Sarco/endoplasmic Reticulum Calcium ATPase Research Articles

Prolongation of Relaxation Time in Extraocular Muscles With Brain Derived Neurotrophic Factor in Adult Rabbit.

PurposeWe tested the hypothesis that short-term treatment with brain derived neurotrophic factor (BDNF) would alter the contractile characteristics of rabbit extraocular muscle (EOM).MethodsOne week after injections of BDNF in adult rabbit superior rectus muscles, twitch properties were determined in treated and control muscles in vitro. Muscles were also examined for changes in mean cross-sectional areas, neuromuscular junction size, and percent of myofibers expressing specific myosin heavy chain isoforms, and sarcoendoplasmic reticulum calcium ATPases (SERCA) 1 and 2.ResultsBrain derived neurotrophic factor–treated muscles had prolonged relaxation times compared with control muscles. Time to 50% relaxation, time to 100% relaxation, and maximum rate of relaxation were increased by 24%, 27%, and 25%, respectively. No significant differences were seen in time to peak force, twitch force, or maximum rate of contraction. Brain derived neurotrophic factor treatment significantly increased mean cross-sectional areas of slow twitch and tonic myofibers, with increased areas ranging from 54% to 146%. Brain derived neurotrophic factor also resulted in an increased percentage of slow twitch myofibers in the orbital layers, ranging from 54% to 77%, and slow-tonic myofibers, ranging from 44% to 62%. No significant changes were seen SERCA1 or 2 expression or in neuromuscular junction size.ConclusionsShort-term treatment with BDNF significantly prolonged the duration and rate of relaxation time and increased expression of both slow-twitch and slow-tonic myosin-expressing myofibers without changes in neuromuscular junctions or SERCA expression. The changes induced by BDNF treatment might have potential therapeutic value in dampening/reducing uncontrolled eye oscillations in nystagmus.

Redox-assisted regulation of Ca2+ homeostasis in the endoplasmic reticulum by disulfide reductase ERdj5

Calcium ion (Ca2+) is an important second messenger that regulates numerous cellular functions. Intracellular Ca2+ concentration ([Ca2+]i) is strictly controlled by Ca2+ channels and pumps on the endoplasmic reticulum (ER) and plasma membranes. The ER calcium pump, sarco/endoplasmic reticulum calcium ATPase (SERCA), imports Ca2+ from the cytosol into the ER in an ATPase activity-dependent manner. The activity of SERCA2b, the ubiquitous isoform of SERCA, is negatively regulated by disulfide bond formation between two luminal cysteines. Here, we show that ERdj5, a mammalian ER disulfide reductase, which we reported to be involved in the ER-associated degradation of misfolded proteins, activates the pump function of SERCA2b by reducing its luminal disulfide bond. Notably, ERdj5 activated SERCA2b at a lower ER luminal [Ca2+] ([Ca2+]ER), whereas a higher [Ca2+]ER induced ERdj5 to form oligomers that were no longer able to interact with the pump, suggesting [Ca2+]ER-dependent regulation. Binding Ig protein, an ER-resident molecular chaperone, exerted a regulatory role in the oligomerization by binding to the J domain of ERdj5. These results identify ERdj5 as one of the master regulators of ER calcium homeostasis and thus shed light on the importance of cross talk among redox, Ca2+, and protein homeostasis in the ER.

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

Effect of gamma-carboxylase inhibition on serum osteocalcin may be partially protective against developing diabetic cardiomyopathy in type 2 diabetic rats.

