Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation.

Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

INTRODUCTION: In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. METHODS: Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." RESULTS: We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 µM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. CONCLUSION: The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

Cellular target identification of Withangulatin A using fluorescent analogues and subsequent chemical proteomics.

Withangulatin A (WA) has been reported to exhibit potent antitumor activity. However, its possible mechanism and direct proteomic targets remain unknown. Herein we report the subcellular localization of WA by designing and synthesizing its fluorescent analogues with coumarin moieties. Furthermore, sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 was identified as the potential target of WA for its antitumor activity by chemical proteomics.

Nanoscale reorganization of sarcoplasmic reticulum in pressure-overload cardiac hypertrophy visualized by dSTORM.

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.

Geriatric Polytrauma-Cardiovascular and Immunologic Response in a Murine Two-Hit Model of Trauma.

BACKGROUND: The aims of the present study were to establish a clinically relevant two-hit model with trauma/hemorrhage followed by sepsis in older mice and investigate age-dependent cardiovascular and immunologic specificities under these conditions. MATERIALS AND METHODS: In aged mice (12, 18, and 24 mo old), a femur fracture followed by hemorrhage was induced. After resuscitation, animals were monitored for 72 h before sepsis was induced. Vital signs were monitored during shock. Systemic interleukin (IL)-6 levels were measured daily. Expression of sarcoplasmic or endoplasmic reticulum calcium ATPase (SERCA) and IL-6 receptor were analyzed in heart, lung, and liver tissues. RESULTS: After induction of shock, mean arterial pressure decreased significantly in all groups (12 mo, P < 0.001; 18 mo, P < 0.001; 24 mo, P = 0.013). Compared with younger animals, 24-mo old mice were not able to adequately compensate for hypovolemia by an increase of heart rate (P = 0.711). Expression of SERCA2 (P = 0.002) and IL-6 receptor on myocytes (P = 0.037), lung (P = 0.005), and liver (P = 0.009) tissues were also lowest in this group. Systemic IL-6 values showed the most distinct posttraumatic response in 24-mo-old mice (P = 0.016). Survival rate decreased significantly with increased age (P = 0.005). CONCLUSIONS: The increased mortality rate in older animals was associated with a limited compensatory physiological response and a more distinct immunologic reaction after trauma and sepsis. A decreased SERCA2 expression and missing feedback loops due to a reduced density of organ bound immune receptors might represent possible explanations for the observed age-dependent differences.

PKCδ silencing alleviates saturated fatty acid induced ER stress by enhancing SERCA activity.

Protein kinase C δ (PKCδ) plays an important role in nonalcoholic fatty liver disease (NAFLD), however, the mechanism remains unknown. The present study explored the role of PKCδ in NAFLD development and investigated the relationships between PKCδ, calcium homeostasis, and endoplasmic reticulum (ER) stress (ERS). Hepatic steatosis cell model was induced by palmitic acid (PA) in L02 cells. Lipid accretion was evaluated using Oil Red O staining and a triglyceride (TG) detection kit. PKCδ was down-regulated by siRNA. RT-PCR and Western blotting were used to detect the expression of ERS markers. The fluorescence of Ca2+ influx was recorded using confocal microscopy. Sarco-ER Ca2+-ATPase (SERCA) activity was measured by ultramicro-ATP enzyme test kit. PA treatment induced lipid accretion in L02 cells, destroyed the ER structure, and increased PKCδ activation in a time-dependent manner. Further, PA treatment significantly increased the expression of ERS markers, Ig heavy chain binding protein (Bip), and homologous proteins of CCAAT-enhancer binding proteins (CHOP). PKCδ silencing down-regulated Bip and CHOP expression, indicating a successful alleviation of ERS. The increased calcium storage induced by PA stimulation was significantly decreased in L02 cells treated with PKCδ siRNA compared with the negative control. Moreover, diminished SERCA activity caused by PA was recovered in PKCδ siRNA transfected cells. To the best of our knowledge, this is the first report demonstrating that the inhibition of PKCδ alleviates ERS by enhancing SERCA activity and stabilizing calcium homeostasis.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats.

