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.     

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.     

The Regulatory Roles of Mitochondrial Calcium and the Mitochondrial Calcium Uniporter in Tumor Cells

Mitochondria, as the main site of cellular energy metabolism and the generation of oxygen free radicals, are the key switch for mitochondria-mediated endogenous apoptosis. Ca²⁺ is not only an important messenger for cell proliferation, but it is also an indispensable signal for cell death. Ca²⁺ participates in and plays a crucial role in the energy metabolism, physiology, and pathology of mitochondria. Mitochondria control the uptake and release of Ca²⁺ through channels/transporters, such as the mitochondrial calcium uniporter (MCU), and influence the concentration of Ca²⁺ in both mitochondria and cytoplasm, thereby regulating cellular Ca²⁺ homeostasis. Mitochondrial Ca²⁺ transport-related processes are involved in important biological processes of tumor cells including proliferation, metabolism, and apoptosis. In particular, MCU and its regulatory proteins represent a new era in the study of MCU-mediated mitochondrial Ca²⁺ homeostasis in tumors. Through an in-depth analysis of the close correlation between mitochondrial Ca²⁺ and energy metabolism, autophagy, and apoptosis of tumor cells, we can provide a valuable reference for further understanding of how mitochondrial Ca²⁺ regulation helps diagnosis and therapy.     

Impaired Intracellular Ca2+ Dynamics,M-Band and Sarcomere Fragility in Skeletal Muscles of Obscurin KO Mice

Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum’s Ca²⁺ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca²⁺ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.     

Confounding Roles of ER Stress and the Unfolded Protein Response in Skeletal Muscle Atrophy

Skeletal muscle is an essential organ, responsible for many physiological functions such as breathing, locomotion, postural maintenance, thermoregulation, and metabolism. Interestingly, skeletal muscle is a highly plastic tissue, capable of adapting to anabolic and catabolic stimuli. Skeletal muscle contains a specialized smooth endoplasmic reticulum (ER), known as the sarcoplasmic reticulum, composed of an extensive network of tubules. In addition to the role of folding and trafficking proteins within the cell, this specialized organelle is responsible for the regulated release of calcium ions (Ca²⁺) into the cytoplasm to trigger a muscle contraction. Under various stimuli, such as exercise, hypoxia, imbalances in calcium levels, ER homeostasis is disturbed and the amount of misfolded and/or unfolded proteins accumulates in the ER. This accumulation of misfolded/unfolded protein causes ER stress and leads to the activation of the unfolded protein response (UPR). Interestingly, the role of the UPR in skeletal muscle has only just begun to be elucidated. Accumulating evidence suggests that ER stress and UPR markers are drastically induced in various catabolic stimuli including cachexia, denervation, nutrient deprivation, aging, and disease. Evidence indicates some of these molecules appear to be aiding the skeletal muscle in regaining homeostasis whereas others demonstrate the ability to drive the atrophy. Continued investigations into the individual molecules of this complex pathway are necessary to fully understand the mechanisms.     

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.     

Low-Dose Empagliflozin Improves Systolic Heart Function after Myocardial Infarction in Rats: Regulation of MMP9, NHE1, and SERCA2a

The effects of the selective sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin in low dose on cardiac function were investigated in normoglycemic rats. Cardiac parameters were measured by intracardiac catheterization 30 min after intravenous application of empagliflozin to healthy animals. Empagliflozin increased the ventricular systolic pressure, mean pressure, and the max dP/dt (p < 0.05). Similarly, treatment with empagliflozin (1 mg/kg, p.o.) for one week increased the cardiac output, stroke volume, and fractional shortening (p < 0.05). Myocardial infarction (MI) was induced by ligation of the left coronary artery. On day 7 post MI, empagliflozin (1 mg/kg, p.o.) improved the systolic heart function as shown by the global longitudinal strain (−21.0 ± 1.1% vs. −16.6 ± 0.7% in vehicle; p < 0.05). In peri-infarct tissues, empagliflozin decreased the protein expression of matrix metalloproteinase 9 (MMP9) and favorably regulated the cardiac transporters sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and sodium hydrogen exchanger 1 (NHE1). In H9c2 cardiac cells, empagliflozin decreased the MMP2,9 activity and prevented apoptosis. Empagliflozin did not alter the arterial stiffness, blood pressure, markers of fibrosis, and necroptosis. Altogether, short-term treatment with low-dose empagliflozin increased the cardiac contractility in normoglycemic rats and improved the systolic heart function in the early phase after MI. These effects are attributed to a down-regulation of MMP9 and NHE1, and an up-regulation of SERCA2a. This study is of clinical importance because it suggests that a low-dose treatment option with empagliflozin may improve cardiovascular outcomes post-MI. Down-regulation of MMPs could be relevant to many remodeling processes including cancer disease.     

