Sarco/endoplasmic Reticulum Ca(2+)-ATPase Activity Research Articles

Role of TG2-Mediated SERCA2 Serotonylation on Hypoxic Pulmonary Vein Remodeling.

Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pumps take up Ca2+ from the cytoplasm to maintain the balance of intracellular Ca2+. A decline in expression or activity of SERCA results in persistent store-operated calcium entry (SOCE). In cardiomyocytes as well as vascular smooth muscle cells (SMCs), SERCA2 acts as an important regulator of calcium cycling. The purpose of this study is to identify and better understand the role of transglutaminases2 (TG2) as a key factor involved in SERCA2 serotonination (s-SERCA2) and to elucidate the underlying mechanism of action. Human pulmonary venous smooth muscle cell in normal pulmonary lobe were isolated and cultured in vitro. Establishment of hypoxic pulmonary hypertension model in wild type and TG2 knockout mice. SERCA2 serotonylation was analyzed by co-(immunoprecipitation) IP when the TG2 gene silenced or overexpressed under normoxia and hypoxia in vivo and in vitro. Intracellular calcium ion was measured by using Fluo-4AM probe under normoxia and hypoxia. Real-time (RT)-PCR and Western blot analyzed expression of TG2, TRPC1, and TRPC6 under normoxia and hypoxia. Bioactivity of cells were analyzed by using Cell Counting Kit (CCK)-8, flow cytometry, wound healing, RT-PCR, and Western blot under PST-2744 and cyclopiazonic acid. We confirmed that 1) hypoxia enhanced the expression and activity of TG2, and 2) hypoxia increased the basal intracellular Ca2+ concentration ([Ca2+]i) and SOCE through activating TRPC6 on human pulmonary vein smooth muscle cells (hPVSMC). Then, we investigated the effects of overexpression and downregulation of the TG2 gene on the activity of SERCA2, s-SERCA2, basal [Ca2+]i, and SOCE under normoxia and hypoxia in vitro, and investigated the activity of SERCA2 and s-SERCA2 in vivo, respectively. We confirmed that SERCA2 serotonylation inhibited the activity of SERCA2 and increased the Ca2+ influx, and that hypoxia induced TG2-mediated SERCA2 serotonylation both in vivo and in vitro. Furthermore, we investigated the effect of TG2 activity on the biological behavior of hPVSMC by using an inhibitor and agonist of SERCA2, respectively. Finally, we confirmed that chronic hypoxia cannot increase vessel wall thickness, the right ventricular systolic pressure (RVSP), and right ventricular hypertrophy index (RVHI) of vascular smooth muscle-specific Tgm2−/− mice. These results indicated that hypoxia promoted TG2-mediated SERCA2 serotonylation, thereby leading to inhibition of SERCA2 activity, which further increased the calcium influx through the TRPC6 channel. Furthermore, tissue-specific conditional TG2 knockout mice prevents the development of pulmonary hypertension caused by hypoxia. In summary, we uncovered a new target (TG2) for treatment of chronic hypoxic pulmonary hypertension (CHPH).

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. While contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This re-uptake of calcium is catalysed by the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. SERCA activity is regulated by small membrane protein subunits, most well-known being phospholamban (PLN) and sarcolipin (SLN).

Significance of bilayer-forming phospholipids for skeletal muscle insulin sensitivity and mitochondrial function.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which make up the bulk of mammalian cell membrane phospholipids, are recognized for their importance in metabolic health. Perturbations in the ratio of PC:PE can affect membrane integrity and function, which thus have serious health consequences. Imbalance in the hepatic PC and PE membrane content can be linked to metabolic disturbances such as ER stress, fatty liver and insulin resistance. Given that impaired insulin sensitivity underlies the pathology of many metabolic disorders and skeletal muscle is a significant regulator of energy metabolism, it is likely that aberrant phospholipid metabolism in skeletal muscle affects whole-body insulin sensitivity. Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) activity and mitochondrial function respond to alterations in PC:PE ratio and are associated with glucose homeostasis. Moreover, PC and PE content within the mitochondrial membrane influence mitochondrial respiration and biogenesis and thus, metabolic function. As skeletal muscle phospholipids respond to stimuli such as diet and exercise, understanding the implications of imbalances in PC:PE ratio is of great importance in the face of the rising epidemic of obesity related diseases. This review will summarize the current state of knowledge signifying the links between skeletal muscle PC:PE ratio and insulin sensitivity with respects to PC and PE metabolism, SERCA activity, mitochondrial function and exercise.

