Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle

We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1beta may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1beta on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1beta (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1beta significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1beta-treated cells. IL-1beta modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.     

Expression of estrogen receptors alpha and beta in term human myometrium

We used immunohistochemistry to compare the expression of estrogen receptors (ERalpha and ERbeta) in term myometria of 32 pregnant women divided in two groups. Group I comprised of 16 women in labour and group II included 16 non-laboring gravidas. We observed cytoplasmatic localization of both ER isoforms and no differences in the ER expression between the two groups of patients. The abundance and specific localization of ERs in human term myometrium seems to be independent of its contractile activity which may point to the specific role of those receptors in late pregnancy myometrium.     

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.     

Presence of SERCA and calcineurin during fetal development of porcine skeletal muscle.

Mechanisms involved in skeletal myofiber differentiation during fetal development of large animals are poorly understood. Studies in small animals suggest that the calcineurin (Cn) pathway is involved in myofiber differentiation. Neural activity is a prerequisite for Cn activity, implying maintenance of sustained low intracellular Ca(2+) concentrations. To study the role of Cn in fetal myofiber differentiation, we monitored the temporal and spatial distribution of Cn subunits, sarcoplasmic reticulum Ca(2+) ATPase (SERCA), phospholamban (PLB), and myosin heavy chain (MyHC) isoforms in relation to ingrowing nerves in porcine semitendinosus muscle (m. semitendinosus) at 55 and 75 days of gestation (dg) and at term. Immunofluorescence analysis revealed the presence of Cn subunits and SERCA isoforms at all analyzed stages. Cn distribution was not fiber-type specific, but expression became more prominent at term. At 75 dg, differential SERCA2 expression was accompanied by perinuclear PLB in primary fibers. SERCA1 was expressed in all fiber types at all stages. No specific MyHC isoform distribution was seen in relation to neuromuscular contacts, although neuromuscular contacts were present. From these results we speculate that in porcine m. semitendinosus differential SERCA2 expression precedes differential Cn expression. The question whether the Cn pathway is involved in prenatal myofiber differentiation needs further studies.     

Functional comparisons between isoforms of the sarcoplasmic or endoplasmic reticulum family of calcium pumps.

ATP-dependent calcium pumps that reside in intracellular organelles are encoded by a family of structurally related enzymes, termed the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPases (SERCA), which each have a distinct pattern of tissue-specific and developmentally regulated expression. A COS-1 cell expression system was used to examine the biochemical properties of the isoforms: SERCA1 (fast-twitch skeletal muscle). SERCA2a (cardiac/slow-twitch skeletal muscle), SERCA2b (ubiquitous smooth- and non-muscle), and SERCA3 (non-muscle). Each isoform was expressed efficiently and appeared to be targeted to the endoplasmic reticulum. All isoforms displayed qualitatively similar enzymatic properties and were activated by calcium in a cooperative manner with a Hill coefficient of 2. The quantitative properties of SERCA1 and SERCA2a (the muscle isoforms) were identical in all respects. SERCA2b, however, appeared to have a lower turnover rate for both calcium transport and ATP hydrolysis. SERCA3 displayed a reduced apparent affinity for calcium, an increased apparent affinity for vanadate, and an altered pH dependence when compared with the other isoforms. These properties are consistent with an enzyme in which the equilibrium between the E1 and E2 conformations is shifted toward the E2 state.     

Conditional increase in SERCA2a protein is able to reverse contractile dysfunction and abnormal calcium flux in established diabetic cardiomyopathy

Diabetic cardiomyopathy is characterized by reduced cardiac contractility independent of vascular disease. A contributor to contractile dysfunction in the diabetic heart is impaired sarcoplasmic reticulum function with reduced sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) pump activity, leading to disturbed intracellular calcium handling. It is currently unclear whether increasing SERCA2a activity in hearts with existing diabetic cardiomyopathy could still improve calcium flux and contractile performance. To test this hypothesis, we generated a cardiac-specific tetracycline-inducible double transgenic mouse, which allows for doxycycline (DOX)-based inducible SERCA2a expression in which DOX exposure turns on SERCA2a expression. Isolated cardiomyocytes and Langendorff perfused hearts from streptozotocin-induced diabetic mice were studied. Our results show that total SERCA2a protein levels were decreased in the diabetic mice by 60% compared with control. SERCA2a increased above control values in the diabetic mice after DOX. Dysfunctional contractility in the diabetic cardiomyocyte was restored to normal by induction of SERCA2a expression. Calcium transients from diabetic cardiomyocytes showed a delayed rate of diastolic calcium decay of 66%, which was reverted toward normal after SERCA2a expression induced by DOX. Global cardiac function assessed in the diabetic perfused heart showed diminished left ventricular pressure, rate of contraction, and relaxation. These parameters were returned to control values by SERCA2a expression. In conclusion, we have used mice allowing for inducible expression of SERCA2a and could demonstrate that increased expression of SERCA2a leads to improved cardiac function in mice with an already established diabetic cardiomyopathy in absence of detrimental effects.     

Sarco(endo)plasmic reticulum calcium pump isoforms in paralyzed rat slow muscle

To assess the influence of paralysis on the expression of phenotypic protein isoforms related to muscle relaxation, the effects of spinal cord transection (ST) on sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump isoform protein levels in the slow rat soleus were measured. Western blotting using SERCA isoform specific antibodies demonstrated a rapid up-regulation (7 days post ST) of the fast fiber type-specific isoform (SERCA1). In contrast, the slow fiber type-specific isoform, SERCA2, was decreased with a slower time-course. The up-regulation of SERCA1 protein preceded the up-regulation of fast myosin heavy chain (MyHC) (i.e., MyHC-II). Immunohistochemical analyses of single muscle fibers showed that 15 days after ST there was a pronounced increase in the proportion of slow MyHC fibers with SERCA1 confirming that SERCA1 was up-regulated in the slow fibers of the soleus prior to MyHC-II. These data suggest that the expression of the SERCA isoforms (particularly SERCA1) may serve as more sensitive markers of phenotypic adaptation in response to altered levels of contractile activity than the MyHC isoforms. In addition, since the expression of SERCA isoforms was dissociated from MyHC isoforms, regulation of gene expression for these two different protein systems must involve different signaling events and/or synthetic processes.     

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.     

Down-regulation of the alpha- and beta-subunits of the calcium-activated potassium channel in human myometrium with parturition

Large-conductance, calcium-dependent potassium (BKCa) channels are implicated in maintaining uterine quiescence during pregnancy. The mechanisms whereby calcium sensitivity of the BKCa channel is dramatically removed at parturition remain unknown. The aim of the present study was to investigate whether this loss of calcium sensitivity of the BKCa channel with the onset of labor is associated with changes in the protein expression of the alpha- and/or beta-subunit or arises from a physical dissociation of the alpha-subunit from the beta-subunit. The beta-subunit is a key determinant of BKCa-channel Ca2+ sensitivity. Western blot analysis, using alpha- and beta-subunit-specific antibodies, detected bands of 110-125 and 36 kDa, respectively. Protein expression levels of the alpha-subunit in term labor myometrium were significantly reduced compared with term pregnancy without labor. Furthermore, alpha-subunit levels at term pregnancy were significantly increased relative to the nonpregnant state, whereas levels at preterm gestations were unchanged. Densitometric analysis demonstrated significantly decreased beta-subunit levels in term and preterm labor samples compared with term nonlabor samples. Immunoprecipitation studies revealed the presence of both the alpha- and beta-subunits in samples taken before or after the onset of labor. We conclude that during labor, the alpha-subunit is not physically uncoupled from the beta-subunit, but a decline occurs in the level of beta-subunit protein, which may underlie the loss of calcium and voltage sensitivity of the BKCa channel with labor. Furthermore, reduced beta-subunit protein in preterm labor myometrium implies that ion channels may also contribute to pathophysiological labor.     

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.     

Doxycycline inducible expression of SERCA2a improves calcium handling and reverts cardiac dysfunction in pressure overload-induced cardiac hypertrophy

Delayed cardiac relaxation in failing hearts has been attributed to reduced activity and/or expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a). Although constitutive overexpression of SERCA2a has proven effective in preventing cardiac dysfunction, it is unclear whether increasing SERCA2a expression in hearts with preexisting hypertrophy will be therapeutic. To test this hypothesis, we generated a binary transgenic (BTG) system that allows tetracycline-inducible, cardiac-specific SERCA2a expression. In this system (tet-on SERCA2a), a FLAG-tagged SERCA2a transgene is expressed in the presence of doxycycline (Dox) but not in the absence of Dox (2.3-fold more mRNA, 45% more SERCA2a protein). Calcium transients measured in isolated cardiac myocytes from nonbanded Dox-treated BTG mice showed an accelerated calcium decline and an increased systolic Ca2+ peak. Sarcoplasmic reticulum (SR) calcium loading was increased by 45% in BTG mice. In the presence of pressure overload (aortic banding), echocardiographic analysis revealed that expression of SERCA2a-FLAG caused an improvement in fractional shortening. SERCA2a-FLAG expression alleviated the resultant cardiac dysfunction. This was illustrated by an increase in the rate of decline of the calcium transient. Cell shortening and SR calcium loading were also improved in cardiac myocytes isolated from banded BTG mice after SERCA2a overexpression. In conclusion, we generated a novel transgenic mouse that conditionally overexpresses SERCA2a. This model is suitable for both long- and short-term studies of the effects of controlled SERCA2a expression on cardiac function. In addition, inducible overexpression of SERCA2a improved cardiac function and calcium handling in mice with established contractile dysfunction.     

SERCA pump activity is physiologically regulated by presenilin and regulates amyloid beta production

In addition to disrupting the regulated intramembraneous proteolysis of key substrates, mutations in the presenilins also alter calcium homeostasis, but the mechanism linking presenilins and calcium regulation is unresolved. At rest, cytosolic Ca(2+) is maintained at low levels by pumping Ca(2+) into stores in the endoplasmic reticulum (ER) via the sarco ER Ca(2+)-ATPase (SERCA) pumps. We show that SERCA activity is diminished in fibroblasts lacking both PS1 and PS2 genes, despite elevated SERCA2b steady-state levels, and we show that presenilins and SERCA physically interact. Enhancing presenilin levels in Xenopus laevis oocytes accelerates clearance of cytosolic Ca(2+), whereas higher levels of SERCA2b phenocopy PS1 overexpression, accelerating Ca(2+) clearance and exaggerating inositol 1,4,5-trisphosphate-mediated Ca(2+) liberation. The critical role that SERCA2b plays in the pathogenesis of Alzheimer's disease is underscored by our findings that modulating SERCA activity alters amyloid beta production. Our results point to a physiological role for the presenilins in Ca(2+) signaling via regulation of the SERCA pump.     

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.     

Expression of Stretch-Activated Two-Pore Potassium Channels in Human Myometrium in Pregnancy and Labor

Background

We tested the hypothesis that the stretch-activated, four-transmembrane domain, two pore potassium channels (K2P), TREK-1 and TRAAK are gestationally-regulated in human myometrium and contribute to uterine relaxation during pregnancy until labor.

Methodology

We determined the gene and protein expression of K2P channels in non-pregnant, pregnant term and preterm laboring myometrium. We employed both molecular biological and functional studies of K2P channels in myometrial samples taken from women undergoing cesarean delivery of a fetus.

Principal Findings

TREK-1, but not TREK-2, channels are expressed in human myometrium and significantly up-regulated during pregnancy. Down-regulation of TREK-1 message was seen by Q-PCR in laboring tissues consistent with a role for TREK-1 in maintaining uterine quiescence prior to labor. The TRAAK channel was unregulated in the same women. Blockade of stretch-activated channels with a channel non-specific tarantula toxin (GsMTx-4) or the more specific TREK-1 antagonist L-methionine ethyl ester altered contractile frequency in a dose-dependent manner in pregnant myometrium. Arachidonic acid treatment lowered contractile tension an effect blocked by fluphenazine. Functional studies are consistent with a role for TREK-1 in uterine quiescence.

Conclusions

We provide evidence supporting a role for TREK-1 in contributing to uterine quiescence during gestation and hypothesize that dysregulation of this mechanism may underlie certain cases of spontaneous pre-term birth.     

Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.     

Up-regulation of myometrial RHO effector proteins (PKN1 and DIAPH1) and CPI-17 (PPP1R14A) phosphorylation in human pregnancy is associated with increased GTP-RHOA in spontaneous preterm labor

RHO GTP-binding proteins are important regulators of actin-myosin interactions in uterine smooth muscle cells. Active (GTP-bound) RHOA binds to RHO-associated protein kinase (ROCK1), which inhibits the myosin-binding subunit (PPP1R12A) of myosin light chain phosphatase, leading to calcium-independent increases in myosin light chain phosphorylation and tension, which are termed "calcium sensitization." The RHO effector protein kinase N (PKN1) also increases calcium sensitization by phosphorylating the protein kinase C (PRKCB)-dependent protein CPI-17 (PPP1R14A) to inhibit the PPP1c subunit of myosin phosphatase. Moreover, other RHO proteins, such as RHOB, RHOD, and their effectors (DIAPH1 and DIAPH2), may modulate PKN1/ ROCK1 signaling to effect changes in myosin phosphatase activity and myosin light chain phosphorylation. The increases in contractile activity observed in term and preterm labor may be due to an increase in RHO activity and/or changes in RHO-related proteins. We found that the RHOA and RHOB mRNA levels in the myometrium were increased in pregnancy, although the expression levels of the RHOA and RHOB proteins did not change with pregnancy or labor. GTP-bound RHOA was increased in pregnancy, and this increase was significant in spontaneous preterm labor myometrium. PKN1 expression and PPP1R14A phosphorylation were dramatically increased in the pregnant myometrium. We also observed increases in DIAPH1 expression in spontaneous term and preterm labor myometrial tissues. The present study shows that human pregnancy is characterized by increases in PKN1 expression and PPP1R14A phosphorylation in the myometrium. Moreover, increases in GTP-bound RHOA and DIAPH1 expression may contribute to the increase in uterine activity in idiopathic preterm labor.     

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.