Calsequestrins in skeletal and cardiac muscle from adult Danio rerio

Calsequestrin (Casq) is a high capacity, low affinity Ca2+-binding protein, critical for Ca2+-buffering in cardiac and skeletal muscle sarcoplasmic reticulum. All vertebrates have multiple genes encoding for different Casq isoforms. Increasing interest has been focused on mammalian and human Casq genes since mutations of both cardiac (Casq2) and skeletal muscle (Casq1) isoforms cause different, and sometime severe, human pathologies. Danio rerio (zebrafish) is a powerful model for studying function and mutations of human proteins. In this work, expression, biochemical properties cellular and sub-cellular localization of D. rerio native Casq isoforms are investigated. By quantitative PCR, three mRNAs were detected in skeletal muscle and heart with different abundances. Three zebrafish Casqs: Casq1a, Casq1b and Casq2 were identified by mass spectrometry (Data are available via ProteomeXchange with identifier PXD002455). Skeletal and cardiac zebrafish calsequestrins share properties with mammalian Casq1 and Casq2. Skeletal Casqs were found primarily, but not exclusively, at the sarcomere Z-line level where terminal cisternae of sarcoplasmic reticulum are located

Role of the JP45-Calsequestrin Complex on Calcium Entry in Slow Twitch Skeletal Muscles

We exploited a variety of mouse models to assess the roles of JP45-CASQ1 (CASQ, calsequestrin) and JP45-CASQ2 on calcium entry in slow twitch muscles. In flexor digitorum brevis (FDB) fibers isolated from JP45-CASQ1-CASQ2 triple KO mice, calcium transients induced by tetanic stimulation rely on calcium entry via La3+- and nifedipine-sensitive calcium channels. The comparison of excitation-coupled calcium entry (ECCE) between FDB fibers from WT, JP45KO, CASQ1KO, CASQ2KO, JP45-CASQ1 double KO, JP45-CASQ2 double KO, and JP45-CASQ1-CASQ2 triple KO shows that ECCE enhancement requires ablation of both CASQs and JP45. Calcium entry activated by ablation of both JP45-CASQ1 and JP45-CASQ2 complexes supports tetanic force development in slow twitch soleus muscles. In addition, we show that CASQs interact with JP45 at Ca2+ concentrations similar to those present in the lumen of the sarcoplasmic reticulum at rest, whereas Ca2+ concentrations similar to those present in the SR lumen after depolarization-induced calcium release cause the dissociation of JP45 from CASQs. Our results show that the complex JP45-CASQs is a negative regulator of ECCE and that tetanic force development in slow twitch muscles is supported by the dynamic interaction between JP45 and CASQs

Functional characterization of orbicularis oculi and extraocular muscles

The orbicularis oculi are the sphincter muscles of the eyelids and are involved in modulating facial expression. They differ from both limb and extraocular muscles (EOMs) in their histology and biochemistry. Weakness of the orbicularis oculi muscles is a feature of neuromuscular disorders affecting the neuromuscular junction, and weakness of facial muscles and ptosis have also been described in patients with mutations in the ryanodine receptor gene. Here, we investigate human orbicularis oculi muscles and find that they are functionally more similar to quadriceps than to EOMs in terms of excitation-contraction coupling components. In particular, they do not express the cardiac isoform of the dihydropyridine receptor, which we find to be highly expressed in EOMs where it is likely responsible for the large depolarization-induced calcium influx. We further show that human orbicularis oculi and EOMs express high levels of utrophin and low levels of dystrophin, whereas quadriceps express dystrophin and low levels of utrophin. The results of this study highlight the notion that myotubes obtained by explanting satellite cells from different muscles are not functionally identical and retain the physiological characteristics of their muscle of origin. Furthermore, our results indicate that sparing of facial and EOMs in patients with Duchenne muscular dystrophy is the result of the higher levels of utrophin expression

Modulation of PGC-1α activity as a treatment for metabolic and muscle-related diseases

Physical inactivity is a predisposing factor for various disease states including obesity, cardiovascular disease, as well as for certain types of cancer. Regular endurance exercise mediates several beneficial effects such as increased energy expenditure and improved skeletal muscle function, and has been suggested as a therapeutic strategy for both metabolic and muscle‐related disorders. "Exercise mimetic" is a collective term for compounds that can pharmacologically activate pathways which are normally induced during skeletal muscle contraction, and that could be used in the treatment of metabolic or muscle related diseases. Two such experimental "exercise mimetics" are AICAR and resveratrol, which have both been extensively studied in the context of metabolic dysfunction and muscle wasting in rodent disease models. These compounds have been postulated to activate AMP‐activated protein kinase (AMPK) and sirtuin 1 (SIRT1), respectively, in skeletal muscle, and to increase the activation of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α). PGC‐1α can mediate several metabolic and functional adaptations in skeletal muscle in response to physical exercise and is therefore an interesting target for the development of new "exercise mimetic" drugs

