Sarcoplasmic/endoplasmic Reticulum Ca2+-ATPase Research Articles

The Sarcoplasmic/Endoplasmic reticulum Ca2+-ATPase (SERCA) is present in pig sperm and modulates their physiology over liquid preservation

Liquid storage is the primary preservation method in the swine breeding industry because of its advantages over cryopreservation. Calcium (Ca2+), a key regulator of cell physiology, plays a crucial role during liquid preservation. Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPases (SERCA) belong to a family of P-type ATPases that regulate Ca2+ homeostasis within cells and have been previously described to play a function in the sperm of various mammalian species. Herein, we hypothesized that SERCA2 is present in pig sperm and is involved in the resilience of this cell to liquid preservation at 17 °C. For this purpose, sperm were incubated with different concentrations of thapsigargin (Thg; 0, 5, 25, and 50 µM) and stored at 17 °C for ten days. The presence and localization of SERCA2 were evaluated using immunoblotting and immunofluorescence, respectively. On days 0, 4, and 10, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system, and sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), and intracellular levels of Ca2+, superoxides and total reactive oxygen species (ROS) were evaluated by flow cytometry. We localized SERCA2 in the acrosome and midpiece of pig sperm. Furthermore, inhibition of SERCA with Thg resulted in reduced sperm viability and membrane stability, and increased MMP, and Ca2+ and ROS levels. In conclusion, the activity of SERCA prevents the accumulation of intracellular Ca2+ in sperm, which is detrimental to sperm quality and function during liquid storage at 17 °C. We thus suggest that the function of SERCA is crucial for the preservation of pig semen.

The juxtamembrane sequence of small ankyrin 1 mediates the binding of its cytoplasmic domain to SERCA1 and is required for inhibitory activity.

Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase1 (SERCA1) is responsible for the clearance of cytosolic Ca2+ in skeletal muscle. Due to its vital importance in regulating Ca2+ homeostasis, the regulation of SERCA1 has been intensively studied. Small ankyrin 1 (sAnk1, Ank1.5), a 17 kDa muscle-specific isoform of ANK1, binds to SERCA1 directly via both its transmembrane and cytoplasmic domains and inhibits SERCA1's ATPase activity. Here we characterize the interaction between the cytoplasmic domain of sAnk1 (sAnk1(29-155)) and SERCA1. The binding affinity for sAnk1 (29-155) to SERCA1 was 444 nM by blot overlay, about 7-fold weaker than the binding of sAnk1(29-155) to obscurin, a giant protein of the muscle cytoskeleton. Site-directed mutagenesis identified K38, H39, and H41, in the juxtamembrane region, as residues likely to mediate binding to SERCA1. These residues are not required for obscurin binding. Residues R64-K73, which do contribute to obscurin binding, are also required for binding to SERCA1, but only the hydrophobic residues in this sequence are required, not the positively charged residues necessary for obscurin binding. Circular dichroism analysis of sAnk1(29-155) indicates that most mutants show significant structural changes, with the exception of those containing alanines in place of K38, H39 and H41. Although the cytoplasmic domain of sAnk1 does not inhibit SERCA1's Ca2+-ATPase activity, with or without mutations in the juxtamembrane sequence, the inhibitory activity of full-length sAnk1 requires the WT juxtamembrane sequence. We used these data to model sAnk1 and the sAnk1-SERCA1 complex. Our results suggest that, in addition to its transmembrane domain, sAnk1 uses its juxtamembrane sequence and perhaps part of its obscurin binding site to bind to SERCA1, and that this binding contributes to their robust association in situ, as well as regulation of SERCA1's activity.

Role of voltage-gated Ca2+ channels and Ano1 Ca2+-activated Cl- channels in M2 muscarinic receptor-dependent contractions of murine airway smooth muscle.

