RESUMEN
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.
Asunto(s)
Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Modelos Teóricos , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Sarcómeros/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Simulación por Computador , Cinética , Ratones , Mitocondrias/metabolismo , Parvalbúminas/metabolismo , Retículo Sarcoplasmático/metabolismo , Troponina/metabolismoRESUMEN
The skeletal muscle and myocardial cells present highly specialized structures; for example, the close interaction between the sarcoplasmic reticulum (SR) and mitochondria-responsible for excitation-metabolism coupling-and the junction that connects the SR with T-tubules, critical for excitation-contraction (EC) coupling. The mechanisms that underlie EC coupling in these two cell types, however, are fundamentally distinct. They involve the differential expression of Ca2+ channel subtypes: CaV1.1 and RyR1 (skeletal), vs. CaV1.2 and RyR2 (cardiac). The CaV channels transform action potentials into elevations of cytosolic Ca2+, by activating RyRs and thus promoting SR Ca2+ release. The high levels of Ca2+, in turn, stimulate not only the contractile machinery but also the generation of mitochondrial reactive oxygen species (ROS). This forward signaling is reciprocally regulated by the following feedback mechanisms: Ca2+-dependent inactivation (of Ca2+ channels), the recruitment of Na+/Ca2+ exchanger activity, and oxidative changes in ion channels and transporters. Here, we summarize both well-established concepts and recent advances that have contributed to a better understanding of the molecular mechanisms involved in this bidirectional signaling.
Asunto(s)
Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Sarcolema/metabolismo , Retículo Sarcoplasmático/metabolismo , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo L/fisiología , Citosol/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Humanos , Músculo Esquelético/metabolismo , Miocitos Cardíacos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcolema/fisiología , Retículo Sarcoplasmático/fisiología , Transducción de SeñalRESUMEN
[Ca2+] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca2+ release of extrinsic Ca2+ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca2+ buffer is added to the myoplasm, part of the released Ca2+ binds to it, reducing the Ca2+ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca2+ released. Monitoring release by measuring [Ca2+] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca2+] in the SR ([Ca2+]SR) continuously from a starting value close to 400 µM reaching the range of 100 µM in about half an hour. The effect of reducing resting [Ca2+]SR on the Ca2+ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca2+] ([Ca2+]myo) transients were simultaneously recorded with Rhod2. The Ca2+ release flux was calculated from [Ca2+]myo and divided by [Ca2+]SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026 ± 0.005 ms-1 at resting [Ca2+]SR = 372 ± 5 µM to 0.021 ± 0.004 ms-1 at resting [Ca2+]SR = 120 ± 16 µM (n = 4, p = 0.03). The time averaged permeability was not significantly changed (0.009 ± 0.003 and 0.010 ± 0.003 ms-1, at the higher and lower [Ca2+]SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca2+]SR (Δ[Ca2+]SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca2+]SR = 30.2 ± 3.5 µM from resting [Ca2+]SR = 88.8 ± 13.6 µM, n = 5) than in 40 mM EGTA (Δ[Ca2+]SR = 72.2 ± 10.4 µM from resting [Ca2+]SR = 98.2 ± 15.6 µM, n = 4) suggesting that a Ca2+ activated component of release was suppressed by BAPTA.
Asunto(s)
Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Fibras Musculares Esqueléticas/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , RanidaeRESUMEN
Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/µF meanwhile the Ca2+ release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 µM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Músculo Esquelético/metabolismo , Técnicas de Placa-Clamp/métodos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , AnurosRESUMEN
The aim of this study was to investigate the effects of the ageing process in the electromechanical delay (EMD), rate of torque development (RTD) and peak torque (PT) of the knee extensor muscles. The volunteers were assigned to three groups: young group (YG - 23·44 ± 4·74 years, 78·14 ± 15·11 kg, 1·72 ± 0·05 m), middle-aged group (MAG - 49·56 ± 6·06 years, 72·01 ± 14·07 kg, 1·67 ± 0·06 m) and elderly group (EG - 68·67 ± 9·06 years, 67·96 ± 7·60 kg, 1·64 ± 0·07 m). The PT and RTD were assessed during maximal voluntary ballistic isometric contractions (MVBIC) in the isokinetic dynamometer. Muscle electrical activity was recorded (EMG) during MVBIC in the vastus lateralis (VL), vastus medialis (VM) and rectus femoris (RF) muscles. The EMD was calculated during the MVBIC, through the time interval between the EMG onset and torque onset. The PT and RTD were higher in the YG than in the MAG (P = 0·02; P = 0·01, respectively) and in the EG (P = 0·002; P = 0·0004, respectively). There were no significant differences in EMD among the three age groups for the VL, VM and RF (P>0·05) muscles. We conclude that age affects the PT and RTD, but not EMD of the VL, VM and RF muscles.