To investigate the possible protective effect of elevated undercarboxylated osteocalcin on diabetic cardiomyopathy mechanisms and risk factors. In all, 32 male rats were divided into four groups: control, diabetic, diabetic warfarin and normal warfarin-treated groups. Isolated heart functions were assessed; fasting serum insulin, glucose and glycosylated haemoglobin, homeostasis model assessment insulin resistance and lipid profile were investigated. Serum undercarboxylated osteocalcin and adiponectin were also measured. In cardiac tissue, malondialdehyde content, acyl-CoA dehydrogenase gene expression, Bax/Bcl2 ratio, sarcoendoplasmic reticulum calcium ATPase and osteocalcin receptor (G protein-coupled receptor family C group 6 member A) genes expression were investigated. Prophylactic elevation of undercarboxylated osteocalcin was accompanied by improved insulin sensitivity and lipid profile, increased serum adiponectin, upregulated myocardial osteocalcin receptor with preserved left ventricular function, decreased cardiac malondialdehyde content, acyl-CoA dehydrogenase and Bax/Bcl2 ratio. Undercarboxylated osteocalcin was suggested to have protective effects against diabetic cardiomyopathy, possibly through direct action on upregulated G protein-coupled receptor family C group 6 member A and indirectly via adiponectin. These effects may be mediated through antagonizing oxidative stress and apoptosis.

How Important Is SERCA In Neuronal Calcium Homeostasis?

Maintaining intracellular calcium homeostasis is critical for excitation‐contraction coupling in the heart, and is essentially dependent on primary and secondary active transport systems. However, upregulating sarcoendoplasmic reticulum calcium ATPase (SERCA) in cardiomyocytes from patients with heart failure has recently proved inconclusive (CUPID2 trial). Neuronal calcium impairment is also seen in cardiovascular diseases resulting in heightened sympathetic responsiveness that further exacerbate the disease phenotype. We tested whether overexpression of SERCA in cardiac sympathetic neurons could modulate intracellular calcium transients. Isolated neurons were incubated with either Ad‐MCherry‐SERCA2A or Ad‐MCherry‐Empty (control) for 24 hours immediately post isolation. Calcium imaging using fura‐2AM (2.5μM) was carried out three days post transfection. SERCA2A transgene neurons showed greater depolarisation induced intracellular calcium transients, compared to control (Δ 380/340 ratio (au): SERCA 0.75±0.03, n=68; Empty 0.64±0.03, n=57. P<0.01). SERCA2A expressing neurons also showed greater SR calcium storage compared to control, as demonstrated by significantly higher calcium responses to caffeine (10mM: Δ 380/340 ratio (au): SERCA 0.15±0.01, n=45; Empty 0.03±0.01, n=35. P<0.0001) and thapsigargin stimulation (1μM: Δ 380/340 ratio (au): SERCA 0.12±0.02, n=42; Empty 0.03±0.01, n=33. P<0.0001). Additionally mitochondrial calcium uncoupling with FCCP (1μM) resulted in a greater intracellular calcium change in the SERCA2A transgenic neurons (Δ 380/340 ratio (au): SERCA 0.13±0.01, n=22; Empty 0.05±0.01, n=22. P<0.0001), indicating that altering neuronal calcium handling with upregulation of SERCA has also effected mitochondrial calcium handling and potentially metabolic function in these neurons. Within this study we demonstrated that a SERCA2A gene construct can be transfected in isolated sympathetic neurons from the stellate ganglia. Moreover, this transduction caused an increase in intracellular calcium transients that would be expected to facilitate neurotransmission and be potentially deleterious for cardiac function.Support or Funding InformationBritish Heart Foundation