Cholinergic tone is elevated in obstructive lung conditions such as COPD and asthma, but the cellular mechanisms underlying cholinergic contractions of airway smooth muscle (ASM) are still unclear. Some studies report an important role for L-type Ca2+ channels (LTCC) and Ano1 Ca2+-activated Cl™ channels (CACC) in these responses, but others dispute their importance. Cholinergic contractions of ASM involve activation of M3Rs, however stimulation of M2Rs exerts a profound hypersensitisation of these responses. Here we show that M2R-dependent potentiation of cholinergic nerve-evoked contractions of ASM was reversed by the LTCC blocker nifedipine and the Ano1 CACC inhibitors Ani9 and CaCCinh-A01. Carbachol induced sustained contractions of ASM which were converted into oscillatory contractions when M3Rs were blocked with 4-DAMP. The 4-DAMP resistant contractions were absent in preparations taken from M2R knock out mice. The remaining M2R-dependent responses, observed in wild-type mice, were abolished by nifedipine and Ani9. Inhibition of sarcoplasmic endoplasmic reticulum Ca2+ ATPases (SERCA) with thapsigargin increased the amplitude of contractions induced by EFS and these effects were also reversed by nifedipine and Ani9. Thapsigargin also potentiated contractions of ASM induced by the LTCC activator FPL64176. Therefore, contractions of ASM that involved Ca2+ influx via LTCC were enhanced by inhibition of SERCA. Immunocytochemistry experiments revealed prominent SERCA staining around the periphery of ASM cells. These data indicate that M2R-dependent contractions of ASM involves Ano1 CACC and LTCC by a mechanism involving inhibition of buffering of Ca2+ influx by SERCA.

The SGLT2 inhibitor remogliflozin induces vasodilation in the femoral artery of rabbits via activation of a Kv channel, the SERCA pump, and the cGMP signaling pathway.

This study explored the vasodilatory mechanisms of the sodium-glucose cotransporter-2 inhibitor remogliflozin using femoral arteries of rabbits. Remogliflozin dilated femoral arterial rings pre-contracted with phenylephrine in a concentration-dependent manner. Pretreatment with the Ca2+-sensitive K+ channel inhibitor (paxilline), the ATP-sensitive K+ channel inhibitor (glibenclamide), or the inwardly rectifying K+ channel inhibitor (Ba2+) did not alter the vasodilatory effect. However, vasodilation was significantly reduced by pretreatment with the voltage-dependent K+ (Kv) channel inhibitor (4-AP) and with the Kv1.5 subtype inhibitor (DPO-1) but not with Kv2.1 or Kv7 subtype inhibitor. Neither endothelium removal nor the inhibition of nitric oxide production altered the vasodilatory effect of remogliflozin. However, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid effectively reduced the remogliflozin effect, as did pretreatment with cGMP/PKG-related but not cAMP/PKA-related signaling pathway inhibitors. These results indicate that remogliflozin-mediated dilation of the femoral artery occurs via the activation of Kv channels, mainly the Kv1.5 subtype, SERCA pump, and cGMP/PKG-related signaling pathways.

Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM.

An ellagitannin-derived metabolite, Urolithin A (UA), has emerged as a potential therapeutic agent for metabolic disorders due to its antioxidant, anti-inflammatory, and mitochondrial function-improving properties, but its efficacy in protecting against ER stress remains underexplored. The endoplasmic reticulum (ER) is a cellular organelle involved in protein folding, lipid synthesis, and calcium regulation. Perturbations in these functions can lead to ER stress, which contributes to the development and progression of metabolic disorders such as metabolic-associated fatty liver disease (MAFLD). In this study, we identified a novel target protein of UA and elucidated its mechanism for alleviating palmitic acid (PA)-induced ER stress. Cellular thermal shift assay (CETSA)-LC-MS/MS analysis revealed that UA binds directly to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), an important regulator of calcium homeostasis in mitochondria-associated ER membranes (MAMs). As an agonist of SERCA, UA attenuates abnormal calcium fluctuations and ER stress in PA-treated liver cells, thereby contributing to cell survival. The lack of UA activity in SERCA knockdown cells suggests that UA regulates cellular homeostasis through its interaction with SERCA. Collectively, our results demonstrate that UA protects against PA-induced ER stress and enhances cell survival by regulating calcium homeostasis in MAMs through SERCA. This study highlights the potential of UA as a therapeutic agent for metabolic disorders associated with ER stress.

SERCA Modulators Reveal Distinct Signaling and Functional Roles of T Lymphocyte Ca2+ Stores.

The allosteric SERCA (Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase) activator CDN1163 has been recently added to the group of pharmacological tools for probing SERCA function. We chose to investigate the effects of the compound on T lymphocyte Ca2+ stores, using the well-described Jurkat T lymphocyte as a reliable cell system for Ca2+ signaling pathways. Our study identified the lowest concentrations of the SERCA inhibitors thapsigargin (TG) and 2,5-di-(tert butyl)-1,4-benzohydroquinone (tBHQ) capable of releasing Ca2+, permitting the differentiation of the TG-sensitive SERCA 2b Ca2+ store from the tBHQ-sensitive SERCA 3 Ca2+ store. We proceeded to test the effects of CDN1163 on Ca2+ stores, examining specific actions on the SERCA 2b and SERCA 3 Ca2+ pools using our low-dose SERCA blocker regimen. In contrast to previous work, we find CDN1163 exerts complex time-sensitive and SERCA isoform-specific actions on Ca2+ stores. Surprisingly, short-term exposure (0-30 min) to CDN1163 perturbs T cell Ca2+ stores by suppressing Ca2+ uptake with diminished Ca2+ release from the SERCA 2b-controlled store. Concomitantly, we find evidence for a SERCA-activating effect of CDN1163 on the SERCA-3 regulated store, given the observation of increased Ca2+ release inducible by low-dose tBHQ. Intriguingly, longer-term (>12 h) CDN1163 exposure reversed this pattern, with increased Ca2+ release from SERCA 2b-regulated pools yet decreased Ca2+ release responses from the tBHQ-sensitive SERCA 3 pool. Indeed, this remodeling of SERCA 2b Ca2+ stores with longer-term CDN1163 exposure also translated into the compound's ability to protect Jurkat T lymphocytes from TG but not tBHQ-induced growth suppression.

Ca2+ signals are essential for T-cell proliferation, while Zn2+ signals are necessary for T helper cell 1 differentiation

The regulation of T-cell fate is crucial for the balance between infection control and tolerance. Calcium (Ca2+) and zinc (Zn2+) signals are both induced after T-cell stimulation, but their specific roles in the fate of activation and differentiation remain to be elucidated. Are Zn2+- and Ca2+ signals responsible for different aspects in T-cell activation and differentiation and do they act in concert or in opposition? It is crucial to understand the interplay of the intracellular signals to influence the fate of T cells in diseases with undesirable T-cell activities or in Zn2+-deficient patients. Human peripheral blood mononuclear cells were stimulated with the Zn2+ ionophore pyrithione and thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). Intracellular Zn2+ and Ca2+ signals were monitored by flow cytometry and ELISA, quantitative PCR and western blot were used to evaluate T-cell differentiation and the underlying molecular mechanism. We found that Zn2+ signals upregulated the early T-cell activation marker CD69, interferon regulatory factor 1 (IRF-1), and Krüppel-like factor 10 (KLF-10) expression, which are important for T helper cell (Th) 1 differentiation. Ca2+ signals, on the other hand, increased T-bet and Forkhead box P3 (FoxP3) expression and interleukin (IL)-2 release. Most interestingly, the combination of Zn2+ and Ca2+ signals was indispensable to induce interferon (IFN)-γ expression and increased the surface expression of CD69 by several-fold. These results highlight the importance of the parallel occurrence of Ca2+ and Zn2+ signals. Both signals act in concert and are required for the differentiation into Th1 cells, for the stabilization of regulatory T cells, and induces T-cell activation by several-fold. This provides further insight into the impaired immune functions of patients with zinc deficiency.

