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.

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.

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.

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.

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.

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.

Overexpression of catalase targeted to mitochondria attenuates murine cardiac aging.

BACKGROUND: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%. METHODS AND RESULTS: We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin-nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%. CONCLUSIONS: This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species-related cardiovascular diseases.

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.

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.

Calsequestrin content and SERCA determine normal and maximal Ca2+ storage levels in sarcoplasmic reticulum of fast- and slow-twitch fibres of rat.

Whilst calsequestrin (CSQ) is widely recognized as the primary Ca2+ buffer in the sarcoplasmic reticulum (SR) in skeletal muscle fibres, its total buffering capacity and importance have come into question. This study quantified the absolute amount of CSQ isoform 1 (CSQ1, the primary isoform) present in rat extensor digitorum longus (EDL) and soleus fibres, and related this to their endogenous and maximal SR Ca2+ content. Using Western blotting, the entire constituents of minute samples of muscle homogenates or segments of individual muscle fibres were compared with known amounts of purified CSQ1. The fidelity of the analysis was proven by examining the relative signal intensity when mixing muscle samples and purified CSQ1. The CSQ1 contents of EDL fibres, almost exclusively type II fibres, and soleus type I fibres [SOL (I)] were, respectively, 36 +/- 2 and 10 +/- 1 micromol (l fibre volume)(-1), quantitatively accounting for the maximal SR Ca2+ content of each. Soleus type II [SOL (II)] fibres (approximately 20% of soleus fibres) had an intermediate amount of CSQ1. Every SOL (I) fibre examined also contained some CSQ isoform 2 (CSQ2), which was absent in every EDL and other type II fibre except for trace amounts in one case. Every EDL and other type II fibre had a high density of SERCA1, the fast-twitch muscle sarco(endo)plasmic reticulum Ca2+-ATPase isoform, whereas there was virtually no SERCA1 in any SOL (I) fibre. Maximal SR Ca2+ content measured in skinned fibres increased with CSQ1 content, and the ratio of endogenous to maximal Ca2+ content was inversely correlated with CSQ1 content. The relative SR Ca2+ content that could be maintained in resting cytoplasmic conditions was found to be much lower in EDL fibres than in SOL (I) fibres (approximately 20 versus >60%). Leakage of Ca2+ from the SR in EDL fibres could be substantially reduced with a SR Ca2+ pump blocker and increased by adding creatine to buffer cytoplasmic [ADP] at a higher level, both results indicating that at least part of the Ca2+ leakage occurred through SERCA. It is concluded that CSQ1 plays an important role in EDL muscle fibres by providing a large total pool of releasable Ca2+ in the SR whilst maintaining free [Ca2+] in the SR at sufficiently low levels that Ca2+ leakage through the high density of SERCA1 pumps does not metabolically compromise muscle function.

Localization of ank1.5 in the sarcoplasmic reticulum precedes that of SERCA and RyR: relationship with the organization of obscurin in developing sarcomeres.

Ank1.5 is a muscle-specific isoform of ankyrin1 localized on the sarcoplasmic reticulum (SR) membrane that has been shown to interact with obscurin, a sarcomeric protein. We report here studies on the localization of obscurin and ank1.5 in embryonic and postnatal rodent skeletal muscles. Using two antibodies against epitopes in the N- and C-terminus of obscurin, two distinct patterns of localization were observed. Before birth, the antibodies against the N- and the C-terminus of obscurin stained the Z-disk and M-band, respectively. At the same time, ank1.5 was detected at the Z-disk, rising the possibility that obscurin molecules at M-band may not be able to interact with ank1.5. Localization of ank1.5 at Z-disks in E14 muscle fibers revealed that ank1.5 is among the earliest SR proteins to assemble, since its organization preceded that of other SR proteins, like SERCA and RyR. After birth, the antibody against the N-terminus of obscurin stained the M-band while that against the C-terminus stained both M-bands and the Z-disks. Starting from postnatal day 1, ank1.5 was found at the level of both M-bands and Z-disks. Altogether, from these results we infer that exposure of some obscurin epitopes changes during skeletal muscle development, resulting in distinct, antibody-specific, localization pattern. Why this occurs is not clear, yet these data indicate that the organization of obscurin at different locations in the sarcomere changes during muscle development and that this might affect the interaction with ank1.5.

