Sarco/endoplasmic Reticulum Ca2+-ATPase Research Articles

A hydroalcoholic extract of Senecio nutans SCh. Bip (Asteraceae); its effects on cardiac function and chemical characterization

Ethnopharmacology relevanceThe plant Senecio nutans SCh. Bip. is used by Andean communities to treat altitude sickness. Recent evidence suggests it may produce vasodilation and negative cardiac inotropy, though the cellular mechanisms have not been elucidated. PurposeTo determinate the mechanisms action of S. nutans on cardiovascular function in normotensive animals. MethodsThe effect of the extract on rat blood pressure was measured with a transducer in the carotid artery and intraventricular pressure by a Langendorff system. The effects on sheep ventricular intracellular calcium handling and contractility were evaluated using photometry. Ultra-high-performance liquid-chromatography with diode array detection coupled with heated electrospray-ionization quadrupole-orbitrap mass spectrometric detection (UHPLC-DAD-ESI-Q-OT-MSn) was used for extract chemical characterization. ResultsIn normotensive rats, S. nutans (10 mg/kg) reduced mean arterial pressure (MAP) by 40% (p < 0.05), causing a dose-dependent coronary artery dilation and decreased left ventricular pressure. In isolated cells, S. nutans extract (1 μg/ml) rapidly reduced the [Ca2+]i transient amplitude and sarcomere shorting by 40 and 49% (p < 0.001), respectively. The amplitude of the caffeine evoked [Ca2+]i transient was reduced by 24% (p < 0.001), indicating reduced sarcoplasmic reticulum (SR) Ca2+ content. Sodium-calcium exchanger (NCX) activity increased by 17% (p < 0.05), while sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) activity was decreased by 21% (p < 0.05). LC-MS results showed the presence of vitamin C, malic acid, and several antioxidant phenolic acids reported for the first time. Dihydroeuparin and 4-hydroxy-3-(3-methylbut-2-enyl) acetophenone were abundant in the extract. ConclusionIn normotensive animals, S. nutans partially reduces MAP by decreasing heart rate and cardiac contractility. This negative inotropy is accounted for by decreased SERCA activity and increased NCX activity which reduces SR Ca2+ content. These results highlight the plant's potential as a source of novel cardio-active phytopharmaceuticals or nutraceuticals.

Altered skeletal muscle sarco-endoplasmic reticulum Ca2+-ATPase calcium transport efficiency after a thermogenic stimulus.

Exposure to predator threat induces a rapid and robust increase in skeletal muscle thermogenesis in rats. The central nervous system relays threat information to skeletal muscle through activation of the sympathetic nervous system, but muscle mechanisms mediating this thermogenesis remain unidentified. Given the relevance of sarcolipin-mediated futile calcium cycling through the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump to mammalian muscle nonshivering thermogenesis, we hypothesized that this plays a role in contextually induced muscle thermogenesis as well. This was assessed by measuring enzymatic activity of SERCA and sarcoplasmic reticulum Ca2+ transport, where the apparent coupling ratio (Ca2+ uptake rate divided by ATPase activity rate at a standard Ca2+ concentration) was predicted to decrease in association with muscle thermogenesis. Sprague-Dawley rats exposed to predator (ferret) odor (PO) showed a rapid decrease in the apparent coupling ratio in the soleus muscle, indicating SERCA uncoupling compared with control-odor-exposed rats. A rat model of high aerobic fitness and elevated muscle thermogenesis also demonstrated soleus muscle SERCA uncoupling relative to their obesity-prone, low-fitness counterparts. Both the high- and low-aerobic fitness rats showed soleus SERCA uncoupling with exposure to PO. Finally, no increase in sarcolipin expression in soleus muscle was detected with PO exposure. This dataset implicates muscle uncoupling of SERCA Ca2+ transport and ATP hydrolysis, likely through altered SERCA or sarcolipin function outside of translational regulation, as one contributor to the muscle thermogenesis provoked by exposure to predator threat. These data support the involvement of SERCA uncoupling in both muscle thermogenic induction and enhanced aerobic capacity.