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump, SERCA

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn4-Ala and Thr5-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.

SERCA2a tyrosine nitration coincides with impairments in maximal SERCA activity in left ventricles from tafazzin‐deficient mice

The sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) is imperative for normal cardiac function regulating both muscle relaxation and contractility. SERCA2a is the predominant isoform in cardiac muscles and is inhibited by phospholamban (PLN). Under conditions of oxidative stress, SERCA2a may also be impaired by tyrosine nitration. Tafazzin (Taz) is a mitochondrial‐specific transacylase that regulates mature cardiolipin (CL) formation, and its absence leads to mitochondrial dysfunction and excessive production of reactive oxygen/nitrogen species (ROS/RNS). In the present study, we examined SERCA function, SERCA2a tyrosine nitration, and PLN expression/phosphorylation in left ventricles (LV) obtained from young (3‐5 months) and old (10‐12 months) wild‐type (WT) and Taz knockdown (TazKD) male mice. These mice are a mouse model for Barth syndrome, which is characterized by mitochondrial dysfunction, excessive ROS/RNS production, and dilated cardiomyopathy (DCM). Here, we show that maximal SERCA activity was impaired in both young and old TazKD LV, a result that correlated with elevated SERCA2a tyrosine nitration. In addition PLN protein was decreased, and its phosphorylation was increased in TazKD LV compared with control, which suggests that PLN may not contribute to the impairments in SERCA function. These changes in expression and phosphorylation of PLN may be an adaptive response aimed to improve SERCA function in TazKD mice. Nonetheless, we demonstrate for the first time that SERCA function is impaired in LVs obtained from young and old TazKD mice likely due to elevated ROS/RNS production. Future studies should determine whether improving SERCA function can improve cardiac contractility and pathology in TazKD mice.

Parkinson\u2019s disease-associated LRRK2-G2019S mutant acts through regulation of SERCA activity to control ER stress in astrocytes

Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a common feature of Parkinson’s disease (PD) and further suggests that several PD-related genes are responsible for ER dysfunction. However, the underlying mechanisms are largely unknown. Here, we defined the mechanism by which LRRK2-G2019S (LRRK2-GS), a pathogenic mutation in the PD-associated gene LRRK2, accelerates ER stress and cell death. Treatment of cells with α-synuclein increased the expression of ER stress proteins and subsequent cell death in LRRK2-GS astrocytes. Intriguingly, we found that LRRK2-GS localizes to the ER membrane, where it interacts with sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and suppress its activity by preventing displacement of phospholamban (PLN). LRRK2-GS–mediated SERCA malfunction leads to ER Ca2+ depletion, which induces the formation of mitochondria-ER contacts and subsequent Ca2+ overload in mitochondria, ultimately resulting in mitochondrial dysfunction. Collectively, our data suggest that, in astrocytes, LRRK2-GS impairs ER Ca2+ homeostasis, which determines cell survival, and as a result, could contribute to the development of PD.

Familial Alzheimer’s disease-linked presenilin mutants and intracellular Ca2+ handling: A single-organelle, FRET-based analysis

