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Background: Ventricular fibrillation (VF) is associated with dysfunctional cardiac calcium cycling and poor survival. We hypothesized dantrolene improves survival following VF by stabilizing calcium dysregulation. Methods: VF was induced in 26 healthy Yorkshire pigs and left untreated for 4 min followed by 3 min of CPR and defibrillation. Dantrolene was infused during CPR. Rabbit hearts (n=14) were studied to evaluate the effect of dantrolene on VF-induced calcium cycling dysfunction. Results: Survival was higher in the dantrolene group. (85% vs. 39%, P=0.01) Dantrolene-treated pigs required significantly lower defibrillation energy level. (150J vs. 650J, P<0.05) Systolic pressure was significantly higher during the post-defibrillation period in the dantrolene group. (P=0.001) In rabbit hearts, dantrolene significantly mitigated the amplitude of VF-induced diastolic calcium elevations and increased the calcium alternans threshold. (P<0.05) Conclusion: Our findings suggest dantrolene facilitates successful defibrillation, prevents myocardial stunning and improves survival following VF. The effects are mediated through normalizing the VF-induced dysfunctional calcium cycling.

Background: Ventricular fibrillation (VF) is associated with dysfunctional cardiac calcium cycling and poor survival. We hypothesized dantrolene improves survival following VF by stabilizing calcium dysregulation. Methods: VF was induced in 26 healthy Yorkshire pigs and left untreated for 4 min followed by 3 min of CPR and defibrillation. Dantrolene was infused during CPR. Rabbit hearts (n=14) were studied to evaluate the effect of dantrolene on VF-induced calcium cycling dysfunction. Results: Survival was higher in the dantrolene group. (85% vs. 39%, P=0.01) Dantrolene-treated pigs required significantly lower defibrillation energy level. (150J vs. 650J, P<0.05) Systolic pressure was significantly higher during the post-defibrillation period in the dantrolene group. (P=0.001) In rabbit hearts, dantrolene significantly mitigated the amplitude of VF-induced diastolic calcium elevations and increased the calcium alternans threshold. (P<0.05) Conclusion: Our findings suggest dantrolene facilitates successful defibrillation, prevents myocardial stunning and improves survival following VF. The effects are mediated through normalizing the VF-induced dysfunctional calcium cycling.

Ryanodine receptor 1 (RyR1) is an isoform of ryanodine receptor predominantly expressed in skeletal muscle. It is a ~2MDa homotetrameric Ca²⁺ release channel with a large cytosolic domain and is in the membrane of the sarcoplasmic reticulum in skeletal muscle cells. RyR1 plays a key role in coordinating excitation-contraction (EC) coupling in skeletal muscle. The activity of the RyR1 channel is regulated by multiple factors, including phosphorylation, oxidation, and ligand binding, all of which tightly control the channel function. The cytosolic domain of RyR1 contains binding sites for these ligands, enabling allosteric regulation. Malignant hyperthermia susceptibility (MHS) is a condition that predisposes individuals to an episode of malignant hyperthermia (MH), a pharmacogenetic shock syndrome triggered by the administration of volatile inhalational anesthetics such as halothane, isoflurane, and succinylcholine. Variants in the RYR1 gene is responsible for over 50% of MHS cases. To treat the rapid metabolic shock that occurs during an MH episode, dantrolene must be administered quickly. Dantrolene, the only approved drug for MH treatment, inhibits RyR1 and reduces Ca²⁺ influx into the cytoplasm of skeletal muscle cells. However, the detailed molecular mechanism by which dantrolene inhibits RyR1 has not been fully elucidated. I purified rabbit RyR1 reconstituted in detergent micelles and subjected the vitrified protein-ligand samples to cryo-electron microscopy (cryoEM) in the presence of dantrolene and other RyR1 agonists, such as ATP, ADP, caffeine, and 4-chloro-m-cresol (4CmC; an MH-triggering molecule). I identified dantrolene binding in complex with ATP or ADP at the RY12 domain on RyR1. Additionally, multiple binding sites for 4CmC on RyR1 were identified. Following the initial characterization of the novel dantrolene and adenosine phosphate binding site in the RY12 domain, purified RyR1 was reconstituted in liposomes for single-channel planar lipid bilayer assays. These assays confirmed that either ATP or ADP is required at the dantrolene binding site for RyR1 inhibition. These findings led us to hypothesize that the novel drug and ATP/ADP binding site in the RY12 domain may also play a physiological role in sensing an increased ADP concentrations in skeletal muscle cells, particularly in the cytosolic compartment during muscle fatigue and pathological conditions. During EC COUPLING, ATP hydrolysis for muscle contraction increases cytosolic ADP concentrations above resting levels. I found that the RY12 site preferentially binds ADP rather than ATP when neither dantrolene nor 4CmC is present. I also discovered that RyR1 forms an endogenous complex with calstabin1 (Cs1, also known as FK506-binding protein 12) and calmodulin (CaM), as observed through cryoEM analysis of rabbit RyR1 solubilized with digitonin and purified by sucrose gradient centrifugation. During these experiments, I noticed that RyR1s in digitonin non-specifically adhere to the air-water interface (AWI) between the sample buffer and atmospheric air on cryoEM grids, resulting in biased orientations of RyR1 particles in the micrographs. To mitigate this effect, glycyrrhizic acid was added to the purified RyR1 sample immediately before vitrification. I applied a similar strategy to prevent the protein from adhering to the AWI when solving the structure of iodinated bovine thyroglobulin (Tg), a ~660 kDa homodimeric soluble protein responsible for thyroid hormone biosynthesis in the thyroid gland. Thyroxines (T4) and iodotyrosines (mono- or di-iodotyrosine) were identified at hormonogenic sites on bovine Tg in the reconstructed EM map, and the analysis around the T4 sites allowed us to hypothesize the molecular mechanism of coupling reactions between two diiodotyrosine residues to synthesize T4 within the Tg molecule.

O complexo distrofina-glicoproteínas associadas (DGC) localiza-se no sarcolema das células musculares esqueléticas e cardíacas e tem como função principal proporcionar ligação mecânica entre o citoesqueleto intracelular e a matriz extracelular. Estudos prévios realizados em nosso laboratório, focalizando o complexo DGC, demonstraram perda de proteínas importantes desse complexo. As situações avaliadas anteriormente foram: infecção experimental por Trypanosoma cruzi (T. cruzi) e sépsis experimental. Em ambas as situações verificou-se a perda da distrofina acompanhada por disfunção contrátil e aumento nos níveis da calpaína, protease dependente de cálcio implicada na proteólise da distrofina. Todavia, o mecanismo responsável pela ativação das calpaínas e proteólise da distrofina na infecção experimental por T. cruzi e na sépsis experimental não está totalmente definido. O objetivo desse trabalho foi avaliar in vitro o mecanismo responsável pela ativação das calpaínas nas culturas de cardiomiócitos desafiadas com o soro dos animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave experimental. Camundongos C57BL/6 foram submetidos à sépsis grave ou infectados com a cepa Y de T. cruzi. No pico de expressão das citocinas pró-inflamatórias, 12 dias após inoculação do parasito ou 6 horas após a indução da sépsis, o sangue foi coletado e o soro separado. Corações de camundongos recém-nascidos foram isolados para o cultivo dos cardiomiócitos. No quinto dia após o início das culturas, as células foram estimuladas com 10% do soro de animais infectados com T. cruzi ou o soro de animais submetidos à sépsis grave durante 24 horas. Após, as células foram coletadas para análises de Western blotting e imunofluorescência para verificar a expressão da distrofina e calpaína-1. Avaliou-se também, por imunofluorescência, a expressão do NF-B. Os cardiomiócitos foram estimulados e tratados com o dantrolene, inibidor da liberação de cálcio do retículo sarcoplasmático, ou ALLN, inibidor da calpaína-1, e após coletados para verificar a expressão da distrofina e calpaína-1 por Western blotting e imunofluorescência. Nossos resultados mostraram uma redução significativa na expressão da distrofina com desarranjo das miofibrilas contráteis e formação de bolhas citoplasmáticas, além de um aumento nos níveis da calpaína-1 e do NF-B. O tratamento com dantrolene nas culturas estimuladas com o soro de animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave, recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. O tratamento com ALLN nos cardiomiócitos estimulados com o soro de animais infectados experimentalmente com T. cruzi recuperou a expressão da distrofina e não alterou os níveis da calpaína-1. Nas culturas estimuladas com o soro dos animais submetidos à sépsis grave, o tratamento com o ALLN recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. Nossos resultados demonstraram que citocinas pró-inflamatórias presentes no soro dos animais infectados experimentalmente com T. cruzi como também no soro dos animais submetidos à sépsis grave induziriam um aumento no influxo de cálcio com consequente ativação das calpaínas, as quais atuariam na ativação do NF-B e na degradação da distrofina. Esse mecanismo poderia ser responsável pela proteólise da distrofina cardíaca observada na infecção experimental por Trypanosoma cruzi como também sépsis experimental. Mais estudos são necessários para elucidar este mecanismo, principalmente em relação a inibidores dos canais de cálcio, das citocinas pró-inflamatórias e das calpaínas, com o objetivo de fornecer novas vias de intervenção na prevenção de alterações cardíacas observadas na doença de Chagas e na sépsis. / The dystrophin-glycoprotein complex (DGC), located in the sarcolemma of cardiac and skeletal muscle cells and concentrated along the plasma membrane in costameric structures provides a framework that connects the intracellular cytoskeleton to the extracellular matrix. Previous studies from our laboratory clearly demonstrated disruption of DGC proteins in experimentally-induced T. cruzi infection and experimental sepsis. Both situation presented dystrophin disruption associated with contractile dysfunction and increased calpain levels, calcium dependent protease responsible for dystrophin proteolysis. However, the mechanism responsible for calpain activation and dystrophin proteolysis in experimentally-induced T. cruzi infection and experimental sepsis is not totally understood. The aim of this study was to evaluate in vitro the mechanism responsible for calpain activation in cultured cardiomyocytes challenged with serum from animals experimentally infected with T. cruzi or subjected to severe sepsis. Mice C57BL/6 were subjected to sepsis