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1.
Proc Natl Acad Sci U S A ; 119(19): e2201136119, 2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35507876

RESUMO

SignificanceVertebrate skeletal muscle excitation-contraction coupling (ECC) is based on Ca2+-influx-independent interchannel cross-talk between DHPR and RyR1. The skeletal muscle DHPR complex consists of the main, voltage-sensing, and pore-forming α1S subunit, the auxiliary ß1a, α2δ-1, γ1 subunits, and Stac3. The DHPRß1a subunit plays an essential role in full triad targeting of DHPRα1S, voltage sensing, and tetrad formation (grouping of four DHPRs)-the three prerequisites for skeletal muscle ECC. Hence, a lack of DHPRß1a results in a lethal phenotype in both ß1-null mice and zebrafish. Here, we identified the nonconserved, distal C terminus of DHPRß1a as playing a pivotal role in the formation of DHPR tetrads, and thus allosteric DHPR-RyR1 coupling, essential for proper skeletal muscle ECC.


Assuntos
Canais de Cálcio Tipo L , Fibras Musculares Esqueléticas , Canal de Liberação de Cálcio do Receptor de Rianodina , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
2.
Protein Sci ; 31(5): e4311, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481653

RESUMO

Excitation-contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage-dependent L-type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine-rich domain-containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II-III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II-III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II-III loop. Therefore, this work demonstrates an isoform-specific interaction between STAC3 and the II-III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.


Assuntos
Canais de Cálcio Tipo L , Canal de Liberação de Cálcio do Receptor de Rianodina , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração/fisiologia , Músculo Esquelético/fisiologia , Isoformas de Proteínas/metabolismo
3.
Cells ; 11(7)2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35406654

RESUMO

Cardiomyocyte calcium-handling is the key mediator of cardiac excitation-contraction coupling. In the healthy heart, calcium controls both electrical impulse propagation and myofilament cross-bridge cycling, providing synchronous and adequate contraction of cardiac muscles. However, calcium-handling abnormalities are increasingly implicated as a cause of cardiac arrhythmias. Due to the complex, dynamic and localized interactions between calcium and other molecules within a cardiomyocyte, it remains experimentally challenging to study the exact contributions of calcium-handling abnormalities to arrhythmogenesis. Therefore, multiscale computational modeling is increasingly being used together with laboratory experiments to unravel the exact mechanisms of calcium-mediated arrhythmogenesis. This article describes various examples of how integrative computational modeling makes it possible to unravel the arrhythmogenic consequences of alterations to cardiac calcium handling at subcellular, cellular and tissue levels, and discusses the future challenges on the integration and interpretation of such computational data.


Assuntos
Cálcio , Miócitos Cardíacos , Arritmias Cardíacas , Simulação por Computador , Acoplamento Excitação-Contração , Humanos , Miócitos Cardíacos/fisiologia
4.
Proc Natl Acad Sci U S A ; 119(16): e2117435119, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35412911

RESUMO

SignificanceExcitation-transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca2+ signals into selective gene transcription related to chemotaxis, leukocyte adhesion, and inflammation. Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.


Assuntos
Canais de Cálcio Tipo L , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Cavéolas , Transcrição Genética , Remodelação Vascular , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Cavéolas/metabolismo , Caveolina 1/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Acoplamento Excitação-Contração , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Neurônios/metabolismo , Fosforilação
5.
J Gen Physiol ; 154(9)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-35482009