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Importance of SERCA2a on early isolated diastolic dysfunction induced by supravalvular aortic stenosis in rats

Cardiac remodeling is defined as changes in shape and function of the heart in response to aggression (pressure overload). The sarcoplasmic reticulum calcium ATPase cardiac isoform 2a (SERCA2a) is a known factor that influences function. A wide spectrum of studies report a decrease in SERCA2a in heart failure, but none evaluate it's the role in early isolated diastolic dysfunction in supravalvular aortic stenosis (AoS). Our hypothesis was that SERCA2a participates in such dysfunction. Thirty-day-old male Wistar rats (60-80 g) were divided into AoS and Sham groups, which were submitted to surgery with or without aorta clipping, respectively. After 6 weeks, the animals were submitted to echocardiogram and functional analysis by isolated papillary muscle (IPM) in basal condition, hypoxia, and SERCA2a blockage with cyclopiazonic acid at calcium concentrations of 0.5, 1.5, and 2.5 mM. Western-blot analyses were used for SERCA2a and phospholamban detection. Data analysis was carried out with Student's t-test and ANOVA. AoS enhanced left atrium and E and A wave ratio, with preserved ejection fraction. Basal condition in IPM showed similar increases in developed tension (DT) and resting tension (RT) in AoS, and hypoxia was similar between groups. After cyclopiazonic acid blockage, final DT was equally decreased and RT was similar between groups, but the speed of relaxation was decreased in the AoS group. Western-blot was uniform in all evaluations. The hypothesis was confirmed, since functional parameters regarding SERCA2a were changed in the AoS group.

Effects of Growth Hormone on Cardiac Remodeling During Resistance Training in Rats / Efeitos do Hormônio do Crescimento na Remodelação Cardíaca Durante Treinamento Resistido em Ratos

Abstract Background: Although the beneficial effects of resistance training (RT) on the cardiovascular system are well established, few studies have investigated the effects of the chronic growth hormone (GH) administration on cardiac remodeling during an RT program. Objective: To evaluate the effects of GH on the morphological features of cardiac remodeling and Ca2+ transport gene expression in rats submitted to RT. Methods: Male Wistar rats were divided into 4 groups (n = 7 per group): control (CT), GH, RT and RT with GH (RTGH). The dose of GH was 0.2 IU/kg every other day for 30 days. The RT model used was the vertical jump in water (4 sets of 10 jumps, 3 bouts/wk) for 30 consecutive days. After the experimental period, the following variables were analyzed: final body weight (FBW), left ventricular weight (LVW), LVW/FBW ratio, cardiomyocyte cross-sectional area (CSA), collagen fraction, creatine kinase muscle-brain fraction (CK-MB) and gene expressions of SERCA2a, phospholamban (PLB) and ryanodine (RyR). Results: There was no significant (p > 0.05) difference among groups for FBW, LVW, LVW/FBW ratio, cardiomyocyte CSA, and SERCA2a, PLB and RyR gene expressions. The RT group showed a significant (p < 0.05) increase in collagen fraction compared to the other groups. Additionally, the trained groups (RT and RTGH) had greater CK-MB levels compared to the untrained groups (CT and GH). Conclusion: GH may attenuate the negative effects of RT on cardiac remodeling by counteracting the increased collagen synthesis, without affecting the gene expression that regulates cardiac Ca2+ transport.

