RESUMO
BACKGROUND: Most patients with malignant hyperthermia susceptibility diagnosed by the in vitro caffeine-halothane contracture test (CHCT) develop excessive force in response to halothane but not caffeine (halothane-hypersensitive). Hallmarks of halothane-hypersensitive patients include high incidence of musculoskeletal symptoms at rest and abnormal calcium events in muscle. By measuring sensitivity to halothane of myotubes and extending clinical observations and cell-level studies to a large group of patients, we reach new insights into the pathological mechanism of malignant hyperthermia susceptibility. METHODS: Patients with malignant hyperthermia susceptibility were classified into subgroups HH and HS (positive to halothane only and positive to both caffeine and halothane). The effects on [Ca2+]cyto of halothane concentrations between 0.5 and 3 % were measured in myotubes and compared with CHCT responses of muscle. A clinical index that summarises patient symptoms was determined for 67 patients, together with a calcium index summarising resting [Ca2+]cyto and spontaneous and electrically evoked Ca2+ events in their primary myotubes. RESULTS: Halothane-hypersensitive myotubes showed a higher response to halothane 0.5% than the caffeine-halothane hypersensitive myotubes (P<0.001), but a lower response to higher concentrations, comparable with that used in the CHCT (P=0.055). The HH group had a higher calcium index (P<0.001), but their clinical index was not significantly elevated vs the HS. Principal component analysis identified electrically evoked Ca2+ spikes and resting [Ca2+]cyto as the strongest variables for separation of subgroups. CONCLUSIONS: Enhanced sensitivity to depolarisation and to halothane appear to be the primary, mutually reinforcing and phenotype-defining defects of halothane-hypersensitive patients with malignant hyperthermia susceptibility.
Assuntos
Hipertermia Maligna , Humanos , Hipertermia Maligna/diagnóstico , Halotano/farmacologia , Cálcio , Fibras Musculares Esqueléticas , Suscetibilidade a Doenças/complicações , Cafeína/farmacologia , Contração MuscularRESUMO
Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle. Two UNet-type semantic segmentation models were built: "Locations" classified pixels as belonging to different regions in the cell; "Granules" identified pixels within granules. From their joint output, a pixel fraction pf was calculated for images from patients positive (MHS) or negative (MHN) to a test for malignant hyperthermia susceptibility. pf was used to derive vf, the volume fraction occupied by granules. The relationship vf (pf) was derived from a simulation of volumes ("baskets") containing virtual granules at realistic concentrations. The simulated granules had diameters matching the real ones, which were measured by adapting a utility devised for calcium sparks. Applying this relationship to the pf measured in images, vf was calculated for every region and patient, and from them a glycogen concentration. The intermyofibrillar spaces and the sarcomeric I band had the highest granular content. The measured glycogen concentration was low enough to allow for a substantial presence of non-granular glycogen. The MHS samples had an approximately threefold lower concentration (significant in a hierarchical test), consistent with earlier evidence of diminished glucose processing in MHS. The AI models and the approach to infer three-dimensional magnitudes from two-dimensional images should be adaptable to other tasks on a variety of images from patients and animal models and different disease conditions.
Assuntos
Glicogênio , Músculo Esquelético , Humanos , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/diagnóstico por imagem , Inteligência Artificial , Microscopia Eletrônica/métodosRESUMO
Calcium ion movements between cellular stores and the cytosol govern muscle contraction, the most energy-consuming function in mammals, which confers skeletal myofibers a pivotal role in glycemia regulation. Chronic myoplasmic calcium elevation ("calcium stress"), found in malignant hyperthermia-susceptible (MHS) patients and multiple myopathies, has been suggested to underlie the progression from hyperglycemia to insulin resistance. What drives such progression remains elusive. We find that muscle cells derived from MHS patients have increased content of an activated fragment of GSK3ß - a specialized kinase that inhibits glycogen synthase, impairing glucose utilization and delineating a path to hyperglycemia. We also find decreased content of junctophilin1, an essential structural protein that colocalizes in the couplon with the voltage-sensing CaV1.1, the calcium channel RyR1 and calpain1, accompanied by an increase in a 44 kDa junctophilin1 fragment (JPh44) that moves into nuclei. We trace these changes to activated proteolysis by calpain1, secondary to increased myoplasmic calcium. We demonstrate that a JPh44-like construct induces transcriptional changes predictive of increased glucose utilization in myoblasts, including less transcription and translation of GSK3ß and decreased transcription of proteins that reduce utilization of glucose. These effects reveal a stress-adaptive response, mediated by the novel regulator of transcription JPh44.
Assuntos
Hiperglicemia , Hipertermia Maligna , Animais , Humanos , Cálcio/metabolismo , Cálcio da Dieta , Suscetibilidade a Doenças , Glucose/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Hiperglicemia/metabolismo , Hipertermia Maligna/metabolismo , Mamíferos/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Triadin, a protein of the sarcoplasmic reticulum (SR) of striated muscles, anchors the calcium-storing protein calsequestrin to calcium release RyR channels at the junction with t-tubules, and modulates these channels by conformational effects. Triadin ablation induces structural SR changes and alters the expression of other proteins. Here we quantify alterations of calcium signaling in single skeletal myofibers of constitutive triadin-null mice. We find higher resting cytosolic and lower SR-luminal [Ca2+], 40% lower calsequestrin expression, and more CaV1.1, RyR1 and SERCA1. Despite the increased CaV1.1, the mobile intramembrane charge was reduced by ~20% in Triadin-null fibers. The initial peak of calcium release flux by pulse depolarization was minimally altered in the null fibers (revealing an increase in peak calcium permeability). The "hump" phase that followed, attributable to calcium detaching from calsequestrin, was 25% lower, a smaller change than expected from the reduced calsequestrin content and calcium saturation. The exponential decay rate of calcium transients was 25% higher, consistent with the higher SERCA1 content. Recovery of calcium flux after a depleting depolarization was faster in triadin-null myofibers, consistent with the increased uptake rate and lower SR calsequestrin content. In sum, the triadin knockout determines an increased RyR1 channel openness, which depletes the SR, a substantial loss of calsequestrin and gains in other couplon proteins. Powerful functional compensations ensue: activation of SOCE that increases [Ca2+]cyto; increased SERCA1 activity, which limits the decrease in [Ca2+]SR and a restoration of SR calcium storage of unknown substrate. Together, they effectively limit the functional loss in skeletal muscles.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas Musculares/deficiência , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Mutantes , Proteínas Musculares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genéticaRESUMO
This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes τ1 and τ2 derived from a two-exponential fit in freshly dissected mouse flexor digitorum brevis (FDB) and soleus muscles. While τ1 values changed following a bimodal behavior between muscles, the distribution of τ2 is shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes of flavin mononucleotide and dinucleotide (FMN/FAD) in cryosections, where excitation was set at 440 nm and emission at a bandwidth of between 500 and 570 nm, and paired them with immunofluorescence images of myosin heavy chain isoforms, which allowed identification of fiber types. In soleus, τ2 was 3.16 ns for type I (SD 0.11, 97 fibers), 3.45 ns for IIA (0.10, 69), and 3.46 ns for IIX (0.12, 65). In FDB muscle, τ2 was 3.17 ns for type I (0.08, 22), 3.46 ns for IIA (0.16, 48), and 3.66 ns for IIX (0.15, 43). From τ2 distributions, it follows that an FDB fiber with τ2 > 3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first and second kind errors estimated at 0.02 and 0.10, which can be lowered by reducing the threshold for identification of type I and increasing it for type II. Lifetime maps of autofluorescence, therefore, constitute a tool to identify fiber types that, for being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.
Assuntos
Fibras Musculares Esqueléticas , Cadeias Pesadas de Miosina , Animais , Camundongos , Microscopia de Fluorescência , Músculo Esquelético , Isoformas de ProteínasRESUMO
Dengue virus (DENV) infection elevates intracellular Ca2+ concentration ([Ca2+]i), but it is unknown whether Ca2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca2+]i examining the effects of DENV infection and drugs that alter Ca2+/CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity.
Assuntos
Calmodulina/metabolismo , Núcleo Celular/metabolismo , Dantroleno/farmacologia , Vírus da Dengue/fisiologia , Mioblastos Cardíacos/virologia , Transporte Ativo do Núcleo Celular , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citosol/metabolismo , Vírus da Dengue/efeitos dos fármacos , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Fosforilação , Poli I-C/farmacologia , Ratos , Fator de Transcrição STAT2/metabolismo , Regulação para Cima , Proteínas Virais/metabolismoRESUMO
Most glucose is processed in muscle, for energy or glycogen stores. Malignant Hyperthermia Susceptibility (MHS) exemplifies muscle conditions that increase [Ca2+]cytosol. 42% of MHS patients have hyperglycemia. We show that phosphorylated glycogen phosphorylase (GPa), glycogen synthase (GSa) - respectively activated and inactivated by phosphorylation - and their Ca2+-dependent kinase (PhK), are elevated in microsomal extracts from MHS patients' muscle. Glycogen and glucose transporter GLUT4 are decreased. [Ca2+]cytosol, increased to MHS levels, promoted GP phosphorylation. Imaging at ~100 nm resolution located GPa at sarcoplasmic reticulum (SR) junctional cisternae, and apo-GP at Z disk. MHS muscle therefore has a wide-ranging alteration in glucose metabolism: high [Ca2+]cytosol activates PhK, which inhibits GS, activates GP and moves it toward the SR, favoring glycogenolysis. The alterations probably cause these patients' hyperglycemia. For basic studies, MHS emerges as a variable stressor, which forces glucose pathways from the normal to the diseased range, thereby exposing novel metabolic links.
Animals and humans move by contracting the skeletal muscles attached to their bones. These muscles take up a type of sugar called glucose from food and use it to fuel contractions or store it for later in the form of glycogen. If muscles fail to use glucose it can lead to excessive sugar levels in the blood and a condition called diabetes. Within muscle cells are stores of calcium that signal the muscle to contract. Changes in calcium levels enhance the uptake of glucose that fuel these contractions. However, variations in calcium have also been linked to diabetes, and it remained unclear when and how these 'signals' become harmful. People with a condition called malignant hyperthermia susceptibility (MHS for short) have genetic mutations that allow calcium to leak out from these stores. This condition may result in excessive contractions causing the muscle to over-heat, become rigid and break down, which can lead to death if left untreated. A clinical study in 2019 found that out of hundreds of patients who had MHS, nearly half had high blood sugar and were likely to develop diabetes. Now, Tammineni et al. including some of the researchers involved in the 2019 study have set out to find why calcium leaks lead to elevated blood sugar levels. The experiments showed that enzymes that help convert glycogen to glucose are more active in patients with MHS, and found in different locations inside muscle cells. Whereas the enzymes that change glucose into glycogen are less active. This slows down the conversion of glucose into glycogen for storage and speeds up the breakdown of glycogen into glucose. Patients with MHS also had fewer molecules that transport glucose into muscle cells and stored less glycogen. These changes imply that less glucose is being removed from the blood. Next, Tammineni et al. used a microscopy technique that is able to distinguish finely separated objects with a precision not reached before in living muscle. This revealed that when the activity of the enzyme that breaks down glycogen increased, it moved next to the calcium store. This effect was also observed in the muscle cells of MHS patients that leaked calcium from their stores. Taken together, these observations may explain why patients with MHS have high levels of sugar in their blood. These findings suggest that MHS may start decades before developing diabetes and blood sugar levels in these patients should be regularly monitored. Future studies should investigate whether drugs that block calcium from leaking may help prevent high blood sugar in patients with MHS or other conditions that cause a similar calcium leak.
Assuntos
Cálcio/metabolismo , Diabetes Mellitus/etiologia , Glucose/metabolismo , Hiperglicemia/etiologia , Hipertermia Maligna/complicações , Músculo Esquelético/metabolismo , Adulto , Idoso , Animais , Glicemia/metabolismo , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase Muscular/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/metabolismo , Hipertermia Maligna/sangue , Hipertermia Maligna/metabolismo , Hipertermia Maligna/patologia , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Fosforilase Quinase/metabolismo , FosforilaçãoRESUMO
The auxiliary ß4 subunit of the cardiac Cav1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the ß4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-ß (IFN-ß)] and of the IFN-ß-related genes Ddx58, Ifitm3, Irf7, Stat2, Ifih1, and Mx1, as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the ß4 subunit in H9c2 cells reduced the expression of IFN-ß-stimulated genes. In response to inhibition of the kinase JAK1, the abundances of ß4 subunit mRNA and protein were decreased. ß4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-ß, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of ß4 subunit proteins to nuclei in response to spreading infection. We showed that the ß4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter-driven reporter was increased in cells overexpressing the ß4 subunit. Last, overexpressing ß4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca2+ in these cells. These findings suggest that the ß4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection.
Assuntos
Canais de Cálcio/metabolismo , Interferon beta/imunologia , Miócitos Cardíacos/imunologia , Animais , Antivirais/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Células Cultivadas , Dengue/imunologia , Dengue/patologia , Dengue/prevenção & controle , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection.