An improved DNA isolation method and PCR protocol for efficient detection of multicomponents of begomovirus in legumes.

A relatively inexpensive protocol for the detection of genomic components of whitefly-transmitted begomoviruses in symptomatic legumes in the field is described. The method involves extraction with a modified CTAB buffer containing beta-mercaptoethanol upto 5% and sodium chloride concentration from 1.4 to 2.0M. Using this method PCR amplifiable DNA could be extracted from mature leaves of legume hosts rich in polyphenols, tannins and polysaccharides. The non-coding region and full-length DNA A, DNA B components of yellow mosaic viruses were consistently amplifiable from 97 samples, out of 136 tested in PCR reaction, employing primers specific for intergenic regions and full-length genome. The system is robust and the protocol is useful for the detection and identification of begomoviruses infecting grain legumes.

Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration.

The AV promoter expressing AV1 and AV2 genes and AC1 promoter expressing AC1 gene are present in opposite orientation in the intergenic region of Mungbean yellow mosaic India virus (MYMIV). Transient Agrobacterium-mediated delivery of putative promoter constructs into Nicotiana benthamiana and different legumes, followed by reporter gene (beta-d-glucuronidase, GUS) assay, identified the promoter region of both AC1 and AV genes that is necessary for transcriptional initiation. Transcription activator protein-independent activity of AV promoter and differential regulation of AC1 promoter are unique to MYMIV. The AV promoter is a composite core promoter having both TATA box and Initiator elements (TATA(+)Inr(+)). Many transcription factor binding sites were identified in the upstream promoter sequences of both virion and complementary sense genes, which might be used in the transcription regulation studies of the host plant as well as the virus.

Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line.

The kinetics of smooth muscle are thought to be partially determined by the level of the expression of the 7 amino acid insert, SMB, in the myosin heavy chain, as SMB is generally expressed at higher levels in faster smooth muscle. In this study, we determined the role of this insert on shortening velocity and force regeneration following rapid reduction in muscle length (k(step)) in bladder tissue from a transgenic mouse line expressing the insert at three different levels: wild type (WT, +/+, SMB/SMB), an SMA homozygous type (SMB KO, -/-), and a heterozygous type (+/-, SMB/SMA). Smooth muscle from +/+ bladder shorten faster than both the +/- and -/- bladder smooth muscle when activated with Ca2+, consistent with SMB determining the shortening velocity of smooth muscle. The addition of Pi to the fully activated skinned bladder strips did not affect the rate of shortening for either the +/+ or -/- bladder types but did significantly decrease the rate of shortening for the +/- type. In contrast, the addition of ADP to fully Ca2+ activated bladder strips increased the rate of shortening for all three bladder types. However after thiophosphorylation, ADP slowed the shortening velocity. These data are consistent with shortening velocity being determined by the level of activation (or crossbridge attachment) in smooth muscle. The rates of force regeneration according to the k(step) protocol showed no differences between bladder types and also proved insensitive to either Pi or ADP. These data suggest that the rates of force regeneration were determined not only by the kinetics of the crossbridge cycle, but also by factors outside the contractile apparatus.

Replacement of the muscle-specific sarcoplasmic reticulum Ca(2+)-ATPase isoform SERCA2a by the nonmuscle SERCA2b homologue causes mild concentric hypertrophy and impairs contraction-relaxation of the heart.

The cardiac sarco(endo)plasmic reticulum Ca(2+)-ATPase gene (ATP2A2) encodes the following two different protein isoforms: SERCA2a (muscle-specific) and SERCA2b (ubiquitous). We have investigated whether this isoform specificity is required for normal cardiac function. Gene targeting in mice successfully disrupted the splicing mechanism responsible for generating the SERCA2a isoform. Homozygous SERCA2a(-/-) mice displayed a complete loss of SERCA2a mRNA and protein resulting in a switch to the SERCA2b isoform. The expression of SERCA2b mRNA and protein in hearts of SERCA2a(-/-) mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a(-/-) mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a approximately 20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a(-/-) animals (adult phenotype) showed a reduced spontaneous nocturnal activity and developed a mild compensatory concentric cardiac hypertrophy with impaired cardiac contractility and relaxation, but preserved beta-adrenergic response. Ca(2+) uptake levels in SERCA2a(-/-) cardiac homogenates were reduced by approximately 50%. In isolated cells, relaxation and Ca(2+) removal by the SR were significantly reduced. Comparison of our data with those obtained in mice expressing similar cardiac levels of SERCA2a instead of SERCA2b indicate the importance of the muscle-specific SERCA2a isoform for normal cardiac development and for the cardiac contraction-relaxation cycle.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

SERCA pump level is a critical determinant of Ca(2+)homeostasis and cardiac contractility.