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca²⁺) binding site that is non-functional, thus displaying large variance in Ca²⁺ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca²⁺ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca²⁺ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins.     

The Effect of Uridine on the State of Skeletal Muscles and the Functioning of Mitochondria in Duchenne Dystrophy

Duchenne muscular dystrophy is caused by the loss of functional dystrophin that secondarily causes systemic metabolic impairment in skeletal muscles and cardiomyocytes. The nutraceutical approach is considered as a possible complementary therapy for this pathology. In this work, we have studied the effect of pyrimidine nucleoside uridine (30 mg/kg/day for 28 days, i.p.), which plays an important role in cellular metabolism, on the development of DMD in the skeletal muscles of dystrophin deficient mdx mice, as well as its effect on the mitochondrial dysfunction that accompanies this pathology. We found that chronic uridine administration reduced fibrosis in the skeletal muscles of mdx mice, but it had no effect on the intensity of degeneration/regeneration cycles and inflammation, pseudohypetrophy, and muscle strength of the animals. Analysis of TEM micrographs showed that uridine also had no effect on the impaired mitochondrial ultrastructure of mdx mouse skeletal muscle. The administration of uridine was found to lead to an increase in the expression of the Drp1 and Parkin genes, which may indicate an increase in the intensity of organelle fission and the normalization of mitophagy. Uridine had little effect on OXPHOS dysfunction in mdx mouse mitochondria, and moreover, it was suppressed in the mitochondria of wild type animals. At the same time, uridine restored the transport of potassium ions and reduced the production of reactive oxygen species; however, this had no effect on the impaired calcium retention capacity of mdx mouse mitochondria. The obtained results demonstrate that the used dose of uridine only partially prevents mitochondrial dysfunction in skeletal muscles during Duchenne dystrophy, though it mitigates the development of destructive processes in skeletal muscles.     

Repeated and Interrupted Resistance Exercise Induces the Desensitization and Re-Sensitization of mTOR-Related Signaling in Human Skeletal Muscle Fibers

The acute resistance exercise (RE)-induced phosphorylation of mTOR-related signaling proteins in skeletal muscle can be blunted after repeated RE. The time frame in which the phosphorylation (_p) of mTOR^S2448, p70S6k^T421/S424, and rpS6^S235/236 will be reduced during an RE training period in humans and whether progressive (PR) loading can counteract such a decline has not been described. (1) To enclose the time frame in which _pmTOR^S2448, _prpS6^S235/236, and _pp70S6k^T421/S424 are acutely reduced after RE occurs during repeated RE. (2) To test whether PR will prevent that reduction compared to constant loading (CO) and (3) whether 10 days without RE may re-increase blunted signaling. Fourteen healthy males (24 ± 2.8 yrs.; 1.83 ± 0.1 cm; 79.3 ± 8.5 kg) were subjected to RE with either PR (n = 8) or CO (n = 6) loading. Subjects performed RE thrice per week, conducting three sets with 10–12 repetitions on a leg press and leg extension machine. Muscle biopsies were collected at rest (T0), 45 min after the first (T1), seventh (T7), 13th (T13), and 14th (X-T14) RE session. No differences were found between PR and CO for any parameter. Thus, the groups were combined, and the results show the merged values. _prpS6^S235/236 and _pp70s6k^T421/S424 were increased at T1, but were already reduced at T7 and up to T13 compared to T1. Ten days without RE re-increased _prpS6^S235/236 and _pp70S6k^T421/S424 at X-T14 to a level comparable to that of T1. _pmTOR^S2448 was increased from T1 to X-T14 and did not decline over the training period. Single-fiber immunohistochemistry revealed a reduction in _prpS6^S235/236 in type I fibers from T1 to T13 and a re-increase at X-T14, which was more augmented in type II fibers at T13 (p < 0.05). The entity of myofibers revealed a high heterogeneity in the level of _prpS6^S235/236, possibly reflecting individual contraction-induced stress during RE. The type I and II myofiber diameter increased from T0 and T1 to T13 and X-T14 (p < 0.05) _prpS6^S235/236 and _pp70s6k^T421/S424 reflect RE-induced states of desensitization and re-sensitization in dependency on frequent loading by RE, but also by its cessation.     

Structure,Function and Regulation of the Plasma Membrane Calcium Pump in Health and Disease

In this review, I summarize the present knowledge of the structural and functional properties of the mammalian plasma membrane calcium pump (PMCA). It is outlined how the cellular expression of the different spliced isoforms of the four genes are regulated under normal and pathological conditions.     

Distinct Roles of Soluble and Transmembrane Adenylyl Cyclases in the Regulation of Flagellar Motility in Ciona Sperm

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca²⁺-dependent chemotactic movement of sperm.     