Reduced sarcoplasmic reticulum Ca2+ pump activity is antiarrhythmic in ischemic cardiomyopathy

We have described an arrhythmic mechanism seen only in cardiomyopathy that involves increased mitochondrial Ca2+ handling and selective transfer of Ca2+ to the sarcoplasmic reticulum (SR). Modeling suggested that mitochondrial Ca2+ transfer to the SR via type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a crucial element of this arrhythmic mechanism. We tested the role of SERCA2a in arrhythmias during ischemic cardiomyopathy. Myocardial infarction (MI) was induced in wild-type (Wt) and SERCA2a heterozygous knockdown (SERCA+/-) mice. Compared with Wt MI mice, SERCA2a heterozygous knockdown (SERCA+/-) MI mice had a substantially lower mortality after 3 weeks of MI without a significant change in MI area. Aside from a significant delay of the cytoplasmic Ca2+ transient decay existed in SERCA+/- compared with Wt, SERCA+/- did not affect cardiac systolic and diastolic function at the whole organ or single cell levels either before or after MI. After MI, SERCA+/- mice had reduced SERCA2a expression in the MI border zone compared with Wt MI mice. SERCA+/- mice had significantly decreased corrected QT intervals and less ventricular tachycardia compared with Wt MI mice. SERCA+/- cardiomyocytes from MI mice showed a reduced action potential duration and reduced triggered activity compared with Wt MI cardiomyocytes. Reduction in arrhythmic risk was accompanied by reduced diastolic SR Ca2+ sparks, reduced SR Ca2+ content, reduced oxidized ryanodine receptor, and increased calsequestrin 2 in SERCA+/- MI mice. SERCA2a knockdown was antiarrhythmic after MI without affecting overall systolic performance. Possible antiarrhythmic mechanisms included reduced SR free Ca2+ and reduced diastolic SR Ca2+ release.

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.

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated ER stress crosstalk with autophagy is involved in tris(2-chloroethyl) phosphate stress-induced cardiac fibrosis

Excessive organophosphate flame retardant (OPFR) use in consumer products has been reported to increase human disease susceptibility. However, the adverse effects of tris(2-chloroethyl) phosphate (TCEP) (a chlorinated alkyl OPFR) on the heart remain unknown. In this study, we tested whether cardiac fibrosis occurred in animal models of TCEP (10 mg/kg b.w./day) administered continuously by gavage for 30 days and evaluated the specific role of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). First, we confirmed that TCEP could trigger cardiac fibrosis by histopathological observation and cardiac fibrosis markers. We further verified that cardiac fibrosis occurred in animal models of TCEP exposure accompanied by SERCA2a, SERCA2b and SERCA2c downregulation. Notably, inductively coupled plasma–mass spectrometry (ICP–MS) analysis revealed that the cardiac concentrations of Ca2+ increased by 45.3% after TCEP exposure. Using 4-Isopropoxy-N-(2-methylquinolin-8-yl)benzamide (CDN1163, a small molecule SERCA activator), we observed that Ca2+ overload and subsequent cardiac fibrosis caused by TCEP were both alleviated. Simultaneously, the protein levels of endoplasmic reticulum (ER) markers (protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1α (IRE1α), eukaryotic initiation factor 2 α (eIF2α)) were upregulated by TCEP, which could be abrogated by CDN1163 pretreatment. Furthermore, we observed that CDN1163 supplementation prevented overactive autophagy induced by TCEP in the heart. Mechanistically, TCEP could lead to Ca2+ overload by inhibiting the expression of SERCA, thereby triggering ER stress and overactive autophagy, eventually resulting in cardiac fibrosis. Together, our results suggest that the Ca2+ overload/ER stress/autophagy axis can act as a driver of cardiotoxicity induced by TCEP.

Natural Polyphenols as SERCA Activators: Role in the Endoplasmic Reticulum Stress-Related Diseases.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a key protein responsible for transporting Ca2+ ions from the cytosol into the lumen of the sarco/endoplasmic reticulum (SR/ER), thus maintaining Ca2+ homeostasis within cells. Accumulating evidence suggests that impaired SERCA function is associated with disruption of intracellular Ca2+ homeostasis and induction of ER stress, leading to different chronic pathological conditions. Therefore, appropriate strategies to control Ca2+ homeostasis via modulation of either SERCA pump activity/expression or relevant signaling pathways may represent a useful approach to combat pathological states associated with ER stress. Natural dietary polyphenolic compounds, such as resveratrol, gingerol, ellagic acid, luteolin, or green tea polyphenols, with a number of health-promoting properties, have been described either to increase SERCA activity/expression directly or to affect Ca2+ signaling pathways. In this review, potential Ca2+-mediated effects of the most studied polyphenols on SERCA pumps or related Ca2+ signaling pathways are summarized, and relevant mechanisms of their action on Ca2+ regulation with respect to various ER stress-related states are depicted. All data were collected using scientific search tools (i.e., Science Direct, PubMed, Scopus, and Google Scholar).

