Tight interplay between the Ca2+ affinity of the cardiac SERCA2 Ca2+ pump and the SERCA2 expression level

A reduced activity of the sarcoplasmic reticulum Ca2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2b/b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy. Here, the interplay was studied between the increased Ca2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2b/b mice were crossed with SERCA2b transgenes to boost cardiac SERCA2b expression. However, the enforced expression of SERCA2b was spontaneously countered by an increased inhibition by phospholamban (PLB), reducing the pump's Ca2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2b/WT mice, which also express lower SERCA2 levels compared to wild-type. Hypertrophy was not observed. In heterozygotes, SERCA2b expression was specifically suppressed, explaining the reduced SERCA2 content. The SERCA2b/WT model strikingly differs from the homozygote models because SERCA2a (not SERCA2b) is the major isoform and because the inhibition of the pump by PLB is decreased instead of being increased. Thus, a tight correlation exists between the SERCA2 levels and Ca2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.     

Improving cardiac Ca⁺² transport into the sarcoplasmic reticulum in heart failure: lessons from the ubiquitous SERCA2b Ca⁺² pump

As a major Ca2+ pump in the sarcoplasmic reticulum of the cardiomyocyte, SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) controls the relaxation and contraction of the cardiomyocyte. It is meticulously regulated by adapting its expression levels and affinity for Ca2+ ions to the physiological demand of the heart. Dysregulation of the SERCA2a activity entails poor cardiomyocyte contractility, resulting in heart failure. Conversely, improving cardiac SERCA2a activity, e.g. by boosting its expression level or by increasing its affinity for Ca2+, is a promising strategy to rescue contractile dysfunction of the failing heart. The structures of the related SERCA1a Ca2+ pump and the Na+/K+-ATPase of the plasma membrane exposed the pumping mechanism and conserved domain architecture of these ion pumps. However, how the Ca2+ affinity of SERCA2a is regulated at the molecular level remained unclear. A structural and functional analysis of the closely related SERCA2b Ca2+ pump, i.e. the housekeeping Ca2+ pump found in the endoplasmic reticulum and the only SERCA isoform characterized by a high Ca2+ affinity, aimed to fill this gap. We demonstrated the existence of a novel and highly conserved site on the SERCA2 pump mediating Ca2+ affinity regulation by the unique C-terminus of SERCA2b (2b-tail). It differs from the earlier-described target site of the affinity regulator phospholamban. Targeting this novel site may provide a new approach to improve SERCA2a function in the failing heart. Strikingly, the intramembrane interaction site of the 2b-tail in SERCA2b shares sequence and structural homology with the binding site of the β-subunit on the α Na+/K+-ATPase. Thus P-type ATPases seem to have developed related mechanisms of regulation, and it is a future challenge for us to discover these general principles of P-type regulation.     

Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase.

Chronic excitation, at 2 Hz for 6-7 weeks, of the predominantly fast-twitch canine latissimus dorsi muscle promoted the expression of phospholamban, a protein found in sarcoplasmic reticulum (SR) from slow-twitch and cardiac muscle but not in fast-twitch muscle. At the same time that phospholamban was expressed, there was a switch from the fast-twitch (SERCA1) to the slow-twitch (SERCA2a) Ca(2+)-ATPase isoform. Antibodies against Ca(2+)-ATPase (SERCA2a) and phospholamban were used to assess the relative amounts of the slow-twitch/cardiac isoform of the Ca(2+)-ATPase and phospholamban, which were found to be virtually the same in SR vesicles from the slow-twitch muscle, vastus intermedius; cardiac muscle; and the chronically stimulated fast-twitch muscle, latissimus dorsi. The phospholamban monoclonal antibody 2D12 was added to SR vesicles to evaluate the regulatory effect of phospholamban on calcium uptake. The antibody produced a strong stimulation of calcium uptake into cardiac SR vesicles, by increasing the apparent affinity of the Ca2+ pump for calcium by 2.8-fold. In the SR from the conditioned latissimus dorsi, however, the phospholamban antibody produced only a marginal effect on Ca2+ pump calcium affinity. These different effects of phospholamban on calcium uptake suggest that phospholamban is not tightly coupled to the Ca(2+)-ATPase in SR vesicles from slow-twitch muscles and that phospholamban may have some other function in slow-twitch and chronically stimulated fast-twitch muscle.     

Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.     

Modulating sarco(endo)plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological implications

Of the three mammalian members belonging to the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) family, SERCA2 is evolutionary the oldest and shows the most wide tissue-expression pattern. Two major SERCA2 splice variants are well-characterized: the muscle-specific isoform SERCA2a and the housekeeping isoform SERCA2b. Recently, several interacting proteins and post-translational modifications of SERCA2 were identified which may modulate the activity of the Ca2+ pump. This review aims to give an overview of the vast literature concerning the cell biological implications of the SERCA2 isoform diversity and the factors regulating SERCA2. Proteins reported to interact with SERCA2 from the cytosolic domain involve the anti-apoptotic Bcl-2, the insulin receptor substrates IRS1/2, the EF-hand Ca2+-binding protein S100A1 and acylphosphatase. We will focus on the very particular position of SERCA2 as an enzyme functioning in a thin, highly fluid, leaky and cholesterol-poor membrane. Possible differential interactions of SERCA2b and SERCA2a with calreticulin, calnexin and ERp57, which could occur within the lumen of the endoplasmic reticulum will be discussed. Reported post-translational modifications possibly affecting pump activity involve N-glycosylation, glutathionylation and Ca2+/calmodulin kinase II-dependent phosphorylation. Finally, the pronounced vulnerability to oxidative damage of SERCA2 appears to be pivotal in the etiology of various pathologies.     

Ca2+-ATPases in non-failing and failing heart: evidence for a novel cardiac sarco/endoplasmic reticulum Ca2+-ATPase 2 isoform (SERCA2c)

We recently documented the expression of a novel human mRNA variant encoding a yet uncharacterized SERCA [SR (sarcoplasmic reticulum)/ER (endoplasmic reticulum) Ca2+-ATPase] protein, SERCA2c [Gélébart, Martin, Enouf and Papp (2003) Biochem. Biophys. Res. Commun. 303, 676-684]. In the present study, we have analysed the expression and functional characteristics of SERCA2c relative to SERCA2a and SERCA2b isoforms upon their stable heterologous expression in HEK-293 cells (human embryonic kidney 293 cells). All SERCA2 proteins induced an increased Ca2+ content in the ER of intact transfected cells. In microsomes prepared from transfected cells, SERCA2c showed a lower apparent affinity for cytosolic Ca2+ than SERCA2a and a catalytic turnover rate similar to SERCA2b. We further demonstrated the expression of the endogenous SERCA2c protein in protein lysates isolated from heart left ventricles using a newly generated SERCA2c-specific antibody. Relative to the known uniform distribution of SERCA2a and SERCA2b in cardiomyocytes of the left ventricle tissue, SERCA2c was only detected in a confined area of cardiomyocytes, in close proximity to the sarcolemma. This finding led us to explore the expression of the presently known cardiac Ca2+-ATPase isoforms in heart failure. Comparative expression of SERCAs and PMCAs (plasma-membrane Ca2+-ATPases) was performed in four nonfailing hearts and five failing hearts displaying mixed cardiomyopathy and idiopathic dilated cardiomyopathies. Relative to normal subjects, cardiomyopathic patients express more PMCAs than SERCA2 proteins. Interestingly, SERCA2c expression was significantly increased (166+/-26%) in one patient. Taken together, these results demonstrate the expression of the novel SERCA2c isoform in the heart and may point to a still unrecognized role of PMCAs in cardiomyopathies.     