RESUMO

Each heartbeat begins with the generation of an action potential in pacemaking cells in the sinoatrial node. This signal triggers contraction of cardiac muscle through a process termed excitation-contraction (EC) coupling. EC coupling is initiated in dyadic structures of cardiac myocytes, where ryanodine receptors in the junctional sarcoplasmic reticulum come into close apposition with clusters of CaV1.2 channels in invaginations of the sarcolemma. Cooperative activation of CaV1.2 channels within these clusters causes a local increase in intracellular Ca2+ that activates the juxtaposed ryanodine receptors. A salient feature of healthy cardiac function is the reliable and precise beat-to-beat pacemaking and amplitude of Ca2+ transients during EC coupling. In this review, we discuss recent discoveries suggesting that the exquisite reproducibility of this system emerges, paradoxically, from high variability at subcellular, cellular, and network levels. This variability is attributable to stochastic fluctuations in ion channel trafficking, clustering, and gating, as well as dyadic structure, which increase intracellular Ca2+ variance during EC coupling. Although the effects of these large, local fluctuations in function and organization are sometimes negligible at the macroscopic level owing to spatial-temporal summation within and across cells in the tissue, recent work suggests that the "noisiness" of these intracellular Ca2+ events may either enhance or counterintuitively reduce variability in a context-dependent manner. Indeed, these noisy events may represent distinct regulatory features in the tuning of cardiac contractility. Collectively, these observations support the importance of incorporating experimentally determined values of Ca2+ variance in all EC coupling models. The high reproducibility of cardiac contraction is a paradoxical outcome of high Ca2+ signaling variability at subcellular, cellular, and network levels caused by stochastic fluctuations in multiple processes in time and space. This underlying stochasticity, which counterintuitively manifests as reliable, consistent Ca2+ transients during EC coupling, also allows for rapid changes in cardiac rhythmicity and contractility in health and disease.


Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Cálcio/metabolismo , Acoplamento Excitação-Contração , Miócitos Cardíacos/metabolismo , Reprodutibilidade dos Testes , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
6.
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
7.
Methods Mol Biol ; 2475: 215-222, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35451760

RESUMO

Calcium Ca2+ regulation is a key component of numerous cellular functions. In cardiomyocytes, Ca2+ regulates excitation-contraction coupling and influences signaling cascades involved in cell metabolism and cell survival. Prolonged dysregulation of mitochondrial Ca2+ leads to dysfunctional cardiomyocytes, apoptosis and ultimately heart failure. VEGF promotes cardiomyocyte contractility by increasing calcium transients to control the strength of the heartbeat. Here, we describe a method to measure mitochondrial Ca2+ fluxes in human ventricular cardiomocytes after inducing stretch-mediated hypertrophy in vitro.


Assuntos
Cálcio , Miócitos Cardíacos , Cálcio/metabolismo , Sinalização do Cálcio , Acoplamento Excitação-Contração , Ventrículos do Coração , Humanos , Hipertrofia/metabolismo , Miócitos Cardíacos/metabolismo
8.
J Gen Physiol ; 154(9)2022 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-35349630

RESUMO

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, ß3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents.


Assuntos
Processamento Alternativo , Cálcio , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Acoplamento Excitação-Contração , Células HEK293 , Humanos
10.
J Gen Physiol ; 154(3)2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35179560

RESUMO

JGP study reveals that lower troponin expression in the right ventricle underlies interventricular differences in excitation-contraction coupling.


Assuntos
Acoplamento Excitação-Contração , Troponina , Ventrículos do Coração/metabolismo , Contração Miocárdica , Troponina/metabolismo
11.
Clin Neurophysiol ; 135: 30-36, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35026538

RESUMO

OBJECTIVE: This study aimed to develop a simple and reliable technique to assess excitation-contraction (E-C) coupling for early diagnosis of critical illness myopathy (CIM). METHODS: We prospectively performed clinical and electrophysiological examinations on patients admitted to intensive care unit (ICU). In addition to full neurological examinations and routine nerve conduction study, motor related potential (MRP) was recorded using an accelerometer attached to the base of hallux after tibial nerve stimulation, and E-C coupling time (ECCT) was measured from the latency difference between soleus compound muscle action potential (CMAP) and MRP. RESULTS: Of 41 patients evaluated, 25 met the criteria for ICU-acquired weakness, 23 of whom had CIM. The time to the first electrophysiological examination (time to first test) correlated negatively with CMAP and with MRP. Conversely, a positive correlation was observed between the time to first test and ECCT. E-C coupling impairment occurred in most of our patients with CIM by the third day of ICU admission, and prolonged ECCT could be the earliest detectable abnormality. CONCLUSIONS: The ECCT measurement is an easy and reliable technique to detect reduced muscle membrane excitability in the early stage of CIM. SIGNIFICANCE: The ECCT measured by our method using an accelerometer may be a parameter that predicts the development of CIM.


