Exploring the Effects of Greek Yogurt Supplementation and Exercise Training on Serum Lithium and Its Relationship With Musculoskeletal Outcomes in Men

Dairy products can act as a dietary source of lithium (Li), and a recent study in university-aged males demonstrated that Greek yogurt (GY) supplementation augmented gains in fat free mass, strength and bone formation after 12 weeks of resistance exercise training compared to carbohydrate (CHO) pudding supplementation. Here, we performed secondary analyses to explore whether GY would alter serum Li levels and whether changes in serum Li would associate with changes in body composition, strength, and bone turnover markers. Results show that the GY group maintained serum Li levels after exercise training, whereas the CHO group did not. Maintaining/elevating serum Li levels was also associated with greater gains in strength and reductions in bone resorption. However, controlling for other dietary factors in GY such as protein and calcium weakened these associations. Thus, future studies should assess the causative role, if any, of dietary Li alone on strength and bone resorption in humans.Brock Library Open Access Publishing Fun

SERCA2a tyrosine nitration coincides with impairments in maximal SERCA activity in left ventricles from tafazzin deficient mice

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is imperative for normal cardiac function regulating both muscle relaxation and contractility. SERCA2a is the predominant isoform in cardiac muscles and is inhibited by phospholamban (PLN). Under conditions of oxidative stress, SERCA2a may also be impaired by tyrosine nitration. Tafazzin (Taz) is a mitochondrial specific transacylase that regulates mature cardiolipin (CL) formation, and its absence leads to mitochondrial dysfunction and excessive production of reactive oxygen/nitrogen species (ROS/RNS). In the present study, we examined SERCA function, SERCA2a tyrosine nitration, and PLN expression/phosphorylation in left ventricles (LV) obtained from young (3-5 months) and old (10-12 months) wild-type (WT) and Taz knockdown (TazKD) male mice. These mice are a mouse model for Barth syndrome, which is characterized by mitochondrial dysfunction, excessive ROS/RNS production, and dilated cardiomyopathy (DCM). Here, we show that maximal SERCA activity was impaired in both young and old TazKD LV, a result that correlated with elevated SERCA2a tyrosine nitration. In addition PLN protein was decreased, and its phosphorylation was increased in TazKD LV compared with control, which suggests that PLN may not contribute to the impairments in SERCA function. These changes in expression and phosphorylation of PLN may be an adaptive response aimed to improve SERCA function in TazKD mice. Nonetheless, we demonstrate for the first time that SERCA function is impaired in LVs obtained from young and old TazKD mice likely due to elevated ROS/RNS production. Future studies should determine whether improving SERCA function can improve cardiac contractility and pathology in TazKD miceBrock University Library Open Access Publishing Fun

Characterizing SERCA Function in Murine Skeletal Muscles after 35–37 Days of Spaceflight

It is well established that microgravity exposure causes significant muscle weakness and atrophy via muscle unloading. On Earth, muscle unloading leads to a disproportionate loss in muscle force and size with the loss in muscle force occurring at a faster rate. Although the exact mechanisms are unknown, a role for Ca2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump actively brings cytosolic Ca2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca2+ ([Ca2+]i). SERCA dysfunction contributes to elevations in [Ca2+]i, leading to cellular damage, and may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content, and reactive oxygen/nitrogen species (RONS) protein adduction in murine skeletal muscle after 35–37 days of spaceflight. In male and female soleus muscles, spaceflight led to drastic impairments in Ca2+ uptake despite significant increases in SERCA1a protein content. We attribute this impairment to an increase in RONS production and elevated total protein tyrosine (T) nitration and cysteine (S) nitrosylation. Contrarily, in the tibialis anterior (TA), we observed an enhancement in Ca2+ uptake, which we attribute to a shift towards a faster muscle fiber type (i.e., increased myosin heavy chain IIb and SERCA1a) without elevated total protein T-nitration and S-nitrosylation. Thus, spaceflight affects SERCA function differently between the soleus and TA.Brock University Unrestricted Gran

