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1.
Nat Commun ; 13(1): 2185, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35449169

RESUMO

Cardiac excitation-contraction coupling requires dyads, the nanoscopic microdomains formed adjacent to Z-lines by apposition of transverse tubules and junctional sarcoplasmic reticulum. Disruption of dyad architecture and function are common features of diseased cardiomyocytes. However, little is known about the mechanisms that modulate dyad organization during cardiac development, homeostasis, and disease. Here, we use proximity proteomics in intact, living hearts to identify proteins enriched near dyads. Among these proteins is CMYA5, an under-studied striated muscle protein that co-localizes with Z-lines, junctional sarcoplasmic reticulum proteins, and transverse tubules in mature cardiomyocytes. During cardiac development, CMYA5 positioning adjacent to Z-lines precedes junctional sarcoplasmic reticulum positioning or transverse tubule formation. CMYA5 ablation disrupts dyad architecture, dyad positioning at Z-lines, and junctional sarcoplasmic reticulum Ca2+ release, leading to cardiac dysfunction and inability to tolerate pressure overload. These data provide mechanistic insights into cardiomyopathy pathogenesis by demonstrating that CMYA5 anchors junctional sarcoplasmic reticulum to Z-lines, establishes dyad architecture, and regulates dyad Ca2+ release.


Assuntos
Acoplamento Excitação-Contração , Retículo Sarcoplasmático , Cálcio/metabolismo , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo
2.
Methods Mol Biol ; 2442: 663-683, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320552

RESUMO

Galectin-1 is a small (14.5 kDa) multifunctional protein with cell-cell and cell-ECM adhesion due to interactions with the carbohydrate recognition domain (CRD). In two types of muscular dystrophies, this lectin protein has shown therapeutic properties, including positive regulation of skeletal muscle differentiation and regeneration. Both Duchenne and limb-girdle muscular dystrophy 2B (LGMD2B) are subtypes of muscular dystrophies characterized by deficient membrane repair, muscle weakness, and eventual loss of ambulation. This chapter explains confocal techniques such as laser injury, calcium imaging, and galectin-1 localization to examine the effects of galectin-1 on membrane repair in injured LGMD2B models.


Assuntos
Galectina 1 , Distrofia Muscular do Cíngulo dos Membros , Sarcolema , Galectina 1/metabolismo , Galectina 1/farmacologia , Galectina 1/uso terapêutico , Humanos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/tratamento farmacológico , Sarcolema/efeitos dos fármacos , Sarcolema/fisiologia
3.
Am J Physiol Cell Physiol ; 322(3): C546-C553, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35138177

RESUMO

We aimed to determine the combined effects of overexpressing plasma membrane fatty acid binding protein (FABPpm) and fatty acid translocase (CD36) on skeletal muscle fatty acid transport to establish if these transport proteins function collaboratively. Electrotransfection with either FABPpm or CD36 increased their protein content at the plasma membrane (+75% and +64%), increased fatty acid transport rates by +24% for FABPpm and +62% for CD36, resulting in a calculated transport efficiency of ∼0.019 and ∼0.053 per unit protein change for FABPpm and CD36, respectively. We subsequently used these data to determine if increasing both proteins additively or synergistically increased fatty acid transport. Cotransfection of FABPpm and CD36 simultaneously increased protein content in whole muscle (FABPpm, +46%; CD36, +45%) and at the sarcolemma (FABPpm, +41%; CD36, +42%), as well as fatty acid transport rates (+50%). Since the relative effects of changing FABPpm and CD36 content had been independently determined, we were able to a predict a change in fatty acid transport based on the overexpression of plasmalemmal transporters in the cotransfection experiments. This prediction yielded an increase in fatty acid transport of +0.984 and +1.722 pmol/mg prot/15 s for FABPpm and CD36, respectively, for a total increase of +2.96 pmol/mg prot/15 s. This calculated determination was remarkably consistent with the measured change in transport, namely +2.89 pmol/mg prot/15 s. Altogether, these data indicate that increasing CD36 and FABPpm alters fatty acid transport rates additively, but not synergistically, suggesting an independent mechanism of action within muscle for each transporter. This conclusion was further supported by the observation that plasmalemmal CD36 and FABPpm did not coimmunoprecipitate.