Resumo Fundamento: Apesar de os efeitos benéficos do treinamento resistido (TR) sobre o sistema cardiovascular estarem bem estabelecidos, poucos estudos têm investigado os efeitos crônicos da administração de hormônio do crescimento (GH) sobre a remodelação cardíaca durante um programa de TR. Objetivo: avaliar os efeitos do GH sobre a remodelação cardíaca em suas características morfológicas e na expressão dos genes do trânsito de Ca2+ em ratos submetidos ao TR. Métodos: Ratos Wistar machos foram divididos em 4 grupos (n = 7 por grupo): controle (CT), GH, TR e TR com GH (TRGH). A dose de GH foi de 0,2 UI/kg, a cada dois dias, por 30 dias. O modelo de TR utilizado foi o salto vertical em água (4 séries de 10 saltos, 3 vezes/semana) durante 30 dias consecutivos. Após o período experimental, as seguintes variáveis foram analisadas: peso corporal final (PCF), peso do ventrículo esquerdo (PVE), razão PVE/PCF, área seccional de cardiomiócitos (ASC), fração de colágeno, creatina quinase fração músculo-cérebro (CK-MB) e expressão gênica de SERCA2a, fosfolambam (PLB) e rianodina (RyR). Resultados: Não houve diferença significativa (p > 0,05) entre os grupos para PCF, PVE, razão PVE/PCF, ASC, e expressão gênica de SERCA2a, PLB e RyR. O grupo TR mostrou um significativo aumento (p < 0,05) da fração de colágeno em comparação aos outros. Além disso, os grupos treinados (TR e TRGH) apresentaram maiores níveis de CK-MB em comparação aos não treinados (CT e GH). Conclusão: Esses resultados indicam que o GH pode atenuar os efeitos negativos do TR na remodelação cardíaca por contrabalançar o aumento da síntese de colágeno, sem afetar a expressão de genes que regulam o trânsito de Ca2+ cardíaco.

Effects of Growth Hormone on Cardiac Remodeling During Resistance Training in Rats.

BACKGROUND: Although the beneficial effects of resistance training (RT) on the cardiovascular system are well established, few studies have investigated the effects of the chronic growth hormone (GH) administration on cardiac remodeling during an RT program. OBJECTIVE: To evaluate the effects of GH on the morphological features of cardiac remodeling and Ca2+ transport gene expression in rats submitted to RT. METHODS: Male Wistar rats were divided into 4 groups (n = 7 per group): control (CT), GH, RT and RT with GH (RTGH). The dose of GH was 0.2 IU/kg every other day for 30 days. The RT model used was the vertical jump in water (4 sets of 10 jumps, 3 bouts/wk) for 30 consecutive days. After the experimental period, the following variables were analyzed: final body weight (FBW), left ventricular weight (LVW), LVW/FBW ratio, cardiomyocyte cross-sectional area (CSA), collagen fraction, creatine kinase muscle-brain fraction (CK-MB) and gene expressions of SERCA2a, phospholamban (PLB) and ryanodine (RyR). RESULTS: There was no significant (p > 0.05) difference among groups for FBW, LVW, LVW/FBW ratio, cardiomyocyte CSA, and SERCA2a, PLB and RyR gene expressions. The RT group showed a significant (p < 0.05) increase in collagen fraction compared to the other groups. Additionally, the trained groups (RT and RTGH) had greater CK-MB levels compared to the untrained groups (CT and GH). CONCLUSION: GH may attenuate the negative effects of RT on cardiac remodeling by counteracting the increased collagen synthesis, without affecting the gene expression that regulates cardiac Ca2+ transport.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP.

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Regulation of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) activity by phosphodiesterase 3A (PDE3A) in human myocardium: phosphorylation-dependent interaction of PDE3A1 with SERCA2.