Sarcalumenin alleviates stress-induced cardiac dysfunction by improving Ca2+ handling of the sarcoplasmic reticulum.

AIMS: Sarcalumenin (SAR) is a Ca(2+)-binding protein expressed in the longitudinal sarcoplasmic reticulum (SR) of striated muscle cells. Although its Ca(2+)-binding property is similar to that of calsequestrin, its role in the regulation of Ca(2+) cycling remains unclear. METHODS AND RESULTS: To investigate whether SAR plays an important role in maintaining cardiac function under pressure overload stress, SAR-knockout (SAR-KO) mice were subjected to transverse aortic constriction (TAC). To examine the relation of SAR with cardiac type of SR Ca(2+) pump, SERCA2a, we designed cDNA expression using cultured cells. We found that SAR expression was significantly downregulated in hypertrophic hearts from three independent animal models. SAR-KO mice experienced higher mortality than did wild-type (WT) mice after TAC. TAC significantly downregulated SERCA2a protein but not mRNA in the SAR-KO hearts, whereas it minimally did so in hearts from WT mice. Accordingly, SR Ca(2+) uptake and cardiac function were significantly reduced in SAR-KO mice after TAC. Then we found that SAR was co-immunoprecipitated with SERCA2a in cDNA-transfected HEK293T cells and mouse ventricular muscles, and that SERCA2a-mediated Ca(2+) uptake was augmented when SAR was co-expressed in HEK293T cells. Furthermore, SAR significantly prolonged the half-life of SERCA2a protein in HEK293T cells. CONCLUSION: These findings suggest that functional interaction between SAR and SERCA2a enhances protein stability of SERCA2a and facilitates Ca(2+) sequestration into the SR. Thus the SAR-SERCA2a interaction plays an essential role in preserving cardiac function under biomechanical stresses such as pressure overload.

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.

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 mechanisms of reduced sarcoplasmic reticulum Ca2+ content in L-thyroxin induced rat ventricular hypertrophy.

AIM: To examine how the sarcoplasmic reticulum (SR) Ca2+ content changes and the underlying mechanism in L-thyroxin-induced cardiac hypertrophy. METHODS: Echocardiography was used to confirm the establishment of the cardiac hypertrophy model. The confocal microscopy and fluorescent indicator Fluo-3 was applied to examine the intracellular Ca2+ concentration ([Ca2+]i), the Ca2+ sparks, and the caffeine-induced Ca2+ transient in freshly isolated cardiac ventricular myocytes. The activity of sarcolemmal and SR Ca2+-ATPase 2a (SERCA2a) in the ventricular tissue was also measured, respectively. RESULTS: L-thyroxin (1 mg/kg injection for 10 d) induces left ventricular cardiac hypertrophy with normal myocardial function. The decreased caffeine-induced Ca2+ transient in the Ca2+-free solution was detected. The spontaneous Ca2+ sparks in hypertrophied myocytes occurred more frequently than in normal cells, with similar duration and spatial spread, but smaller amplitude. Then the basal [Ca2+]i increase was observed in quiescent left ventricular myocytes from hyperthyroidism rats. The activity of sarcolemmal and SR Ca2+-ATPase was decreased in the hypertrophied ventricle tissue. CONCLUSION: The results suggested that the reduced SR Ca2+ content may be associated with an increased Ca2+ leak and reduced SERCA2a activity, contributing to abnormal intracellular Ca2+ handling during hypertrophy in hyperthyroidism rats.

Functional, structural and molecular aspects of diastolic heart failure in the diabetic (mRen-2)27 rat.