High Glucose Alters Endoplasmic Reticulum Ca2+ Stores in T Lymphocytes Revealing Potential Novel Therapeutic Targets for Diabetic-Induced Immune Dysfunction

The objective of this study was to examine the effects of high glucose stress on endoplasmic reticulum (ER) Ca2+ stores in T lymphocytes. Our hypothesis is that T lymphocytes exposed to chronic high glucose undergo an ER “stress” response due to impairment of Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump-mediated Ca2+ transport and dysregulated T cell ER Ca2+ signaling. To examine ER Ca2+ signaling events we conducted both intact and permeabilized cell Ca2+ assays using Ca2+ sensitive fluorescence dyes. Western Blot and RT-qPCR experiments were performed to detect SERCA expression levels. Our study found: 1. Incubation of T cells for an “early” phase (7-10 days) in high glucose resulted in differential changes in the SERCA 2b and SERCA 3 regulated Ca2+ stores, as revealed by our specific Ca2+ release protocol using thapsigargin and 2,5-di-( tert-butyl)-1,4-hydroquinone SERCA inhibition. 2. The early phase high glucose exposure increases Ca2+ release from the SERCA 2b-regulated Ca2+ pool whereas no change is observed in the SERCA 3-regulated pool. 3. We observed an increase in SERCA 2b expression levels during the early phase high glucose exposure but no change in SERCA 3 expression. 4. Long term high glucose exposure (>11 days) resulted in the loss of the augmented Ca2+ release response from SERCA 2b sensitive Ca2+ stores. 5. Treatment of T cells with the SERCA activator CDN1163 restored the augmented Ca2+ release response from the SERCA 2b store. 6. Treatment of T cells in the ER stress phase with the chemical chaperone TUDCA protected T cells from high glucose induced loss in viability. Summary: High glucose exposure (as an in vitro mimic of a diabetic hyperglycemia state) perturbs ER Ca2+ stores in T lymphocytes resulting in increased expression of SERCA 2b and augmented Ca2+ storage whereas no apparent change is observable in the SERCA 3 Ca2+ pool. Longer-term high glucose exposure results in the loss of the augmented SERCA 2b-sensitive store response, yet pharmacological SERCA 2b activation was able to restore enhanced Ca2+ release from this pool. Conclusion: Our results are consistent with our hypothesis that high glucose exposure imposes a stress condition on T cell ER Ca2+ stores with changes in SERCA-mediated Ca2+ uptake activity. The findings in this study reveal an intriguing differential effect with the SERCA 2b pump isoform targeted for increased expression and augmented Ca2+ storage whereas the SERCA 3-regulated Ca2+ pools appear unaffected. Our experiments with the SERCA activator CDN1163 suggest a potential novel strategy to restore critical ER Ca2+ signaling and T lymphocyte function in patients suffering from diabetic hyperglycemia. This work was supported by a SEED grant from the Jie Du Center, University of the Pacific. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Prolyl isomerase Pin1 in skeletal muscles contributes to systemic energy metabolism and exercise capacity through regulating SERCA activity

The skeletal muscle is a pivotal organ involved in the regulation of both energy metabolism and exercise capacity. There is no doubt that exercise contributes to a healthy life through the consumption of excessive energy or the release of myokines. Skeletal muscles exhibit insulin sensitivity and can rapidly uptake blood glucose. In addition, they can undergo non-shivering thermogenesis through actions of both the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and small peptide, sarcolipin, resulting in systemic energy metabolism. Accordingly, the maintenance of skeletal muscles is important for both metabolism and exercise. Prolyl isomerase Pin1 is an enzyme that converts the cis-trans form of proline residues and controls substrate function. We have previously reported that Pin1 plays important roles in insulin release, thermogenesis, and lipolysis. However, the roles of Pin1 in skeletal muscles remains unknown. To clarify this issue, we generated skeletal muscle-specific Pin1 knockout mice.Pin1 deficiency had no effects on muscle weights, morphology and ratio of fiber types. However, they showed exacerbated obesity or insulin resistance when fed with a high-fat diet. They also showed a lower ability to exercise than wild type mice did. We also found that Pin1 interacted with SERCA and elevated its activity, resulting in the upregulation of oxygen consumption. Overall, our study reveals that Pin1 in skeletal muscles contributes to both systemic energy metabolism and exercise capacity.

Calcium signaling mediates proliferation of the precursor cells that give rise to the ciliated left-right organizer in the zebrafish embryo.