State of the Art in the Current Management and Future Directions of Targeted Therapy for Differentiated Thyroid Cancer

Two-thirds of differentiated thyroid cancer (DTC) patients with distant metastases would be classified as radioactive iodine-refractory (RAIR-DTC), evolving into a poor outcome. Recent advances underlying DTC molecular mechanisms have shifted the therapy focus from the standard approach to targeting specific genetic dysregulations. Lenvatinib and sorafenib are first-line, multitargeted tyrosine kinase inhibitors (TKIs) approved to treat advanced, progressive RAIR-DTC. However, other anti-angiogenic drugs, including single targeted TKIs, are currently being evaluated as alternative or salvage therapy after the failure of first-line TKIs. Combinatorial therapy of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signalling cascade inhibitors has become a highly advocated strategy to improve the low efficiency of the single agent treatment. Recent studies pointed out targetable alternative pathways to overcome the resistance to MAPK and PI3K pathways’ inhibitors. Because radioiodine resistance originates in DTC loss of differentiation, redifferentiation therapies are currently being explored for efficacy. The present review will summarize the conventional management of DTC, the first-line and alternative TKIs in RAIR-DTC, and the approaches that seek to overcome the resistance to MAPK and PI3K pathways’ inhibitors. We also aim to emphasize the latest achievements in the research of redifferentiation therapy, immunotherapy, and agents targeting gene rearrangements in advanced DTC.     

Molecular Mechanisms for the Regulation of Insulin-Stimulated Glucose Uptake by Small Guanosine Triphosphatases in Skeletal Muscle and Adipocytes

Insulin is a hormone that regulates the blood glucose level by stimulating various physiological responses in its target tissues. In skeletal muscle and adipose tissue, insulin promotes membrane trafficking of the glucose transporter GLUT4 from GLUT4 storage vesicles to the plasma membrane, thereby facilitating the uptake of glucose from the circulation. Detailed mechanisms underlying insulin-dependent intracellular signal transduction for glucose uptake remain largely unknown. In this article, I give an overview on the recently identified signaling network involving Rab, Ras, and Rho family small guanosine triphosphatases (GTPases) that regulates glucose uptake in insulin-responsive tissues. In particular, the regulatory mechanisms for these small GTPases and the cross-talk between protein kinase and small GTPase cascades are highlighted.     

Molecular Biomarkers in Idiopathic Pulmonary Fibrosis: State of the Art and Future Directions

Idiopathic pulmonary fibrosis (IPF), the most lethal form of interstitial pneumonia of unknown cause, is associated with a specific radiological and histopathological pattern (the so-called “usual interstitial pneumonia” pattern) and has a median survival estimated to be between 3 and 5 years after diagnosis. However, evidence shows that IPF has different clinical phenotypes, which are characterized by a variable disease course over time. At present, the natural history of IPF is unpredictable for individual patients, although some genetic factors and circulating biomarkers have been associated with different prognoses. Since in its early stages, IPF may be asymptomatic, leading to a delayed diagnosis. Two drugs, pirfenidone and nintedanib, have been shown to modify the disease course by slowing down the decline in lung function. It is also known that 5–10% of the IPF patients may be affected by episodes of acute and often fatal decline. The acute worsening of disease is sometimes attributed to identifiable conditions, such as pneumonia or heart failure; but many of these events occur without an identifiable cause. These idiopathic acute worsenings are termed acute exacerbations of IPF. To date, clinical biomarkers, diagnostic, prognostic, and theranostic, are not well characterized. However, they could become useful tools helping facilitate diagnoses, monitoring disease progression and treatment efficacy. The aim of this review is to cover molecular mechanisms underlying IPF and research into new clinical biomarkers, to be utilized in diagnosis and prognosis, even in patients treated with antifibrotic drugs.     

Uptake of Phosphate,Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein–umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.     

Liquid Biopsies in the Clinical Management of Germ Cell Tumor Patients: State-of-the-Art and Future Directions

Liquid biopsies constitute a minimally invasive means of managing cancer patients, entailing early diagnosis, follow-up and prediction of response to therapy. Their use in the germ cell tumor field is invaluable since diagnostic tissue biopsies (which are invasive) are often not performed, and therefore only a presumptive diagnosis can be made, confirmed upon examination of the surgical specimen. Herein, we provide an overall review of the current liquid biopsy-based biomarkers of this disease, including the classical, routinely used serum tumor markers—the promising microRNAs rapidly approaching the introduction into clinical practice—but also cell-free DNA markers (including DNA methylation) and circulating tumor cells. Finally, and importantly, we also explore novel strategies and challenges for liquid biopsy markers and methodologies, providing a critical view of the future directions for liquid biopsy tests in this field, highlighting gaps and unanswered questions.