An imbalance in Ca2+ homeostasis represents an early event in the pathogenesis of Alzheimer’s disease (AD). Presenilin-1 and -2 (PS1 and PS2) mutations, the major cause of familial AD (FAD), have been extensively associated with alterations in different Ca2+ signaling pathways, in particular those handled by storage compartments. However, FAD-PSs effect on organelles Ca2+ content is still debated and the mechanism of action of mutant proteins is unclear.To fulfil the need of a direct investigation of intracellular stores Ca2+ dynamics, we here present a detailed and quantitative single-cell analysis of FAD-PSs effects on organelle Ca2+ handling using specifically targeted, FRET (Fluorescence/Förster Resonance Energy Transfer)-based Ca2+ indicators. In SH-SY5Y human neuroblastoma cells and in patient-derived fibroblasts expressing different FAD-PSs mutations, we directly measured Ca2+ concentration within the main intracellular Ca2+ stores, e.g., Endoplasmic Reticulum (ER) and Golgi Apparatus (GA) medial- and trans-compartment. We unambiguously demonstrate that the expression of FAD-PS2 mutants, but not FAD-PS1, in either SH-SY5Y cells or FAD patient-derived fibroblasts, is able to alter Ca2+ handling of ER and medial-GA, but not trans-GA, reducing, compared to control cells, the Ca2+ content within these organelles by partially blocking SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) activity. Moreover, by using a cytosolic Ca2+ probe, we show that the expression of both FAD-PS1 and -PS2 reduces the Ca2+ influx activated by stores depletion (Store-Operated Ca2+ Entry; SOCE), by decreasing the expression levels of one of the key molecules, STIM1 (STromal Interaction Molecule 1), controlling this pathway.Our data indicate that FAD-linked PSs mutants differentially modulate the Ca2+ content of intracellular stores yet leading to a complex dysregulation of Ca2+ homeostasis, which represents a common disease phenotype of AD.

Farnesoid X Receptor Activation Modulates Calcium Homeostasis in Rat Aortic Vascular Smooth Muscle Cells.

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is expressed in the vasculature; our previous work showed that FXR regulated vascular reactivity through NO mechanism. The underlying mechanism for the regulation of vascular tension by FXR remains unclear. The present work was designed to investigate whether FXR regulates calcium homeostasis in aortic vascular smooth muscle cells (VSMCs). Protein abundances of angiotensin II type 1 and 2 receptors (AT₁R, AT₂R), bradykinin type 1 and 2 receptors (B₁R, B₂R), and the inositol 1,4,5-trisphosphate receptor (IP₃R) were analyzed by western blotting. Kallikrein activity and bradykinin content were assayed by using spectrophotometry and a bradykinin assay kit, respectively. Aortic contraction, intracellular Ca²⁺ concentrations ([Ca²⁺]i), sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) activities, and IP₃-evoked Ca²⁺ release were investigated, following FXR activation in the presence or absence of AT₂R and B₂R blockade. We found that the FXR agonists GW4064 and INT-747 increased the protein abundance of AT₂R and B₂R in rat aortic VSMCs. AT₂R blockade with PD123319 reversed the effects of FXR agonists on kallikrein activity, B₂R, and bradykinin levels. Moreover, we found that GW4064 and INT-747 decreased intracellular [Ca²⁺], increased SERCA activity, downregulated IP₃R₁ expression, and attenuated IP₃-induced Ca²⁺ release. These effects were partially reversed by AT₂R and B₂R blockade with PD123319 and HOE140, respectively. Our data suggest that FXR regulates vascular tension by modulating extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the sarcoplasmic reticulum via activation of an AT₂R-B₂R pathway in rat aortic VSMCs.

Genetic deletion of muscle RANK or selective inhibition of RANKL is not as effective as full-length OPG-fc in mitigating muscular dystrophy

Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that regulates synchronously bone and skeletal muscle physiopathology is still elusive. Receptor-activator of nuclear factor κB (RANK), its ligand RANKL and the soluble decoy receptor osteoprotegerin (OPG) are the key regulators of osteoclast differentiation and bone remodelling. We thus hypothesized that RANK/RANKL/OPG, which is a key pathway for bone regulation, is involved in Duchenne muscular dystrophy (DMD) physiopathology. Our results show that muscle-specific RANK deletion (mdx-RANKmko) in dystrophin deficient mdx mice improves significantly specific force [54% gain in force] of EDL muscles with no protective effect against eccentric contraction-induced muscle dysfunction. In contrast, full-length OPG-Fc injections restore the force of dystrophic EDL muscles [162% gain in force], protect against eccentric contraction-induced muscle dysfunction ex vivo and significantly improve functional performance on downhill treadmill and post-exercise physical activity. Since OPG serves a soluble receptor for RANKL and as a decoy receptor for TRAIL, mdx mice were injected with anti-RANKL and anti-TRAIL antibodies to decipher the dual function of OPG. Injections of anti-RANKL and/or anti-TRAIL increase significantly the force of dystrophic EDL muscle [45% and 17% gains in force, respectively]. In agreement, truncated OPG-Fc that contains only RANKL domains produces similar gains, in terms of force production, than anti-RANKL treatments. To corroborate that full-length OPG-Fc also acts independently of RANK/RANKL pathway, dystrophin/RANK double-deficient mice were treated with full-length OPG-Fc for 10 days. Dystrophic EDL muscles exhibited a significant gain in force relative to untreated dystrophin/RANK double-deficient mice, indicating that the effect of full-length OPG-Fc is in part independent of the RANKL/RANK interaction. The sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity is significantly depressed in dysfunctional and dystrophic muscles and full-length OPG-Fc treatment increased SERCA activity and SERCA-2a expression. These findings demonstrate the superiority of full-length OPG-Fc treatment relative to truncated OPG-Fc, anti-RANKL, anti-TRAIL or muscle RANK deletion in improving dystrophic muscle function, integrity and protection against eccentric contractions. In conclusion, full-length OPG-Fc represents an efficient alternative in the development of new treatments for muscular dystrophy in which a single therapeutic approach may be foreseeable to maintain both bone and skeletal muscle functions.

Effect of sevoflurane preconditioning on function of sarcoplasmic reticulum in cardiomyocytes during ischemia-reperfusion in isolated rat hearts

Objective To evaluate the effect of sevoflurane preconditioning on the function of sarcoplasmic reticulum in cardiomyocytes during ischemia-reperfusion (I/R) in isolated rat hearts. Methods Healthy adult male Sprague-Dawley rats, weighing 270-300 g, were anesthetized with intraperitoneal pentobarbital.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃.Twenty-four isolated rat hearts successfully perfused in the Langendorff apparatus were divided into 3 groups (n=8 each) using a random number table: control group (group C), I/R group and sevoflurane preconditioning group (group SP). Myocardial ischemia was induced by interrupting perfusion for 30 min followed by 120 min reperfusion.In group SP, the hearts were perfused with 2.4% sevoflurane for 10 min starting from 10 min before ischemia.Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded at 5, 10, 15, 30 and 60 min of reperfusion.Coronary effluent was collected at 10 min before reperfusion for measurement of the levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTnI). Myocardial specimens were obtained at 120 min of reperfusion for examination of the pathological changes (using HE staining) and for determination of myocardial infarct size (by TTC staining), sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA2a) activity (with a spectrophotometer) and expression of Bcl-2, Bax and SERCA2a (by Western blot). Results Compared with group C, LVDP was significantly decreased and LVEDP was increased at each time point, myocardial infarct size was increased, the levels of LDH and cTnI in coronary effluent were increased, the expression of Bax was up-regulated, the expression of Bcl-2 and SERCA2a was down-regulated, and the activity of SERCA2a was decreased in group I/R (P<0.01). Compared with group I/R, LVDP was significantly increased and LVEDP was decreased at each time point, myocardial infarct size was decreased, the levels of LDH and cTnI in coronary effluent were decreased, the expression of Bax was down-regulated, the expression of Bcl-2 and SERCA2a was up-regulated, the activity of SERCA2a was increased (P<0.01), and the pathological changes were significantly attenuated in group SP. Conclusion The mechanism by which sevoflurane preconditioning reduces I/R injury in isolated rat hearts may be related to improving the function of sarcoplasmic reticulum in cardiomyocytes. Key words: Anesthetics, inhalation; Ischemic preconditioning; Myocardial reperfusion injury; Sarcoplasmic reticulum

Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

Unregulated increases in cellular Ca2+ homeostasis are a hallmark of pathophysiological conditions and a key trigger of cell death. Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.

β-Arrestin2 Improves Post-Myocardial Infarction Heart Failure via Sarco(endo)plasmic Reticulum Ca2+-ATPase-Dependent Positive Inotropy in Cardiomyocytes.