Cyclic nucleotide phosphodiesterase 3A (PDE3) regulates cAMP-mediated signaling in the heart, and PDE3 inhibitors augment contractility in patients with heart failure. Studies in mice showed that PDE3A, not PDE3B, is the subfamily responsible for these inotropic effects and that murine PDE3A1 associates with sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2), phospholamban (PLB), and AKAP18 in a multiprotein signalosome in human sarcoplasmic reticulum (SR). Immunohistochemical staining demonstrated that PDE3A co-localizes in Z-bands of human cardiac myocytes with desmin, SERCA2, PLB, and AKAP18. In human SR fractions, cAMP increased PLB phosphorylation and SERCA2 activity; this was potentiated by PDE3 inhibition but not by PDE4 inhibition. During gel filtration chromatography of solubilized SR membranes, PDE3 activity was recovered in distinct high molecular weight (HMW) and low molecular weight (LMW) peaks. HMW peaks contained PDE3A1 and PDE3A2, whereas LMW peaks contained PDE3A1, PDE3A2, and PDE3A3. Western blotting showed that endogenous HMW PDE3A1 was the principal PKA-phosphorylated isoform. Phosphorylation of endogenous PDE3A by rPKAc increased cAMP-hydrolytic activity, correlated with shift of PDE3A from LMW to HMW peaks, and increased co-immunoprecipitation of SERCA2, cav3, PKA regulatory subunit (PKARII), PP2A, and AKAP18 with PDE3A. In experiments with recombinant proteins, phosphorylation of recombinant human PDE3A isoforms by recombinant PKA catalytic subunit increased co-immunoprecipitation with rSERCA2 and rat rAKAP18 (recombinant AKAP18). Deletion of the recombinant human PDE3A1/PDE3A2 N terminus blocked interactions with recombinant SERCA2. Serine-to-alanine substitutions identified Ser-292/Ser-293, a site unique to human PDE3A1, as the principal site regulating its interaction with SERCA2. These results indicate that phosphorylation of human PDE3A1 at a PKA site in its unique N-terminal extension promotes its incorporation into SERCA2/AKAP18 signalosomes, where it regulates a discrete cAMP pool that controls contractility by modulating phosphorylation-dependent protein-protein interactions, PLB phosphorylation, and SERCA2 activity.

Measuring Ca2+ pump activity in overexpression systems and cardiac muscle preparations.

Sarco-/endoplasmic reticulum (SR/ER) Ca(2+) pumps (SERCAs) build up vital Ca(2+) gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca(2+) transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca(2+)-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive (45)Ca(2+). Here, we first provide an overview of the mechanism of Ca(2+)-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca(2+) pump activity.

Expression of sarcoplasmic-endoplasmic reticulum Ca-ATPase isoforms in masticatory muscles.

The aim of this study was to characterize the sarcoplasmic-endoplasmic reticulum Ca-ATPase (SERCA) isoforms in rabbit masticatory muscles compared with those in fast-twitch muscle. It was hypothesized that combined expression of the SERCA isoforms in fast- and slow-twitch muscles accounts for lower Ca-ATPase activity. SERCA was isolated by differential centrifugation, the isoforms were determined by ELISA, and the activity of each isoform was measured using a colorimetric method. Activity was tested for significance by anova, and the distribution of isoforms was assessed using the chi-square test (P < 0.05) and correlated to SERCA activity using Spearman's rank correlation. SERCA1 was predominant (90.5%) in fast-twitch muscle, whereas a mixture of SERCA isoforms was found in masticatory muscles: 62-78% was SERCA2, 20-37% was SERCA1, and the SERCA3 content was negligible. Depressor muscles showed a significantly higher content (77.8%) of SERCA2, and elevator muscles showed a higher content (35.4%) of SERCA1. Elevator muscles showed higher expression of SERCA2a (58%), and depressor muscles showed higher expression of SERCA2b (20%). The SERCA1 content was mainly SERCA1a and significantly higher for elevator muscles (33%), whereas depressor muscles showed a higher content of SERCA1b (4%). The SERCA1 content of fast-twitch muscle was mainly SERCA1a (88.5%). It is concluded that the mixture of different SERCA isoforms, along with a substantial content of SERCA2b, in masticatory muscles would support lower Ca-ATPase activity and calcium transport.

