Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease.

Mucus plugging and non-resolving inflammation are inherent features of cystic fibrosis (CF) that may lead to progressive lung disease and exercise intolerance, which are the main causes of morbidity and mortality for people with CF. Therefore, understanding the influence of mucus on basic mechanisms underlying the inflammatory response and identifying strategies to resolve mucus-driven airway inflammation and consequent morbidity in CF are of wide interest. Here, we investigated the effects of the proresolving lipid mediator resolvin (Rv) D1 on mucus-related inflammation as a proof-of-concept to alleviate the burden of lung disease and restore exercise intolerance in CF. We tested the effects of RvD1 on inflammatory responses of human organotypic airways and leukocytes to CF mucus and of humanized mice expressing the epithelial Na + channel (ßENaC-Tg) having CF-like mucus obstruction, lung disease, and physical exercise intolerance. RvD1 reduced pathogenic phenotypes of CF-airway supernatant (ASN)-stimulated human neutrophils, including loss of L-selectin shedding and CD16. RNASeq analysis identified select transcripts and pathways regulated by RvD1 in ASN-stimulated CF bronchial epithelial cells that are involved in sugar metabolism, NF-κB activation and inflammation, and response to stress. In in vivo inflammation using ßENaC TG mice, RvD1 reduced total leukocytes, PMN, and interstitial Siglec-MΦ when given at 6-8 weeks of age, and in older mice at 10-12 weeks of age, along with the decrease of pro-inflammatory chemokines and increase of anti-inflammatory IL-10. Furthermore, RvD1 treatment promoted the resolution of pulmonary exacerbation caused by Pseudomonas aeruginosa infection and significantly enhanced physical activity and energy expenditure associated with mucus obstruction, which was impaired in ßENaC-Tg mice compared with wild-type. These results demonstrate that RvD1 can rectify features of CF and offer proof-of-concept for its therapeutic application in this and other muco-obstructive lung diseases.

Structural Adaptation of the Excitation-Contraction Coupling Apparatus in Calsequestrin1-Null Mice during Postnatal Development.

The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.

Searching for Mechanisms Underlying the Assembly of Calcium Entry Units: The Role of Temperature and pH.

Store-operated Ca2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca2+, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

Ablation of Calsequestrin-1, Ca2+ unbalance, and susceptibility to heat stroke.

Introduction: Ca2+ levels in adult skeletal muscle fibers are mainly controlled by excitation-contraction (EC) coupling, a mechanism that translates action potentials in release of Ca2+ from the sarcoplasmic reticulum (SR) release channels, i.e. the ryanodine receptors type-1 (RyR1). Calsequestrin (Casq) is a protein that binds large amounts of Ca2+ in the lumen of the SR terminal cisternae, near sites of Ca2+ release. There is general agreement that Casq is not only important for the SR ability to store Ca2+, but also for modulating the opening probability of the RyR Ca2+ release channels. The initial studies: About 20 years ago we generated a mouse model lacking Casq1 (Casq1-null mice), the isoform predominantly expressed in adult fast twitch skeletal muscle. While the knockout was not lethal as expected, lack of Casq1 caused a striking remodeling of membranes of SR and of transverse tubules (TTs), and mitochondrial damage. Functionally, CASQ1-knockout resulted in reduced SR Ca2+ content, smaller Ca2+ transients, and severe SR depletion during repetitive stimulation. The myopathic phenotype of Casq1-null mice: After the initial studies, we discovered that Casq1-null mice were prone to sudden death when exposed to halogenated anaesthetics, heat and even strenuous exercise. These syndromes are similar to human malignant hyperthermia susceptibility (MHS) and environmental-exertional heat stroke (HS). We learned that mechanisms underlying these syndromes involved excessive SR Ca2+ leak and excessive production of oxidative species: indeed, mortality and mitochondrial damage were significantly prevented by administration of antioxidants and reduction of oxidative stress. Though, how Casq1-null mice could survive without the most important SR Ca2+ binding protein was a puzzling issue that was not solved. Unravelling the mystery: The mystery was finally solved in 2020, when we discovered that in Casq1-null mice the SR undergoes adaptations that result in constitutively active store-operated Ca2+ entry (SOCE). SOCE is a mechanism that allows skeletal fibers to use external Ca2+ when SR stores are depleted. The post-natal compensatory mechanism that allows Casq1-null mice to survive involves the assembly of new SR-TT junctions (named Ca2+ entry units) containing Stim1 and Orai1, the two proteins that mediate SOCE.

Oxygen Consumption and Basal Metabolic Rate as Markers of Susceptibility to Malignant Hyperthermia and Heat Stroke.

Calsequestrin 1 (CASQ1) and Ryanodine receptor 1 (RYR1) are two of the main players in excitation-contraction (EC) coupling. CASQ1-knockout mice and mice carrying a mutation in RYR1 (Y522S) linked to human malignant hyperthermia susceptibility (MHS) both suffer lethal hypermetabolic episodes when exposed to halothane (MHS crises) and to environmental heat (heat stroke, HS). The phenotype of Y522S is more severe than that of CASQ1-null mice. As MHS and HS are hypermetabolic responses, we studied the metabolism of adult CASQ1-null and Y522S mice using wild-type (WT) mice as controls. We found that CASQ1-null and Y522S mice have increased food consumption and higher core temperature at rest. By indirect calorimetry, we then verified that CASQ1-null and Y522S mice show an increased oxygen consumption and a lower respiratory quotient (RQ). The accelerated metabolism of CASQ1-null and Y522S mice was also accompanied with a reduction in body fat. Moreover, both mouse models displayed increased oxygen consumption and a higher core temperature during heat stress. The results collected suggest that metabolic rate, oxygen consumption, and body temperature at rest, all more elevated in Y522S than in CASQ1-null mice, could possibly be used as predictors of the level of susceptibility to hyperthermic crises of mice (and possibly humans).

High-Fat Diet Impairs Muscle Function and Increases the Risk of Environmental Heatstroke in Mice.

Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.

Store-Operated Ca2+ Entry in Skeletal Muscle Contributes to the Increase in Body Temperature during Exertional Stress.

Exertional heat stroke (HS) is a hyperthermic crisis triggered by an excessive accumulation of Ca2+ in skeletal muscle fibers. We demonstrated that exercise leads to the formation of calcium entry units (CEUs), which are intracellular junctions that reduce muscle fatigue by promoting the recovery of extracellular Ca2+ via store-operated Ca2+ entry (SOCE). Here, we tested the hypothesis that exercise-induced assembly of CEUs may increase the risk of HS when physical activity is performed in adverse environmental conditions (high temperature and humidity). Adult mice were: (a) first, divided into three experimental groups: control, trained-1 month (voluntary running in wheel cages), and acutely exercised-1 h (incremental treadmill run); and (b) then subjected to an exertional stress (ES) protocol, a treadmill run in an environmental chamber at 34 °C and 40% humidity. The internal temperature of the mice at the end of the ES was higher in both pre-exercised groups. During an ES ex-vivo protocol, extensor digitorum longus(EDL) muscles from the trained-1 month and exercised-1 h mice generated greater basal tension than in the control and were those that contained a greater number of CEUs, assessed by electron microscopy. The data collected suggest that the entry of Ca2+ from extracellular space via CEUs could contribute to exertional HS when exercise is performed in adverse environmental conditions.

Aerobic Training Prevents Heatstrokes in Calsequestrin-1 Knockout Mice by Reducing Oxidative Stress.

Calsequestrin-1 knockout (CASQ1-null) mice suffer lethal episodes when exposed to strenuous exercise and environmental heat, crises known as exertional/environmental heatstroke (EHS). We previously demonstrated that administration of exogenous antioxidants (N-acetylcysteine and trolox) reduces CASQ1-null mortality during exposure to heat. As aerobic training is known to boost endogenous antioxidant protection, we subjected CASQ1-null mice to treadmill running for 2 months at 60% of their maximal speed for 1 h, 5 times/week. When exposed to heat stress protocol (41°C/1 h), the mortality rate of CASQ1-null mice was significantly reduced compared to untrained animals (86% versus 16%). Protection from heatstrokes was accompanied by a reduced increase in core temperature during the stress protocol and by an increased threshold of response to caffeine of isolated extensor digitorum longus muscles during in vitro contracture test. At cellular and molecular levels, aerobic training (i) improved mitochondrial function while reducing their damage and (ii) lowered calpain activity and lipid peroxidation in membranes isolated from sarcoplasmic reticulum and mitochondria. Based on this evidence, we hypothesize that the protective effect of aerobic training is essentially mediated by a reduction in oxidative stress during exposure of CASQ1-null mice to adverse environmental conditions.