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content

Mitochondrial efficiency : focus on dietary nitrate, hypoxia and exercise

Metabolic efficiency affects weight control, generation of heat, exercise performance and health. The tiny radical nitric oxide (NO) targets several cellular components that can influence metabolic efficiency. NO is produced endogenously from L-arginine and molecular oxygen by specific NO-synthases. In addition, extensive research during the past two decades shows that the inorganic anions nitrate and nitrite, which are oxidation products from endogenous NO generation, can be reduced back to NO and other nitrogen oxides. Apart from reflecting endogenous NOS-activity, circulating nitrate and nitrite are dependent on dietary intake, where green leafy vegetables in particular contain high amounts of nitrate. Circulating nitrate is actively taken up by the salivary glands and excreted in saliva. Oral commensal bacteria then reduce salivary nitrate into nitrite, which after swallowing and effective uptake in the gut, reaches the systemic circulation. In blood and tissues several enzymatic and non-enzymatic pathways are able to reduce nitrite to NO. These pathways are potentiated under acidic and hypoxic conditions. The nitratenitrite- NO pathway is considered a back-up system during conditions when NO-synthases are failing. Prior to the inception of this thesis, our group had shown that dietary nitrate was able to decrease oxygen cost during exercise and we wanted to further explore its metabolic effects With this background, we investigated the underlying mechanisms behind the oxygen sparing effect of dietary nitrate during exercise (Paper I). Moreover, we explored the effects of dietary nitrate on muscular function in mice (Paper II), oxygen consumption in a human model of global hypoxia (Paper III) and on resting metabolic rate in humans (Paper IV). In the last two papers we wanted to investigate if metabolic efficiency can affect exercise tolerance in hypoxia (Paper V) and how cytochrome c oxidase (COX) subunit IV isoform composition affects resting metabolic rate (Paper VI). Respirometric analysis of isolated mitochondria from healthy humans revealed that dietary nitrate improves mitochondrial efficiency (P/O ratio) and this effect correlated strongly with the reduction in oxygen consumption during cycling ergometry. In addition we found respirometric support for less uncoupling which was supported by reduced expression of adenine nucleotide transporter (ANT), a major determinant of proton conductance (Paper I). In muscle from mice fed for 7 days with nitrate, electric stimulation led to increased contractile force and speed of force development in fast twitch muscle compared to controls. This was accompanied by higher Ca2+ levels and increased expression of the Ca2+- handling proteins dihydropyridine receptor and calsequestrin-1 (Paper II). In the human model of global hypoxia a reduction in arterial oxygen saturation was achieved during prolonged breath-holding by experienced free divers after nitrate or placebo. In contrast to our hypothesis, nitrate during resting apnea increased pulmonary oxygen uptake, reduced arterial oxygen saturation and shortened maximal breathholding time. This was probably related to a NO-mediated attenuation of the oxygen conserving diving response as showed by less bradycardia and indications of an attenuation of the increase in blood pressure after nitrate (Paper III). In healthy humans we could demonstrate that dietary nitrate reduces resting metabolic rate by 4% and that acute administration of nitrite in vitro reduces respiration by 40% in primary myotubes from the same individuals (Paper IV). We found that healthy subjects with a high metabolic efficiency in normoxia had higher tolerance to exercise in hypoxia. Interestingly, these subjects acutely reduced their metabolic efficiency during hypoxia in order to maintain power output. On the other hand, isolated mitochondria, which work in the lower efficiency range, acutely increased their efficiency during a steady state hypoxic challenge in order to maintain ATP production (Paper V). There is a largely unexplained variation in resting metabolic rate between seemingly similar individuals. We found that the inter-individual variation in resting metabolic rate seems to depend on the composition of COX subunit IV isoforms. We could show that COX IV-2 isoform is present in human skeletal muscle and that a high COX IV-2/COX IV-1 ratio showed a strong negative correlation to resting metabolic rate. Moreover, concurrent overexpression of COX IV-2 and knock down of COX IV-1 in primary human myotubes significantly reduced basal cell respiration and ROS generation without affecting the COX activity (Paper VI). In conclusion, this thesis demonstrates profound effects of dietary nitrate on mitochondrial efficiency, muscle function and metabolism. In addition, metabolic efficiency plays a role in exercise tolerance during hypoxia and adapts to obtain optimal power. Finally, mitochondrial COX subunit IV isoform composition seems to affect resting metabolic rate. The physiological, therapeutic and nutritional aspects of these findings create a platform for further studies on dietary nitrate, mitochondrial function and metabolism

Gene expression profiling in slow-Type calf soleus muscle of 30 days space-flown mice

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight