Dysregulation of skeletal muscle morphology and metabolism is associated with chronic diseases such as obesity and type 2 diabetes. The enzyme glycogen synthase kinase 3 (GSK3) is highly involved in skeletal muscle physiology and metabolism, acting as a negative regulator of muscle size, strength, adaptive thermogenesis, and glucose homeostasis. Correspondingly, we have shown that partial knockdown (∼40%) of GSK3 specifically in skeletal muscle increases lean mass, reduces fat mass, and activates muscle-based adaptive thermogenesis via sarco(endo)plasmic reticulum Ca
Mast cells are granulocytic immune sentinels present in vascularized tissues that drive chronic inflammatory mechanisms characteristic of allergic pathologies. IgE-mediated mast cell activation leads to a rapid mobilization of Ca2+ from intracellular stores, which is essential for the release of preformed mediators via degranulation and de novo synthesized proinflammatory cytokines and chemokines. Given its potent signaling capacity, the dynamics of Ca2+ localization are highly regulated by various pumps and channels controlling cytosolic Ca2+ concentrations. Among these is sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), which functions to maintain low cytosolic Ca2+ concentrations by actively transporting cytosolic Ca2+ ions into the endoplasmic reticulum. In this study, we characterized the role of SERCA in allergen-activated mast cells using IgE-sensitized bone marrow-derived mast cells (BMMCs) treated with the SERCA activating compound, CDN1163, and simultaneously stimulated with allergen through FcεRI under stem cell factor (SCF) potentiation. Acute treatment with CDN1163 was found to attenuate early phase mast cell degranulation along with reactive oxygen species (ROS) production. Additionally, treatment with CDN1163 significantly reduced secretion of IL-6, IL-13, and CCL3, suggesting a role for SERCA in the late phase mast cell response. The protective effects of SERCA activation via CDN1163 treatment on the early and late phase mast cell response may be driven by the selective suppression of p38 MAPK signaling. Together, these findings implicate SERCA as an important regulator of the mast cell response to allergen and suggest SERCA activity may offer therapeutic potential targeting allergic pathologies, warranting further investigation.
Introduction The kynurenine (KYN) pathway has been implicated in depression and neurotoxicity. Derived from tryptophan, KYN can be further degraded along one of two distinct branches. The KYN-KYNA branch is regulated by the enzyme kynurenine aminotransferase (KAT) and is considered neuroprotective, as it degrades KYN into the non-blood brain barrier (BBB) transportable metabolite kynurenic acid (KYNA). The KYN-NAD branch is regulated by the enzyme kynurenine monooxygenase (KMO) and is considered neurotoxic as it degrades KYN into the BBB transportable metabolite 3-hydroxykynurenine (3-HK) and, further in the cascade, quinolinic acid (QUIN). Recent studies have shown the importance of muscle health on directing kynurenine metabolism towards the neuroprotective branch, highlighting a novel muscle-to-brain axis. Specifically, exercise induced increases in the transcription factor PCG-1⍺ amplifies the content of KAT enzymes that convert KYNA from KYN. Duchenne muscular dystrophy (DMD) is an X-linked severe muscle disorder caused by a loss of dystrophin leading to muscle fragility, wasting, and weakness. In light of recent evidence revealing the cognitive and depressive behaviours in DMD patients and in the preclinical mdx mouse, we sought to determine whether KYN metabolism as well as PGC-1α and KAT content would be altered in the mdx model. Methods 8-10 week old male mdx and wild-type (DBA/2J) mice were purchased from Jackson laboratories. Behavioural changes (ie., grooming activity, food and water intake) were measured using a Promethion metabolic cage system along with fear and anxiety-like behaviour during a novel object recognition test (NORT). Mice were euthanized and serum KYN and KYNA were measured using commercially available ELISA kits. Extensor digitorum longus muscle was collected, homogenized, and Western blotting was performed for PGC-1⍺, KAT1, and KAT3. Results Metabolic cage results showed that fine activity (-5%), water intake (-25%), and food intake (-60%) were lower across light and dark stages in mdx mice compared to WT mice (main effect of genotype, p<0.0001 for all measures). The mdx mice also spent more time in the corners of the NORT arenas compared to their WT counterparts (+270s, p<0.0001). Though the change in serum KYN was insignificant across mdx and WT mice, the concentration of serum KYNA was lower in the mdx mice (-57%, p = 0.01), therefore causing a lower KYN:KYNA ratio in mdx mice compared with WT (-56%, p = 0.01). Western blotting demonstrated a reduction in PGC-1⍺ (-65%, p = 0.002) and KAT1 (-35%, p = 0.02) content in mdx mice compared to WT mice, whereas the KAT3 content was elevated in mdx mice (1.5-fold, p = 0.05). Conclusion Our results of lowered serum KYN:KYNA concentrations from mdx mice (compared to WT) correspond well with changes in affective and anxiety-related behaviours. The observed reduction in muscle PGC-1⍺ and KAT1 content likely contributes to these changes in KYN:KYNA ratio. Though KAT3 was upregulated in mdx muscle compared to WT, this could represent a failed compensatory response.
Summary The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca 2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca 2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca 2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca 2+ uptake were unattainable due to the severe impairments in their ability to transport Ca 2+ . Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice.
Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca2+-ATPase pumps. In a recent study, we discovered that Pln overexpression (PlnOE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the PlnOE mice. To this end, we crossed Sln-null (SlnKO) mice with PlnOE mice to generate a PlnOE/SlnKO mouse colony and assessed SERCA function, CNM pathology, in vitro contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of Sln did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the PlnOE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target.
Abstract 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. Though the exact mechanisms are unknown, a role for Ca 2+ dysregulation has been suggested. The sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) pump actively brings cytosolic Ca 2+ into the SR, eliciting muscle relaxation and maintaining low intracellular Ca 2+ ([Ca 2+ ] i ). SERCA dysfunction contributes to elevations in [Ca 2+ ] i , leading to cellular damage and thus may contribute to the muscle weakness and atrophy observed with spaceflight. Here, we investigated SERCA function, SERCA regulatory protein content (sarcolipin, phospholamban, and neuronatin), 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 Ca 2+ 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 Ca 2+ 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. As the soleus is severely affected by spaceflight, future studies should determine whether improving SERCA function in this muscle can mitigate muscle atrophy and weakness.
