citologia , Animais , Ciclo Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Medicina Regenerativa/tendências
10.
J Clin Invest ; 128(3): 1043-1056, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29431731

RESUMO

Congenital long QT syndrome (LQTS) is an inherited channelopathy associated with life-threatening arrhythmias. LQTS type 2 (LQT2) is caused by mutations in KCNH2, which encodes the potassium channel hERG. We hypothesized that modifier genes are partly responsible for the variable phenotype severity observed in some LQT2 families. Here, we identified contributors to variable expressivity in an LQT2 family by using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and whole exome sequencing in a synergistic manner. We found that iPSC-CMs recapitulated the clinical genotype-phenotype discordance in vitro. Importantly, iPSC-CMs derived from the severely affected LQT2 patients displayed prolonged action potentials compared with cells from mildly affected first-degree relatives. The iPSC-CMs derived from all patients with hERG R752W mutation displayed lower IKr amplitude. Interestingly, iPSC-CMs from severely affected mutation-positive individuals exhibited greater L-type Ca2+ current. Whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2, providing biologically plausible explanations for this variable expressivity. Genome editing to correct a REM2 variant reversed the enhanced L-type Ca2+ current and prolonged action potential observed in iPSC-CMs from severely affected individuals. Thus, our findings showcase the power of combining complementary physiological and genomic analyses to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. Furthermore, we propose that this strategy can be deployed to unravel myriad confounding pathologies displaying variable expressivity.


Assuntos
Síndrome do QT Longo/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Canais de Potássio de Domínios Poros em Tandem/genética , Potenciais de Ação , Adolescente , Adulto , Animais , Arritmias Cardíacas/metabolismo , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Exoma , Saúde da Família , Feminino , Genes Modificadores , Estudos de Associação Genética , Genoma , Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/citologia , Linhagem , Fenótipo , Análise de Sequência de DNA
11.
Nat Commun ; 8(1): 2077, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29233994

RESUMO

Fast opening and closing of voltage-gated sodium channels are crucial for proper propagation of the action potential through excitable tissues. Unlike potassium channels, sodium channel α-subunits are believed to form functional monomers. Yet, an increasing body of literature shows inconsistency with the traditional idea of a single α-subunit functioning as a monomer. Here we demonstrate that sodium channel α-subunits not only physically interact with each other but they actually assemble, function and gate as a dimer. We identify the region involved in the dimerization and demonstrate that 14-3-3 protein mediates the coupled gating. Importantly we show conservation of this mechanism among mammalian sodium channels. Our study not only shifts conventional paradigms in regard to sodium channel assembly, structure, and function but importantly this discovery of the mechanism involved in channel dimerization and biophysical coupling could open the door to new approaches and targets to treat and/or prevent sodium channelopathies.


Assuntos
Proteínas 14-3-3/metabolismo , Canalopatias/patologia , Ativação do Canal Iônico/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Multimerização Proteica/fisiologia , Potenciais de Ação/fisiologia , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sódio/metabolismo
13.
Stem Cell Rev Rep ; 13(5): 631-643, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28623610

RESUMO

The inward rectifier potassium current (IK1) is generally thought to suppress cardiac automaticity by hyperpolarizing membrane potential (MP). We recently observed that IK1 could promote the spontaneously-firing automaticity induced by upregulation of pacemaker funny current (If) in adult ventricular cardiomyocytes (CMs). However, the intriguing ability of IK1 to activate If and thereby promote automaticity has not been explored. In this study, we combined mathematical and experimental assays and found that only IK1 and If, at a proper-ratio of densities, were sufficient to generate rhythmic MP-oscillations even in unexcitable cells (i.e. HEK293T cells and undifferentiated mouse embryonic stem cells [ESCs]). We termed this effect IK1-induced If activation. Consistent with previous findings, our electrophysiological recordings observed that around 50% of mouse (m) and human (h) ESC-differentiated CMs could spontaneously fire action potentials (APs). We found that spontaneously-firing ESC-CMs displayed more hyperpolarized maximum diastolic potential and more outward IK1 current than quiescent-yet-excitable m/hESC-CMs. Rather than classical depolarization pacing, quiescent mESC-CMs were able to fire APs spontaneously with an electrode-injected small outward-current that hyperpolarizes MP. The automaticity to spontaneously fire APs was also promoted in quiescent hESC-CMs by an IK1-specific agonist zacopride. In addition, we found that the number of spontaneously-firing m/hESC-CMs was significantly decreased when If was acutely upregulated by Ad-CGI-HCN infection. Our study reveals a novel role of IK1 promoting the development of cardiac automaticity in m/hESC-CMs through a mechanism of IK1-induced If activation and demonstrates a synergistic interaction between IK1 and If that regulates cardiac automaticity.