Assuntos
Proteínas de Ligação a Ácido Graxo , Ácidos Graxos , Transporte Biológico/fisiologia , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Sarcolema/metabolismo
4.
J Physiol ; 600(8): 1953-1968, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35156706

RESUMO

Dysferlin is an integral membrane protein of the transverse tubules of skeletal muscle that is mutated or absent in limb girdle muscular dystrophy 2B and Miyoshi myopathy. Here we examine the role of dysferlin's seven C2 domains, C2A through C2G, in membrane repair and Ca2+ release, as well as in targeting dysferlin to the transverse tubules of skeletal muscle. We report that deletion of either domain C2A or C2B inhibits membrane repair completely, whereas deletion of C2C, C2D, C2E, C2F or C2G causes partial loss of membrane repair that is exacerbated in the absence of extracellular Ca2+ . Deletion of C2C, C2D, C2E, C2F or C2G also causes significant changes in Ca2+ release, measured as the amplitude of the Ca2+ transient before or after hypo-osmotic shock and the appearance of Ca2+ waves. Most deletants accumulate in endoplasmic reticulum. Only the C2A domain can be deleted without affecting dysferlin trafficking to transverse tubules, but Dysf-ΔC2A fails to support normal Ca2+ signalling after hypo-osmotic shock. Our data suggest that (i) every C2 domain contributes to repair; (ii) all C2 domains except C2B regulate Ca2+ signalling; (iii) transverse tubule localization is insufficient for normal Ca2+ signalling; and (iv) Ca2+ dependence of repair is mediated by C2C through C2G. Thus, dysferlin's C2 domains have distinct functions in Ca2+ signalling and sarcolemmal membrane repair and may play distinct roles in skeletal muscle. KEY POINTS: Dysferlin, a transmembrane protein containing seven C2 domains, C2A through C2G, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients and participates in sarcolemmal membrane repair. Each of dysferlin's C2 domains except C2B regulate Ca2+ signalling. Localization of dysferlin variants to the transverse tubules is not sufficient to support normal Ca2+ signalling or membrane repair. Each of dysferlin's C2 domains contributes to sarcolemmal membrane repair. The Ca2+ dependence of membrane repair is mediated by C2C through C2G. Dysferlin's C2 domains therefore have distinct functions in Ca2+ signalling and sarcolemmal membrane repair.


Assuntos
Domínios C2 , Proteínas de Membrana , Disferlina/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Sarcolema/metabolismo
5.
Biochem Biophys Res Commun ; 595: 89-95, 2022 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-35121232

RESUMO

In cardiac muscle cells, heterodimeric integrin transmembrane receptors are known to serve as mechanotransducers, translating mechanical force to biochemical signaling. However, the roles of many individual integrins have still not been delineated. In this report, we demonstrate that Itga3b is localized to the sarcolemma of cardiomyocytes from 24 to 96 hpf. We further show that heterozygous and homozygous itga3b/bdf mutant embryos display a cardiomyopathy phenotype, with decreased cardiac contractility and reduced cardiomyocyte number. Correspondingly, proliferation of ventricular and atrial cardiomyoctyes and ventricular epicardial cells is decreased in itga3b mutant hearts. The contractile dysfunction of itga3b mutants can be attributed to cardiomyocyte sarcomeric disorganization, including thin myofilaments with blurred and shortened Z-discs. Together, our results reveal that Itga3b localizes to the myocardium sarcolemma, and it is required for cardiac contractility and cardiomyocyte proliferation.


Assuntos
Integrina alfa3/genética , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Apoptose/genética , Proliferação de Células/genética , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização In Situ , Integrina alfa3/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Contração Miocárdica/genética , Miocárdio/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/ultraestrutura , Sarcolema/metabolismo , Sarcolema/ultraestrutura , Sarcômeros/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
6.
NMR Biomed ; 35(3): e4688, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35060211

RESUMO

The article of Lopez et al describes the use of a multi-parametric MR approach to study muscle T2 relaxation times and 31 P-MRS indices of energetics and sarcolemma integrity in a mouse model of DMD, the mdx mouse. Muscular dystrophies have a multi-factorial disease cascade, and there are several MR methods used to assess these. Aspects that reflect disease progression are outlined on the left, while features that are more related to disease activity are outlined on the right.