Asunto(s)
Envejecimiento/fisiología , Acoplamiento Excitación-Contracción/fisiología , Contracción Isométrica/fisiología , Articulación de la Rodilla/fisiología , Músculo Esquelético/fisiología , Resistencia Física/fisiología , Adulto , Anciano , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Torque , Adulto JovenRESUMEN
The sarcoplasmic reticulum (SR) is the main source of contraction-activating Ca2+ in the adult mammalian myocardium. The fraction of the SR Ca2+ content released at a twitch (fractional SR Ca2+ release, FR) is an important parameter for assessing the efficiency of excitation-contraction coupling under physiological and pathophysiological conditions, as well as for identification of modulators of this process. We here describe and propose an approach for FR quantitation based on the estimation of integrated Ca2+ fluxes mediated by different transporters that remove the ion from the cytosol. These fluxes may be calculated solely from the measurement of cytosolic free Ca2+ concentration ([Ca2+]i) during Ca2+ transients evoked under selective inhibition of the transporters, and from the cell Ca2+ buffering parameters available in the literature. The FR values obtained with this approach in intact rat ventricular myocytes (0.63 ± 0.04; n = 12) were comparable to those estimated in the same cell type with an already established method, based on electrophysiological measurements with the patch-clamp technique, in addition to [Ca2+]i measurement (0.69 ± 0.05; n = 6; p > 0.40). We conclude that the proposed method might be a suitable and technically simpler alternative to the electrophysiological method for FR estimation.
Asunto(s)
Algoritmos , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Modelos Cardiovasculares , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Retículo Sarcoplasmático/fisiología , Animales , Células Cultivadas , Simulación por Computador , Masculino , Tasa de Depuración Metabólica , Ratas , Ratas WistarRESUMEN
We use laser diffraction in the analysis of the transversal deformation that the papillary muscle of the female and male Wistar rat may undergo when is subjected to different tension (tension range, 0-30 mN) in the longitudinal plane. Papillary muscles from the right ventricle were illuminated at normal incidence with a He-Ne laser lasing at 594 nm at room temperature. The far-field diffraction pattern projected to a screen was recorded with a digital camera for its analysis. The analysis of the stress-strain curves from the two experimental groups shows that the papillary muscles from male rats exhibit a higher stiffness in the transversal axis compared to the female rats.
Asunto(s)
Músculos Papilares/fisiología , Animales , Fenómenos Biomecánicos , Acoplamiento Excitación-Contracción/fisiología , Femenino , Técnicas In Vitro , Rayos Láser , Masculino , Contracción Miocárdica/fisiología , Ratas , Ratas Wistar , Estrés MecánicoRESUMEN
Calcitonin gene related peptide (CGRP) is a vasodilator; its plasma levels are altered in several human diseases, including migraine, hypertension and diabetes. CGRP is locally released by motor neurons, and is overexpressed in response to surgical or pharmacological blockage of neuromuscular transmission. Additionally to a brief discussion with regard to the clinical relevance of CGRP, this review focuses on the effects of CGRP on skeletal muscle excitation-contraction (EC) coupling, as well as the corresponding pathophysiological consequences. EC coupling involves activation of 2 different types of calcium channels: dihydropyridine receptors (DHPRs) located at the sarcolemma, and ryanodine receptors (RyR1s) located at the sarcoplasmic reticulum (SR). In response to electrical depolarization, DHPRs activate nearby and physically bound RyR1s, allowing Ca(2+) from the SR to move into the cytosol (termed voltage-gated Ca(2+) release, or VGCR). We recently found that CGRP stimulates VGCR by 350 % in as short as 1h. This effect, which lasts for at least 48 h, is due to activation of the CGRP receptor, and requires activation of the cAMP/PKA signaling pathway. CGRP also increases the amplitude of caffeine-induced Ca(2+) release (400 %); suggesting increased SR Ca(2+) content underlies stimulation of VGCR. Interestingly, in the long-term CGRP also increases the density of sarcolemmal DHPRs (up to 30%, within 24-48 h). We propose that these CGRP effects may contribute to prevent and/or restore symptoms in central core disease (CCD); a congenital myopathy that is linked to mutations in the gene encoding RyR1.