Several of our internal organs, including heart, lungs, stomach, and spleen, develop asymmetrically along the left-right (LR) body axis. Errors in establishing LR asymmetry, or laterality, of internal organs during early embryonic development can result in birth defects. In several vertebrates-including humans, mice, frogs, and fish-cilia play a central role in establishing organ laterality. Motile cilia in a transient embryonic structure called the "left-right organizer" (LRO) generate a directional fluid flow that has been proposed to be detected by mechanosensory cilia to trigger asymmetric signaling pathways that orient the LR axis. However, the mechanisms that control the form and function of the ciliated LRO remain poorly understood. In the zebrafish embryo, precursor cells called dorsal forerunner cells (DFCs) develop into a transient ciliated structure called Kupffer's vesicle (KV) that functions as the LRO. DFCs can be visualized and tracked in the embryo, thereby providing an opportunity to investigate mechanisms that control LRO development. Previous work revealed that proliferation of DFCs via mitosis is a critical step for developing a functional KV. Here, we conducted a targeted pharmacological screen to identify mechanisms that control DFC proliferation. Small molecule inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) were found to reduce DFC mitosis. The SERCA pump is involved in regulating intracellular calcium ion (Ca2+) concentration. To visualize Ca2+ in living embryos, we generated transgenic zebrafish using the fluorescent Ca2+ biosensor GCaMP6f. Live imaging identified dynamic cytoplasmic Ca2+ transients ("flux") that occur unambiguously in DFCs. In addition, we report Ca2+ flux events that occur in the nucleus of DFCs. Nuclear Ca2+ flux occurred in DFCs that were about to undergo mitosis. We find that SERCA inhibitor treatments during DFC proliferation stages alters Ca2+ dynamics, reduces the number of ciliated cells in KV, and alters embryo laterality. Mechanistically, SERCA inhibitor treatments eliminated both cytoplasmic and nuclear Ca2+ flux events, and reduced progression of DFCs through the S/G2 phases of the cell cycle. These results identify SERCA-mediated Ca2+ signaling as a mitotic regulator of the precursor cells that give rise to the ciliated LRO.

CDN1163, a SERCA activator, causes intracellular Ca2+ leak, mitochondrial hyperpolarization and cell cycle arrest in mouse neuronal N2A cells

ObjectiveActivity or expression of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) is diminished in some disease states such as cardiac failure and diabetes mellitus. A newly developed activator of SERCA, CDN1163, reportedly rescued or alleviated pathological conditions attributed to dysfunctional SERCA. We examined whether CDN1163 could relieve mouse neuronal N2A cell growth inhibition caused by cyclopiazonic acid (CPA, SERCA inhibitor). We also examined how CDN1163 affected cytosolic Ca2+, mitochondrial Ca2+ and mitochondrial membrane potential. MethodsCell viability was measured by MTT assay and trypan blue exclusion test. Cytosolic Ca2+, mitochondrial Ca2+ and mitochondrial membrane potential were measured using fura 2, Rhod-2 and JC-1, respectively, as fluorescent probes. ResultsCDN1163 (10 μM) itself suppressed cell proliferation, and did not alleviate CPA’s inhibitory effect (and vice versa). Cell cycle was arrested at the G1 phase after CDN1163 treatment. CDN1163 treatment caused a slow yet persistent cytosolic [Ca2+] elevation partly due to Ca2+ release from an internal store other than the CPA-sensitive endoplasmic reticulum (ER). Treatment with CDN1163 for 3 h raised mitochondrial Ca2+ level and such increase was suppressed by MCU-i4 (an inhibitor of mitochondria Ca2+ uniporter, MCU), suggesting Ca2+ entered the mitochondrial matrix through MCU. Treatment of cells with CDN1163 up to 2 days resulted in mitochondrial hyperpolarization. ConclusionCDN1163 caused internal Ca2+ leak, cytosolic Ca2+ overload, mitochondrial Ca2+ elevation and hyperpolarization, cell cycle arrest and cell growth inhibition.

Isolation and Biological Activity of Iezoside and Iezoside B, SERCA Inhibitors from Floridian Marine Cyanobacteria.

Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside (1) and its C-31 O-demethyl analogue, iezoside B (2), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC50 of 1.5 and 3.0 μΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 μΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC50 values of 0.3 and 0.6 μΜ in A549 cells and 0.1 and 0.5 μΜ, respectively, in HeLa cells, near the IC50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.

Therapeutic targeting of organelles for inhibition of Zika virus replication in neurons

Zika virus (ZIKV) is an arbovirus belonging to the family Flaviviridae. Since 2015, ZIKV infection has emerged as a leading cause of virus-induced placental insufficiency, microcephaly and other neuronal complications. Currently, no therapeutics have been approved to treat ZIKV infection. In this study, we examined how targeted inhibition of cellular organelles or trafficking processes affected ZIKV infection and replication in neural progenitor cells. We found that blocking endocytosis, Golgi function or structural filaments like actin or microtubules had moderate effects on virus replication. However, inducing endoplasmic reticulum (ER) stress by treatment with Thapsigargin substantially inhibited virus production, suggesting the ER might be a candidate cellular target. Further analysis showed that sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) was important for ZIKV inhibition. Collectively, these studies indicate that targeting the SERCA-dependent ER stress pathway may be useful to develop antivirals to inhibit ZIKV replication.

The antidiabetic drug teneligliptin induces vasodilation via activation of PKG, Kv channels, and SERCA pumps in aortic smooth muscle

Diabetes mellitus (DM) is a metabolic disease closely related to cardiovascular disease. The dipeptidyl peptidase-4 inhibitor teneligliptin is used to treat DM and has recently been shown to have a cardiovascular protective effect against diseases such as hypertension and heart failure. The present study demonstrates the vasodilatory effect of teneligliptin using aortic rings pre-contracted with phenylephrine. Teneligliptin induced a vasodilatory effect in a dose-dependent manner, with and without endothelium. In addition, pretreatment with the nitric oxide synthase inhibitor L-NAME and small-conductance Ca2+-activated K+ channel inhibitor apamin did not alter the teneligliptin-induced vasodilatory effect. Although the adenylyl cyclase inhibitor SQ 22536 and protein kinase A (PKA) inhibitor KT 5720 did not modulate the vasodilatory effect of teneligliptin, the guanylyl cyclase inhibitor ODQ and protein kinase G (PKG) inhibitor KT 5823 effectively reduced the effect of teneligliptin. Similarly, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) also reduced teneligliptin-induced vasodilation. However, pretreatment with the inward rectifier K+ (Kir) channel inhibitor Ba2+, large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline, and ATP-sensitive K+ (KATP) channel inhibitor glibenclamide did not alter the vasodilatory effect of teneligliptin. Our data suggest that Kv7.X, but not Kv1.5 or Kv2.1, is one of the major Kv subtypes involved in teneligliptin-induced vasodilation. Furthermore, pretreatment with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin and CPA inhibited the vasodilation induced by teneligliptin. Our results suggest that teneligliptin-induced vasodilation occurs via activation of PKG, SERCA pumps and Kv channels, but not the PKA signaling pathway, other K+ channels, or endothelium.

Cathelicidin-related antimicrobial peptide mediates skeletal muscle degeneration caused by injury and Duchenne muscular dystrophy in mice.