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

Multiple effects of SERCA2b mutations associated with Darier's disease

Darier's disease (DD) is an autosomal dominant disorder caused by mutations in the ATP2A2 gene, encoding sarco/endoplasmic reticulum Ca2+-ATPase pump type 2b isoform (SERCA2b). Although >100 mutations in the ATP2A2 gene were identified, no apparent relation between genotype/phenotype emerged. In this work, we analyzed 12 DD-associated mutations from all of the regions of SERCA2b to study the underlying pathologic mechanism of DD and to elucidate the role of dimerization in SERCA2b activity. Most mutations markedly affected protein expression, partially because of enhanced proteasome-mediated degradation. All of the mutants showed lower activity than the wild type pump. Notably, several mutants that cause relatively severe phenotype of DD inhibited the activity of the endogenous and the co-expressed wild type SERCA2b. Importantly, these effects were not attributed to changes in passive Ca2+ leak, inositol 1,4,5-trisphosphate receptor activity, or sensitivity to inositol 1,4,5-trisphosphate. Rather, co-immunoprecipitation experiments showed that SERCA2b monomers interact to influence the activity of each other. These findings reveal multiple molecular mechanisms to account for the plethora of pathologic states observed in DD and provide the first evidence for the importance of SERCA2b dimerization in pump function in vivo.     

Three novel sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 3 isoforms. Expression, regulation, and function of the membranes of the SERCA3 family

Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a-c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a-e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.     

Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT-PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+ pools may play distinct roles in cell proliferation and differentiation.     

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach

Heart tissue contains large amounts of the protein encoded by the Ca²⁺ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms byin situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca²⁺ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca²⁺ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.Deceased August 14, 1992     

Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 (2000) 24722]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca2+ handling observed in SERCA2(b/b), we wanted to compare SERCA2b distribution in SERCA2(b/b) with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2(b/b) mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca2+ handling and the development of hypertrophy in adult SERCA2(b/b) mice do not result from different SERCA2b targeting.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 2 isoforms and characterization of Darier disease (SERCA2) mutants by steady-state and transient kinetic analyses

Steady-state and rapid kinetic studies were conducted to functionally characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms, SERCA2a and SERCA2b, and 10 Darier disease (DD) mutants upon heterologous expression in HEK-293 cells. SERCA2b displayed a 10-fold decrease in the rate of Ca2+ dissociation from E1Ca2 relative to SERCA2a (i.e. SERCA2b enzyme manifests true high affinity at cytosolic Ca2+ sites) and a lower rate of dephosphorylation. These fundamental kinetic differences explain the increased apparent affinity for activation by cytosolic Ca2+ and the reduced catalytic turnover rate in SERCA2b. Relative to SERCA1a, both SERCA2 isoforms displayed a 2-fold decrease of the rate of E2 to E1Ca2 transition. Furthermore, seven DD mutants were expressed at similar levels as wild type. The expression level was 2-fold reduced for Gly23 --> Glu and Ser920 --> Tyr and 10-fold reduced for Gly749 --> Arg. Uncoupling between Ca2+ translocation and ATP hydrolysis and/or changes in the rates of partial reactions account for lack of function for 7 of 10 mutants: Gly23 --> Glu (uncoupling), Ser186 --> Phe, Pro602 --> Leu, and Asp702 --> Asn (block of E1 approximately P(Ca2) to E2-P transition), Cys318 --> Arg (uncoupling and 3-fold reduction of E2-P to E2 transition rate), and Thr357 --> Lys and Gly769 --> Arg (lack of phosphorylation). A 2-fold decrease in the E1 approximately P(Ca2) to E2-P transition rate is responsible for the 2-fold decrease in activity for Pro895 --> Leu. Ser920 --> Tyr is a unique DD mutant showing an enhanced molecular Ca2+ transport activity relative to wild-type SERCA2b. In this case, the disease may be a consequence of the low expression level and/or reduction of Ca2+ affinity and sensitivity to inhibition by lumenal Ca2+.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves.

Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.