Assuntos
Acoplamento Excitação-Contração , Doenças Musculares/fisiopatologia , Acelerometria/instrumentação , Acelerometria/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estado Terminal , Diagnóstico Precoce , Eletromiografia/instrumentação , Eletromiografia/métodos , Potencial Evocado Motor , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Doenças Musculares/diagnóstico
12.
Pflugers Arch ; 474(1): 33-61, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34978597

RESUMO

Diabetic cardiomyopathy is defined as the myocardial dysfunction that suffers patients with diabetes mellitus (DM) in the absence of hypertension and structural heart diseases such as valvular or coronary artery dysfunctions. Since the impact of DM on cardiac function is rather silent and slow, early stages of diabetic cardiomyopathy, known as prediabetes, are poorly recognized, and, on many occasions, cardiac illness is diagnosed only after a severe degree of dysfunction was reached. Therefore, exploration and recognition of the initial pathophysiological mechanisms that lead to cardiac dysfunction in diabetic cardiomyopathy are of vital importance for an on-time diagnosis and treatment of the malady. Among the complex and intricate mechanisms involved in diabetic cardiomyopathy, Ca2+ mishandling and mitochondrial dysfunction have been described as pivotal early processes. In the present review, we will focus on these two processes and the molecular pathway that relates these two alterations to the earlier stages and the development of diabetic cardiomyopathy.


Assuntos
Cálcio/metabolismo , Cardiomiopatias Diabéticas/etiologia , Mitocôndrias Cardíacas/metabolismo , Estado Pré-Diabético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Citosol/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Acoplamento Excitação-Contração , Humanos
13.
Elife ; 112022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34985413

RESUMO

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than -30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below -21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.


Assuntos
Potenciais de Ação/fisiologia , Acoplamento Excitação-Contração , Potenciais da Membrana , Músculo Esquelético/fisiologia , Animais , Camundongos
14.
J Cachexia Sarcopenia Muscle ; 13(1): 454-466, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35001540

RESUMO

BACKGROUND: The role of Numb, a protein that is important for cell fate and development and that, in human muscle, is expressed at reduced levels with advanced age, was investigated; adult mice skeletal muscle and its localization and function within myofibres were determined. METHODS: Numb expression was evaluated by western blot. Numb localization was determined by confocal microscopy. The effects of conditional knock out (cKO) of Numb and the closely related gene Numb-like in skeletal muscle fibres were evaluated by in situ physiology, transmission and focused ion beam scanning electron microscopy, three-dimensional reconstruction of mitochondria, lipidomics, and bulk RNA sequencing. Additional studies using primary mouse myotubes investigated the effects of Numb knockdown on cell fusion, mitochondrial function, and calcium transients. RESULTS: Numb protein expression was reduced by ~70% (P < 0.01) at 24 as compared with 3 months of age in gastrocnemius and tibialis anterior muscle. Numb was localized within muscle fibres as bands traversing fibres at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36% (P < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (P < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (P < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1, and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acids through lipoxygenases were up-regulated in Numb cKO skeletal muscle: 12-HEPE was increased by ~250% (P < 0.05) and 17,18-EpETE by ~240% (P < 0.05). In mouse primary myotubes, Numb knockdown reduced cell fusion (~20%, P < 0.01) and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (P < 0.01). CONCLUSIONS: These findings implicate Numb as a critical factor in skeletal muscle structure and function and suggest that Numb is critical for calcium release. We therefore speculate that Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.


Assuntos
Proteínas de Membrana , Músculo Esquelético , Proteínas do Tecido Nervoso , Retículo Sarcoplasmático , Animais , Cálcio/metabolismo , Acoplamento Excitação-Contração , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Retículo Sarcoplasmático/metabolismo
15.
Pflugers Arch ; 474(3): 267-279, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34820713

RESUMO

14-3-3 proteins (14-3-3 s) are a family of highly conserved proteins that regulate many cellular processes in eukaryotes by interacting with a diverse array of client proteins. The 14-3-3 proteins have been implicated in several disease states and previous reviews have condensed the literature with respect to their structure, function, and the regulation of different cellular processes. This review focuses on the growing body of literature exploring the important role 14-3-3 proteins appear to play in regulating the biochemical and biophysical events associated with excitation-contraction coupling (ECC) in muscle. It presents both a timely and unique analysis that seeks to unite studies emphasizing the identification and diversity of 14-3-3 protein function and client protein interactions, as modulators of muscle contraction. It also highlights ideas within these two well-established but intersecting fields that support further investigation with respect to the mechanistic actions of 14-3-3 proteins in the modulation of force generation in muscle.


Assuntos
Proteínas 14-3-3 , Acoplamento Excitação-Contração , Proteínas 14-3-3/metabolismo , Cálcio/metabolismo , Acoplamento Excitação-Contração/fisiologia , Humanos , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo
16.
Cardiovasc Res ; 118(2): 503-516, 2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33624748

RESUMO

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare laminopathy caused by expression of progerin, a lamin A variant, also present at low levels in non-HGPS individuals. HGPS patients age and die prematurely, predominantly from cardiovascular complications. Progerin-induced cardiac repolarization defects have been described previously, although the underlying mechanisms are unknown. METHODS AND RESULTS: We conducted studies in heart tissue from progerin-expressing LmnaG609G/G609G (G609G) mice, including microscopy, intracellular calcium dynamics, patch-clamping, in vivo magnetic resonance imaging, and electrocardiography. G609G mouse cardiomyocytes showed tubulin-cytoskeleton disorganization, t-tubular system disruption, sarcomere shortening, altered excitation-contraction coupling, and reductions in ventricular thickening and cardiac index. G609G mice exhibited severe bradycardia, and significant alterations of atrio-ventricular conduction and repolarization. Most importantly, 50% of G609G mice had altered heart rate variability, and sinoatrial block, both significant signs of premature cardiac aging. G609G cardiomyocytes had electrophysiological alterations, which resulted in an elevated action potential plateau and early afterdepolarization bursting, reflecting slower sodium current inactivation and long Ca+2 transient duration, which may also help explain the mild QT prolongation in some HGPS patients. Chronic treatment with low-dose paclitaxel ameliorated structural and functional alterations in G609G hearts. CONCLUSIONS: Our results demonstrate that tubulin-cytoskeleton disorganization in progerin-expressing cardiomyocytes causes structural, cardiac conduction, and excitation-contraction coupling defects, all of which can be partially corrected by chronic treatment with low dose paclitaxel.


Assuntos
Antiarrítmicos/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Citoesqueleto/efeitos dos fármacos , Acoplamento Excitação-Contração/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Paclitaxel/farmacologia , Progéria/tratamento farmacológico , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Masculino , Camundongos Mutantes , Mutação , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Progéria/genética , Progéria/metabolismo , Progéria/fisiopatologia , Período Refratário Eletrofisiológico/efeitos dos fármacos , Suínos , Porco Miniatura , Tubulina (Proteína)/metabolismo
17.
Int J Mol Sci ; 22(22)2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34830262

RESUMO

Mag-Fluo-4 has revealed differences in the kinetics of the Ca2+ transients of mammalian fiber types (I, IIA, IIX, and IIB). We simulated the changes in [Ca2+] through the sarcomere of these four fiber types, considering classical (troponin -Tn-, parvalbumin -Pv-, adenosine triphosphate -ATP-, sarcoplasmic reticulum Ca2+ pump -SERCA-, and dye) and new (mitochondria -MITO-, Na+/Ca2+ exchanger -NCX-, and store-operated calcium entry -SOCE-) Ca2+ binding sites, during single and tetanic stimulation. We found that during a single twitch, the sarcoplasmic peak [Ca2+] for fibers type IIB and IIX was around 16 µM, and for fibers type I and IIA reached 10-13 µM. The release rate in fibers type I, IIA, IIX, and IIB was 64.8, 153.6, 238.8, and 244.5 µM ms-1, respectively. Both the pattern of change and the peak concentrations of the Ca2+-bound species in the sarcoplasm (Tn, PV, ATP, and dye), the sarcolemma (NCX, SOCE), and the SR (SERCA) showed the order IIB ≥ IIX > IIA > I. The capacity of the NCX was 2.5, 1.3, 0.9, and 0.8% of the capacity of SERCA, for fibers type I, IIA, IIX, and IIB, respectively. MITO peak [Ca2+] ranged from 0.93 to 0.23 µM, in fibers type I and IIB, respectively, while intermediate values were obtained in fibers IIA and IIX. The latter numbers doubled during tetanic stimulation. In conclusion, we presented a comprehensive mathematical model of the excitation-contraction coupling that integrated most classical and novel Ca2+ handling mechanisms, overcoming the limitations of the fast- vs. slow-fibers dichotomy and the use of slow dyes.


Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração/fisiologia , Modelos Teóricos , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Sarcômeros/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Simulação por Computador , Cinética , Camundongos , Mitocôndrias/metabolismo , Parvalbuminas/metabolismo , Retículo Sarcoplasmático/metabolismo , Troponina/metabolismo
18.
FEBS Lett ; 595(22): 2756-2767, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34693525

RESUMO

Neuronatin (NNAT) is a transmembrane protein in the endoplasmic reticulum involved in metabolic regulation. It shares sequence homology with sarcolipin (SLN), which negatively regulates the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) that maintains energy homeostasis in muscles. Here, we examined whether NNAT could uncouple the Ca2+ transport activity of SERCA from ATP hydrolysis, similarly to SLN. NNAT significantly reduced Ca2+ uptake without altering SERCA activity, ultimately lowering the apparent coupling ratio of SERCA. This effect of NNAT was reversed by the adenylyl cyclase activator forskolin. Furthermore, soleus muscles from high fat diet (HFD)-fed mice showed a significant downregulation in NNAT content compared with chow-fed mice, whereas an upregulation in NNAT content was observed in fast-twitch muscles from HFD- versus chow- fed mice. Therefore, NNAT is a SERCA uncoupler in cells and may function in adaptive thermogenesis.


Assuntos
Acoplamento Excitação-Contração , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Animais , Sinalização do Cálcio , Colforsina/farmacologia , Dieta Hiperlipídica , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Proteínas do Tecido Nervoso/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
19.
Circulation ; 144(21): 1694-1713, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34648376

RESUMO

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.


Assuntos
Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/etiologia , Síndrome de Barth/complicações , Síndrome de Barth/genética , Canais de Cálcio/deficiência , Contração Miocárdica/genética , Trifosfato de Adenosina/biossíntese , Animais , Síndrome de Barth/metabolismo , Biomarcadores , Encéfalo/metabolismo , Cálcio/metabolismo , Diástole , Modelos Animais de Doenças , Suscetibilidade a Doenças , Acoplamento Excitação-Contração/genética , Testes de Função Cardíaca , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Volume Sistólico , Sístole
20.
J Gen Physiol ; 153(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34636893

RESUMO

One of the most important functions of skeletal muscle is to respond to nerve stimuli by contracting. This function ensures body movement but also participates in other important physiological roles, like regulation of glucose homeostasis. Muscle activity is closely regulated to adapt to different demands and shows a plasticity that relies on both transcriptional activity and nerve stimuli. These two processes, both dependent on depolarization of the plasma membrane, have so far been regarded as separated and independent processes due to a lack of evidence of common protein partners or molecular mechanisms. In this study, we reveal intimate functional interactions between the process of excitation-induced contraction and the process of excitation-induced transcriptional activity in skeletal muscle. We show that the plasma membrane voltage-sensing protein CaV1.1 and the ATP-releasing channel Pannexin-1 (Panx1) regulate each other in a reciprocal manner, playing roles in both processes. Specifically, knockdown of CaV1.1 produces chronically elevated extracellular ATP concentrations at rest, consistent with disruption of the normal control of Panx1 activity. Conversely, knockdown of Panx1 affects not only activation of transcription but also CaV1.1 function on the control of muscle fiber contraction. Altogether, our results establish the presence of bidirectional functional regulations between the molecular machineries involved in the control of contraction and transcription induced by membrane depolarization of adult muscle fibers. Our results are important for an integrative understanding of skeletal muscle function and may impact our understanding of several neuromuscular diseases.


Assuntos
Canais de Cálcio Tipo L , Acoplamento Excitação-Contração , Canais de Cálcio Tipo L/metabolismo , Contração Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo
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