Cathelicidin, an antimicrobial peptide, plays a key role in regulating bacterial killing and innate immunity; however, its role in skeletal muscle function is unknown. We investigated the potential role of cathelicidin in skeletal muscle pathology resulting from acute injury and Duchenne muscular dystrophy (DMD) in mice. Expression changes and muscular localization of mouse cathelicidin-related antimicrobial peptide (Cramp) were examined in the skeletal muscle of normal mice treated with chemicals (cardiotoxin and BaCl2 ) or in dystrophic muscle of DMD mouse models (mdx, mdx/Utrn+/- and mdx/Utrn-/- ). Cramp penetration into myofibres and effects on muscle damage were studied by treating synthetic peptides to mouse skeletal muscles or C2C12 myotubes. Cramp knockout (KO) mice and mdx/Utrn/Cramp KO lines were used to determine whether Cramp mediates muscle degeneration. Muscle pathophysiology was assessed by histological methods, serum analysis, grip strength and lifespan. Molecular factors targeted by Cramp were identified by the pull-down assay and proteomic analysis. In response to acute muscle injury, Cramp was activated in muscle-infiltrating neutrophils and internalized into myofibres. Cramp treatments of mouse skeletal muscles or C2C12 myotubes resulted in muscle degeneration and myotube damage, respectively. Genetic ablation of Cramp reduced neutrophil infiltration and ameliorated muscle pathology, such as fibre size (P<0.001; n=6) and fibrofatty infiltration (P<0.05). Genetic reduction of Cramp in mdx/Utrn+/- mice not only attenuated muscle damage (35%, P<0.05; n=9-10), myonecrosis (53%, P<0.05), inflammation (37-65%, P<0.01) and fibrosis (14%, P<0.05) but also restored muscle fibre size (14%, P<0.05) and muscle force (18%, P<0.05). Reducing Cramp levels led to a 63% (male, P<0.05; n=10-14) and a 124% (female, P<0.001; n=20) increase in the lifespan of mdx/Utrn-/- mice. Proteomic and mechanistic studies revealed that Cramp cross-talks with Ca2+ signalling in skeletal muscle through sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase1 (SERCA1). Cramp binds and inactivates SERCA1, leading to the activation of Ca2+ -dependent calpain proteases that exacerbate DMD progression. These findings identify Cramp as an immune cell-derived regulator of skeletal muscle degeneration and provide a potential therapeutic target for DMD.

Suppression of Ca2+ oscillations by SERCA inhibition in human alveolar type 2 A549 cells: rescue by ochratoxin A but not CDN1163

AimsLung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca2+-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca2+ signaling in human alveolar type 2 A549 cells. Main methodsCa2+ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye. Key findingsCa2+ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca2+ release and store-operated Ca2+ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca2+ oscillation frequency and the percentage of cells exhibiting Ca2+ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca2+ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca2+. SignificanceDifferent modes of activation of SERCA may determine the outcome of rescue of Ca2+ oscillations in case of SERCA inhibition in alveolar type 2 cells.

Synergistic and Attenuating Effect of Electroacupuncture on Aconitine in Improving Heart Failure and Its Calcium Regulation Mechanism.

Objective The objective is to observe the synergistic and attenuating effect of electroacupuncture (EA) on aconitine (ACO) in improving heart failure (HF) and to explore its underlying mechanism for calcium regulation. Methods Twenty-four male Sprague-Dawley rats were randomly divided into four groups: normal control (NC) (n = 6), HF(n = 6), ACO (n = 6), and ACO + EA (n = 6). The maximum rates of left ventricular pressure rising and declining (±dp/dtmax), arrhythmia, the left ventricular systolic pressure (LVSP), ejection fraction (LVEF), and fractional shortening (LVFS) were measured by physiological recorder and ultrasound, respectively. Protein expressions of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a), phospholamban (PLB), and Na+-Ca2+ exchange (NCX1) in the left ventricle tissue were detected by fluorescence immunoblotting. Results Compared with the NC group, LVSP, ±dp/dtmax, LVEF, and LVFS were decreased in the HF group; compared with the HF group, LVSP, ±dp/dtmax, LVEF, and LVFS were significantly increased in the ACO + EA group. Compared with the ACO group, the incidence and the degree of arrhythmia were significantly reduced in the ACO + EA group. Compared with the NC group, the activity of SERCA2a was decreased, and the expression of PLB and NCX1 was enhanced in the HF group; compared with the HF group and ACO group, the activity of SERCA2a was increased, and the expression of PLB and NCX1 was significantly attenuated in the ACO + EA group. Conclusions EA plays a synergistic and attenuated role in ACO improving HF, and the mechanism may be related to the enhancement of the SERCA2a activity and the decrease of the expression of PLB and NCX1 in cardiomyocytes.

SLC8 family of sodium/calcium exchangers in GtoPdb v.2021.3

The sodium/calcium exchangers (NCX) use the extracellular sodium concentration to facilitate the extrusion of calcium out of the cell. Alongside the plasma membrane Ca2+-ATPase (PMCA) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the sodium/potassium/calcium exchangers (NKCX, SLC24 family), NCX allow recovery of intracellular calcium back to basal levels after cellular stimulation. When intracellular sodium ion levels rise, for example, following depolarisation, these transporters can operate in the reverse direction to allow calcium influx and sodium efflux, as an electrogenic mechanism. Structural modelling suggests the presence of 9 TM segments, with a large intracellular loop between the fifth and sixth TM segments [1].

