An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle ...fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate Ins(1,4,5)P₃-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P₃ production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P₃ was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.
Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca²⁺ release in skeletal muscle and a potentially fatal rise in ...perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca v1.1, the principal subunit of the L-type Ca²⁺ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca²⁺ entry. The Ca v1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Cav channels Carpenter D, et al. (2009) BMC Med Genet 10: 104-115.. To define the functional consequences of this mutation, we expressed it in dysgenic (Cav 1.1 null) myotubes. Unlike previously described MH-linked mutations in Cav1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca²⁺ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Cav1.1 R174W-expressing myotubes, resting myoplasmic Ca²⁺ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Cav1.1. Our results indicate that Cav1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca²⁺ leak from the SR, and that perturbation of Cav1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.
Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and ...progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased Ca(2+)r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the Ca(2+)r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced Ca(2+)r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces Ca(2+)r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The recognition that oxidative stress is a major component of several chronic diseases has engendered numerous trials of antioxidant therapies with minimal or no direct benefits. Nanomolar quantities ...of nitric oxide released into the circulation by pharmacologic stimulation of eNOS have antioxidant properties but physiologic stimulation as through increased pulsatile shear stress of the endothelium has not been assessed. The present study utilized a non-invasive technology, periodic acceleration (pGz) that increases pulsatile shear stress such that upregulation of cardiac eNOS occurs, We assessed its efficacy in normal mice and mouse models with high levels of oxidative stress, e.g. Diabetes type 1 and mdx (Duchene Muscular Dystrophy). pGz increased protein expression and upregulated eNOS in hearts. Application of pGz was associated with significantly increased expression of endogenous antioxidants (Glutathioneperoxidase-1(GPX-1), Catalase (CAT), Superoxide, Superoxide Dismutase 1(SOD1). This led to an increase of total cardiac antioxidant capacity along with an increase in the antioxidant response element transcription factor Nrf2 translocation to the nucleus. pGz decreased reactive oxygen species in both mice models of oxidative stress. Thus, pGz is a novel non-pharmacologic method to harness endogenous antioxidant capacity.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this study, different strategies used to count vehicles and people in different image areas at a street intersection were analyzed to obtain counts at appropriate times suitable for real-time ...control of a traffic light. To achieve this, video recordings of cameras placed at the intersection were used to test and verify image processing algorithms and deep learning using the YOLOv3 network implemented on a 4 GB RAM Jetson Nano card. We counted the vehicles and people that stopped and crossed the polygons to delimit the different areas of interest, with a maximum error of ±2 in the validation tests for all cases. In addition, as a strategy, we combined the images from both cameras into a single one, thereby allowing us to make a single detection and subsequently determine if they are inside or outside the polygons used in separating the areas of interest with the respective counts. Furthermore, this enabled us to obtain information on vehicles and people stopped and crossing in a time of 0.73 s on average. Hence, it was established that the inclusion of the control algorithm is appropriate for real-time control of traffic lights.
Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The ...mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP3) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of β-myosin heavy chain, α-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP3/Ca2+ and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.
Reactive oxygen species (ROS) participate as signaling molecules in response to exercise in skeletal muscle. However, the source of ROS and the molecular mechanisms involved in these phenomena are ...still not completely understood. The aim of this work was to study the role of skeletal muscle NADPH oxidase isoform 2 (NOX2) in the molecular response to physical exercise in skeletal muscle. BALB/c mice, pre-treated with a NOX2 inhibitor, apocynin, (3 mg/kg) or vehicle for 3 days, were swim-exercised for 60 min. Phospho-p47(phox) levels were significantly upregulated by exercise in flexor digitorum brevis (FDB). Moreover, exercise significantly increased NOX2 complex assembly (p47(phox)-gp91(phox) interaction) demonstrated by both proximity ligation assay and co-immunoprecipitation. Exercise-induced NOX2 activation was completely inhibited by apocynin treatment. As expected, exercise increased the mRNA levels of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx), citrate synthase (CS), mitochondrial transcription factor A (tfam) and interleukin-6 (IL-I6) in FDB muscles. Moreover, the apocynin treatment was associated to a reduced activation of p38 MAP kinase, ERK 1/2, and NF-κB signaling pathways after a single bout of exercise. Additionally, the increase in plasma IL-6 elicited by exercise was decreased in apocynin-treated mice compared with the exercised vehicle-group (p < 0.001). These results were corroborated using gp91-dstat in an in vitro exercise model. In conclusion, NOX2 inhibition by both apocynin and gp91dstat, alters the intracellular signaling to exercise and electrical stimuli in skeletal muscle, suggesting that NOX2 plays a critical role in molecular response to an acute exercise.
Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca2+ dysregulation. NCX is a bidirectional transporter that effluxes (forward ...mode) or influxes (reverse mode) Ca2+ depending on cellular activity. Resting intracellular calcium (Ca2+r) and sodium (Na+r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering Na+e elevates Ca2+r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca2+ or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced Ca2+r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of Ca2+r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces Ca2+r in MHS muscle fibers and decreases the amplitude of Ca2+r rise triggered by halothane, but had no effect on Ca2+r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca2+ transient elicited by high K+e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced Ca2+r and the Ca2+ transient area induced by high K+e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca2+ transients associated with K+-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.
Background: Dysregulation of Ca2+ homeostasis have been described in malignant hyperthermia (MH).
Results: Na+/Ca2+ exchanger (NCX3) reverse mode activity is enhanced in MH muscles and it contributes to resting intracellular calcium concentration and Ca2+ transients induced by high K+e and by halothane.
Conclusion: NCX3 reverse mode activity is increased in MH muscle.
Significance: Understanding mechanisms influencing Ca2+ dynamics in MH muscle.
Myocardial infarction (MI) may produce significant inflammatory changes and adverse ventricular remodeling leading to heart failure and premature death. Pharmacologic, stem cell transplantation, and ...exercise have not halted the inexorable rise in the prevalence and great economic costs of heart failure despite extensive investigations of such treatments. New therapeutic modalities are needed. Whole Body Periodic Acceleration (pGz) is a non-invasive technology that increases pulsatile shear stress to the endothelium thereby producing several beneficial cardiovascular effects as demonstrated in animal models, normal humans and patients with heart disease. pGz upregulates endothelial derived nitric oxide synthase (eNOS) and its phosphorylation (p-eNOS) to improve myocardial function in models of myocardial stunning and preconditioning. Here we test whether pGz applied chronically after focal myocardial infarction in rats improves functional outcomes from MI. Focal MI was produced by left coronary artery ligation. One day after ligation animals were randomized to receive daily treatments of pGz for four weeks (MI-pGz) or serve as controls (MI-CONT), with an additional group as non-infarction controls (Sham). Echocardiograms and invasive pressure volume loop analysis were carried out. Infarct transmurality, myocardial fibrosis, and markers of inflammatory and anti-inflammatory cytokines were determined along with protein analysis of eNOS, p-eNOS and inducible nitric oxide synthase (iNOS).At four weeks, survival was 80% in MI-pGz vs 50% in MI-CONT (p< 0.01). Ejection fraction and fractional shortening and invasive pressure volume relation indices of afterload and contractility were significantly better in MI-pGz. The latter where associated with decreased infarct transmurality and decreased fibrosis along with increased eNOS, p-eNOS. Additionally, MI-pGz had significantly lower levels of iNOS, inflammatory cytokines (IL-6, TNF-α), and higher level of anti-inflammatory cytokine (IL-10). pGz improved survival and contractile performance, associated with improved myocardial remodeling. pGz may serve as a simple, safe, non-invasive therapeutic modality to improve myocardial function after MI.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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