Assuntos
Potenciais de Ação/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Miócitos Cardíacos/metabolismo , Periodicidade , Canais de Potássio Corretores do Fluxo de Internalização/genética , Potenciais de Ação/efeitos dos fármacos , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Marca-Passo Artificial , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Transgenes
14.
PLoS One ; 12(4): e0175221, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28384221

RESUMO

Pathologic electrical remodeling and attenuated cardiac contractility are featured characteristics of heart failure. Coinciding with these remodeling events is a loss of the K+ channel interacting protein, KChIP2. While, KChIP2 enhances the expression and stability of the Kv4 family of potassium channels, leading to a more pronounced transient outward K+ current, Ito,f, the guinea pig myocardium is unique in that Kv4 expression is absent, while KChIP2 expression is preserved, suggesting alternative consequences to KChIP2 loss. Therefore, KChIP2 was acutely silenced in isolated guinea pig myocytes, which led to significant reductions in the Ca2+ transient amplitude and prolongation of the transient duration. This change was reinforced by a decline in sarcomeric shortening. Notably, these results were unexpected when considering previous observations showing enhanced ICa,L and prolonged action potential duration following KChIP2 loss, suggesting a disruption of fundamental Ca2+ handling proteins. Evaluation of SERCA2a, phospholamban, RyR, and sodium calcium exchanger identified no change in protein expression. However, assessment of Ca2+ spark activity showed reduced spark frequency and prolonged Ca2+ decay following KChIP2 loss, suggesting an altered state of RyR activity. These changes were associated with a delocalization of the ryanodine receptor activator, presenilin, away from sarcomeric banding to more diffuse distribution, suggesting that RyR open probability are a target of KChIP2 loss mediated by a dissociation of presenilin. Typically, prolonged action potential duration and enhanced Ca2+ entry would augment cardiac contractility, but here we see KChIP2 fundamentally disrupts Ca2+ release events and compromises myocyte contraction. This novel role targeting presenilin localization and RyR activity reveals a significance for KChIP2 loss that reflects adverse remodeling observed in cardiac disease settings.


Assuntos
Cálcio/metabolismo , Proteínas Interatuantes com Canais de Kv/fisiologia , Células Musculares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Cobaias , Imuno-Histoquímica , Proteínas Interatuantes com Canais de Kv/genética
15.
Am J Physiol Heart Circ Physiol ; 312(6): H1144-H1153, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28341634

RESUMO

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias.NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.


Assuntos
Potenciais de Ação , Diferenciação Celular , Ventrículos do Coração/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Estudos de Casos e Controles , Linhagem Celular , Feminino , Perfilação da Expressão Gênica/métodos , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Isquemia Miocárdica/complicações , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatologia , Canais de Potássio de Domínios Poros em Tandem/genética , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Transfecção
16.
Elife ; 62017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28263709

RESUMO

Arrhythmogenesis from aberrant electrical remodeling is a primary cause of death among patients with heart disease. Amongst a multitude of remodeling events, reduced expression of the ion channel subunit KChIP2 is consistently observed in numerous cardiac pathologies. However, it remains unknown if KChIP2 loss is merely a symptom or involved in disease development. Using rat and human derived cardiomyocytes, we identify a previously unobserved transcriptional capacity for cardiac KChIP2 critical in maintaining electrical stability. Through interaction with genetic elements, KChIP2 transcriptionally repressed the miRNAs miR-34b and miR-34c, which subsequently targeted key depolarizing (INa) and repolarizing (Ito) currents altered in cardiac disease. Genetically maintaining KChIP2 expression or inhibiting miR-34 under pathologic conditions restored channel function and moreover, prevented the incidence of reentrant arrhythmias. This identifies the KChIP2/miR-34 axis as a central regulator in developing electrical dysfunction and reveals miR-34 as a therapeutic target for treating arrhythmogenesis in heart disease.