induction or infected with Y strain from T. cruzi. At the peak of proinflammatory cytokines expression, 12 days after parasite inoculation or 6 hours after sepsis induction, the blood was collected and the serum separated. Hearts from newborn mice were isolated for culture of cardiomyocytes. After 5 days of incubation, the cardiac cells were stimulated with 10% of serum from animals experimentally infected with T. cruzi or subjected to severe sepsis during 24 hours, and collected for Western blotting and immunofluorescence analysis to verify dystrophin and calpain-1 expression. The expression of NF-B was evaluated by immunofluorescence. The treatments with dantrolene, inhibitor of calcium release from sarcoplasmic reticulum, or ALLN, calpain-1 inhibitor, were performed in cultured cardiomyocytes stimulated during 24 hours with serum from animals infected with T. cruzi or subjected to severe sepsis, and dystrophin and calpain-1 expression were analyzed by Western blotting and immunofluorescence. Our results demonstrated loss of dystrophin associated with myofibers derangement and presence of cytoplasmic blebs as well increase of calpain-1 and NF-B expression. The dantrolene treatment in cultures stimulated with serum from animals infected with T. cruzi or subjected to severe sepsis recovey dystrophin expression and reduced calpain-1 levels. The ALLN treatment in cardiomyocytes stimulated with serum from animals infected with T. cruzi recovery dystrophin expression and preserved calpain-1 levels. In cultures stimulated with serum from animals subjected to severe sepsis, the ALLN treatment recovery dystrophin expression and decreased calpain-1 levels. Our results demonstrated that proinflammatory cytokines in serum from mice infected with T. cruzi or subjected to severe sepsis could induce an increase calcium influx with calpain activation, which could act in NF-B activation and dystrophin disruption. Possibly, this mechanism could be responsible to dystrophin proteolysis observed in experimentally-induced acute T. cruzi infection and experimental sepsis. More studies are needed to elucidate this mechanism, especially in relation to calcium channel blockers and inhibitors of pro-inflammatory cytokines and calpains, which may provide new routes for intervention to prevent cardiac damage in Chagas disease and sepsis.

O complexo distrofina-glicoproteínas associadas (DGC) localiza-se no sarcolema das células musculares esqueléticas e cardíacas e tem como função principal proporcionar ligação mecânica entre o citoesqueleto intracelular e a matriz extracelular. Estudos prévios realizados em nosso laboratório, focalizando o complexo DGC, demonstraram perda de proteínas importantes desse complexo. As situações avaliadas anteriormente foram: infecção experimental por Trypanosoma cruzi (T. cruzi) e sépsis experimental. Em ambas as situações verificou-se a perda da distrofina acompanhada por disfunção contrátil e aumento nos níveis da calpaína, protease dependente de cálcio implicada na proteólise da distrofina. Todavia, o mecanismo responsável pela ativação das calpaínas e proteólise da distrofina na infecção experimental por T. cruzi e na sépsis experimental não está totalmente definido. O objetivo desse trabalho foi avaliar in vitro o mecanismo responsável pela ativação das calpaínas nas culturas de cardiomiócitos desafiadas com o soro dos animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave experimental. Camundongos C57BL/6 foram submetidos à sépsis grave ou infectados com a cepa Y de T. cruzi. No pico de expressão das citocinas pró-inflamatórias, 12 dias após inoculação do parasito ou 6 horas após a indução da sépsis, o sangue foi coletado e o soro separado. Corações de camundongos recém-nascidos foram isolados para o cultivo dos cardiomiócitos. No quinto dia após o início das culturas, as células foram estimuladas com 10% do soro de animais infectados com T. cruzi ou o soro de animais submetidos à sépsis grave durante 24 horas. Após, as células foram coletadas para análises de Western blotting e imunofluorescência para verificar a expressão da distrofina e calpaína-1. Avaliou-se também, por imunofluorescência, a expressão do NF-B. Os cardiomiócitos foram estimulados e tratados com o dantrolene, inibidor da liberação de cálcio do retículo sarcoplasmático, ou ALLN, inibidor da calpaína-1, e após coletados para verificar a expressão da distrofina e calpaína-1 por Western blotting e imunofluorescência. Nossos resultados mostraram uma redução significativa na expressão da distrofina com desarranjo das miofibrilas contráteis e formação de bolhas citoplasmáticas, além de um aumento nos níveis da calpaína-1 e do NF-B. O tratamento com dantrolene nas culturas estimuladas com o