The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere−1 s−1. The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.
Tetanic electrical stimulation induces two separate calcium signals in rat skeletal myotubes, a fast one, dependent on Cav 1.1 or dihydropyridine receptors (DHPRs) and ryanodine receptors and related to contraction, and a slow signal, dependent on DHPR and inositol trisphosphate receptors (IP3Rs) and related to transcriptional events. We searched for slow calcium signals in adult muscle fibers using isolated adult flexor digitorum brevis fibers from 5–7-wk-old mice, loaded with fluo-3. When stimulated with trains of 0.3-ms pulses at various frequencies, cells responded with a fast calcium signal associated with muscle contraction, followed by a slower signal similar to one previously described in cultured myotubes. Nifedipine inhibited the slow signal more effectively than the fast one, suggesting a role for DHPR in its onset. The IP3R inhibitors Xestospongin B or C (5 µM) also inhibited it. The amplitude of post-tetanic calcium transients depends on both tetanus frequency and duration, having a maximum at 10–20 Hz. At this stimulation frequency, an increase of the slow isoform of troponin I mRNA was detected, while the fast isoform of this gene was inhibited. All three IP3R isoforms were present in adult muscle. IP3R-1 was differentially expressed in different types of muscle fibers, being higher in a subset of fast-type fibers. Interestingly, isolated fibers from the slow soleus muscle did not reveal the slow calcium signal induced by electrical stimulus. These results support the idea that IP3R-dependent slow calcium signals may be characteristic of distinct types of muscle fibers and may participate in the activation of specific transcriptional programs of slow and fast phenotype.
The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere−1 s−1. The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.
Our objective was to investigate in cardiac muscle the contribution of NADPH oxidase to (a) ryanodine receptor-2 (RyR2) S-glutathionylation and (b) the preconditioning effects of exercise and tachycardia on infarct size following coronary artery occlusion.We measured NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release kinetics in sarcoplasmic reticulum (SR) vesicles isolated from dog ventricular muscle after exercise and tachycardia, plus or minus prior administration of the NADPH oxidase inhibitor apocynin. In ventricular muscle sections, we studied the colocalization of NADPH oxidase and RyR2 by confocal microscopy using fluorescent antibodies. We determined the effect of apocynin on the infarct size produced by occlusion of the descendent anterior coronary artery in animals preconditioned by exercise or tachycardia. Exercise and tachycardia increased NADPH oxidase activity, RyR2 S-glutathionylation, and calcium release rates in isolated SR vesicles. Cardiac muscle sections displayed significant colocalization of NADPH oxidase and RyR2, suggesting direct and specific effects of reactive oxygen species (ROS) produced by NADPH oxidase on RyR2 activation. The NADPH oxidase inhibitor apocynin prevented the increase in RyR2 S-glutathionylation, reduced calcium release activity, and completely prevented the protective effects of exercise and tachycardia on infarct size.The loss of cardioprotection induced by the NADPH oxidase inhibitor suggests that ROS generated by this enzyme are important mediators of the preconditioning response, which presumably involves NADPH oxidase-induced RyR2 S-glutathionylation.
Antecedentes de la investigacion: El preacondicionamiento hipoxico se ha utilizado como medida de proteccion para el miocardio, sin embargo es clinicamente inaplicable. Las especies reactivas de oxigeno (ERO), estarian involucradas en la produccion de modificaciones estructurales y electricas, que hacen susceptible al tejido auricular a arritmias y disfuncion contractil. Por lo tanto, un reforzamiento del sistema de defensa antioxidante protegeria al corazon del dano hipoxico. Objetivo: Determinar el efecto de la suplementacion de Omega 3 y vitaminas antioxidantes C y E, en el estado antioxidante de pacientes sometidos a cirugia cardiaca con circulacion extracorporea (CEC). Metodos: Un total de 50 pacientes se incluyo en el protocolo. Se randomizaron 15 pacientes a recibir Omega 3, siete dias antes de la cirugia, y vitaminas C y E, dos dias antes de la cirugia, ambos hasta el post-operatorio y 15 a placebo. Los valores controles se obtuvieron en 20 pacientes sin intervencion farmacologica preacondicionante. Se obtuvieron muestras de sangre en condiciones basales, antes del inicio de las vitaminas, 6-8 h post-operatorio y al alta para la determinacion del estado antioxidante (capacidad antioxidante del plasma, FRAP; relacion glutation reducido (GSH)Zoxidado (GSSG) y enzimas antioxidantes en eritrocito y dano oxidativo (malondialdehido y F2 - isoprostanos en plasma). En tejido se midio por Western Blot la expresion de la subunidad catalitica de la NADPH oxidasa (NOX-2) y la citosolica p47phox. La localizacion de la subunidad de membrana NOX-2/gp91phox se evidencio mediante inmunofluorescencia. Como indice de dano oxidativo en tejido se utilizaron los niveles de malondialdehido. Se utilizo analisis multivariado ANOVA con post-test de una via de Bonferroni. Resultados: Al momento de la cirugia, los pacientes que habian recibido tratamiento, presentaban niveles de GSH/GSSG un 56% mayor (p<0.03); en el postoperatorio inmediato tenian valores mayores de FRAP (30%; p<0.04) y glutation peroxidasa (25%; p<0.05) que el grupo placebo. En el tejido auricular habia menor expresion de la subunidad NOX-2/gp91 phox y de la subunidad citosolica p47phox en los tratados. La presencia de gp91phox en la membrana plasmatica de los cardiomiocitos se confirmo por inmunofluorescencia. En cuanto al dano oxidativo en plasma y tejido, los niveles de malondialdehido fueron 21 (p<0.03) y 46 % (p<0.05) menores y los niveles plasmaticos de F2-isoprostano 27% (p<0.04) menores en el grupo que recibio tratamiento. Conclusiones: La suplementacion con Omega 3 y vitaminas antioxidantes, atenua el dano oxidativo en pacientes sometidos a cirugia cardiaca con CEC. Se sugiere que este efecto podria obedecer al menos en parte a una regulacion negativa de la actividad de la NADPH oxidasa de los cardiomiocitos.
'/%3e%3cuse xlink:href='%23a' transform='scale(-1 1)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(72)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(-72)'/%3e%3cuse xlink:href='%23c' transform='rotate(144)'/%3e%3c/svg%3e)