Assuntos
Distrofia Muscular de Duchenne , Sarcolema , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos mdx , Músculos
7.
NMR Biomed ; 35(3): e4659, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34841594

RESUMO

31 Phosphorus magnetic resonance spectroscopy (31 P-MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid-base status, and free intracellular magnesium ([Mg2+ ]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine 31 P-MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild-type and to evaluate the effects of downhill running to induce muscle damage on 31 P-MRS indices in dystrophic muscle. In vivo 31 P-MRS spectra were acquired from the posterior hindlimb muscles in young (4-10 weeks of age) mdx (C57BL/10ScSn-DMDmdx) and wild-type (C57BL/10ScSnJ) mice using an 11.1-T MR system. The flux of phosphate from PCr to ATP was estimated by 31 P-MRS saturation transfer experiments. Relative concentrations of high-energy phosphates were measured, and intracellular pH and [Mg2+ ] were calculated. 1 H2 O-T2 was measured using single-voxel 1 H-MRS from the gastrocnemius and soleus using a 4.7-T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated 1 H2 O-T2 (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg2+ ] compared with wild-type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild-type mice compared with baseline, and a decrease (p < 0.001) in [Mg2+ ] and a lower (p = 0.048) intracellular [H+ ] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that 31 P-MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild-type mice, and these indices are exacerbated following downhill running.


Assuntos
Metabolismo Energético , Distrofia Muscular Animal/metabolismo , Sarcolema/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fosfocreatina/metabolismo , Fósforo , Condicionamento Físico Animal
8.
Annu Rev Physiol ; 84: 229-255, 2022 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-34780259

RESUMO

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.


Assuntos
Insuficiência Cardíaca , Sarcolema , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Humanos , Mamíferos , Miócitos Cardíacos/fisiologia , Sarcolema/metabolismo
9.
Int J Mol Sci ; 22(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34884867

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.


Assuntos
Distrofina/genética , Éxons , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Animais , Animais Geneticamente Modificados , Dependovirus/genética , Modelos Animais de Doenças , Proteínas Associadas à Distrofina/genética , Proteínas Associadas à Distrofina/metabolismo , Feminino , Deleção de Genes , Masculino , Fibras Musculares Esqueléticas/patologia , Técnicas de Transferência Nuclear , Oligonucleotídeos Antissenso/genética , Sarcolema/metabolismo , Suínos , Porco Miniatura
10.
Neuromuscul Disord ; 31(10): 1013-1020, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34736624

RESUMO

Duchenne muscular dystrophy (DMD) is a severe X-linked disease characterized by progressive muscle weakness. It is caused by a variety of DMD gene pathogenic variations (large deletions or duplications, and small mutations) which leads to the absence or to a decreased amount of dystrophin protein. The allelic Becker muscular dystrophy is characterized by later onset and milder muscle involvement, and other rarer phenotypes might also be associated, such as dilated cardiomyopathy, cognitive impairment, and other neurological signs. Following the identification of the genetic cause and the disease pathophysiology, innovative personalized therapies emerged. These can be categorized into two main groups: (1) therapies aiming at the restoration of dystrophin at the sarcolemma; (2) therapeutics dealing with secondary consequences of dystrophin deficiency. In this review we provide an overview about DMD genotype-phenotype correlation, and on main approaches to restore dystrophin as stop codon read-through, exon skipping, vector-mediated gene therapy, and genome-editing strategies, some of these are based on approved orphan drugs. Finally, we present the clinical potential of novel strategies combining therapies to correct the genetic defect and other approaches, targeting secondary downstream pathological cascade due to dystrophin deficiency.


Assuntos
Distrofina/metabolismo , Distrofia Muscular de Duchenne/terapia , Éxons , Terapia Genética , Humanos , Mutação , Fenótipo , Sarcolema/metabolismo
11.
J Cell Biol ; 220(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34633413

RESUMO

The cavin proteins are essential for caveola biogenesis and function. Here, we identify a role for the muscle-specific component, Cavin4, in skeletal muscle T-tubule development by analyzing two vertebrate systems, mouse and zebrafish. In both models, Cavin4 localized to T-tubules, and loss of Cavin4 resulted in aberrant T-tubule maturation. In zebrafish, which possess duplicated cavin4 paralogs, Cavin4b was shown to directly interact with the T-tubule-associated BAR domain protein Bin1. Loss of both Cavin4a and Cavin4b caused aberrant accumulation of interconnected caveolae within the T-tubules, a fragmented T-tubule network enriched in Caveolin-3, and an impaired Ca2+ response upon mechanical stimulation. We propose a role for Cavin4 in remodeling the T-tubule membrane early in development by recycling caveolar components from the T-tubule to the sarcolemma. This generates a stable T-tubule domain lacking caveolae that is essential for T-tubule function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sarcolema/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Cavéolas/metabolismo , Linhagem Celular , Embrião não Mamífero/metabolismo , Imageamento Tridimensional , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Ligação Proteica , Sarcolema/ultraestrutura , Peixe-Zebra/embriologia
12.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638854

RESUMO

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, affecting more than 33 million people worldwide. Despite important advances in therapy, AF's incidence remains high, and treatment often results in recurrence of the arrhythmia. A better understanding of the cellular and molecular changes that (1) trigger AF and (2) occur after the onset of AF will help to identify novel therapeutic targets. Over the past 20 years, a large body of research has shown that intracellular Ca2+ handling is dramatically altered in AF. While some of these changes are arrhythmogenic, other changes counteract cellular arrhythmogenic mechanisms (Calcium Signaling Silencing). The intracellular Na+ concentration ([Na+])i is a key regulator of intracellular Ca2+ handling in cardiac myocytes. Despite its importance in the regulation of intracellular Ca2+ handling, little is known about [Na+]i, its regulation, and how it might be changed in AF. Previous work suggests that there might be increases in the late component of the atrial Na+ current (INa,L) in AF, suggesting that [Na+]i levels might be high in AF. Indeed, a pharmacological blockade of INa,L has been suggested as a treatment for AF. Here, we review calcium signaling silencing and changes in intracellular Na+ homeostasis during AF. We summarize the proposed arrhythmogenic mechanisms associated with increases in INa,L during AF and discuss the evidence from clinical trials that have tested the pharmacological INa,L blocker ranolazine in the treatment of AF.


Assuntos
Fibrilação Atrial/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Homeostase/fisiologia , Miócitos Cardíacos/metabolismo , Sódio/metabolismo , Animais , Fibrilação Atrial/fisiopatologia , Humanos , Miócitos Cardíacos/citologia , Sarcolema/metabolismo , Trocador de Sódio e Cálcio/metabolismo
13.
Nat Commun ; 12(1): 4900, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385433

RESUMO

Skeletal muscle subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria subpopulations have distinct metabolic activity and sensitivity, though the mechanisms that localize SSM to peripheral areas of muscle fibers are poorly understood. A protein interaction study and complexome profiling identifies PERM1 interacts with the MICOS-MIB complex. Ablation of Perm1 in mice reduces muscle force, decreases mitochondrial membrane potential and complex I activity, and reduces the numbers of SSM in skeletal muscle. We demonstrate PERM1 interacts with the intracellular adaptor protein ankyrin B (ANKB) that connects the cytoskeleton to the plasma membrane. Moreover, we identify a C-terminal transmembrane helix that anchors PERM1 into the outer mitochondrial membrane. We conclude PERM1 functions in the MICOS-MIB complex and acts as an adapter to connect the mitochondria with the sarcolemma via ANKB.