Assuntos
Proteínas Interatuantes com Canais de Kv/metabolismo , Miócitos Cardíacos/fisiologia , Proteínas Repressoras/metabolismo , Transcrição Genética , Animais , Células Cultivadas , Humanos , MicroRNAs/biossíntese , Ratos
17.
Am J Physiol Heart Circ Physiol ; 312(5): H886-H895, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28283549

RESUMO

Acute cardiac ischemia induces conduction velocity (CV) slowing and conduction block, promoting reentrant arrhythmias leading to sudden cardiac arrest. Previously, we found that mild hypothermia (MH; 32°C) attenuates ischemia-induced conduction block and CV slowing in a canine model of early global ischemia. Acute ischemia impairs cellular excitability and the gap junction (GJ) protein connexin (Cx)43. We hypothesized that MH prevented ischemia-induced conduction block and CV slowing by preserving GJ expression and localization. Canine left ventricular preparations at control (36°C) or MH (32°C) were subjected to no-flow prolonged (30 min) ischemia. Optical action potentials were recorded from the transmural left ventricular wall, and CV was measured throughout ischemia. Cx43 and Na+ channel (NaCh) remodeling was assessed using both confocal immunofluorescence (IF) and/or Western blot analysis. Cellular excitability was determined by microelectrode recordings of action potential upstroke velocity (dV/dtmax) and resting membrane potential (RMP). NaCh current was measured in isolated canine myocytes at 36 and 32°C. As expected, MH prevented conduction block and mitigated ischemia-induced CV slowing during 30 min of ischemia. MH maintained Cx43 at the intercalated disk (ID) and attenuated ischemia-induced Cx43 degradation by both IF and Western blot analysis. MH also preserved dV/dtmax and NaCh function without affecting RMP. No difference in NaCh expression was seen at the ID by IF or Western blot analysis. In conclusion, MH preserves myocardial conduction during prolonged ischemia by maintaining Cx43 expression at the ID and maintaining NaCh function. Hypothermic preservation of GJ coupling and NaCh may be novel antiarrhythmic strategies during resuscitation.NEW & NOTEWORTHY Therapeutic hypothermia is now a class I recommendation for resuscitation from cardiac arrest. This study determined that hypothermia preserves gap junction coupling as well as Na+ channel function during acute cardiac ischemia, attenuating conduction slowing and preventing conduction block, suggesting that induced hypothermia may be a novel antiarrhythmic strategy in resuscitation.


Assuntos
Comunicação Celular , Junções Comunicantes , Sistema de Condução Cardíaco , Hipotermia Induzida/métodos , Isquemia Miocárdica/terapia , Canais de Sódio , Potenciais de Ação/fisiologia , Animais , Conexinas/metabolismo , Cães , Masculino , Microeletrodos , Microscopia Confocal , Células Musculares/metabolismo , Função Ventricular Esquerda
18.
J Physiol ; 595(7): 2229-2252, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-27808412

RESUMO

This is the second of the two White Papers from the fourth UC Davis Cardiovascular Symposium Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias (3-4 March 2016), a biennial event that brings together leading experts in different fields of cardiovascular research. The theme of the 2016 symposium was 'K+ channels and regulation', and the objectives of the conference were severalfold: (1) to identify current knowledge gaps; (2) to understand what may go wrong in the diseased heart and why; (3) to identify possible novel therapeutic targets; and (4) to further the development of systems biology approaches to decipher the molecular mechanisms and treatment of cardiac arrhythmias. The sessions of the Symposium focusing on the functional roles of the cardiac K+ channel in health and disease, as well as K+ channels as therapeutic targets, were contributed by Ye Chen-Izu, Gideon Koren, James Weiss, David Paterson, David Christini, Dobromir Dobrev, Jordi Heijman, Thomas O'Hara, Crystal Ripplinger, Zhilin Qu, Jamie Vandenberg, Colleen Clancy, Isabelle Deschenes, Leighton Izu, Tamas Banyasz, Andras Varro, Heike Wulff, Eleonora Grandi, Michael Sanguinetti, Donald Bers, Jeanne Nerbonne and Nipavan Chiamvimonvat as speakers and panel discussants. This article summarizes state-of-the-art knowledge and controversies on the functional roles of cardiac K+ channels in normal and diseased heart. We endeavour to integrate current knowledge at multiple scales, from the single cell to the whole organ levels, and from both experimental and computational studies.