soro de animais infectados experimentalmente com T. cruzi ou com o soro dos animais submetidos à sépsis grave, recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. O tratamento com ALLN nos cardiomiócitos estimulados com o soro de animais infectados experimentalmente com T. cruzi recuperou a expressão da distrofina e não alterou os níveis da calpaína-1. Nas culturas estimuladas com o soro dos animais submetidos à sépsis grave, o tratamento com o ALLN recuperou a expressão da distrofina e reduziu os níveis da calpaína-1. Nossos resultados demonstraram que citocinas pró-inflamatórias presentes no soro dos animais infectados experimentalmente com T. cruzi como também no soro dos animais submetidos à sépsis grave induziriam um aumento no influxo de cálcio com consequente ativação das calpaínas, as quais atuariam na ativação do NF-B e na degradação da distrofina. Esse mecanismo poderia ser responsável pela proteólise da distrofina cardíaca observada na infecção experimental por Trypanosoma cruzi como também sépsis experimental. Mais estudos são necessários para elucidar este mecanismo, principalmente em relação a inibidores dos canais de cálcio, das citocinas pró-inflamatórias e das calpaínas, com o objetivo de fornecer novas vias de intervenção na prevenção de alterações cardíacas observadas na doença de Chagas e na sépsis. / The dystrophin-glycoprotein complex (DGC), located in the sarcolemma of cardiac and skeletal muscle cells and concentrated along the plasma membrane in costameric structures provides a framework that connects the intracellular cytoskeleton to the extracellular matrix. Previous studies from our laboratory clearly demonstrated disruption of DGC proteins in experimentally-induced T. cruzi infection and experimental sepsis. Both situation presented dystrophin disruption associated with contractile dysfunction and increased calpain levels, calcium dependent protease responsible for dystrophin proteolysis. However, the mechanism responsible for calpain activation and dystrophin proteolysis in experimentally-induced T. cruzi infection and experimental sepsis is not totally understood. The aim of this study was to evaluate in vitro the mechanism responsible for calpain activation in cultured cardiomyocytes challenged with serum from animals experimentally infected with T. cruzi or subjected to severe sepsis. Mice C57BL/6 were subjected to sepsis induction or infected with Y strain from T. cruzi. At the peak of proinflammatory cytokines expression, 12 days after parasite inoculation or 6 hours after sepsis induction, the blood was collected and the serum separated. Hearts from newborn mice were isolated for culture of cardiomyocytes. After 5 days of incubation, the cardiac cells were stimulated with 10% of serum from animals experimentally infected with T. cruzi or subjected to severe sepsis during 24 hours, and collected for Western blotting and immunofluorescence analysis to verify dystrophin and calpain-1 expression. The expression of NF-B was evaluated by immunofluorescence. The treatments with dantrolene, inhibitor of calcium release from sarcoplasmic reticulum, or ALLN, calpain-1 inhibitor, were performed in cultured cardiomyocytes stimulated during 24 hours with serum from animals infected with T. cruzi or subjected to severe sepsis, and dystrophin and calpain-1 expression were analyzed by Western blotting and immunofluorescence. Our results demonstrated loss of dystrophin associated with myofibers derangement and presence of cytoplasmic blebs as well increase of calpain-1 and NF-B expression. The dantrolene treatment in cultures stimulated with serum from animals infected with T. cruzi or subjected to severe sepsis recovey dystrophin expression and reduced calpain-1 levels. The ALLN treatment in cardiomyocytes stimulated with serum from animals infected with T. cruzi recovery dystrophin expression and preserved calpain-1 levels. In cultures stimulated with serum from animals subjected to severe sepsis, the ALLN treatment recovery dystrophin expression and decreased calpain-1 levels. Our results demonstrated that proinflammatory cytokines in serum from mice infected with T. cruzi or subjected to severe sepsis could induce an increase calcium influx with calpain activation, which could act in NF-B activation and dystrophin disruption. Possibly, this mechanism could be responsible to dystrophin proteolysis observed in experimentally-induced acute T. cruzi infection and experimental sepsis. More studies are needed to elucidate this mechanism, especially in relation to calcium channel blockers and inhibitors of pro-inflammatory cytokines and calpains, which may provide new routes for intervention to prevent cardiac damage in Chagas disease and sepsis.