ALL-043: Selective Blockade of Oncogenic NOTCH1 with the New SERCA Inhibitor CAD204520 in T-cell Acute Lymphoblastic Leukemia

The discovery of the P-type ATPase sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggests an innovative approach for treating T-cell acute lymphoblastic leukemia (T-ALL). In fact, SERCA inhibition preferentially affects the maturation and activity of the most common class of oncogenic NOTCH1 mutants. The aim of this study was to identify inhibitors with better drug-like properties and reduced off-target toxicity for SERCA inhibition. We developed a novel oral SERCA inhibitor, CAD204520, through medicinal chemistry optimization and crystal structure-oriented analysis describing its anti-leukemic effects in vitro and in vivo to support a SERCA-based therapeutic modality in T-ALL. From a 191,000 small-molecule screening targeting P-type ATPase, we identified CAD204520 as a selective inhibitor of human SERCA compared to Na+/K+ and H+-ATPase. Crystal structure analysis showed that CAD204520 binds the transmembrane interface of SERCA between helices M1, M2, M3, and M4. CAD204520 minimally alters Ca2+ shift and fails to trigger Ca2+-dependent programs, such as the unfolded protein response. We demonstrated that CAD204520 impairs the proliferation of T-ALL cell lines carrying activating mutations of NOTCH1. Importantly, clinical samples carrying NOTCH1 mutations, including PEST deletions, were more sensitive to CAD204520 compared to normal lymphocytes or wild-type NOTCH1 ALL cells. Mechanistically, CAD204520 treatment reduces the levels of the activated form of NOTCH1 as a consequence of a defect in NOTCH1 trafficking. Next, we demonstrated that somatic hotspot mutations in the SERCA2 ATPase pocket that confer resistance to known SERCA modulators (e.g., thapsigargin) do not interfere with CAD204520 binding, suggesting that the activity of CAD204520 will be unlikely affected by recurrent resistant genetic variants. Finally, we showed that 30 mg/kg BID for 21 days is well-tolerated in vivo in CD1 mice without causing loss of weight and cardiac toxicity. In a xenograft SKW-3/KE-37 T-ALL model, CAD204520 reduces circulating and tissue-infiltrating human leukemia T-ALL cells with no heart-related or gastrointestinal toxicities. In conclusion, we present CAD204520 as a novel orally bioavailable SERCA inhibitor with tolerable off-target toxicity in NOTCH1-dependent tumors. This work provides a foundation for the further development of novel drugs targeting Notch-dependent hematopoietic malignancies.

Leishmania spp.-Infected Dogs Have Circulating Anti-Skeletal Muscle Autoantibodies Recognizing SERCA1.

Leishmania spp. infection is associated with an inflammatory myopathy (IM) in dogs. The pathomechanism underlying this disorder is still elusive, however, the pattern of cellular infiltration and MHC I and II upregulation indicate an immune-mediated myositis. This study aimed to investigate the presence of autoantibodies targeting the skeletal muscle in sera of leishmania-infected dogs and individuate the major autoantigen. We tested sera from 35 leishmania-infected dogs and sera from 10 negative controls for the presence of circulating autoantibodies with indirect immunofluorescence. Immunoblot and mass spectrometry were used to identify the main target autoantigen. Immunocolocalization and immunoblot on immunoprecipitated muscle proteins were performed to confirm the individuated major autoantigen. We identified circulating autoantibodies that recognize skeletal muscle antigen(s) in sera of leishmania-infected dogs. The major antigen was identified as the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1). We also found that canine SERCA1 presents several identical traits to the calcium-translocating P-type ATPase of Leishmania infantum. In the present study, we defined circulating anti-SERCA1 autoantibodies as part of the pathogenesis of the leishmania-associated IM in dogs. Based on our data, we hypothesize that antigen mimicry is the mechanism underlying the production of these autoantibodies in leishmania-infected dogs.