RESUMO
The skeletal muscle dihydropyridine receptor (DHPR) ß1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitationcontraction (EC) coupling. Previously, we showed that all four vertebrate ß isoforms (ß1ß4) facilitate α1S triad targeting and, except for ß3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 74887493 (2013)]. Consequently, ß3 failed to restore EC coupling despite the fact that both ß3 and ß1a restore tetrads. Thus, all ß-subunits are able to restore triad targeting, but only ß1a restores both tetrads and proper DHPRRyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 74887493 (2013)]. To investigate the molecular region(s) of ß1a responsible for the tetradic arrangement of DHPRs and thus DHPRRyR1 coupling, we expressed loss- and gain-of-function chimeras between ß1a and ß4, with systematically swapped domains in zebrafish strain relaxed (ß1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. ß1a/ß4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from ß4 showed complete restoration of SR Ca2+ release. However, chimera ß1a/ß4(C) with ß4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera ß4/ß1a(C) with ß1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of ß1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPRRyR1 interaction, essential for skeletal muscle EC coupling.
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/metabolismoRESUMO
Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222CâA, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598CâT) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).
Assuntos
Mutação em Linhagem Germinativa , Transtornos do Crescimento/genética , Hipoglicemia/genética , Fator 1 Induzível por Hipóxia/deficiência , Mitocôndrias/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Expressão Gênica , Crescimento/genética , Humanos , Masculino , Metaboloma/genética , Metaboloma/fisiologia , Síndrome , Adulto JovemRESUMO
Mitochondria respond to stress and undergo fusion and fission at variable rates, depending on cell status. To understand mitochondrial behavior during muscle fatigue, we investigated mitochondrial ultrastructure and expression levels of a fission- and stress-related protein in fast-twitch muscle fibers of mice subjected to fatigue testing. Mice were subjected to running at increasing speed until exhaustion at 45â min-1â h. In further experiments, high-intensity muscle stimulation through the sciatic nerve simulated the forced treadmill exercise. We detected a rare phenotype characterized by elongated mitochondrial constrictions (EMCs) connecting two separate segments of the original organelles. EMCs are rare in resting muscles and their frequency increases, albeit still at low levels, in stimulated muscles. The constrictions are accompanied by elevated phosphorylation of Drp1 (Dnm1l) at Ser 616, indicating an increased translocation of Drp1 to the mitochondrial membrane. This is indicative of a mitochondrial stress response, perhaps leading to or facilitating a long-lasting fission event. A close apposition of sarcoplasmic reticulum (SR) to the constricted areas, detected using both transmission and scanning electron microscopy, is highly suggestive of SR involvement in inducing mitochondrial constrictions.
Assuntos
Fadiga Muscular/fisiologia , Músculo Esquelético/metabolismo , Animais , Camundongos , Mitocôndrias/metabolismoRESUMO
NEW FINDINGS: What is the topic for this review? This review summarizes recent discoveries in mitochondrial development and morphology studied with electron microscopy. What advances does it highlight? Although mitochondria are generally considered to be isolated from each other, this review highlights recently discovered evidence for the presence of intermitochondrial communication structures in cardiac and skeletal muscle, in animal models and humans. Within striated muscles, the means of intermitochondrial exchange and the reaction of mitochondria to external stimuli are uniquely dependent on the tissue, and we clearly differentiate between nanotunnels, the active protrusion of cardiac mitochondria, and the connecting ducts of skeletal muscle derived from fusion-fission and elongation events. ABSTRACT: This review focuses on recent discoveries in skeletal and cardiac muscles indicating that mitochondria behave as an interactive cohort with inter-organelle communication and specific reactions to stress signals. Our new finding is that intermitochondrial communications in cardiac and skeletal muscles rely on two distinct methods. In cardiac muscle, mitochondria are discrete entities and are fairly well immobilized in a structural context. The organelles have developed a unique method of communication, via nanotunnels, which allow temporary connection from one mitochondrion to another over distances of up to several micrometres, without overall movement of the individual organelles and loss of their identity. Skeletal muscle mitochondria, in contrast, are dynamic. Through fusion, fission and elongation, they form connections that include constrictions and connecting ducts (distinct from nanotunnels) and lose individual identity in the formation of extensive networks. Connecting elements in skeletal muscle are distinct from nanotunnels in cardiac muscle.
Assuntos
Coração/fisiologia , Mitocôndrias Cardíacas/fisiologia , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/fisiologia , Animais , Humanos , MiocárdioRESUMO
Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR--measured with a calsequestrin-fused biosensor--was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.
Assuntos
Cálcio/metabolismo , Calsequestrina/metabolismo , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Canais de Cálcio/metabolismo , Camundongos , Miocárdio/metabolismoRESUMO
Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Canais de Cálcio Tipo L/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Geneticamente Modificados , Cafeína/farmacologia , Cálcio , Embrião não Mamífero , Microscopia Eletrônica , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/ultraestrutura , Mutação , Miotonia Congênita , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.
Assuntos
Sinalização do Cálcio/fisiologia , Mitocôndrias Cardíacas/fisiologia , Animais , Acoplamento Excitação-Contração/fisiologia , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/ultraestrutura , Dinâmica Mitocondrial/fisiologia , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Canal de Liberação de Cálcio do Receptor de Rianodina/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Taquicardia Ventricular/genéticaRESUMO
Stromal interacting molecule (STIM) and Orai proteins constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize STIM1 and Orai1 at endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HEK 293 cells. Compared with control cells, thin sections of STIM1-transfected cells possessed far more ER elements, which took the form of complex stackable cisternae and labyrinthine structures adjoining the PM at junctional couplings (JCs). JC formation required STIM1 expression but not store depletion, induced here by thapsigargin (TG). Extended molecules, indicative of STIM1, decorated the cytoplasmic surface of ER, bridged a 12-nm ER-PM gap, and showed clear rearrangement into small clusters following TG treatment. Freeze-fracture replicas of the PM of Orai1-transfected cells showed extensive domains packed with characteristic "particles"; TG treatment led to aggregation of these particles into sharply delimited "puncta" positioned upon raised membrane subdomains. The size and spacing of Orai1 channels were consistent with the Orai crystal structure, and stoichiometry was unchanged by store depletion, coexpression with STIM1, or an Orai1 mutation (L273D) affecting STIM1 association. Although the arrangement of Orai1 channels in puncta was substantially unstructured, a portion of channels were spaced at â¼15 nm. Monte Carlo analysis supported a nonrandom distribution for a portion of channels spaced at â¼15 nm. These images offer dramatic, direct views of STIM1 aggregation and Orai1 clustering in store-depleted cells and provide evidence for the interaction of a single Orai1 channel with small clusters of STIM1 molecules.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cálcio/genética , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Inibidores Enzimáticos/farmacologia , Técnica de Fratura por Congelamento , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Microscopia Eletrônica , Microscopia de Fluorescência/métodos , Método de Monte Carlo , Mutação , Proteínas de Neoplasias/genética , Proteína ORAI1 , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Molécula 1 de Interação Estromal , Tapsigargina/farmacologia , Gravação de VideoteipeRESUMO
Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.
Assuntos
Cadeias Pesadas de Miosina/fisiologia , Sequência de Aminoácidos , Animais , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , SuínosRESUMO
Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca(2+) handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na(+)/Ca(2+) exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.
Assuntos
Insuficiência Cardíaca/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Estresse Fisiológico , Animais , Cálcio/metabolismo , Insuficiência Cardíaca/fisiopatologia , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Pressão VentricularRESUMO
The dihydropyridine receptor (DHPR) ß1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform ß3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in ß1-null zebrafish relaxed myotubes, unlike the other three vertebrate ß-isoforms (ß1a, ß2a, and ß4). Thus, we used ß3 for chimerization with ß1a to investigate whether any of the five distinct molecular regions of ß1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between ß1a and ß3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of ß1a in charge movement restoration. More interestingly, ß1a SH3 domain and C terminus, when simultaneously engineered into ß3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of ß1a (also of ß2a and ß4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which ß subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Músculo Esquelético/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Domínios de Homologia de src , Motivos de Aminoácidos , Animais , Acoplamento Excitação-Contração , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Mitochondria in many types of cells are dynamically interconnected through constant fusion and fission, allowing for exchange of mitochondrial contents and repair of damaged mitochondria. However, constrained by the myofibril lattice, the â¼6,000 mitochondria in the adult mammalian cardiomyocyte display little motility, and it is unclear how, if at all, they communicate with each other. By means of target-expressing photoactivatable green fluorescent protein (PAGFP) in the mitochondrial matrix or on the outer mitochondrial membrane, we demonstrated that the local PAGFP signal propagated over the entire population of mitochondria in cardiomyocytes on a time scale of â¼10 h. Two elemental steps of intermitochondrial communications were manifested as either a sudden PAGFP transfer between a pair of adjacent mitochondria (i.e., "kissing") or a dynamic nanotubular tunnel (i.e., "nanotunneling") between nonadjacent mitochondria. The average content transfer index (fractional exchange) was around 0.5; the rate of kissing was 1 s(-1) per mitochondrial pair, and that of nanotunneling was about 14 times smaller. Electron microscopy revealed extensive intimate contacts between adjacent mitochondria and elongated nanotubular protrusions, providing a structural basis for the kissing and nanotunneling, respectively. We propose that, through kissing and nanotunneling, the otherwise static mitochondria in a cardiomyocyte form one dynamically continuous network to share content and transfer signals.
Assuntos
Mitocôndrias Cardíacas/fisiologia , Animais , Proteínas de Fluorescência Verde/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/ultraestrutura , RatosRESUMO
After complete nerve transection, a major challenge for regenerating peripheral axons is to traverse the injury site and navigate toward their original trajectory. Denervated Schwann cells distal to the lesion site secrete factors promoting axonal growth and serve as an axonal substrate, yet whether Schwann cells also actively direct axons toward their original trajectory is unclear. Using live-cell imaging in zebrafish, we visualize for the first time how in response to nerve transection distal Schwann cells change morphology as axons fragment, and how Schwann cell morphology reverses once regenerating growth cones have crossed the injury site and have grown along distal Schwann cells outlining the original nerve path. In mutants lacking Schwann cells, regenerating growth cones extend at rates comparable with wild type yet frequently fail to cross the injury site and instead stray along aberrant trajectories. Providing growth-permissive yet Schwann cell-less scaffolds across the injury site was insufficient to direct regenerating growth cones toward the original path, providing compelling evidence that denervated Schwann cells actively direct regenerating axons across the injury site toward their original trajectory. To identify signals that guide regenerating axons in vivo, we examined mutants lacking the deleted in colorectal carcinoma (DCC) guidance receptor. In these dcc mutants, a significant fraction of regenerating motor axons extended along aberrant trajectories, similar to what we observe in mutants lacking Schwann cells. Thus, Schwann cell and dcc-mediated guidance are critical early during regeneration to direct growth cones across the transection gap and onto their original axonal trajectory.
Assuntos
Axônios/metabolismo , Movimento Celular/fisiologia , Neurônios Motores/metabolismo , Regeneração Nervosa/fisiologia , Receptores de Superfície Celular/metabolismo , Células de Schwann/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células/fisiologia , Receptor DCC , Cones de Crescimento/metabolismo , Receptores de Superfície Celular/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
The ß1a subunit is a cytoplasmic component of the dihydropyridine receptor (DHPR) complex that plays an essential role in skeletal muscle excitation-contraction (EC) coupling. Here we investigate the role of the C-terminal end of this auxiliary subunit in the functional and structural communication between the DHPR and the Ca(2+) release channel (RyR1). Progressive truncation of the ß1a C terminus showed that deletion of amino acid residues Gln(489) to Trp(503) resulted in a loss of depolarization-induced Ca(2+) release, a severe reduction of L-type Ca(2+) currents, and a lack of tetrad formation as evaluated by freeze-fracture analysis. However, deletion of this domain did not affect expression/targeting or density (Qmax) of the DHPR-α1S subunit to the plasma membrane. Within this motif, triple alanine substitution of residues Leu(496), Leu(500), and Trp(503), which are thought to mediate direct ß1a-RyR1 interactions, weakened EC coupling but did not replicate the truncated phenotype. Therefore, these data demonstrate that an amino acid segment encompassing sequence (489)QVQVLTSLRRNLSFW(503) of ß1a contains critical determinant(s) for the physical link of DHPR and RyR1, further confirming a direct correspondence between DHPR positioning and DHPR/RyR functional interactions. In addition, our data strongly suggest that the motif Leu(496)-Leu(500)-Trp(503) within the ß1a C-terminal tail plays a nonessential role in the bidirectional DHPR/RyR1 signaling that supports skeletal-type EC coupling.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células Cultivadas , Acoplamento Excitação-Contração , Interações Hidrofóbicas e Hidrofílicas , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Subunidades Proteicas , Transporte ProteicoRESUMO
In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca(2+) homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 (ADA/ADA) ) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca(2+) sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344-1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 (ADA/ADA) hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 (ADA/ADA) mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Calmodulina/genética , Camundongos , Camundongos Mutantes , Contração Miocárdica , Miocárdio/patologia , Miócitos Cardíacos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/genéticaRESUMO
Cardiomyocyte contraction and relaxation are controlled by Ca(2+) handling, which can be regulated to meet demand. Indeed, major reduction in sarcoplasmic reticulum (SR) function in mice with Serca2 knockout (KO) is compensated by enhanced plasmalemmal Ca(2+) fluxes. Here we investigate whether altered Ca(2+) fluxes are facilitated by reorganization of cardiomyocyte ultrastructure. Hearts were fixed for electron microscopy and enzymatically dissociated for confocal microscopy and electrophysiology. SR relative surface area and volume densities were reduced by 63% and 76%, indicating marked loss and collapse of the free SR in KO. Although overall cardiomyocyte dimensions were unaltered, total surface area was increased. This resulted from increased T-tubule density, as revealed by confocal images. Fourier analysis indicated a maintained organization of transverse T-tubules but an increased presence of longitudinal T-tubules. This demonstrates a remarkable plasticity of the tubular system in the adult myocardium. Immunocytochemical data showed that the newly grown longitudinal T-tubules contained Na(+)/Ca(2+)-exchanger proximal to ryanodine receptors in the SR but did not contain Ca(2+)-channels. Ca(2+) measurements demonstrated a switch from SR-driven to Ca(2+) influx-driven Ca(2+) transients in KO. Still, SR Ca(2+) release constituted 20% of the Ca(2+) transient in KO. Mathematical modeling suggested that Ca(2+) influx via Na(+)/Ca(2+)-exchange in longitudinal T-tubules triggers release from apposing ryanodine receptors in KO, partially compensating for reduced SERCA by allowing for local Ca(2+) release near the myofilaments. T-tubule proliferation occurs without loss of the original ordered transverse orientation and thus constitutes the basis for compensation of the declining SR function without structural disarrangement.
Assuntos
ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Proliferação de Células , Eletrofisiologia/métodos , Análise de Fourier , Imuno-Histoquímica/métodos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/fisiologiaRESUMO
Calsequestrin (CASQ) is the major component of the sarcoplasmic reticulum (SR) lumen in skeletal and cardiac muscles. This calcium-binding protein localizes to the junctional SR (jSR) cisternae, where it is responsible for the storage of large amounts of Ca(2+), whereas it is usually absent, at least in its polymerized form, in the free SR. The retention of CASQ inside the jSR is due partly to its association with other jSR proteins, such as junctin and triadin, and partly to its ability to polymerize, in a high Ca(2+) environment, into an intricate gel that holds the protein in place. In this work, we shed some light on the still poorly described in situ structure of polymerized CASQ using detailed EM images from thin sections, with and without tilting, and from deep-etched rotary-shadowed replicas. The latter directly illustrate the fundamental network nature of polymerized CASQ, revealing repeated nodal points connecting short segments of the linear polymer.
Assuntos
Proteínas de Anfíbios/química , Cálcio/química , Calsequestrina/química , Multimerização Proteica/fisiologia , Proteínas de Anfíbios/metabolismo , Animais , Cálcio/metabolismo , Calsequestrina/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Estrutura Quaternária de Proteína , Rana pipiens , SerpentesRESUMO
Many snakes swallow large prey whole, and this process requires large displacements of the unfused tips of the mandibles and passive stretching of the soft tissues connecting them. Under these conditions, the intermandibular muscles are highly stretched but subsequently recover normal function. In the highly stretched condition we observed in snakes, sarcomere length (SL) increased 210% its resting value (SL0), and actin and myosin filaments no longer overlapped. Myofibrils fell out of register and triad alignment was disrupted. Following passive recovery, SLs returned to 82% SL0, creating a region of double-overlapping actin filaments. Recovery required recoil of intracellular titin filaments, elastic cytoskeletal components for realigning myofibrils, and muscle activation. Stretch of whole muscles exceeded that of sarcomeres as a result of extension of folded terminal tendon fibrils, stretching of extracellular elastin and independent slippage of muscle fibers. Snake intermandibular muscles thus provide a unique model of how basic components of vertebrate skeletal muscle can be modified to permit extreme extensibility.
Assuntos
Citoesqueleto de Actina/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Miofibrilas/fisiologia , Sarcômeros/fisiologia , Serpentes/fisiologia , Citoesqueleto de Actina/ultraestrutura , Animais , Citoesqueleto , Mandíbula , Miofibrilas/ultraestrutura , Sarcômeros/ultraestruturaRESUMO
This presentation reviews images of electron micrographs from various skeletal muscles identifying a consistent association of diydropyridine receptors (DHPR) tetrads with alternate ryanodine receptors. Imaging of the junctional gap in triads from various sources provide direct evidence for the association of four diydropyridine receptors (DHPRs), clustered into tetrads, with alternate ryanodine receptors (RyRs). It is not clear whether firing of all four components of a tetrad is necessary to fully activate the opening of the RyR channel.
RESUMO
In vertebrates, the peripheral nervous system has retained its regenerative capacity, enabling severed axons to reconnect with their original synaptic targets. While it is well documented that a favorable environment is critical for nerve regeneration, the complex cellular interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves and macrophages in live intact zebrafish. We show that macrophages arrive at the lesion site long before axon fragmentation, much earlier than previously thought. Moreover, we find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wld(s) model does not alter macrophage recruitment but induces a previously unknown 'nerve scanning' behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms. Finally, we demonstrate that macrophage recruitment, thought to be dependent on Schwann cell-derived signals, occurs independently of Schwann cells. Thus, live cell imaging defines novel cellular and functional interactions between injured nerves and immune cells.