RESUMEN

Our laboratory previously showed lipid hydroperoxides and oxylipin levels are elevated in response to loss of skeletal muscle innervation and are associated with muscle pathologies. To elucidate the pathological impact of lipid hydroperoxides, we overexpressed glutathione peroxidase 4 (GPx4), an enzyme that targets reduction of lipid hydroperoxides in membranes, in adult CuZn superoxide dismutase knockout (Sod1KO) mice that show accelerated muscle atrophy associated with loss of innervation. The gastrocnemius muscle from Sod1KO mice shows reduced mitochondrial respiration and elevated oxidative stress (F2 -isoprostanes and hydroperoxides) compared to wild-type (WT) mice. Overexpression of GPx4 improved mitochondrial respiration and reduced hydroperoxide generation in Sod1KO mice, but did not attenuate the muscle loss that occurs in Sod1KO mice. In contrast, contractile force generation is reduced in EDL muscle in Sod1KO mice relative to WT mice, and overexpression of GPx4 restored force generation to WT levels in Sod1KO mice. GPx4 overexpression also prevented loss of muscle contractility at the single fibre level in fast-twitch fibres from Sod1KO mice. Muscle fibres from Sod1KO mice were less sensitive to both depolarization and calcium at the single fibre level and exhibited a reduced activation by S-glutathionylation. GPx4 overexpression in Sod1KO mice rescued the deficits in both membrane excitability and calcium sensitivity of fast-twitch muscle fibres. Overexpression of GPx4 also restored the sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO gastrocnemius muscles. These data suggest that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and in maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia. KEY POINTS: Knockout of CuZn superoxide dismutase (Sod1KO) induces elevated oxidative stress with accelerated muscle atrophy and weakness. Glutathione peroxidase 4 (GPx4) plays a fundamental role in the reduction of lipid hydroperoxides in membranes, and overexpression of GPx4 improves mitochondrial respiration and reduces hydroperoxide generation in Sod1KO mice. Muscle contractile function deficits in Sod1KO mice are alleviated by the overexpression of GPx4. GPx4 overexpression in Sod1KO mice rescues the impaired muscle membrane excitability of fast-twitch muscle fibres and improves their calcium sensitivity. Sarco/endoplasmic reticulum Ca2+ -ATPase activity in Sod1KO muscles is decreased, and it is restored by the overexpression of GPx4. Our results confirm that GPx4 plays an important role in preserving excitation-contraction coupling function and Ca2+ homeostasis, and maintaining muscle and mitochondrial function in oxidative stress-induced sarcopenia.

Asunto(s)

Sarcopenia , Animales , Ratones , Adenosina Trifosfatasas/genética , Calcio , Glutatión , Glutatión Peroxidasa/genética , Peróxido de Hidrógeno , Lípidos , Ratones Noqueados , Músculo Esquelético/fisiología , Fenotipo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Superóxido Dismutasa , Superóxido Dismutasa-1/genética

RESUMEN

Heart failure is the final stage of various cardiovascular diseases and seriously threatens human health. Increasing mediators have been found to be involved in the pathogenesis of heart failure, including the RNA binding protein RBFox2. It participates in multiple aspects of the regulation of cardiac function and plays a critical role in the process of heart failure. However, how RBFox2 itself is regulated remains unclear. Here, we dissected transcriptomic signatures, including mRNAs and miRNAs, in a mouse model of heart failure after TAC surgery. A global analysis showed that an asymmetric alternation in gene expression and a large-scale upregulation of miRNAs occurred in heart failure. An association analysis revealed that the latter not only contributed to the degradation of numerous mRNA transcripts, but also suppressed the translation of key proteins such as RBFox2. With the aid of Ago2 CLIP-seq data, luciferase assays verified that RBFox2 was targeted by multiple miRNAs, including Let-7, miR-16, and miR-200b, which were significantly upregulated in heart failure. The overexpression of these miRNAs suppressed the RBFox2 protein and its downstream effects in cardiomyocytes, which was evidenced by the suppressed alternative splicing of the Enah gene and impaired E-C coupling via the repression of the Jph2 protein. The inhibition of Let-7, the most abundant miRNA family targeting RBFox2, could restore the RBFox2 protein as well as its downstream effects in dysfunctional cardiomyocytes induced by ISO treatment. In all, these findings revealed the molecular mechanism leading to RBFox2 depression in heart failure, and provided an approach to rescue RBFox2 through miRNA inhibition for the treatment of heart failure.