ATP consumption by sarcoplasmic reticulum Ca²âº pumps accounts for 40-50% of resting metabolic rate in mouse fast and slow twitch skeletal muscle.

The main purpose of this study was to directly quantify the relative contribution of Ca²âº cycling to resting metabolic rate in mouse fast (extensor digitorum longus, EDL) and slow (soleus) twitch skeletal muscle. Resting oxygen consumption of isolated muscles (VO2, µL/g wet weight/s) measured polarographically at 30°C was ~20% higher (P<0.05) in soleus (0.326 ± 0.022) than in EDL (0.261 ± 0.020). In order to quantify the specific contribution of Ca²âº cycling to resting metabolic rate, the concentration of MgCl2 in the bath was increased to 10 mM to block Ca²âº release through the ryanodine receptor, thus eliminating a major source of Ca²âº leak from the sarcoplasmic reticulum (SR), and thereby indirectly inhibiting the activity of the sarco(endo) plasmic reticulum Ca²âº-ATPases (SERCAs). The relative (%) reduction in muscle VO2 in response to 10 mM MgCl2 was similar between soleus (48.0±3.7) and EDL (42.4±3.2). Using a different approach, we attempted to directly inhibit SERCA ATPase activity in stretched EDL and soleus muscles (1.42x optimum length) using the specific SERCA inhibitor cyclopiazonic acid (CPA, up to 160 µM), but were unsuccessful in removing the energetic cost of Ca²âº cycling in resting isolated muscles. The results of the MgCl2 experiments indicate that ATP consumption by SERCAs is responsible for 40-50% of resting metabolic rate in both mouse fast- and slow-twitch muscles at 30°C, or 12-15% of whole body resting VO2. Thus, SERCA pumps in skeletal muscle could represent an important control point for energy balance regulation and a potential target for metabolic alterations to oppose obesity.

Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

Differential calcium handling in two canine models of right ventricular pressure overload.

BACKGROUND: The purpose of this investigation was to characterize differential right atrial (RA) and ventricular (RV) molecular changes in Ca(2+)-handling proteins consequent to RV pressure overload and hypertrophy in two common, yet distinct models of pulmonary hypertension: dehydromonocrotaline (DMCT) toxicity and pulmonary artery (PA) banding. METHODS: A total of 18 dogs underwent sternotomy in four groups: (1) DMCT toxicity (n = 5), (2) mild PA banding over 10 wk to match the RV pressure rise with DMCT (n = 5); (3) progressive PA banding to generate severe RV overload (n = 4); and (4) sternotomy only (n = 4). RESULTS: In the right ventricle, with DMCT, there was no change in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) or phospholamban (PLB), but we saw a trend toward down-regulation of phosphorylated PLB at serine-16 (p[Ser-16]PLB) (P = 0.07). Similarly, with mild PA banding, there was no change in SERCA or PLB, but p(Ser-16)PLB was down-regulated by 74% (P < 0.001). With severe PA banding, there was no change in PLB, but SERCA fell by 57% and p(Ser-16)PLB fell by 67% (P < 0.001). In the right atrium, with DMCT, there were no significant changes. With both mild and severe PA banding, p(Ser-16)PLB fell (P < 0.001), but SERCA and PLB did not change. CONCLUSIONS: Perturbations in Ca(2+)-handling proteins depend on the degree of RV pressure overload and the model used to mimic the RV effects of pulmonary hypertension. They are similar, but blunted, in the atrium compared with the ventricle.

Three-dimensional and molecular analysis of the venous pole of the developing human heart.

BACKGROUND: Various congenital malformations and many abnormal rhythms originate from the venous pole of the heart. Because of rapid changes during morphogenesis, lack of molecular and lineage data, and difficulties in presenting complex morphogenetic changes in the developing heart in a clear fashion, the development of this region in human has been difficult to grasp. METHODS AND RESULTS: To gain insight into the development of the different types of myocardium forming the venous pole of the human heart, we performed an immunohistochemical and 3-dimensional analysis of serial sections of human embryos ranging from 22 through 40 days of development. Three-dimensional models were prepared in a novel interactive portable format providing crucial spatial information and facilitating interpretation. As in the mouse, the systemic venous myocardium expresses the transcription factor TBX18, whereas the pulmonary venous myocardium expresses NKX2-5. In contrast to the mouse, a systemic venous sinus is identified upstream from the atrial chambers, albeit initially with nonmyocardial walls. From the outset, as in the mouse, the pulmonary vein empties to a chamber with atrial, rather than systemic venous, characteristics. Compared with the mouse, the vestibular spine is a more prominent structure. CONCLUSIONS: The similarities in gene expression in the distinctive types of myocardium surrounding the systemic and pulmonary venous tributaries in man and mouse permit extrapolation of the conclusions drawn from transgenic and lineage studies in the mouse to the human, showing that the systemic and pulmonary venous myocardial sleeves are derived from distinct developmental lineages.

Soybean oil increases SERCA2a expression and left ventricular contractility in rats without change in arterial blood pressure.

BACKGROUND: Our aim was to evaluate the effects of soybean oil treatment for 15 days on arterial and ventricular pressure, myocardial mechanics and proteins involved in calcium handling. METHODS: Wistar rats were divided in two groups receiving 100 microL of soybean oil (SB) or saline (CT) i.m. for 15 days. Ventricular performance was analyzed in male 12-weeks old Wistar rats by measuring left ventricle diastolic and systolic pressure in isolated perfused hearts according to the Langendorff technique. Protein expression was measured by Western blot analysis. RESULTS: Systolic and diastolic arterial pressures did not differ between CT and SB rats. However, heart rate was reduced in the SB group. In the perfused hearts, left ventricular isovolumetric systolic pressure was higher in the SB hearts. The inotropic response to extracellular Ca2+ and isoproterenol was higher in the soybean-treated animals than in the control group. Myosin ATPase and Na(+)-K(+)ATPase activities, the expression of sarcoplasmic reticulum calcium pump (SERCA2a) and sodium calcium exchanger (NCX) were increased in the SB group. Although the phosfolamban (PLB) expression did not change, its phosphorylation at Ser16 was reduced while the SERCA2a/PLB ratio was increased. CONCLUSIONS: In summary, soybean treatment for 15 days in rats increases the left ventricular performance without affecting arterial blood pressure. These changes might be associated with an increase in the myosin ATPase activity and SERCA2a expression.

Regulation of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) in turtle muscle and liver during acute exposure to anoxia.

The freshwater turtle Trachemys scripta elegans naturally tolerates extended periods of anoxia during winter hibernation at the bottom of ice-locked ponds. Survival in this anoxic state is facilitated by a profound depression of metabolic rate. As calcium levels are known to be elevated in anoxic turtles, and ion pumping is an ATP-expensive process, we proposed that activity of the sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) would be reduced in muscle and liver of T. s. elegans during acute (up to 20 h) exposure to anoxia. SERCA activity decreased approximately 30% in liver and approximately 40% in muscle after 1 h anoxia exposure and was approximately 50% lower after 20 h of anoxia exposure in both tissues, even though SERCA protein levels did not change. SERCA kinetic parameters (increased substrate K(m) values, increased Arrhenius activation energy) were indicative of a less active enzyme form under anoxic conditions. Interestingly, the less active SERCA in anoxic turtles featured greater stability than the enzyme from normoxic animals as determined by both kinetic analysis (effect of low pH and low temperatures on K(m) MgATP) and conformational resistance to urea denaturation. The quick time course of deactivation and the stable changes in kinetic parameters that resulted suggested that SERCA was regulated by a post-translational mechanism. In vitro experiments indicated that SERCA activity could be blunted by protein phosphorylation and enhanced by dephosphorylation in a tissue-specific manner.