Klotho relieves inflammation and exerts a cardioprotective effect during renal ischemia/reperfusion-induced cardiorenal syndrome

BackgroundRenal ischemia and reperfusion injury (IRI) is the main cause of acute kidney injury (AKI). AKI induces the development of cardiac hypertrophy (CH) during cardiorenal syndrome (CRS), and cardiomyocyte calcium mishandling though systemic inflammation after 8 days of renal IRI. Klotho has recently been described as an anti-inflammatory component. Given this, Klotho treatment could prevent or attenuate the inflammation, thereby also preventing electrical cardiac outcomes incurred by CRS. The aim of this study was to investigate the therapeutic role of Klotho in CRS after unilateral renal IRI through its anti-inflammatory action. MethodsWe examined renal tissue structure and function, intracellular Ca2+ dynamics in adult ventricular cardiomyocytes and serum cytokine levels from C57BL/6 mice that suffered unilateral renal IRI by occluding the left pedicle for 60 min and reperfusion for 8 days. The animals were treated with recombinant Klotho protein starting from the day of the surgery, then daily for 8 days. ResultsAfter Klotho treatment for 8 days, the left renal tissue remained damaged, however the renal function was restored due to the right kidney tissue preservation. In parallel, Klotho also prevented an increase in serum interleukin (IL-) 6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels. CH and low cell contraction were also prevented, as well as a decrease in systolic Ca2+ transients and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity measured as Ca2+ transient decay, an increase in spontaneous Ca2+ release and the incidence of pro-arrhythmic events. ConclusionsThe Klotho treatment showed promise, playing an important role in the pathophysiology of CRS. We were unable to observe a total renoprotective role of the compound in the model; in turn, a cardioprotective role of Klotho was demonstrated through the prevention of hypertrophy and normalization of the Ca2+ cycle dysfunction of cardiomyocytes. We propose that Klotho acts in the cardiorenal syndrome by systematically preventing inflammation and increased FGF23, alleviating cardiac outcomes.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Molecular cloning and functional characterization of sarco/endoplasmic reticulum Ca 2+ ‐ ATPase from Chinese mitten crab ( Eriocheir sinensis )

Ca2+ plays an important role in signal transduction and regulates various physiological functions of cells. The active transport of Ca2+ from the cytosol to the lumen of the sarcoplasmic or endoplasmic reticulum occurs via sarco/endoplasmic reticulum Ca2+-ATPases (SERCA). However, the function of this protein during moulting cycles remains unclear in Chinese mitten crab (Eriocheir sinensis). In this study, we cloned a cDNA encoding a putative SERCA protein from the Y-organs (moulting glands) in E. sinensis. The cloned Es-SERCA cDNA (4552 bp) included an open reading frame of 3003 bp encoding a 1000-residue protein with a predicted molecular mass of 109.60 kDa. The predicted Es-SERCA protein was clustered with those from other arthropods in phylogenetic analysis. Further, qPCR revealed that Es-SERCA was widely expressed in in all tested tissues with the highest expression in the muscle, followed by the eyestalks, intestines, hepatopancreas, Y-organs and gills. The stage-specific fluctuations among different tissues indicated a functional role of SERCA in calcium homeostasis. The relative mRNA level of the Es-SERCA in Y-organs during the moulting cycle was similar to the level trend of ecdysteroid concentration in haemolymph, indicating that Ca2+ signalling stimulated ecdysteroid synthesis in Y-organs. Altogether, our study provides a reference for further research on the molecular mechanisms of the moulting in E. sinensis.