Asunto(s)

Insuficiencia Cardíaca , MicroARNs , Ratones , Animales , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Factores de Empalme de ARN/genética , Insuficiencia Cardíaca/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , ARN Mensajero/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo

RESUMEN

In this study, we report static and perfused models of human myocardial-microvascular interaction. In static culture, we observe distinct regulation of electrophysiology of human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in co-culture with human cardiac microvascular endothelial cells (hCMVECs) and human left ventricular fibroblasts (hLVFBs), including modification of beating rate, action potential, calcium handling, and pro-arrhythmic substrate. Within a heart-on-a-chip model, we subject this three-dimensional (3D) co-culture to microfluidic perfusion and vasculogenic growth factors to induce spontaneous assembly of perfusable myocardial microvasculature. Live imaging of red blood cells within myocardial microvasculature reveals pulsatile flow generated by beating hiPSC-CMs. This study therefore demonstrates a functionally vascularized in vitro model of human myocardium with widespread potential applications in basic and translational research.

Asunto(s)

Células Endoteliales , Células Madre Pluripotentes Inducidas , Humanos , Miocardio , Miocitos Cardíacos , Técnicas de Cocultivo

RESUMEN

Ryanodine receptor 2 (RyR2) is an ion channel in the heart responsible for releasing into the cytosol most of the Ca2+ required for contraction. Proper regulation of RyR2 is critical, as highlighted by the association between channel dysfunction and cardiac arrhythmia. Lower RyR2 expression is also observed in some forms of heart disease; however, there is limited information on the impact of this change on excitation-contraction (e-c) coupling, Ca2+-dependent arrhythmias, and cardiac performance. We used a constitutive knock-out of RyR2 in rabbits (RyR2-KO) to assess the extent to which a stable decrease in RyR2 expression modulates Ca2+ handling in the heart. We found that homozygous knock-out of RyR2 in rabbits is embryonic lethal. Remarkably, heterozygotes (KO+/-) show ~50% loss of RyR2 protein without developing an overt phenotype at the intact animal and whole heart levels. Instead, we found that KO+/- myocytes show (1) remodeling of RyR2 clusters, favoring smaller groups in which channels are more densely arranged; (2) lower Ca2+ spark frequency and amplitude; (3) slower rate of Ca2+ release and mild but significant desynchronization of the Ca2+ transient; and (4) a significant decrease in the basal phosphorylation of S2031, likely due to increased association between RyR2 and PP2A. Our data show that RyR2 deficiency, although remarkable at the molecular and subcellular level, has only a modest impact on global Ca2+ release and is fully compensated at the whole-heart level. This highlights the redundancy of RyR2 protein expression and the plasticity of the e-c coupling apparatus.

Asunto(s)

Adrenérgicos , Canal Liberador de Calcio Receptor de Rianodina , Animales , Arritmias Cardíacas/metabolismo , Calcio/metabolismo , Señalización del Calcio , Acoplamiento Excitación-Contracción , Miocitos Cardíacos/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo

RESUMEN

Defects in cardiac contractility and heart failure (HF) are common following doxorubicin (DOX) administration. Different miRs play a role in HF, and their targeting was suggested as a promising therapy. We aimed to target miR-24, a suppressor upstream of junctophilin-2 (JP-2), which is required to affix the sarcoplasmic reticulum to T-tubules, and hence the release of Ca2+ in excitation-contraction coupling using pachymic acid (PA) and/or losartan (LN). HF was induced with DOX (3.5 mg/kg, i.p., six doses, twice weekly) in 24 rats. PA and LN (10 mg/kg, daily) were administered orally for four weeks starting the next day of the last DOX dose. Echocardiography, left ventricle (LV) biochemical and histological assessment and electron microscopy were conducted. DOX increased serum BNP, HW/TL, HW/BW, mitochondrial number/size and LV expression of miR-24 but decreased EF, cardiomyocyte fiber diameter, LV content of JP-2 and ryanodine receptors-2 (RyR2). Treatment with either PA or LN reversed these changes. Combined PA + LN attained better results than monotherapies. In conclusion, HF progression following DOX administration can be prevented or even delayed by targeting miR-24 and its downstream JP-2. Our results, therefore, suggest the possibility of using PA alone or as an adjuvant therapy with LN to attain better management of HF patients, especially those who developed tolerance toward LN.

Asunto(s)

Doxorrubicina/efectos adversos , Regulación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Proteínas de la Membrana/genética , MicroARNs/genética , Triterpenos/farmacología , Animales , Cardiomegalia/diagnóstico , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/etiología , Cardiomegalia/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Pruebas de Función Cardíaca , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/ultraestructura , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal

RESUMEN

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 µM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

Asunto(s)

Calcio/metabolismo , Colinérgicos/farmacología , Citosol/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Sistema Respiratorio/metabolismo , Animales , Citosol/efectos de los fármacos , Femenino , Masculino , Músculo Liso/efectos de los fármacos , Sistema Respiratorio/efectos de los fármacos , Porcinos

RESUMEN

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation-contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

Asunto(s)

Contracción Muscular/fisiología , Unión Neuromuscular/fisiología , Octopodiformes/fisiología , Animales , Calcio , Proteínas Musculares

RESUMEN

Junctophilins (JPH1-JPH4) are expressed in excitable and nonexcitable cells, where they tether endoplasmic/sarcoplasmic reticulum (ER/SR) and plasma membranes (PM). These ER/SR-PM junctions bring Ca-release channels in the ER/SR and Ca as well as Ca-activated K channels in the PM to within 10-25 nm. Such proximity is critical for excitation-contraction coupling in muscles, Ca modulation of excitability in neurons, and Ca homeostasis in nonexcitable cells. JPHs are anchored in the ER/SR through the C-terminal transmembrane domain (TMD). Their N-terminal Membrane-Occupation-Recognition-Nexus (MORN) motifs can bind phospholipids. Whether MORN motifs alone are sufficient to stabilize JPH-PM binding is not clear. We investigate whether S-palmitoylation of cysteine (Cys), a critical mechanism controlling peripheral protein binding to PM, occurs in JPHs. We focus on JPH2 that has four Cys residues: three flanking the MORN motifs and one in the TMD. Using palmitate-alkyne labeling, Cu(I)-catalyzed alkyne-azide cycloaddition reaction with azide-conjugated biotin, immunoblotting, proximity-ligation-amplification, and various imaging techniques, we show that JPH2 is S-palmitoylatable, and palmitoylation is essential for its ER/SR-PM tether function. Palmitoylated JPH2 binds to lipid-raft domains in PM, whereas palmitoylation of TMD-located Cys stabilizes JPH2's anchor in the ER/SR membrane. Binding to lipid-raft domains protects JPH2 from depalmitoylation. Unpalmitoylated JPH2 is largely excluded from lipid rafts and loses the ability to form stable ER/SR-PM junctions. In adult ventricular myocytes, native JPH2 is S-palmitoylatable, and palmitoylated JPH2 forms distinct PM puncta. Sequence alignment reveals that the palmitoylatable Cys residues in JPH2 are conserved in other JPHs, suggesting that palmitoylation may also enhance ER/SR-PM tethering by these proteins.