Heart failure is the leading cause of death in the Western world, and new and innovative treatments are needed. The GPCR (G protein-coupled receptor) adapter proteins βarr (β-arrestin)-1 and βarr-2 are functionally distinct in the heart. βarr1 is cardiotoxic, decreasing contractility by opposing β1AR (adrenergic receptor) signaling and promoting apoptosis/inflammation post-myocardial infarction (MI). Conversely, βarr2 inhibits apoptosis/inflammation post-MI but its effects on cardiac function are not well understood. Herein, we sought to investigate whether βarr2 actually increases cardiac contractility. Via proteomic investigations in transgenic mouse hearts and in H9c2 rat cardiomyocytes, we have uncovered that βarr2 directly interacts with SERCA2a (sarco[endo]plasmic reticulum Ca2+-ATPase) in vivo and in vitro in a β1AR-dependent manner. This interaction causes acute SERCA2a SUMO (small ubiquitin-like modifier)-ylation, increasing SERCA2a activity and thus, cardiac contractility. βarr1 lacks this effect. Moreover, βarr2 does not desensitize β1AR cAMP-dependent procontractile signaling in cardiomyocytes, again contrary to βarr1. In vivo, post-MI heart failure mice overexpressing cardiac βarr2 have markedly improved cardiac function, apoptosis, inflammation, and adverse remodeling markers, as well as increased SERCA2a SUMOylation, levels, and activity, compared with control animals. Notably, βarr2 is capable of ameliorating cardiac function and remodeling post-MI despite not increasing cardiac βAR number or cAMP levels in vivo. In conclusion, enhancement of cardiac βarr2 levels/signaling via cardiac-specific gene transfer augments cardiac function safely, that is, while attenuating post-MI remodeling. Thus, cardiac βarr2 gene transfer might be a novel, safe positive inotropic therapy for both acute and chronic post-MI heart failure.

Remarkable plasticity of Na+, K+-ATPase, Ca2+-ATPase and SERCA contributes to muscle disuse atrophy resistance in hibernating Daurian ground squirrels

We investigated cytosolic calcium (Ca2+) and sarcoplasmic reticulum Ca2+ regulation in skeletal muscle fibers of hibernating Daurian ground squirrels (Spermophilus dauricus), non-hibernating hindlimb-unloaded (HLU) squirrels, and HLU rats to clarify the molecular mechanisms involved in preventing muscle atrophy in hibernators. The Na+, K+-ATPase and Ca2+-ATPase activities in the soleus muscle (SOL) of squirrels were maintained in hibernation, decreased during interbout arousal (IB-A), and increased to autumn/pre-hibernation (AUT/Pre-H) levels in torpor after interbout arousal (Post-IBA), whereas activities in the extensor digitorum longus muscle (EDL) were stable during hibernation, but increased during post-hibernation (Post-H). Activities increased in the SOL of HLU rats, but were stable in HLU squirrels. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity in the SOL decreased in IB-A squirrels, but returned to AUT/Pre-H levels in the Post-IBA group; no significant changes were found in the EDL. SERCA activity increased in the EDL of HLU squirrels and SOL of HLU rats. Compared with AUT/Pre-H, SERCA type 2 protein expression increased in the SOL and EDL of IB-A and Post-IBA squirrels, but increased in the SOL only in HLU animals. We also describe the protein kinase A changes in this paper. Thus, hibernating ground squirrels displayed remarkable Na+, K+-ATPase, Ca2+-ATPase, and SERCA plasticity.

Comparative Histological Study on the Effect of Stem Cells, and Gene Modified Stem Cells in Experimentally-Induced Diabetes Type 1 Cardiomyopathy