Structural Determination, Total Synthesis, and Biological Activity of Iezoside, a Highly Potent Ca2+-ATPase Inhibitor from the Marine Cyanobacterium Leptochromothrix valpauliae.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein on the endoplasmic reticulum (ER) that transports Ca2+ from the cytosol into the ER. As its function is associated with various biological phenomena, SERCA has been recognized as a promising druggable target. Here, we report the second-strongest SERCA-inhibitory compound known to date, which we isolated from the marine cyanobacterium Leptochromothrix valpauliae and named iezoside (1). The structure of iezoside (1) is fundamentally different from that of any other SERCA inhibitor, and its potency is the strongest among marine natural products (Ki 7.1 nM). In this article, we report our comprehensive analysis of iezoside (1), which covers its isolation, structural characterization supported by density functional theory (DFT) calculations and statistical analysis, total synthesis, and clarification of the mode of action of its potent antiproliferative activity (IC50 6.7 ± 0.4 nM against HeLa cells).

Role of SERCA3-dependent calcium signaling in mouse aortic function

Calcium homeostasis has a major role in vascular reactivity. The Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCAs) contribute to generate intracellular Ca2+-stores that are mobilized during functional responses. The SERCA2 family is described as the most important in vascular cells, but SERCA3 has been reported to be co-expressed, with SERCA3a mainly at the endothelial level and SERCA3b at the smooth muscle level. Our objective is to evaluate the role of the SERCA3-dependent calcium pathway in vascular reactivity. For experiments, aortic rings were collected from wild-type (WT) and SERCA3-deficient (SERCA3-KO) mice (2–3 month-old male) and mounted in an organ bath system. Contractile and relaxant agents were applied to compare the in vitro aortic reactivity in both animal groups. The contractile responses to a depolarizing high-potassium solution or to cumulative increasing concentrations of serotonin (a 5-HT agonist, 10-9 to 10-5 M) or U46619 (a TXA2 analog, 10-10 to 3.10-6 M) were similar in SERCA3-KO and WT aorta. By contrast the response to phenylephrine (an α1-adrenergic agonist, 10-9 to 3.10-5 M) was significantly decreased in SERCA3-KO compared to WT aorta (N = 8-8; P < 0.05, 2-way ANOVA). However, in aorta denuded from endothelium, the contractile response to phenylephrine was similar in SERCA3-KO and WT vessels. In phenylephrine-precontracted aorta, the relaxant responses to acetylcholine (a muscarinic agonist, 10-9 to 3.10-5 M) or to isoproterenol (a b-adrenergic agonist, 10-10 to 10-5 M) were similar in both groups. The concentration-response curve to DEA-NOate (a NO donor, 10-11 to 10-5 M) was shifted to the right in SERCA3-KO compared to WT aortas, with a slight increase in EC50 (N = 9-8; P < 0.05, t -test). When the endothelium was removed, the effect to DEA-NOate was similar in both groups. We observed that SERCA3-KO aorta exhibit a major decrease in the amplitude of the α1-adrenergic contractile response, and in the sensitivity to the relaxant NO donor. These alterations are abolished in the absence of a functional endothelium. These preliminary data suggest that there is an influence of SERCA3 protein on the NO signaling pathway in mouse aorta.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA–micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB–SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB–SERCA regulatory interactions.

SERCA interacts with chitin synthase and participates in cuticular chitin biogenesis in Drosophila

The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral β-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. Here, we identified a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48–81 and aa 247–33) and C-terminal regions (aa 743–783 and aa 824–859) of DmSERCA and two N-terminal regions (aa 121–179 and aa 369–539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to our knowledge on the machinery of chitin biogenesis.

Migratory disposition alters lean mass dynamics and protein metabolism in migratory white-throated sparrows (Zonotrichia albicollis).

Migratory birds seasonally increase fat stores and the capacity to use fat to fuel long-distance migratory flights. However, lean mass loss also occurs during migratory flights and, if adaptive, should exhibit seasonal changes in the capacity for protein metabolism. We conducted a photoperiod manipulation using captive white-throated sparrows (Zonotrichia albicollis) to investigate seasonal changes in protein metabolism between the nonmigratory "winter" condition and after exposure to a long-day "spring" photoperiod to stimulate the migratory condition. After photostimulation, birds in the migratory condition rapidly increased fat mass and activity of fat catabolism enzymes. Meanwhile, total lean mass did not change, but birds increased the activity of protein catabolism enzymes and lost more water and lean mass during water-restricted metabolic testing. These data suggest that more protein may be catabolized during migratory seasons, corresponding with more water loss. Counter to predictions, birds in the migratory condition also showed an approximately 30-fold increase in muscle expression of sarcolipin, which binds to sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and uncouples Ca2+ transport from ATP hydrolysis. Our documented changes to protein catabolism enzymes and whole animal lean mass dynamics may indicate that protein breakdown or increased protein turnover is adaptive during migration in songbirds.

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 H2O2. 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.

SIRT1 Mutation Impairs Ca2+ Buffering in Coronary Smooth Muscle Cells of Ossabaw Miniature Swine

BackgroundMetabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity in coronary smooth muscle (CSM) and is implicated in coronary atherosclerosis in Ossabaw miniature swine. Sirtuin 1 (SIRT1) is a deacetylase that has diverse roles in intracellular Ca2+ signaling, metabolism, and cardiovascular disease. SIRT1 impairment exacerbates MetS and vascular disease, including calcification. After Ca2+ is increased in CSM by Ca2+ influx or release from the sarcoplasmic reticulum (SR), the Ca2+ is then buffered by either Ca2+ efflux from the cell by the plasmalemmal Ca2+ ATPase or sequestration back into the SR via SERCA. SIRT1 inhibition hyper‐acetylates SERCA and decreases activity, thereby impairing the Ca2+ buffering capacity of the cell.HypothesisMetS and SIRT1 mutation will impair Ca2+ buffering in CSM.MethodsUsing CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. CSM cells were enzymatically dispersed and Ca2+ was measured with fura‐2. The main Ca2+ regulation protocol was the maximal release of Ca2+ from the SR by 5 mM caffeine and then assessment of Ca2+ efflux and sequestration. Time to half recovery of Ca2+ to baseline during caffeine exposure was measured to assess Ca2+ efflux (rapid phase Ca2+buffering) and deviation from baseline was measured to assess SERCA function (slow phase Ca2+buffering).ResultsThere was no effect of MetS or SIRT1 mutation on Ca2+ efflux. Two‐way ANOVA showed SIRT1 mutation alone inhibits SERCA buffering of Ca2+ in CSM (p=0.0005). There was no effect of MetS diet (p=0.184) and no interactions of SIRT1 genotype and MetS diet (p=0.113) on SERCA activity.ConclusionSIRT1L100P mutation is likely to contribute to coronary atherosclerosis and SIRT1 activators may be effective therapies.

Effects of Metabolic Syndrome and SIRT1 Mutation on Store‐Operated Ca2+ Entry in Peripheral Blood Mononuclear Cells in Ossabaw Miniature Swine

BackgroundMetabolic syndrome (MetS) impairs sarco‐endoplasmic reticulum Ca2+ ATPase (SERCA) activity that may decrease the ER Ca2+ store and increase store‐operated Ca2+ entry (SOCE) in a variety of cell types. Similar Ca2+ signaling via SOCE causes fibrosis, vascular disease, and may trigger immunogenic (type 1) diabetes by action on peripheral blood mononuclear cells (PBMCs). Sirtuin 1 (SIRT1) is a deacetylase that maintains healthy metabolism and generally attenuates vascular disease. SIRT1 impairment exacerbates MetS and vascular disease by hyper‐acetylation of SERCA, decreasing its activity and thereby potentially increasing SOCE.HypothesisThe SIRT1 mutation made in Ossabaw miniature swine would mimic the mutation found in humans and increase PBMC SOCE.MethodsUsing CRISPR/Cas9 methodology a point mutation (SIRT1L100P) was made in Ossabaw miniature swine to mimic the naturally occurring mutation in humans and decrease SIRT1 activity, thereby resulting in hyperacetylation of Ca2+transporters and impaired function. Four groups of pigs were used to analyze genotype and diet interactions: wild type lean, SIRT1 lean, wild type MetS, and SIRT1 MetS. Pigs were age 4 months at the start and fed normal chow (lean) or atherogenic diet (MetS) for 7 months. PBMCs were isolated and Ca2+ was measured with fura‐2. The ER Ca2+store was depleted by 10‐5M cyclopiazonic acid to inhibit SERCA in a Ca2+‐free extracellular solution to fully deplete the ER Ca2+store and then Ca2+ was returned to the solution to induce maximal SOCE. Cells were recovered in Ca2+‐free solution to partially re‐fill the ER Ca2+ store, then Ca2+ returned to the solution to observe submaximal SOCE.ResultsBoth MetS diet groups (wild type MetS, SIRT1 MetS) showed impaired glucose tolerance. Fasting glucose in SIRT1 mutants was not different from wild type pigs, thus SIRT1 does not cause immunogenic diabetes at this early stage. Robust coronary atherosclerosis was shown by angiography, intravascular ultrasound, and gross imaging in MetS groups, but there was no additive or synergistic effect of SIRT1 mutation. There was no effect of MetS or SIRT1 mutation on maximal SOCE. Two‐way ANOVA showed that MetS diet increased submaximal SOCE (p&lt;0.009), but there was no effect of SIRT1 genotype on submaximal SOCE (p=0.266). The percentage of PBMCs that responded to partial ER Ca2+ depletion was 45% in lean pigs compared to 81% in wild type and 63% in SIRT1 pigs fed MetS diet.ConclusionSIRT1L100P mutation is likely to require longer duration to manifest robust effects on PBMC SOCE that were found with MetS diet. PBMC activation and can be easily studied longitudinally in the SIRT1L100P mutant swine model due to the ease of blood sampling.