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43

Calmodulin (CaM) is an important Ca2+-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca2+ handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor (RyR), and dihydropyridine receptor (DHPR).CaM-regulated targets include genes associated with oxidative metabolism, muscle plasticity, and repair. Despite its importance in muscle, the regulation of CaM—particularly its availability to bind to and activate downstream targets—is an emerging area of research. In this minireview, we discuss recent studies revealing the importance of small IQ motif proteins that bind to CaM to either facilitate (nuclear receptor interacting protein; NRIP) its activation of downstream targets, or sequester (neurogranin, Ng; and growth-associated protein 43, GAP43) CaM away from their downstream targets. Specifically, we discuss recent studies that have begun uncovering the physiological roles of NRIP, Ng, and GAP43 in skeletal and cardiac muscle, thereby highlighting the importance of endogenously expressed CaM-binding proteins and their regulation of CaM in muscle.Brock University Library Open Access Publishing Fun

Characterization of sclerostin’s response within white adipose tissue to an obesogenic diet at rest and in response to acute exercise in male mice

This study examined the effect of a high-fat diet (HFD) on sclerostin content within subcutaneous inguinal visceral white adipose tissue (iWAT), and visceral epididymal WAT (eWAT) depots at rest and in response to acute aerobic exercise. Male C57BL/6 mice (n=40, 18 weeks of age) underwent 10 weeks of either a low-fat diet (LFD) or HFD. Within each diet group, mice were assigned to either remain sedentary (SED) or perform 2h of endurance treadmill exercise at 15 m·min-1 with 5° incline (EX), creating 4 groups: LFD+SED (N=10), LFD+EX (N=10), HFD+SED (N=10), and HFD+EX (N=10). Serum and WAT depots were collected 2h post-exercise. Serum sclerostin showed a diet-by-exercise interaction, reflecting HFD+EX mice having higher concentration than HFD-SED (+31%, p=0.03), and LFD mice being unresponsive to exercise. iWAT sclerostin content decreased post-exercise in both 28 kDa (-31%, p=0.04) and 30 kDa bands (-36%, main effect for exercise, p=0.02). iWAT b-catenin (+44%, p=0.03) and GSK3b content were elevated in HFD mice compared to LFD (+128%, main effect for diet, p=0.005). Monomeric sclerostin content was abolished in eWAT of HFD mice (-96%, main effect for diet, p<0.0001), was only detectable as a 30 kDa band in LFD mice and was unresponsive to exercise. b-catenin and GSK3b were both unresponsive to diet and exercise within eWAT. These results characterized sclerostin’s mobilization to WAT depots in response to acute exercise, which appears to be specific to a reduction in iWAT and identified a differential regulation of sclerostin’s form/post-translational modifications depending on diet and WAT depot.This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC grant to P. Klentrou # 2020-00014). N. Kurgan, B. Baranowski and Joshua Stoikos hold NSERC doctoral scholarships

Muscle RANK is a key regulator of calcium storage, SERCA activity, and function of fast-twitch skeletal muscles

Receptor-activator of nuclear factor kB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG)are the key regulators of osteoclast differentiation and bone remodeling. Here we show that RANK is also expressed in fully differentiated myotubes and skeletal muscle. Muscle RANK deletion (RANKmko) has inotropic effects in denervated, but not in sham, extensor digitorum longus (EDL) muscle preventing the loss of maximum specific force while promoting muscle atrophy, fatigability and increased proportion of fast-twitch fibers. In denervated EDL muscles, RANK deletion markedly increased stromal interaction molecule 1 (Stim1) content, a calcium sensor, and altered activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) modulating Ca2+ storage. Muscle RANK deletion had no significant effects on the sham or denervated slow-twitch soleus (Sol) muscles. These data identify a novel role for RANK as a key regulator of calcium storage and SERCA activity, ultimately affecting denervated skeletal muscle function

Saturation of SERCA's lipid annulus may protect against its thermal inactivation.

