Trpm5 channels encode bistability of spinal motoneurons and ensure motor control of hindlimbs in mice

Bistable motoneurons of the spinal cord exhibit warmth-activated plateau potential driven by Na+ and triggered by a brief excitation. The thermoregulating molecular mechanisms of bistability and their role in motor functions remain unknown. Here, we identify thermosensitive Na+-permeable Trpm5 channels as the main molecular players for bistability in mouse motoneurons. Pharmacological, genetic or computational inhibition of Trpm5 occlude bistable-related properties (slow afterdepolarization, windup, plateau potentials) and reduce spinal locomotor outputs while central pattern generators for locomotion operate normally. At cellular level, Trpm5 is activated by a ryanodine-mediated Ca2+ release and turned off by Ca2+ reuptake through the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump. Mice in which Trpm5 is genetically silenced in most lumbar motoneurons develop hindlimb paresis and show difficulties in executing high-demanding locomotor tasks. Overall, by encoding bistability in motoneurons, Trpm5 appears indispensable for producing a postural tone in hindlimbs and amplifying the locomotor output.

The SarcoEndoplasmic Reticulum Calcium ATPase (SERCA) pump: a potential target for intervention in aging and skeletal muscle pathologies

As a key regulator of cellular calcium homeostasis, the Sarcoendoplasmic Reticulum Calcium ATPase (SERCA) pump acts to transport calcium ions from the cytosol back to the sarcoplasmic reticulum (SR) following muscle contraction. SERCA function is closely associated with muscle health and function, and SERCA activity is susceptible to muscle pathogenesis. For example, it has been well reported that pathological conditions associated with aging, neurodegeneration, and muscular dystrophy (MD) significantly depress SERCA function with the potential to impair intracellular calcium homeostasis and further contribute to muscle atrophy and weakness. As a result, targeting SERCA activity has attracted attention as a therapeutical method for the treatment of muscle pathologies. The interventions include activation of SERCA activity and genetic overexpression of SERCA. This review will focus on SERCA function and regulation mechanisms and describe how those mechanisms are affected under muscle pathological conditions including elevated oxidative stress induced by aging, muscle disease, or neuromuscular disorders. We also discuss the current progress and therapeutic approaches to targeting SERCA in vivo.

SERCA-mediated calcium uptake in the DBA/2J vs C57BL/10 mdx models of Duchenne muscular dystrophy

The C57BL/10ScSn-Dmdmdx/J (C57 mdx) mouse is the most commonly used murine model of Duchenne muscular dystrophy (DMD) but displays a mild phenotype with a late onset, greatly limiting translatability to clinical research. In consequence, the D2.B10-Dmdmdx/J (D2 mdx) mouse was created and produces a more severe, early onset phenotype. Mechanistic insights of the D2 mdx phenotype have yet to be elucidated, specifically related to sarcoplasmic reticulum (SR) calcium (Ca2+) handling. In our study, we aimed to determine if SR Ca2+ handling differences in the D2 mdx versus the C57 mdx mouse could explain model phenotypes. Firstly, analyses determined that D2 mdx mice ambulate less and have weaker muscles, but have greater energy expenditure than C57 counterparts. SR Ca2+ handling measures determined that only D2 mdx mice have impaired SR calcium intake in the gastrocnemius, left ventricle and diaphragm. This was coupled with decrements in maximal sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity and greater activation of the Ca2+-activated protease, calpain, in the gastrocnemius. Overall, our study is the first to determine that SR Ca2+ handling is impaired in the D2 mdx mouse, specifically at the level of the SERCA pump.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis

ABSTRACTSince deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1–sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Thapsigargin—From Traditional Medicine to Anticancer Drug

A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.