Assuntos
Arritmias Cardíacas/fisiopatologia , Coração/fisiologia , Canais de Potássio/fisiologia , Animais , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Coração/fisiopatologia , Humanos , Modelos Biológicos
19.
J Mol Cell Cardiol ; 98: 138-45, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27238412

RESUMO

BACKGROUND: The paracrine action of non-cardiac progenitor cells is robust, but not well understood. Mesenchymal stem cells (MSC) have been shown to enhance calcium (Ca(++)) cycling in myocytes. Therefore, we hypothesized that MSCs can suppress cardiac alternans, an important arrhythmia substrate, by paracrine action on Ca(++) cycling. METHODS AND RESULTS: Human cardiac myocyte monolayers derived from iPS cells (hCM) were cultured without or with human MSCs (hMSC) directly or plated on a transwell insert. Ca(++) transient alternans (Ca(++) ALT) and Ca(++) transient duration (CaD) were measured from hCM monolayers following application of 200µM H2O2. Ca(++) ALT in hCM was significantly decreased when cultured with hMSCs directly (97%, p<0.0001) and when cultured with hMSC in the transwell insert (80%, p<0.0001). When hCM with hMSCs were pretreated with PI3K or eNOS inhibitors, Ca(++) ALT was larger than baseline by 20% (p<0.0001) and 36% (p<0.0001), respectively. In contrast, Ca(++) ALT was reduced by 89% compared to baseline (p<0.0001) when hCM monolayers without hMSCs were pretreated with 20µM GSNO. In all experiments, changes in Ca(++) ALT were mirrored by changes in CaD. Finally, real time quantitative PCR revealed no significant differences in mRNA expression of RyR2, SERCA2a, and phospholamban between hCM cultured with or without hMSCs. CONCLUSION: Ca(++) ALT is suppressed by hMSCs in a paracrine fashion due to activation of a PI3K-mediated nitroso-redox pathway. These findings demonstrate, for the first time, how stem cell therapy might be antiarrhythmic by suppressing cardiac alternans through paracrine action on Ca(++) cycling.


Assuntos
Glucanos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Oxirredução , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Comunicação Celular , Expressão Gênica , Humanos , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
20.
PLoS One ; 11(1): e0146561, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26764482

RESUMO

Cardiac ion channels and their respective accessory subunits are critical in maintaining proper electrical activity of the heart. Studies have indicated that the K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for the channel Kv4, a component of the transient outward K+ channel (Ito), is a Ca2+ binding protein whose regulatory function does not appear restricted to Kv4 modulation. Indeed, the guinea pig myocardium does not express Kv4, yet we show that it still maintains expression of KChIP2, suggesting roles for KChIP2 beyond this canonical auxiliary interaction with Kv4 to modulate Ito. In this study, we capitalize on the guinea pig as a system for investigating how KChIP2 influences the cardiac action potential, independent of effects otherwise attributed to Ito, given the endogenous absence of the current in this species. By performing whole cell patch clamp recordings on isolated adult guinea pig myocytes, we observe that knock down of KChIP2 significantly prolongs the cardiac action potential. This prolongation was not attributed to compromised repolarizing currents, as IKr and IKs were unchanged, but was the result of enhanced L-type Ca2+ current due to an increase in Cav1.2 protein. In addition, cells with reduced KChIP2 also displayed lowered INa from reduced Nav1.5 protein. Historically, rodent models have been used to investigate the role of KChIP2, where dramatic changes to the primary repolarizing current Ito may mask more subtle effects of KChIP2. Evaluation in the guinea pig where Ito is absent, has unveiled additional functions for KChIP2 beyond its canonical regulation of Ito, which defines KChIP2 as a master regulator of cardiac repolarization and depolarization.


Assuntos
Potenciais de Ação , Proteínas Interatuantes com Canais de Kv/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Cães , Cobaias , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Miócitos Cardíacos/fisiologia , Ratos
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