Asunto(s)

Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Ácido Palmítico/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos

RESUMEN

Heart failure remains a major cause of morbidity and mortality in developed countries. There is still a strong need to devise new mechanism-based treatments for heart failure. Numerous studies have suggested the importance of the Ca2+-dependent protease calpain in cardiac physiology and pathology. However, no drugs are currently under development or testing in human patients to target calpain for heart failure treatment. Herein the data demonstrate that inhibition of calpain activity protects against deleterious ultrastructural remodeling and cardiac dysfunction in multiple rodent models of heart failure, providing compelling evidence that calpain inhibition is a promising therapeutic strategy for heart failure treatment.

RESUMEN

Calcium channels are necessary for cardiac excitation-contraction (E-C) coupling, but Ca2+ channel composition of fish hearts is still largely unknown. To this end, we determined transcript expression of Ca2+ channels in the heart of zebrafish (Danio rerio), a popular model species. Altogether, 18 Ca2+ channel α-subunit genes were expressed in both atrium and ventricle. Transcripts for 7 L-type (Cav1.1a, Cav1.1b, Cav1.2, Cav1.3a, Cav1.3b, Cav1.4a, Cav1.4b), 5 T-type (Cav3.1, Cav3.2a, Cav3.2b, Cav3.3a, Cav3.3b) and 6 P/Q-, N- and R-type (Cav2.1a, Cav2.1b, Cav2.2a, Cav2.2b, Cav2.3a, Cav2.3b) Ca2+ channels were expressed. In the ventricle, T-type channels formed 54.9%, L-type channels 41.1% and P/Q-, N- and R-type channels 4.0% of the Ca2+ channel transcripts. In the atrium, the relative expression of T-type and L-type Ca2+ channel transcripts was 64.1% and 33.8%, respectively (others accounted for 2.1%). Thus, at the transcript level, T-type Ca2+ channels are prevalent in zebrafish atrium and ventricle. At the functional level, peak densities of ventricular T-type (ICaT) and L-type (ICaL) Ca2+ current were 6.3±0.8 and 7.7±0.8 pA pF-1, respectively. ICaT mediated a sizeable sarcolemmal Ca2+ influx into ventricular myocytes: the increment in total cellular Ca2+ content via ICaT was 41.2±7.3 µmol l-1, which was 31.7% of the combined Ca2+ influx (129 µmol l-1) via ICaT and ICaL (88.5±20.5 µmol l-1). The diversity of expressed Ca2+ channel genes in zebrafish heart is high, but dominated by the members of the T-type subfamily. The large ventricular ICaT is likely to play a significant role in E-C coupling.

Asunto(s)

Canales de Calcio/metabolismo , Regulación de la Expresión Génica , Pez Cebra/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/metabolismo , Células Musculares/metabolismo , Técnicas de Placa-Clamp , Pez Cebra/genética

RESUMEN

A glutamate-to-lysine substitution at position 1014 within the selectivity filter of the skeletal muscle L-type Ca2+ channel (CaV1.1) abolishes Ca2+ flux through the channel pore. Mice engineered to exclusively express the mutant channel display accelerated muscle fatigue, changes in muscle composition, and altered metabolism relative to wildtype littermates. By contrast, mice expressing another mutant CaV1.1 channel that is impermeable to Ca2+ (CaV1.1 N617D) have shown no detectable phenotypic differences from wildtype mice to date. The major biophysical difference between the CaV1.1 E1014K and CaV1.1 N617D mutants elucidated thus far is that the former channel conducts robust Na+ and Cs+ currents in patch-clamp experiments, but neither of these monovalent conductances seems to be of relevance in vivo Thus, the basis for the different phenotypes of these mutants has remained enigmatic. We now show that CaV1.1 E1014K readily conducts 1,4-dihydropyridine-sensitive K+ currents at depolarizing test potentials, whereas CaV1.1 N617D does not. Our observations, coupled with a large body of work by others regarding the role of K+ accumulation in muscle fatigue, raise the possibility that the introduction of an additional K+ flux from the myoplasm into the transverse-tubule lumen accelerates the onset of fatigue and precipitates the metabolic changes observed in CaV1.1 E1014K muscle. These results, highlighting an unexpected consequence of a channel mutation, may help define the complex mechanisms underlying skeletal muscle fatigue and related dysfunctions.