Abstract 15747: The Sarcoplasmic Reticulum Calcium ATPase Pump Modulates Whole-body Glucose Homeostasis Through a Protein Disulfide Isomerase-dependent Pathway

Introduction: The heart is a very active metabolic organ which exerts endocrine effects by secreting cardiokines. However, its role in regulating whole-body metabolism has received scarce attention. Hypothesis: We hypothesized that cardiac-specific overexpression of the sarcoplasmic reticulum calcium ATPase (SERCA) pump, which tightly regulates cytosolic calcium, modulates whole-body glucose homeostasis by secreting cardiokines during healthy and diabetic states. Methods: Diabetes was induced by streptozotocin in wild type (WT) and transgenic (TG) mice overexpressing the SERCA pump in the heart (n=6-10/group). Translocation of glucose transporters (GLUTs) to the cell surface was measured via a photolabeling biotinylation assay. Glucose and palmitate oxidation rates, cardiac work and ATP production were quantified in isolated working hearts. Results: Blood glucose levels were lower in diabetic TG mice compared to diabetic WT mice (P=0.023). Diabetes induced a downregulation of cell surface GLUT4 protein content in cardiac and skeletal muscle (by 56% and 79%, respectively, P<0.05), as well as in white and brown adipose tissue of WT mice (by 68% and 74%, respectively, P<0.05), which was rescued in TG diabetic mice. WT diabetic hearts had decreased cardiac glucose oxidation rates and cardiac efficiency, and increased palmitate oxidation rates (P<0.05), which were restored in TG diabetic hearts. Using quantitative mass spectrometry, we found protein disulfide isomerase (PDI) upregulated in the heart of TG mice (by 1822%, P<0.0001), which was confirmed in the serum of TG mice via an ELISA assay (by 243%, P=0.03). Inoculation with exogenous PDI reduced blood glucose in WT healthy mice and partially rescued hyperglycemia in WT diabetic mice compared to untreated counterparts (P=0.009 and P=0.044, respectively). Conclusions: These data suggest that cardiac-specific SERCA overexpression rescues hyperglycemia by improving glucose transport in striated muscle and adipose tissue of diabetic TG mice. Identification of the pathways by which the SERCA pump and cardiac secreted proteins (i.e. PDI) are involved in the regulation of whole-body glucose homeostasis could lead to the identification of novel pharmacological targets in diabetic patients.

The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function

Cardiac contractile function is largely mediated by the regulation of Ca2+ cycling throughout the lifespan. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is paramount to cardiac Ca2+ regulation, and it is well established that SERCA dysfunction pathologically contributes to cardiomyopathy and heart failure. Phospholamban (PLN) is a well-known inhibitor of the SERCA pump and its regulation of SERCA2a—the predominant cardiac SERCA isoform—contributes significantly to proper cardiac function. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in several metabolic pathways, and we and others have shown that it regulates SERCA function. In this mini-review, we highlight the underlying mechanisms behind GSK3’s regulation of SERCA function specifically discussing changes in SERCA2a and PLN expression and its potential protection against oxidative stress. Ultimately, these recent findings that we discuss could have clinical implications in the treatment and prevention of cardiomyopathies and heart failure.

Roles of sarcoplasmic reticulum Ca2+ ATPase pump in the impairments of lymphatic contractile activity in a metabolic syndrome rat model

The intrinsic lymphatic contractile activity is necessary for proper lymph transport. Mesenteric lymphatic vessels from high-fructose diet-induced metabolic syndrome (MetSyn) rats exhibited impairments in its intrinsic phasic contractile activity; however, the molecular mechanisms responsible for the weaker lymphatic pumping activity in MetSyn conditions are unknown. Several metabolic disease models have shown that dysregulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump is one of the key determinants of the phenotypes seen in various muscle tissues. Hence, we hypothesized that a decrease in SERCA pump expression and/or activity in lymphatic muscle influences the diminished lymphatic vessel contractions in MetSyn animals. Results demonstrated that SERCA inhibitor, thapsigargin, significantly reduced lymphatic phasic contractile frequency and amplitude in control vessels, whereas, the reduced MetSyn lymphatic contractile activity was not further diminished by thapsigargin. While SERCA2a expression was significantly decreased in MetSyn lymphatic vessels, myosin light chain 20, MLC20 phosphorylation was increased in these vessels. Additionally, insulin resistant lymphatic muscle cells exhibited elevated intracellular calcium and decreased SERCA2a expression and activity. The SERCA activator, CDN 1163 increased phasic contractile frequency in the vessels from MetSyn, thereby, partially restored lymph flow. Thus, our data provide the first evidence that SERCA2a modulates the lymphatic pumping activity by regulating phasic contractile amplitude and frequency, but not the lymphatic tone. Diminished lymphatic contractile activity in the vessels from the MetSyn animal is associated with the decreased SERCA2a expression and impaired SERCA2 activity in lymphatic muscle.