The control of intracellular calcium is central to regulation of cardiac contractility. A defect in SR Ca(2+)transport and SR Ca(2+)ATPase pump activity and expression level has been implicated as a major player in cardiac dysfunction. However, a precise cause-effect relationship between alterations in SERCA pump level and cardiac contractility could not be established from these studies. Progress in transgenic mouse technology and adenoviral gene transfer has provided new tools to investigate the role of SERCA pump level in the heart. This review focuses on how alterations in SERCA level affect Ca(2+)homeostasis and cardiac contractility. It discusses the consequences of altered SERCA pump levels for the expression and activity of other Ca(2+)handling proteins. Furthermore, the use of SERCA pump as a therapeutic target for gene therapy of heart failure is evaluated.

Squamous cell tumors in mice heterozygous for a null allele of Atp2a2, encoding the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump.

Mutations in the human ATP2A2 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2), cause Darier disease, an autosomal dominant skin disease characterized by multiple keratotic papules in the seborrheic regions of the body. Mice with a single functional Atp2a2 allele (the mouse homolog of ATP2A2) were shown previously to have reduced levels of SERCA2 in heart and mildly impaired cardiac contractility and relaxation. Here we show that aged heterozygous mutant (Atp2a2(+/-)) mice develop squamous cell tumors of the forestomach, esophagus, oral mucosa, tongue, and skin. Squamous cell tumors occurred in 13/14 Atp2a2(+/-) mice but were not observed in age- and sex-matched wild-type controls. Hyperkeratinized squamous cell papillomas and carcinomas of the upper digestive tract were the most frequent finding among Atp2a2(+/-) mice, and many animals had multiple tumors. Western blot analyses showed that SERCA2 protein levels were reduced in skin and other affected tissues of heterozygous mice. The development of squamous cell tumors in aged Atp2a2(+/-) mice indicates that SERCA2 haploinsufficiency predisposes murine keratinocytes to neoplasia. These findings provide the first direct demonstration that a perturbation of Ca(2+) homeostasis or signaling can serve as a primary initiating event in cancer.

Nitric oxide regulates smooth-muscle-specific myosin heavy chain gene expression at the transcriptional level-possible role of SRF and YY1 through CArG element.

Nitric oxide (NO) plays an important role in vascular regulation through its vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits platelet adhesion and aggregation and modulates smooth muscle cell (SMC) proliferation and migration. In animals with experimentally induced vascular injury, ec-NOS gene transfection not only restored NO production to normal levels but also increased vascular reactivity of the injured vessels. However, it is unclear whether NO regulates smooth-muscle-specific gene expression. We report here that addition of PDGF-BB to vascular smooth muscle cells suppressed SM-MHC expression but treatment with the NO donors FK409 and SNAP restored SM-MHC mRNA/protein expression. In vitro transfection and subsequent CAT assays demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC promoter activity. Promoter deletion analysis revealed that a CArG-3 box located at -1276 bp in the SM-MHC promoter was important for NO-dependent promoter regulation and as well as high level promoter activity. Gel mobility shift assays showed that CArG-3 contained the SRF binding site and a binding site for YY1, a nuclear factor which acts as a negative regulator on muscle-specific promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3 element but increased SRF binding, suggesting that these two factors bind competitively to the overlapping sites. We also found that mutation to the YY1 binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced suppression of the SM-MHC promoter activity. These findings indicate that NO regulates SM-MHC gene expression at the transcriptional level at least partially through the regulation of transcription factor binding activities on the CArG element. Thus we propose that NO plays a positive role in maintaining the differentiated state of VSMCs.

Disruption of a single copy of the SERCA2 gene results in altered Ca2+ homeostasis and cardiomyocyte function.

A mouse model carrying a null mutation in one copy of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform 2 (SERCA2) gene, in which SERCA2 protein levels are reduced by approximately 35%, was used to investigate the effects of decreased SERCA2 level on intracellular Ca(2+) homeostasis and contractile properties in isolated cardiomyocytes. When compared with wild-type controls, SR Ca(2+) stores and Ca(2+) release in myocytes of SERCA2 heterozygous mice were decreased by approximately 40-60% and approximately 30-40%, respectively, and the rate of myocyte shortening and relengthening were each decreased by approximately 40%. However, the rate of Ca(2+) transient decline (tau) was not altered significantly, suggesting that compensation was occurring in the removal of Ca(2+) from the cytosol. Phospholamban, which inhibits SERCA2, was decreased by approximately 40% in heterozygous hearts, and basal phosphorylation of Ser-16 and Thr-17, which relieves the inhibition, was increased approximately 2- and 2.1-fold. These results indicate that reduced expression and increased phosphorylation of phospholamban provides compensation for decreased SERCA2 protein levels in heterozygous heart. Furthermore, both expression and current density of the sarcolemmal Na(+)-Ca(2+) exchanger were up-regulated. These results demonstrate that a decrease in SERCA2 levels can directly modify intracellular Ca(2+) homeostasis and myocyte contractility. However, the resulting deficit is partially compensated by alterations in phospholamban/SERCA2 interactions and by up-regulation of the Na(+)-Ca(2+) exchanger.

Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy.

Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.

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.

Frequency-dependent changes in calcium cycling and contractile activation in SERCA2a transgenic mice.

OBJECTIVE: This study was undertaken to investigate the mechanism of altered contractility in hearts from transgenic mice overexpressing the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2a). In particular, we sought to determine whether the reported increase in contractility is frequency-dependent, as might be expected if attributable to changes in SR Ca2+ loading. METHODS: Intracellular [Ca2+] and contractile force were measured at room temperature (22 degrees C) simultaneously in fura-2-loaded isometrically-contracting trabeculae dissected from the hearts of FVB/N control (n = 6) or SERCA2a transgenic (n = 6) mice. RESULTS: SERCA transgenics exhibit a positive force-frequency relationship, but this was flat in age- and strain-matched controls. SERCA transgenics exhibit a sizable increase in calcium transient amplitude relative to controls, with a concomitant increase in force generation at higher frequencies of stimulation. Amplitudes of Ca2+ transients (transgenics: 1.56 +/- 0.09 micromol/L, controls: 1.21 +/- 0.14) and twitches (transgenics: 21.71 +/- 0.91 mN/mm2, controls: 13.74 +/- 1.67) were significantly different at 2.0 Hz stimulation (P < 0.05). CONCLUSION: An increase in SERCA expression increases the ability of the sarcoplasmic reticulum to store calcium, such that more calcium is available to be released during each heartbeat at higher stimulation rates.

The expression of SR calcium transport ATPase and the Na(+)/Ca(2+)Exchanger are antithetically regulated during mouse cardiac development and in Hypo/hyperthyroidism.

The mouse has been used extensively for generating transgenic animal models to study cardiovascular disease. Recently, a number of transgenic mouse models have been created to investigate the importance of sarcoplasmic reticulum (SR) Ca(2+)transport proteins in cardiac pathophysiology. However, the expression and regulation of cardiac SR Ca(2+)ATPase and other Ca(2+)transport proteins have not been studied in detail in the mouse. In this study, we used multiplex RNase mapping analysis to determine SERCA2, phospholamban (PLB), and Na(+)/Ca(2+)-exchanger (NCX-1) gene expression throughout mouse heart development and in hypo/hyperthyroid animals. Our results demonstrate that the expression of SERCA2 and PLB mRNA increase eight-fold from fetal to adult stages, indicating that SR function increases with heart development. In contrast, the expression of the Na(+)/Ca(2+)-exchanger gene is two-fold higher in fetal heart compared to adult. Our study also makes the important observation that in hypothyroidic hearts the NCX-1 mRNA and protein levels were upregulated, whereas the SERCA2 mRNA/protein levels were downregulated. In hyperthyroidic hearts, however, an opposite response was identified. These findings are important and point out that the expression of NCX-1 is regulated antithetically to that of SERCA2 during heart development and in response to alterations in thyroid hormone levels.

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates.

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.

The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene promoter activity is decreased in response to severe left ventricular pressure-overload hypertrophy in rat hearts.

The sarcoplasmic reticulum Ca2+-ATPase (SERCA2) pump plays a key role in the contraction-relaxation cycle of the myocardium by controlling the intracellular Ca2+ concentration. SERCA2 protein and mRNA expression levels, as well as, SR Ca2+ uptake function are depressed in hypertrophied and failing myocardium. At this time, the molecular mechanisms regulating SERCA2 gene transcription during hypertrophy and heart failure are not completely understood, especially in vivo. Direct gene transfer into adult cardiac tissue has recently been shown to be a useful technique to study in vivo gene regulation. In this study, SERCA2 promoter-luciferase (Luc) reporter constructs of various lengths were injected into the beating left ventricular apex of adult rats (groups = compensated hypertrophy, heart failure, and controls) and the expression level was analysed. Our SERCA2 promoter analyses revealed three positive regulatory regions between -1810 bp and -1110 bp, -658 bp and -284 bp, and -267 bp and -72 bp and a negative regulatory region between -1110 bp and -658 bp, important for in vivo expression in rat hearts. SERCA2 promoter activity was also assessed in rat hearts with compensated pressure-overload hypertrophy (induced by the DOCA-salt treatment) and heart failure (induced by severe ascending aortic constriction). In the DOCA-salt-induced hypertrophy model, SERCA2 promoter activity was similar to that of sham controls. In contrast, severe constriction of the ascending aorta decreased the expression of the -1810 Luc and -1110 Luc constructs by 92.8% and 64.3%, respectively. This study suggests that only severe pressure-overload hypertrophy produces a significant decrease in SERCA2 promoter activity, and the promoter region extending to -1810 bp is sufficient for the down regulation of SERCA2 gene expression.

Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene.

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure.

We tested the hypothesis that activation of protein kinase C (PKC) isoforms in pressure-overload heart failure was prevented by angiotensin-converting enzyme (ACE) inhibition, resulting in normalization of cardiac sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) 2a and phospholamban protein levels and improvement in intracellular Ca2+ handling. Aortic-banded and control guinea pigs were given ramipril (5 mg. kg-1. day-1) or placebo for 8 wk. Ramipril-treated banded animals had lower left ventricular (LV) and lung weight, improved survival, increased isovolumic LV mechanics, and improved cardiomyocyte Ca2+ transients compared with placebo-treated banded animals. This was associated with maintenance of SERCA2a and phospholamban protein expression. Translocation of PKC-alpha and -epsilon was increased in placebo-treated banded guinea pigs compared with controls and was attenuated significantly by treatment with ramipril. We conclude that ACE inhibition attenuates PKC translocation and prevents downregulation of Ca2+ cycling protein expression in pressure-overload hypertrophy. This represents a mechanism for the beneficial effects of this therapy on LV function and survival in heart failure.

SERCA1a can functionally substitute for SERCA2a in the heart.

We recently generated a transgenic (TG) mouse model in which the fast-twitch skeletal muscle sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) is overexpressed in the heart. Ectopic overexpression of SERCA1a results in remodeling of the cardiac SR containing 80% SERCA1a and 20% endogenous SERCA2a with an approximately 2.5-fold increase in the total amount of SERCA protein (E. Loukianov et al. Circ. Res. 83: 889-897, 1998). We have analyzed the Ca2+ transport properties of membranes from SERCA1a TG hearts in comparison to control hearts. Our data show that the maximal velocity of SR Ca2+ transport was significantly increased ( approximately 1.9-fold) in TG hearts, whereas the apparent affinity of the SERCA pump for Ca2+ was not changed. Addition of phospholamban antibody in the Ca2+ uptake assays increased the apparent affinity for Ca2+ to the same extent in TG and non-TG (NTG) hearts, suggesting that phospholamban regulates the SERCA1a pump in TG hearts. Analysis of SERCA enzymatic properties in TG hearts revealed that the SERCA pump affinity for ATP, the Hill coefficient, the pH dependence of Ca2+ uptake, and the effect of acidic pH on Ca2+ transport were similar to those of NTG hearts. Interestingly, the rate constant of phosphoenzyme decay (turnover rate of SERCA enzyme) was also very similar between TG and NTG hearts. Together these findings suggest that 1) the SERCA1a pump can functionally substitute for SERCA2a and is regulated by endogenous phospholamban in the heart and 2) SERCA1a exhibits several enzymatic properties similar to those of SERCA2a when expressed in a cardiac setting.