Sarcolipin Ablation Impairs Muscle Regeneration After Acute Injury

Sarcolipin (SLN) is a small transmembrane protein that regulates the sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump in muscle. SLN is highly expressed in embryonic muscle and is involved in the regulation of myogenesis. SLN is also upregulated in mouse dystrophic muscles but the role of SLN in muscular dystrophy is unknown. Since muscular dystrophy is characterized by high rates of muscle degeneration/regeneration, one hypothesis is that SLN may be important in the regeneration process but this has not been examined specifically. Here, we used an acute cardiotoxin injury model and Sln‐null mice to determine SLN's role in muscle regeneration. The tibialis anterior of 30 mice, 15 wild type (WT) and 15 Sln‐null mice were injected with cardiotoxin and sacrificed at day 7, 14 and 21 post injury. Muscle homogenates and sections were prepared and used for immunoblotting and immunofluorescence analyses, respectively. Interestingly, ectopic expression of SLN was seen in WT animals only at 21 days post injury. Correspondingly, Sln‐null mice showed elevated centralized nuclei, a marker of regenerating fibers, specifically at day 21 post injury compared to their WT counterparts (2.7 ± 1.0 ×10−4 vs. 3.8 ± 0.6 ×10−4, per regenerating area (μm2), p=0.03). In addition, the average cross‐sectional area of the regenerating myofibers was significantly smaller in the Sln‐null mice only at day 21 post injury compared to WT (1925 ± 168 μm2 vs. 1471 ± 57 μm2, p =0.01 ). Taken together, the failure to clear central nuclei and increase myofiber size at day 21 post injury indicates that SLN ablation delays the later stages of muscle regeneration.Support or Funding InformationThis work was supported by the Canadian Institutes of Health Research (CIHR; MOP 86618 and MOP 47296 to A.R.T).

Investigation of Inhibitory Potency of BHQ Derivatives as SERCA Inhibitors to use as Potential Drugs as well as Tools to Study the SERCA Function: Binding Free Energy Computation using FEP/MD Simulations

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is an integral membrane C2+ ion pump that play a vital role in skeletal muscle contraction/relaxation and signal transduction by pumping Ca2+ ions into intracellular storage compartments while transporting protons in the opposite direction. Most SERCA inhibitors bind to its E2 conformation, thereby blocking the conformational change to the E1 form, which is a step necessary for the catalytic cycle. We investigate the interactions between SERCA and its highly potent allosteric inhibitor: di-tert butylhydroquinone (BHQ) and its derivatives with diverse chemical and structural groups, using molecular dynamics (MD) simulations. We effectively simulate the ligand bound active spherical region with explicit molecular dynamics while the rest of the system is treated with implicit solvent model known as generalized boundary solvent potential (GSBP). Our goal is to compute the absolute binding free energy of BHQ derivatives by using the all-atom free energy perturbation molecular dynamics (FEP/MD) simulations. This FEP/MD method involve a sequence of intermediate alchemical steps that restrain the translation, orientation, and conformation of the ligand as well as sequential steps during which the ligand-surrounding interactions are turned “on” and “off”. These features are implemented to improve the accuracy and enhance the convergence of the free energy calculations. The force field for the BHQ derivatives are automatically generated by the CHARMM additive CGenFF force field and later optimized by using quantum computations to obtain a good compatibility with the CHARMM force field, which simulates biopolymers effectively. BHQ ligand structure files are obtained from PDB without modification. Information gained from these simulations will guide the future synthesis of novel compounds with improved properties for SERCA inhibition.

Resolving Structural Dynamics of the SERCA-PLB Complex by Merging MD Simulation and Orientation-Sensitive EPR Measurement

We are using molecular dynamics (MD) simulation to resolve the structural dynamics of the membrane protein phospholamban (PLB), a binding inhibitor to the protein pump sarco(endo)plasmic reticulum calcium-ATPase (SERCA). Increase of cytosolic calcium concentration drives cell contraction via the actin-myosin mechanism, while subsequent uptake of calcium into the SR by SERCA allows cell relaxation. Over-inhibition of SERCA by PLB has been correlated with dysfunctional contractility and heart disease, but the structural basis of this mechanism is not fully understood. We combine MD simulation with electron paramagnetic resonance (EPR) spectroscopy to elucidate the structural mechanism of the PLB-SERCA complex. Previously, we have used MD simulation to determine the dynamic model of the spin label, TOAC, rigidly attached to PLB at residues 24, 36, and 46. Based on these results, which provided a bimodal structural model for TOAC at each attachment position, we have refined analysis of EPR measurement to determine PLB's orientation relative to the bilayer normal. We now extend this combined MD and EPR approach to study the structural changes of PLB in the presence and absence of SERCA. Furthermore, we have used EPR measurements on the SERCA-PLB complex to constrain MD simulation of the system, thus extending spatial and temporal resolution of membrane-bound PLB in the presence and absence of SERCA. Computations were carried out at the Minnesota Supercomputing Institute and experiments were performed in the Biophysical Technology Center. This work was funded by grants from NIH to DDT (R01 GM27906 and T32 AR007612).

Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells

Aberrant increase in the expression levels of sarco/endoplasmic reticulum calcium ATPase (SERCA), which regulates Ca2+ homeostasis, has been observed in ovarian cancers. In this study, we demonstrated that curcumin increases cytosolic Ca2+ concentration through inhibition of SERCA activity, causing apoptosis in ovarian cancer cells but not in normal cells, including peripheral blood mononuclear cells (PBMCs) and ovarian surface epithelial cells (OSE). Curcumin induced apoptosis in ovarian cancer cells in a concentration- and time-dependent manner. Cytosolic Ca2+ flux was evident after the curcumin treatment (15 µM). Treatment with Ca2+ chelator reduced curcumin-induced apoptosis, confirming the possible involvement of increased cytosolic Ca2+ concentration in this response. Basal mRNA and protein levels of SERCA2 were significantly higher in ovarian cancer cells than in OSE. SERCA activity was suppressed by curcumin, with no effect on protein expression. Forced expression of the SERCA2b gene in ovarian cancer cells prevented curcumin-induced cytosolic Ca2+ elevation and subsequent apoptosis, supporting an important role of SERCA in curcumin-induced apoptosis of ovarian cancer cells. Taken together, inhibition of SERCA activity by curcumin disrupts the Ca2+ homeostasis and thereby promotes apoptosis in ovarian cancer cells.

Small molecule RL71 targets SERCA2 at a novel site in the treatment of human colorectal cancer.

While targeted agents are an important part of the treatment arsenal for colorectal cancer, there is still a lack of efficient small-molecule targeted agents based on the understanding of pathogenic molecular mechanisms. In this study, curcumin analog RL71 displayed potent cytotoxicity towards human colon cancer cells with an IC50 of 0.8 µM in SW480 cells and inhibited xenotransplanted tumor growth in a dose-dependent manner. Using affinity chromatography, we identified sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2 as the binding target of RL71. Most notably, RL71 demonstrated special binding to SERCA2 at a novel site with the lowest estimated free energy -8.89 kcal mol(-1), and the SERCA2 residues critical for RL71 binding were identified. RL71 suppressed the Ca(2+)-ATPase activity of SERCA2 both in vitro and in vivo, accompanied by the induction of endoplasmic reticulum stress leading to apoptosis and G2/M cycle arrest in SW480 cells. In addition, RL71 showed synergistic cytotoxicity with the pan-SERCA inhibitor thapsigargin. These results suggest that RL71 could be a selective small-molecule inhibitor of SERCA2, and that it may serve as a lead compound for the study of targeted colorectal cancer therapy.

Short-term exercise training attenuates acute doxorubicin cardiotoxicity.

Doxorubicin (DOX) is a potent and widely used antineoplastic agent. Despite the efficacy of DOX, its clinical use is limited by a dose-dependent cardiotoxicity. Chronic exercise training has been shown to protect against DOX-induced cardiotoxicity. It is less clear whether short-term exercise can attenuate DOX-induced dysfunction. The purposes of this study were to determine if short-term wheel running and treadmill exercise training can attenuate the cardiac dysfunction that accompanies DOX treatment and to investigate possible mechanisms that may be involved with any protective effects of exercise. Male Sprague-Dawley rats engaged in a short-term 5-day voluntary wheel running (WR) or treadmill exercise (TM) regimen. Following the exercise preconditioning period, animals received either 10 or 15 mg/kg of DOX or an equivalent volume of saline (SAL). Five days after DOX/SAL exposure, cardiac function was examined. Western immunoblotting was used to quantify left ventricular sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a) protein expression. Exercise preconditioning attenuated in vivo and ex vivo cardiac dysfunction observed with DOX treatment alone. Specifically, short-term treadmill exercise (TM + DOX10, 56 ± 4%; TM + DOX15, 48 ± 5%) and voluntary wheel running (WR + DOX10, 51 ± 5%; WR + DOX15, 45 ± 3%) consistently preserved fractional shortening when compared to sedentary (SED) animals treated with DOX (SED + DOX10, 48 ± 4%; SED + DOX15, 39 ± 6%). Likewise, both exercise protocols preserved left ventricular developed pressure (TM + DOX10, 115 ± 6 mmHg; TM + DOX15, 85 ± 5 mmHg; WR + DOX10, 92 ± 12 mmHg; WR + DOX15, 91 ± 8 mmHg) when compared to SED animals treated with DOX (SED + DOX10, 79 ± 6 mmHg; SED + DOX15, 69 ± 7 mmHg). SERCA2a expression was also preserved in TM + DOX and WR + DOX. These findings suggest that short-term exercise prior to DOX treatment may be a valuable adjuvant therapy to offset acute cardiotoxicities and that maintaining calcium handling in cardiomyocytes may be responsible, in part, for the preservation in cardiac function.

Abstract 189: DWORF: Discovery and Characterization of a Cardiac Micropeptide Encoded in a Putative Long Noncoding RNA

Background: Mammalian transcriptomes contain thousands of potential open reading frames (ORFs) with fewer than fifty codons. Most of these ORFs are presumed to be random and therefore noncoding, but recently several genes annotated as long noncoding RNAs (lncRNAs) were shown to encode functional micropeptides. Objective: Identify and characterize novel micropeptides encoded in cardiac-expressed lncRNAs using comparative genomics. Methods and Results: Using codon substitution frequency, a comparative genomics strategy, we identified several previously unknown small proteins, including a 34-amino acid micropeptide that we have named DWORF ( Dw arf O pen R eading F rame). DWORF is a tail-anchored transmembrane protein that localizes to the sarcoplasmic reticulum and is expressed only in cardiac ventricle and slow-twitch skeletal muscle by quantitative PCR. We subsequently derived a custom antibody targeting the N-terminal residues of DWORF. Western blots using this antibody revealed a 4 kDa protein that is expressed in cardiac ventricle and soleus muscle, but not other tissues. We further validated specificity of this signal by disrupting the reading frame of DWORF in mice using CRISPR/Cas9 mutagenesis, conclusively demonstrating that the mRNA encodes a small protein. Transgenic overexpression of DWORF in cardiac myocytes resulted in increased peak amplitude of Ca 2+ transients and improved Ca 2+ resequestration kinetics with no apparent adverse effects. These findings suggest that DWORF specifically enhances activity of the sarco/endoplasmic reticulum calcium ATPase (SERCA). Conclusions: Some putative lncRNAs encode micropeptides that can be identified using bioinformatics strategies. Micropeptides, though likely too small to have enzymatic activity of their own, may have modulatory functions in important cellular processes such as calcium handling, as we have demonstrated in the case of the muscle-restricted micropeptide DWORF.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

The sarcoendoplasmic reticulum Ca2+ ATPase 2b (SERCA2b) pump maintains a steep Ca2+ concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl l-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-d,l-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca2+ imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca2+ and decreased ER Ca2+ levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b protein stability is decreased under inflammatory conditions through NO- and AMPK-dependent pathways and provide novel insight into pathways leading to altered β-cell calcium homeostasis and reduced β-cell survival in diabetes.

Sarcoendoplasmic reticulum Ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity.

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

0192 : Pharmacological modulation of TRPV1, a cation leak channel in mouse cardiomyocytes

The sarcoplasmic reticulum (SR) calcium homeostasis is due to a dynamic balance between the capture of the cytosolic calcium by calcium pumps such as SERCA (Sarco Endoplasmic Reticulum Calcium ATPase) and calcium release, actively across calcium channels such as IP3 (Inositol Tri-phosphate) and ryanodine receptors, or passively via calcium leak channels. Few data are available concerning the functional characterization of leak channels in the SR. These channels are involved in the regulation of the reticular calcium concentration as well as in the exchange of calcium between SR and other intracellular organelles. Therefore, their characterization is important for a better understanding of the physiology of the cells. Recently, we have demonstrated that TRPV1 (Transient Receptor Potential Vanilloid 1), a cationic channel, is a functional calcium leak channel on the SR of mouse skeletal muscle cells. TRPV1 is activated by acidosis, high temperature (>42°C), and by pharmacological molecules such as capsaicin, resiniferatoxin and capsazepin. We are pursuing our investigation of these channels in C57Bl6 mouse cardiomyocytes. Our preliminary results show that TRPV1 is active in mice cardiomyocytes as a calcium leak channel after stimulation or inhibition using pharmacological molecules. These data were confirmed by using a genetic approach: the C57Bl6 KO mice for TRPV1 channel. Lately, reducing cardiac injuries after ischemia reperfusion where calcium dynamics play a crucial role became a major interest. Therefore, the modulation of the TRPV1 calcium leak channel might be a new approach in cardioprotection.

Acute Inotropic and Lusitropic Effects of Cardiomyopathic R9C Mutation of Phospholamban

A naturally occurring R9C mutation of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). The goal of this study was to investigate the acute physiological consequences of the R9C-PLB mutation on cardiomyocyte calcium kinetics and contractility. We measured the physiological consequences of R9C-PLB mutation on calcium transients and sarcomere shortening in adult cardiomyocytes. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we quantified the effect of R9C on PLB oligomerization and PLB-SERCA binding. FRET measurements in live cells revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA and altered the structure of the PLB-SERCA regulatory complex. The structural change after oxidative modification of R9C-PLB was similar to that observed after PLB phosphorylation. We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect but yields negative consequences of impaired frequency potentiation and blunted β-adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure.

Dihydroartemisinin-Induced Apoptosis is Associated with Inhibition of Sarco/Endoplasmic Reticulum Calcium ATPase Activity in Colorectal Cancer.

Dihydroartemisinin (DHA) is a promising anti-cancer compound capable of inhibiting proliferation and inducing apoptosis of various cancer cells, including colorectal cancer. However, the molecular mechanisms have not been well understood. This study aimed to explore the underlying mechanism of DHA-induced apoptosis in HCT-116 cells. Cell counting kit-8 assay and flow cytometry analysis confirmed that DHA inhibited proliferation, arrested cell cycle at G0/G1 phase, and enhanced apoptosis in HCT-116 cells. Fluo-3/AM-stained flow cytometry assay revealed that the intracellular Ca(2+) concentration of HCT-116 cells was increased significantly after DHA treatment. Meanwhile, the activity of sarco/endoplasmic reticulum calcium ATPase (SERCA) was appeared to be reduced in a dose-dependent manner. We further detected the upregulated expression of CAAT/enhancer binding protein homologous protein (CHOP) in DHA-treated HCT-116 cells. Conversely, silencing CHOP resulted in a decrease of DHA-induced apoptosis. In addition, the expression of Bax in cytoplasm was elevated significantly along with the sharply decline of Bcl-2 expression in DHA-treated HCT-116 cells. Moreover, the distributions of Bid on mitochondria were increased, accompanied by the activation of caspase-3 in the presence of DHA. Overall, our data indicated that DHA triggered endoplasmic reticulum (ER) stress through inhibiting SERCA activity to release intracellular Ca(2+) from ER, the upregulated expression of CHOP activated mitochondrial apoptosis pathway to induce apoptosis of HCT-116 cells. Therefore, our findings provide a theoretical foundation for DHA as a potential candidate in treatment of colorectal cancer.

Sarcolipin overexpression improves muscle energetics and reduces fatigue.

Sarcolipin (SLN) is a regulator of sarcoendoplasmic reticulum calcium ATPase in skeletal muscle. Recent studies using SLN-null mice have identified SLN as a key player in muscle thermogenesis and metabolism. In this study, we exploited a SLN overexpression (Sln(OE)) mouse model to determine whether increased SLN level affected muscle contractile properties, exercise capacity/fatigue, and metabolic rate in whole animals and isolated muscle. We found that Sln(OE) mice are more resistant to fatigue and can run significantly longer distances than wild-type (WT). Studies with isolated extensor digitorum longus (EDL) muscles showed that Sln(OE) EDL produced higher twitch force than WT. The force-frequency curves were not different between WT and Sln(OE) EDLs, but at lower frequencies the pyruvate-induced potentiation of force was significantly higher in Sln(OE) EDL. SLN overexpression did not alter the twitch and force-frequency curve in isolated soleus muscle. However, during a 10-min fatigue protocol, both EDL and soleus from Sln(OE) mice fatigued significantly less than WT muscles. Interestingly, Sln(OE) muscles showed higher carnitine palmitoyl transferase-1 protein expression, which could enhance fatty acid metabolism. In addition, lactate dehydrogenase expression was higher in Sln(OE) EDL, suggesting increased glycolytic capacity. We also found an increase in store-operated calcium entry (SOCE) in isolated flexor digitorum brevis fibers of Sln(OE) compared with WT mice. These data allow us to conclude that increased SLN expression improves skeletal muscle performance during prolonged muscle activity by increasing SOCE and muscle energetics.

The regulation of sarco(endo)plasmic reticulum calcium-ATPases (SERCA).

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is responsible for transporting calcium (Ca(2+)) from the cytosol into the lumen of the sarcoplasmic reticulum (SR) following muscular contraction. The Ca(2+) sequestering activity of SERCA facilitates muscular relaxation in both cardiac and skeletal muscle. There are more than 10 distinct isoforms of SERCA expressed in different tissues. SERCA2a is the primary isoform expressed in cardiac tissue, whereas SERCA1a is the predominant isoform expressed in fast-twitch skeletal muscle. The Ca(2+) sequestering activity of SERCA is regulated at the level of protein content and is further modified by the endogenous proteins phospholamban (PLN) and sarcolipin (SLN). Additionally, several novel mechanisms, including post-translational modifications and microRNAs (miRNAs) are emerging as integral regulators of Ca(2+) transport activity. These regulatory mechanisms are clinically relevant, as dysregulated SERCA function has been implicated in the pathology of several disease states, including heart failure. Currently, several clinical trials are underway that utilize novel therapeutic approaches to restore SERCA2a activity in humans. The purpose of this review is to examine the regulatory mechanisms of the SERCA pump, with a particular emphasis on the influence of exercise in preventing the pathological conditions associated with impaired SERCA function.

Preferential cytotoxicity of bortezomib toward highly malignant human liposarcoma cells via suppression of MDR1 expression and function

Liposarcoma is the most common soft tissue sarcoma with a high risk of relapse. Few therapeutic options are available for the aggressive local or metastatic disease. Here, we report that the clinically used proteasome inhibitor bortezomib exhibits significantly stronger cytotoxicity toward highly malignant human liposarcoma SW872-S cells compared with its parental SW872 cells, which is accompanied by enhanced activation of apoptotic signaling both in vitro and in vivo. Treatment of cells with Jun-N-terminal kinase (JNK) inhibitor SP60015 or the translation inhibitor cycloheximide ameliorated this enhanced apoptosis. Bortezomib inhibited MDR1 expression and function more effectively in SW872-S cells than in SW872 cells, indicating that the increased cytotoxicity relies on the degree of proteasome inhibition. Furthermore, the pharmacological or genetic inhibition of sarco/endoplasmic reticulum calcium-ATPase (SERCA) 2, which is highly expressed in SW872-S cells, resulted in partial reversal of cell growth inhibition and increase of MDR1 expression in bortezomib-treated SW872-S cells. These results show that bortezomib exhibits preferential cytotoxicity toward SW872-S cells possibly via highly expressed SERCA2-associated MDR1 suppression and suggest that bortezomib may serve as a potent agent for treating advanced liposarcoma.