Assuntos
Anquirinas/metabolismo , Mitocôndrias Musculares/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Musculares/metabolismo , Sarcolema/metabolismo , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia
14.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34313306

RESUMO

The ratio between Na+-Ca2+ exchange current densities in t-tubular and surface membranes of rat ventricular cardiomyocytes (JNaCa-ratio) estimated from electrophysiological data published to date yields strikingly different values between 1.7 and nearly 40. Possible reasons for such divergence were analysed by Monte Carlo simulations assuming both normal and log-normal distribution of the measured data. The confidence intervals CI95 of the mean JNaCa-ratios computed from the reported data showed an overlap of values between 1 and 3, and between 0.3 and 4.3 in the case of normal and log-normal distribution, respectively. Further analyses revealed that the published high values likely result from a large scatter of data due to transmural differences in JNaCa, dispersion of cell membrane capacitances and variability in incomplete detubulation. Taking into account the asymmetric distribution of the measured data, the reduction of mean current densities after detubulation and the substantially smaller CI95 of lower values of the mean JNaCa-ratio, the values between 1.6 and 3.2 may be considered as the most accurate estimates. This implies that 40 to 60% of Na+-Ca2+ exchanger is located at the t-tubular membrane of adult rat ventricular cardiomyocytes.


Assuntos
Cálcio , Miócitos Cardíacos , Animais , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Sarcolema/metabolismo , Sódio/metabolismo , Trocador de Sódio e Cálcio
15.
Artigo em Inglês | MEDLINE | ID: mdl-34332046

RESUMO

We indirectly assessed if altered transarcolemmal Ca2+ flux accompanies the decreased cardiac activity displayed by Trachemys scripta with anoxia exposure and cold acclimation. Turtles were first acclimated to 21 °C or 5 °C and held under normoxic (21N; 5N) or anoxic conditions (21A; 5A). We then compared the response of intrinsic heart rate (fH) and maximal developed force of spontaneously contracting right atria (Fmax,RA), and maximal developed force of isometrically-contracting ventricular strips (Fmax,V), to Ni2+ (0.1-10 mM), which respectively blocks T-type Ca2+ channels, L-type Ca2+ channels and the Na+-Ca2+-exchanger at the low, intermediate and high concentrations employed. Dose-response curves were established in simulated in vivo normoxic (Sim Norm) or simulated in vivo anoxic extracellular conditions (Sim Anx; 21A and 5A preparations). Ni2+ decreased intrinsic fH, Fmax,RA and Fmax,V of 21N tissues in a concentration-dependent manner, but the responses were blunted in 21A tissues in Sim Norm. Similarly, dose-response curves for Fmax,RA and Fmax,V of 5N tissues were right-shifted, whereas anoxia exposure at 5 °C did not further alter the responses. The influence of Sim Anx was acclimation temperature-, cardiac chamber- and contractile parameter-dependent. Combined, the findings suggest that: (1) reduced transarcolemmal Ca2+ flux in the cardiac pacemaker is a potential mechanism underlying the slowed intrinsic fH of anoxic turtles at 21 °C, but not 5 °C, (2) a downregulation of transarcolemmal Ca2+ flux may aid cardiac anoxia survival at 21 °C and prime the turtle myocardium for winter anoxia and (3) confirm that altered extracellular conditions with anoxia exposure can modify turtle cardiac transarcolemmal Ca2+ flux.


Assuntos
Adaptação Fisiológica/fisiologia , Cálcio/metabolismo , Átrios do Coração/patologia , Ventrículos do Coração/patologia , Hipóxia/fisiopatologia , Marca-Passo Artificial , Sarcolema/metabolismo , Animais , Pressão Sanguínea , Temperatura Baixa , Átrios do Coração/metabolismo , Frequência Cardíaca , Ventrículos do Coração/metabolismo , Tartarugas
16.
Nat Commun ; 12(1): 3596, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155205

RESUMO

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Receptores de Estrogênio/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Receptores de Estrogênio/química , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Transcriptoma/efeitos dos fármacos , Troponina I/genética , Troponina I/metabolismo
17.
J Mol Cell Cardiol ; 160: 1-14, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34175303

RESUMO

Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 µg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Hipotireoidismo/tratamento farmacológico , Tri-Iodotironina/administração & dosagem , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Expressão Gênica , Hipotireoidismo/sangue , Hipotireoidismo/induzido quimicamente , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Sarcômeros/metabolismo , Resultado do Tratamento , Tri-Iodotironina/sangue , Função Ventricular/efeitos dos fármacos
18.
JAMA Neurol ; 78(8): 948-960, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34115106

RESUMO

Importance: Myalgia, increased levels of creatine kinase, and persistent muscle weakness have been reported in patients with COVID-19. Objective: To study skeletal muscle and myocardial inflammation in patients with COVID-19 who had died. Design, Setting, and Participants: This case-control autopsy series was conducted in a university hospital as a multidisciplinary postmortem investigation. Patients with COVID-19 or other critical illnesses who had died between March 2020 and February 2021 and on whom an autopsy was performed were included. Individuals for whom informed consent to autopsy was available and the postmortem interval was less than 6 days were randomly selected. Individuals who were infected with SARS-CoV-2 per polymerase chain reaction test results and had clinical features suggestive of COVID-19 were compared with individuals with negative SARS-CoV-2 polymerase chain reaction test results and an absence of clinical features suggestive of COVID-19. Main Outcomes and Measures: Inflammation of skeletal muscle tissue was assessed by quantification of immune cell infiltrates, expression of major histocompatibility complex (MHC) class I and class II antigens on the sarcolemma, and a blinded evaluation on a visual analog scale ranging from absence of pathology to the most pronounced pathology. Inflammation of cardiac muscles was assessed by quantification of immune cell infiltrates. Results: Forty-three patients with COVID-19 (median [interquartile range] age, 72 [16] years; 31 men [72%]) and 11 patients with diseases other than COVID-19 (median [interquartile range] age, 71 [5] years; 7 men [64%]) were included. Skeletal muscle samples from the patients who died with COVID-19 showed a higher overall pathology score (mean [SD], 3.4 [1.8] vs 1.5 [1.0]; 95% CI, 0-3; P < .001) and a higher inflammation score (mean [SD], 3.5 [2.1] vs 1.0 [0.6]; 95% CI, 0-4; P < .001). Relevant expression of MHC class I antigens on the sarcolemma was present in 23 of 42 specimens from patients with COVID-19 (55%) and upregulation of MHC class II antigens in 7 of 42 specimens from patients with COVID-19 (17%), but neither were found in any of the controls. Increased numbers of natural killer cells (median [interquartile range], 8 [8] vs 3 [4] cells per 10 high-power fields; 95% CI, 1-10 cells per 10 high-power fields; P < .001) were found. Skeletal muscles showed more inflammatory features than cardiac muscles, and inflammation was most pronounced in patients with COVID-19 with chronic courses. In some muscle specimens, SARS-CoV-2 RNA was detected by reverse transcription-polymerase chain reaction, but no evidence for a direct viral infection of myofibers was found by immunohistochemistry and electron microscopy. Conclusions and Relevance: In this case-control study of patients who had died with and without COVID-19, most individuals with severe COVID-19 showed signs of myositis ranging from mild to severe. Inflammation of skeletal muscles was associated with the duration of illness and was more pronounced than cardiac inflammation. Detection of viral load was low or negative in most skeletal and cardiac muscles and probably attributable to circulating viral RNA rather than genuine infection of myocytes. This suggests that SARS-CoV-2 may be associated with a postinfectious, immune-mediated myopathy.


Assuntos
COVID-19/patologia , Músculo Esquelético/patologia , Miocardite/patologia , Miocárdio/patologia , Miosite/patologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Linfócitos T CD8-Positivos/patologia , COVID-19/metabolismo , Teste de Ácido Nucleico para COVID-19 , Teste Sorológico para COVID-19 , Estudos de Casos e Controles , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Células Matadoras Naturais/patologia , Leucócitos/patologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Miocardite/metabolismo , Miocárdio/metabolismo , Miosite/metabolismo , RNA Viral/metabolismo , SARS-CoV-2 , Sarcolema/metabolismo , Fatores de Tempo
19.
Exp Neurol ; 342: 113758, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33991525

RESUMO

To get insight into the mechanism of action of carbonic anhydrase inhibitors (CAI) in neuromuscular disorders, we investigated effects of dichlorphenamide (DCP) and acetazolamide (ACTZ) on ClC-1 chloride channels and skeletal muscle excitability. We performed patch-clamp experiments to test drugs on chloride currents in HEK293T cells transfected with hClC-1. Using the two-intracellular microelectrode technique in current-clamp mode, we measured the effects of drugs on the resting chloride conductance and action potential properties of sarcolemma in rat and mouse skeletal muscle fibers. Using BCECF dye fluorometry, we measured the effects of ACTZ on intracellular pH in single rat muscle fibers. Similarly to ACTZ, DCP (100 µM) increased hClC-1 chloride currents in HEK cells, because of the negative shift of the open probability voltage dependence and the slowing of deactivation kinetics. Bendroflumethiazide (BFT, 100 µM), structurally related to DCP but lacking activity on carbonic anhydrase, had little effects on chloride currents. In isolated rat muscle fibers, 50-100 µM of ACTZ or DCP, but not BFT, induced a ~ 20% increase of the resting chloride conductance. ACTZ reduced action potential firing in mouse muscle fibers. ACTZ (100 µM) reduced intracellular pH to 6.8 in rat muscle fibers. These results suggest that carbonic anhydrase inhibitors can reduce muscle excitability by increasing ClC-1 channel activity, probably through intracellular acidification. Such a mechanism may contribute in part to the clinical effects of these drugs in myotonia and other muscle excitability disorders.


Assuntos
Inibidores da Anidrase Carbônica/farmacologia , Canais de Cloreto/metabolismo , Doenças Musculares/metabolismo , Sarcolema/metabolismo , Animais , Inibidores da Anidrase Carbônica/uso terapêutico , Diclorofenamida/farmacologia , Diclorofenamida/uso terapêutico , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Doenças Musculares/tratamento farmacológico , Ratos , Ratos Wistar , Sarcolema/efeitos dos fármacos
20.
Neurogenetics ; 22(2): 117-125, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811585

RESUMO

We report a multiplex family with extended multisystem neurological phenotype associated with a CRYAB variant. Two affected siblings were evaluated with whole exome sequencing, muscle biopsy, laser microdissection, and mass spectrometry-based proteomic analysis. Both patients and their mother manifested a combination of early-onset cataracts, cardiomyopathy, cerebellar ataxia, optic atrophy, cognitive impairment, and myopathy. Whole exome sequencing identified a heterozygous c.458C>T variant mapped to the C-terminal extension domain of the Alpha-crystallin B chain, disrupting its function as a molecular chaperone and its ability to suppress protein aggregation. In accordance with the molecular findings, muscle biopsies revealed subsarcolemmal deposits that appeared dark with H&E and trichrome staining were negative for the other routine histochemical staining and for amyloid with the Congo-red stain. Electron microscopy demonstrated that the deposits were composed of numerous parallel fibrils. Laser microdissection and mass spectrometry-based proteomic analysis revealed that the inclusions are almost exclusively composed of crystallized chaperones/heat shock proteins. Moreover,  a structural model suggests that Ser153 could be involved in monomer stabilization, dimer association, and possible binding of partner proteins. We propose that our report potentially expands the complex phenotypic spectrum of alpha B-crystallinopathies with possible effect of a CRYAB variant on the central nervous system.


Assuntos
Cardiomiopatia Hipertrófica/genética , Catarata/genética , Ataxia Cerebelar/genética , Disfunção Cognitiva/genética , Atrofia Óptica/genética , Sarcolema/ultraestrutura , Cadeia B de alfa-Cristalina/genética , Sequência de Aminoácidos , Biópsia , Morte Súbita Cardíaca/etiologia , Feminino , Humanos , Corpos de Inclusão/ultraestrutura , Judeus/genética , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Debilidade Muscular/genética , Músculo Esquelético/patologia , Fenótipo , Conformação Proteica , Sequenciamento Completo do Exoma
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