Background and Objectives: Sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA2a) is the ATP-driven pump thattranslocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. 25% of children and adolescents withtype 1 diabetes will develop diastolic dysfunction that results in part from a reduction in the activity of SERCA2a. Thepresent study aimed at investigating and comparing the therapeutic effect of Adipose Mesenchymal Stem Cells (AMSCs)with SERCA2a gene modified AMSCs in diabetes type 1 induced cardiomyopathy of adult male albino rat.Materials and Methods: Twenty-nine adult male albino rats were divided into: Donor group of 2 rats used to obtainAMSCs. Group I (Control group): 6 rats not exposed to diabetes induction. Group II (Diabetic group): 7 rats injectedwith streptozotocin (STZ) 50mg/Kg once. Group III ( (AMSCs group): 7 rats injected with AMSCs following inductionof diabetes. Group IV (SERCA2a Modified AMSCs Group): 7 rats injected with SERCA2a modified AMSCs, followinginduction of diabetes. All rats were sacrificed 8 weeks from start of experiment.Results: Morphological changes, indicating inflammation and degeneration, were found in the cardiac muscle of diabeticrats and regressed remarkably by AMSCs and SERCA2a modified AMSCs. The regression of morphological changes wasconfirmed by histological, immunohistochemical, morphometric and serological studies.Conclusion: The therapeutic effect of SERCA2a modified AMSCs in diabetes type 1 cardiomyopathy was moreremarkable than that of AMSCs.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals.

SummaryCardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min), cyclic strain (5%), and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

Astragaloside IV Attenuates Podocyte Apoptosis Mediated by Endoplasmic Reticulum Stress through Upregulating Sarco/Endoplasmic Reticulum Ca2+-ATPase 2 Expression in Diabetic Nephropathy.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) plays a central role in the pathogenesis of diabetes. This protein has been recognized as a potential target for diabetic therapy. In this study, we identified astragaloside IV (AS-IV) as a potent modulator of SERCA inhibiting renal injury in diabetic status. Increasing doses of AS-IV (2, 6, and 18 mg kg-1 day-1) were administered intragastrically to db/db mice for 8 weeks. Biochemical and histopathological approaches were conducted to evaluate the therapeutic effects of AS-IV. Cultured mouse podocytes were used to further explore the underlying mechanism in vitro. AS-IV dose-dependently increased SERCA activity and SERCA2 expression, and suppressed ER stress-mediated and mitochondria-mediated apoptosis in db/db mouse kidney. AS-IV also normalized glucose tolerance and insulin sensitivity, improved renal function, and ameliorated glomerulosclerosis and renal inflammation in db/db mice. In palmitate stimulated podocytes, AS-IV markedly improved inhibitions of SERCA activity and SERCA2 expression, restored intracellular Ca2+ homeostasis, and attenuated podocyte apoptosis in a dose-dependent manner with a concomitant abrogation of ER stress as evidenced by the downregulation of GRP78, cleaved ATF6, phospho-IRE1α and phospho-PERK, and the inactivation of both ER stress-mediated and mitochondria-mediated apoptotic pathways. Furthermore, SERCA2b knockdown eliminated the effect of AS-IV on ER stress and ER stress-mediated apoptotic pathway, whereas its overexpression exhibited an anti-apoptotic effect. Our data obtained from in vivo and in vitro studies demonstrate that AS-IV attenuates renal injury in diabetes subsequent to inhibiting ER stress-induced podocyte apoptosis through restoring SERCA activity and SERCA2 expression.

A Decoy Peptide Targeted to Protein Phosphatase 1 Attenuates Degradation of SERCA2a in Vascular Smooth Muscle Cells.

Neointimal growth in the injured vasculature is largely facilitated by the proliferation of vascular smooth muscle cells (VSMC), which associates with reduced sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. The gene transfer-mediated restoration of the SERCA2a level thus attenuates neointimal growth and VSMC proliferation. We previously reported that a peptide targeted to protein phosphatase 1, ψPLB-SE, normalizes SERCA2a activity in cardiomyocytes. In this study, we found that ψPLB-SE attenuated neointimal growth in balloon-injured rat carotid arteries, and the proliferation and migration of VSMC cultured in high-serum media (synthetic conditions). In parallel, ψPLB-SE inhibited the degradation of SERCA2a in the injured carotid arteries and VSMC under synthetic conditions. The calpain inhibitor MDL28170 also attenuated SERCA2a degradation and VSMC proliferation under synthetic conditions, indicating that calpain degrades SERCA2a. The Ca2+ ionophore A23187 induced SERCA2a degradation in VSMC, which was blocked by either ψPLB-SE or MDL28170. Additionally, ψPLB-SE normalized the cytosolic Ca2+ level in VSMC that was increased by either A23187 or synthetic stimulation. Collectively, these data indicate that ψPLB-SE corrects the abnormal Ca2+ handling by activating SERCA2a, which further protects SERCA2a from calpain-dependent degradation in VSMC. We conclude that ψPLB-SE may form the basis of a therapeutic strategy for vascular proliferative disorders.

The N-Termini of Sarcolipin and Phospholamban Play an Important Role in Distinct Regulation of Sarco-Endoplasmic Reticulum Ca2+ ATPase (SERCA) Function

The Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) is responsible for intracellular Ca2+ homeostasis in muscle and non-muscle cells. SERCA activity in muscle can be regulated by Phospholamban (PLB), an affinity modulator, and Sarcolipin (SLN), an uncoupler. While PLB gets dislodged from Ca2+-bound SERCA, SLN continues to bind SERCA throughout its kinetic cycle and promotes uncoupling of Ca2+ transport from ATP hydrolysis. To determine the structural regions of SLN that mediate uncoupling of SERCA, we employed mutagenesis and generated chimeras of PLB and SLN. We demonstrated that deletion of SLN N-terminal residues 2ERSTQ leads to loss of uncoupling function even though the truncated peptide can target and constitutively bind SERCA. Further, Molecular Dynamics simulations of SLN and SERCA interaction showed that the deletion of SLN N-terminus introduces structural rearrangement of SERCA residues. Interestingly, transfer of the PLB cytosolic domain to SLN transmembrane (TM) and luminal tail causes the chimeric protein to lose SLN-like function. We also found that swapping the PLB N-terminus with the SLN N-terminus caused the resulting chimera to acquire SLN-like function. These results suggest that domains can be switched between SLN and PLB without losing the ability to regulate SERCA activity; however the resulting chimera acquires a function different from the parent molecule. Importantly, our studies highlight that the N-termini of SLN and PLB affect their respective unique function by proper positioning of the TM domain along with C-terminus on SERCA.

Calcium Uptake in Crude Tissue Preparation.

The various isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) are responsible for the Ca(2+) uptake from the cytosol into the endoplasmic or sarcoplasmic reticulum (ER/SR). In some tissues, the activity of SERCA can be modulated by binding partners, such as phospholamban and sarcolipin. The activity of SERCA can be characterized by its apparent affinity for Ca(2+) as well as maximal enzymatic velocity. Both parameters can be effectively determined by the protocol described here. Specifically, we describe the measurement of the rate of oxalate-facilitated (45)Ca uptake into the SR of crude mouse ventricular homogenates. This protocol can easily be adapted for different tissues and animal models as well as cultured cells.

Lipogenesis mitigates dysregulated sarcoplasmic reticulum calcium uptake in muscular dystrophy

Muscular dystrophy is accompanied by a reduction in activity of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) that contributes to abnormal Ca2+ homeostasis in sarco/endoplasmic reticulum (SR/ER). Recent findings suggest that skeletal muscle fatty acid synthase (FAS) modulates SERCA activity and muscle function via its effects on SR membrane phospholipids. In this study, we examined muscle's lipid metabolism in mdx mice, a mouse model for Duchenne muscular dystrophy (DMD). De novo lipogenesis was ~50% reduced in mdx muscles compared to wildtype (WT) muscles. Gene expressions of lipogenic and other ER lipid-modifying enzymes were found to be differentially expressed between wildtype (WT) and mdx muscles. A comprehensive examination of muscles' SR phospholipidome revealed elevated phosphatidylcholine (PC) and PC/phosphatidylethanolamine (PE) ratio in mdx compared to WT mice. Studies in primary myocytes suggested that defects in key lipogenic enzymes including FAS, stearoyl-CoA desaturase-1 (SCD1), and Lipin1 are likely contributing to reduced SERCA activity in mdx mice. Triple transgenic expression of FAS, SCD1, and Lipin1 (3TG) in mdx myocytes partly rescued SERCA activity, which coincided with an increase in SR PE that normalized PC/PE ratio. These findings implicate a defect in lipogenesis to be a contributing factor for SERCA dysfunction in muscular dystrophy. Restoration of muscle's lipogenic pathway appears to mitigate SERCA function through its effects on SR membrane composition.