Thapsigargin blocks electromagnetic field‐elicited intracellular Ca2+ increase in HEK 293 cells

Biological effects of electromagnetic fields (EMFs) have previously been identified for cellular proliferation and changes in expression and conduction of diverse types of ion channels. The major effect elicited by EMFs seems to be directed toward Ca2+ homeostasis. This is particularly remarkable since Ca2+ acts as a central modulator in various signaling pathways, including, but not limited to, cell differentiation and survival. Despite this, the mechanisms underlying this modulation have yet to be unraveled. Here, we assessed the effect of EMFs on intracellular [Ca2+], by exposing HEK 293 cells to both radio‐frequency electromagnetic fields (RF‐EMFs) and static magnetic fields (SMFs). We detected a constant and significant increase in [Ca2+] subsequent to exposure to both types of fields. Strikingly, the increase was nulled by administration of 10 μM Thapsigargin, a blocker of sarco/endoplasmic reticulum Ca2+‐ATPases (SERCAs), indicating the involvement of the endoplasmic reticulum (ER) in EMF‐related modulation of Ca2+ homeostasis.

Sarco/Endoplasmic Reticulum Ca2+ ATPase 2 Activator Ameliorates Endothelial Dysfunction; Insulin Resistance in Diabetic Mice.

Background: Sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic (db/db) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db/db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db/db mice. In isometric tension measurements of aortic rings from db/db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db/db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db/db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db/db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db/db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

Heart failure in mice induces a dysfunction of the sinus node associated with reduced CaMKII signaling.

Dysfunction of the sinoatrial node (SAN), the natural heart pacemaker, is common in heart failure (HF) patients. SAN spontaneous activity relies on various ion currents in the plasma membrane (voltage clock), but intracellular Ca2+ ([Ca2+]i) release via ryanodine receptor 2 (RYR2; Ca2+ clock) plays an important synergetic role. Whereas remodeling of voltage-clock components has been revealed in HF, less is known about possible alterations to the Ca2+ clock. Here, we analyzed [Ca2+]i handling in SAN from a mouse HF model after transverse aortic constriction (TAC) and compared it with sham-operated animals. ECG data from awake animals showed slower heart rate in HF mice upon autonomic nervous system blockade, indicating intrinsic sinus node dysfunction. Confocal microscopy analyses of SAN cells within whole tissue showed slower and less frequent [Ca2+]i transients in HF. This correlated with fewer and smaller spontaneous Ca2+ sparks in HF SAN cells, which associated with lower RYR2 protein expression level and reduced phosphorylation at the CaMKII site. Moreover, PLB phosphorylation at the CaMKII site was also decreased in HF, which could lead to reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function and lower sarcoplasmic reticulum Ca2+ content, further depressing the Ca2+ clock. The inhibition of CaMKII with KN93 slowed [Ca2+]i transient rate in both groups, but this effect was smaller in HF SAN, consistent with less CaMKII activation. In conclusion, our data uncover that the mechanism of intrinsic pacemaker dysfunction in HF involves reduced CaMKII activation.