Asunto(s)

Canales de Calcio Tipo L/genética , Músculo Esquelético/metabolismo , Mutación , Potasio/metabolismo , Animales , Transporte Biológico , Canales de Calcio Tipo L/metabolismo , Línea Celular , Ratones

RESUMEN

Aberrant Zn2+ homeostasis is associated with dysregulated intracellular Ca2+ release, resulting in chronic heart failure. In the failing heart a small population of cardiac ryanodine receptors (RyR2) displays sub-conductance-state gating leading to Ca2+ leakage from sarcoplasmic reticulum (SR) stores, which impairs cardiac contractility. Previous evidence suggests contribution of RyR2-independent Ca2+ leakage through an uncharacterized mechanism. We sought to examine the role of Zn2+ in shaping intracellular Ca2+ release in cardiac muscle. Cardiac SR vesicles prepared from sheep or mouse ventricular tissue were incorporated into phospholipid bilayers under voltage-clamp conditions, and the direct action of Zn2+ on RyR2 channel function was examined. Under diastolic conditions, the addition of pathophysiological concentrations of Zn2+ (≥2 nm) caused dysregulated RyR2-channel openings. Our data also revealed that RyR2 channels are not the only SR Ca2+-permeable channels regulated by Zn2+ Elevating the cytosolic Zn2+ concentration to 1 nm increased the activity of the transmembrane protein mitsugumin 23 (MG23). The current amplitude of the MG23 full-open state was consistent with that previously reported for RyR2 sub-conductance gating, suggesting that in heart failure in which Zn2+ levels are elevated, RyR2 channels do not gate in a sub-conductance state, but rather MG23-gating becomes more apparent. We also show that in H9C2 cells exposed to ischemic conditions, intracellular Zn2+ levels are elevated, coinciding with increased MG23 expression. In conclusion, these data suggest that dysregulated Zn2+ homeostasis alters the function of both RyR2 and MG23 and that both ion channels play a key role in diastolic SR Ca2+ leakage.

Asunto(s)

Señalización del Calcio , Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Zinc/metabolismo , Mataderos , Animales , Hipoxia de la Célula , Línea Celular , Regulación de la Expresión Génica , Membrana Dobles de Lípidos/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp , Permeabilidad , Ratas , Oveja Doméstica

RESUMEN

Stokes flow motions induced by a beating single cardiac cell (cardiomyocyte) are obtained numerically using the method of fundamental solutions (MFS). A two-dimensional meshfree-Stokeslets computational framework is used to solve the Stokes governing equations around an isolated cardiomyocyte. An approximate beating kinematical model is derived and used to approximate the cell-length shortening over a complete cardiac cycle. The induced flow patterns have been found to be characterized by the presence of counter-rotating vortices at both cell's edges. These vortical flow structures are clearly shown by rendering the velocity streamlines. The static pressure contours are also calculated at different time snapshots during both contraction and relaxation phases of the beating motion. The pressure signal is calculated at a point in the neighborhood of cell surface to capture the induced normal stress (traction) by the cell morphological motions to the surrounding fluid medium. The presented results have shown that, cells with a slightly different shortening/beating profile can induce different flow field. This implies that, each cell is characterized by a unique flow pattern "signature", which potentially can be correlated to the sub-cellular excitation-contraction processes of cardiac cells.

Asunto(s)

Modelos Cardiovasculares , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Velocidad del Flujo Sanguíneo , Humanos