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps are integral membrane proteins that catalyze the active transport of Ca2+ into the sarcoplasmic reticulum, thereby eliciting muscle relaxation. SERCA pumps are highly susceptible to oxidative damage, and cytoprotection of SERCA dampens thermal inactivation and is a viable therapeutic strategy in combating diseases where SERCA activity is impaired, such as muscular dystrophy. Here, we sought to determine whether increasing the percent of saturated fatty acids (SFA) within SERCA's lipid annulus through diet could protect SERCA pumps from thermal inactivation. Female Wistar rats were fed either a semi-purified control diet (AIN93G, 7% soybean oil by weight) or a modified AIN93G diet containing high SFA (20% lard by weight) for 17 weeks. Soleus muscles were extracted and SERCA lipid annulus and activity under thermal stress were analyzed. Our results show that SERCA's lipid annulus is abundant with short-chain (12–14 carbon) fatty acids, which corresponds well with SERCA's predicted bilayer thickness of 21 Å. Under control-fed conditions, SERCA's lipid annulus was already highly saturated (79%), and high-fat feeding did not increase this any further. High-fat feeding did not mitigate the reductions in SERCA activity seen with thermal stress; however, correlational analyses revealed significant and strong associations between % SFA and thermal stability of SERCA activity with greater %SFA being associated with lower thermal inactivation and greater % polyunsaturation and unsaturation index being associated with increased thermal inactivation. Altogether, these findings show that SERCA's lipid annulus may influence its susceptibility to oxidative damage, which could have implications in muscular dystrophy and age-related muscle wasting.Analyses supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to PJL. The in vivo portion of the study was funded by start-up funding from Brock University to WEW. VAF was supported through a Canadian Institutes of Health Research (CIHR) Doctoral Award; NT and PM were both supported through CIHR Master's Awards; LC was supported through an Ontario Graduate Scholarship. WEW holds a Canada Research Chair in Bone and Muscle Development

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+ ]i ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+ ]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2 O2 . Heterozygous deletion of SOD2 (Sod2+/- ) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month-old wild-type (WT) and Sod2+/- female C57BL/6J mice. Ca2+ -dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a- and SERCA1a-specific T-nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2+/- mice showed a significant reduction in SERCA's apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T-nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.The Brock Library Open Access Publishing Fun

The effects of neurogranin knockdown on SERCA pump efficiency in soleus muscles of female mice fed a high fat diet.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for the transport of Ca2+ from the cytosol into the sarcoplasmic reticulum at the expense of ATP, making it a regulator of both muscle relaxation and muscle-based energy expenditure. Neurogranin (Ng) is a small protein that negatively regulates calcineurin signaling. Calcineurin is Ca2+/calmodulin dependent phosphatase that promotes the oxidative fibre type in skeletal muscle and regulates muscle-based energy expenditure. A recent study has shown that calcineurin activation reduces SERCA Ca2+ transport efficiency, ultimately raising energy expenditure. Since the biomedical view of obesity states that it arises as an imbalance between energy intake and expenditure which favors the former, we questioned whether heterozygous Ng deletion (Ng+/- ) would reduce SERCA efficiency and increase energy expenditure in female mice fed a high-fat diet (HFD). Young (3-4-month-old) female wild type (WT) and Ng+/- mice were fed a HFD for 12 weeks with their metabolic profile being analyzed using metabolic cages and DXA scanning, while soleus SERCA efficiency was measured using SERCA specific Ca2+ uptake and ATPase activity assays. Ng+/- mice showed significantly less cage ambulation compared to WT mice but this did not lead to any added weight gain nor changes in daily energy expenditure, glucose or insulin tolerance despite a similar level of food intake. Furthermore, we observed significant reductions in SERCA's apparent coupling ratio which were associated with significant reductions in SERCA1 and phospholamban content. Thus, our results show that Ng regulates SERCA pump efficiency, and future studies should further investigate the potential cellular mechanisms

Characterization of Alzheimer's disease‐like neuropathology in Duchenne's muscular dystrophy using the DBA/2J mdx mouse model

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by a mutation in the dystrophin gene. In addition to muscle pathology, some patients with DMD will exhibit cognitive impairments with severity being linked to age and type of genetic mutation. Likewise, some studies have shown that mdx mice display impairments in spatial memory compared with wild-type (WT) controls, while others have not observed any such effect. Most studies have utilized the traditional C57BL/10 (C57) mdx mouse, which exhibits a mild disease phenotype. Recently, the DBA/2J (D2) mdx mouse has emerged as a more severe and perhaps clinically relevant DMD model; however, studies examining cognitive function in these mice are limited. Thus, in this study we examined cognitive function in age-matched C57 and D2 mdx mice along with their respective WT controls. Our findings show that 8- to 12-week-old C57 mdx mice did not display any differences in exploration time when challenged with a novel object recognition test. Conversely, age-matched D2 mdx mice spent less time exploring objects in total as a well as less time exploring the novel object, suggestive of impaired recognition memory. Biochemical analyses of the D2 mdx brain revealed higher soluble amyloid precursor protein b(APPb) and APP in the prefrontal cortex of mdx mice compared with WT, and lower soluble APPa in the hippocampus, suggestive of a shift towards amyloidogenesis and a similar pathogenesis to Alzheimer’s disease. Furthermore, our study demonstrates the utility of the D2 mdx model in studying cognitive impairment.Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canad