OBJECTIVE: Diabetic cardiomyopathy is an increasingly recognized cause of cardiac failure despite preserved left ventricular systolic function. Given the over-expression of angiotensin II in human diabetic cardiomyopathy, we hypothesized that combining hyperglycaemia with an enhanced tissue renin-angiotensin system would lead to the development of diastolic dysfunction with adverse remodeling in a rodent model. METHODS: Homozygous (mRen-2)27 rats and non-transgenic Sprague Dawley (SD) rats were randomized to receive streptozotocin (diabetic) or vehicle (non-diabetic) and followed for 6 weeks. Prior to tissue collection, animals underwent pressure-volume loop acquisition. RESULTS: Diabetic Ren-2 rats developed impairment of both active and passive phases of diastole, accompanied by reductions in SERCA-2a ATPase and phospholamban along with activation of the fetal gene program. Structural features of diabetic cardiomyopathy in the Ren-2 rat included interstitial fibrosis, cardiac myocyte hypertrophy and apoptosis in conjunction with increased activity of transforming growth factor-beta (p<0.01 compared with non-diabetic Ren-2 rats for all parameters). No significant functional or structural derangements were observed in non-transgenic, SD diabetic rats. CONCLUSION: These findings indicate that the combination of enhanced tissue renin-angiotensin system and hyperglycaemia lead to the development of diabetic cardiomyopathy. Fibrosis, and myocyte hypertrophy, a prominent feature of this model, may be a consequence of activation of the pro-sclerotic cytokine, transforming growth factor-beta, by the diabetic state.

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulum Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period.

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PRL) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to non-pregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (L3). The expression of the sarcoendoplasmic reticulum Ca(2+)-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in beta-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of P19 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Thyroid hormone attenuates cardiac remodeling and improves hemodynamics early after acute myocardial infarction in rats.

OBJECTIVE: Cardiac remodeling of viable myocardium occurs after acute myocardial infarction (AMI) and further contributes to cardiac dysfunction. The present study explored whether thyroid hormone (TH) administered shortly after AMI in rats can attenuate cardiac remodeling and improve cardiac function. TH regulates important structural and regulatory proteins in the myocardium including myosin isoform expression and calcium cycling proteins. METHODS: AMI was induced in Wistar male rats by ligating left coronary artery (AMI, n=10), while sham-operated rats were used as controls (SHAM, n=10). Animals with acute myocardial infarction were also treated with 0.05% thyroid powder in food (AMI-THYR, n=10). Within 2 weeks, cardiac function was impaired as assessed by echocardiography and under isometric conditions in Langendorff preparations. RESULTS: Ejection fraction (EF%) was 71.5 (SEM, 2.7) in SHAM versus 30.0 (2.0) in AMI, P<0.05. +dp/dt was 3886 (566) in SHAM versus 2266 (206) in AMI hearts, P<0.05 and -dp/dt was 1860 (46) in SHAM versus 1633 (120) in AMI hearts, P=ns. Such changes were associated with alterations in myosin isoform expression in the non-infarcted area; AMI hearts expressed 34% alpha-MHC and 66% beta-MHC versus 52% alpha-MHC and 48% beta-MHC in SHAM, P<0.05, while the expression of SERCA and phospholamban (PLB) remained unchanged. Furthermore, a mismatch of left ventricular size and cardiac mass (2*Posterior Wall thickness/LVIDd was decreased) was observed. After TH treatment, AMI-THYR hearts expressed 71% alpha-MHC and 29% beta-MHC, P<0.05 versus SHAM and AMI and the ratio of SERCA/PLB was increased by 2.0-fold, P<0.05 versus SHAM and AMI. These changes corresponded to a marked improvement in cardiac function; EF% was raised to 45.8 (1.7), P<0.05 versus AMI while +dp/dt and -dp/dt were 3800 (435) and 2600 (200), respectively, in AMI-THYR hearts, P<0.05 versus AMI. The ratio of 2*Posterior Wall thickness/LVIDd was normalized. CONCLUSIONS: Thyroid hormone administration early after infarction attenuates cardiac remodeling and significantly improves myocardial performance.