RESUMEN

BACKGROUND: Motor neurons control muscle contraction by initiating action potentials in muscle. Denervation of muscle from motor neurons leads to muscle atrophy, which is linked to mitochondrial dysfunction. It is known that denervation promotes mitochondrial reactive oxygen species (ROS) production in muscle, whereas the initial cause of mitochondrial ROS production in denervated muscle remains elusive. Since denervation isolates muscle from motor neurons and deprives it from any electric stimulation, no action potentials are initiated, and therefore, no physiological Ca2+ transients are generated inside denervated muscle fibers. We tested whether loss of physiological Ca2+ transients is an initial cause leading to mitochondrial dysfunction in denervated skeletal muscle. METHODS: A transgenic mouse model expressing a mitochondrial targeted biosensor (mt-cpYFP) allowed a real-time measurement of the ROS-related mitochondrial metabolic function following denervation, termed "mitoflash." Using live cell imaging, electrophysiological, pharmacological, and biochemical studies, we examined a potential molecular mechanism that initiates ROS-related mitochondrial dysfunction following denervation. RESULTS: We found that muscle fibers showed a fourfold increase in mitoflash activity 24 h after denervation. The denervation-induced mitoflash activity was likely associated with an increased activity of mitochondrial permeability transition pore (mPTP), as the mitoflash activity was attenuated by application of cyclosporine A. Electrical stimulation rapidly reduced mitoflash activity in both sham and denervated muscle fibers. We further demonstrated that the Ca2+ level inside mitochondria follows the time course of the cytosolic Ca2+ transient and that inhibition of mitochondrial Ca2+ uptake by Ru360 blocks the effect of electric stimulation on mitoflash activity. CONCLUSIONS: The loss of cytosolic Ca2+ transients due to denervation results in the downstream absence of mitochondrial Ca2+ uptake. Our studies suggest that this could be an initial trigger for enhanced mPTP-related mitochondrial ROS generation in skeletal muscle.

Asunto(s)

Señalización del Calcio , Mitocondrias Musculares/metabolismo , Músculo Esquelético/inervación , Potenciales de Acción , Animales , Acoplamiento Excitación-Contracción , Ratones , Desnervación Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by ∼6-8 °C. Similar to other ß isoforms, ß1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of ß1a while also providing a conduit for allosteric signaling events.

Asunto(s)

Canales de Calcio Tipo L/química , Guanilato-Quinasas/química , Regulación Alostérica , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Cristalografía por Rayos X , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Ratones , Estructura Secundaria de Proteína , Transducción de Señal , Dominios Homologos src

RESUMEN

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The ß subunit differentially regulates maturation, trafficking, and activity of α-ß heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three ß isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with ß2 in T-tubules of cardiac myocytes, forming α2ß2 heterodimers. We have expressed human α1ß1, α2ß1, α2ß2, and α2ß3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2ß2 and α2ß3 differ significantly from both α2ß1 and α1ß1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2ß2 and α2ß3 over α1ß1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2ß2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2ß2 for cardiac muscle contractility. The high sensitivity of α2ß2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2ß2-selective digoxin derivatives for reducing cardiotoxicity.

Asunto(s)

Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Inhibidores Enzimáticos/química , Miocardio/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/química , Animales , Proteínas de Transporte de Catión/antagonistas & inhibidores , Proteínas de Transporte de Catión/química , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Moléculas de Adhesión Celular Neuronal/química , Dimerización , Inhibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Miocardio/química , Potasio/química , Potasio/metabolismo , Sodio/química , Sodio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética

RESUMEN

Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Contracción Muscular , Músculo Liso/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Vimentina/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Tráquea/fisiología , Quinasa Tipo Polo 1

RESUMEN

Voltage-dependent CaV1.2 L-type Ca2+ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.

Asunto(s)

Aorta/metabolismo , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína ORAI1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Aorta/citología , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteína ORAI1/genética , Serotonina/metabolismo , Canales Catiónicos TRPC/genética , Vasoconstricción/fisiología

RESUMEN

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La(3+)- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca(2+) concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca(2+) concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Calsecuestrina/metabolismo , Proteínas de la Membrana/metabolismo , Fibras Musculares de Contracción Lenta/fisiología , Animales , Proteínas de Unión al Calcio/genética , Calsecuestrina/genética , Técnicas de Inactivación de Genes , Proteínas de la Membrana/genética , Ratones , Contracción Muscular , Músculo Esquelético/fisiología , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismo