Structure and organization of the mouse Atp2a2 gene encoding the sarco(endo)plasmic reticulum Ca2+-ATPase 2 (SERCA2) isoforms.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
M Periasamy
Publications and source records attributed to M Periasamy.
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Animals↗
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.
Angiotensin-Converting Enzyme Inhibitors↗
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.
Adenosine Triphosphate↗
Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.
In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.
Animals↗
Gene amplification and transcriptional upregulation of the sarco/endoplasmic reticulum Ca2+ transport ATPase in thapsigargin-resistant hamster smooth muscle cells.
We have selected a series of cell lines from the parental Syrian hamster smooth muscle cell line DDT1-MF2that are resistant to thapsigargin (TG), a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+transport ATPases (SERCAs). Cells were selected for resistance to TG in the presence or absence of cyclosporin (CSA), which is a competitive inhibitor of the multidrug transporter p-glycoprotein (pgp). Since TG is a known substrate for pgp, selection for TG resistance was carried out in the presence of CSA in an attempt to minimize the contribution of pgp, and to identify the potential range of adaptive responses of the SERCA pump itself, during the development of the TG-resistant phenotype. Irrespective of whether the selection is carried out in the presence or absence of CSA, pgp is overexpressed in the TG-resistant DDT1-MF2cells. SERCA protein is also overproduced in the TG-resistant cell lines, which occurs through one of several mechanisms. Included among these, is amplification of the SERCA gene and enhanced transcription of the gene. Enhanced transcription is observed only upon long-term selection and occurs through the SERCA gene proximal promoter elements. Although SERCA transcription in wild-type cells is dependent upon the -284 to -72 bp region of the SERCA promoter, the TG-resistant cells utilize both the -284 to -72 bp and the -72 to +80 bp promoter regions for enhanced SERCA transcription. That is, additional elements within the -72 to +80 bp region are recruited in the TG-resistant cells to allow for increased SERCA expression. A post-transcriptional step may also be recruited by the TG-resistant cells in their overall strategy to produce increased amounts of the SERCA protein. These studies demonstrate that the DDT1-MF2cells can utilize different mechanisms which lead to increased levels of SERCA protein as the cells adapt to inhibition of the ATPase by TG.
Animals↗
Sarco(endo)plasmic reticulum Ca2+ ATPase isoforms and their role in muscle physiology and pathology.
Recent studies suggest that SR Ca2+ transport function is altered in hypertrophied and failing myocardium. To understand whether alterations in SR Ca2+ ATPase levels affect myocardial contractility, we generated transgenic mice that specifically overexpress SERCA2a or SERCA1 pump in the mouse heart, using the cardiac alpha-MHC promoter. Analysis of SERCA2a transgenic mice show both an increase in mRNA and protein levels (120-150% of the wild type). Isolated work performing heart preparations revealed that SERCA2a mice have improved myocardial performance. On the other hand, SERCA1 overexpression in the heart resulted in isoform replacement without any change in total SERCA protein. Interestingly, SERCA1 transgenic hearts exhibited super contractility with a significant increase in rates of muscle contraction (+dp/dt) and relaxation (-dp/dT). The time to peak pressure and half-time to relaxation were significantly shorter.
Animals↗
Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression.
In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.
Animals↗
A novel E box/AT-rich element is required for muscle-specific expression of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene.
The cardiac/slow twitch sarcoplasmic reticulum (SR) Ca2+-ATPase gene (SERCA2 ) encodes a calcium transport pump whose expression is regulated in a tissue- and development-specific manner. Previously we have identified two distinct positive regulatory regions (bp -284 to -72 and -1815 to -1105) as important for SERCA2 promoter activity. Here we demonstrate that the SERCA2 distal promoter region functions like an enhancer by activating a heterologous promoter (TK) in a muscle cell-specific manner. Through deletion analysis a core enhancer region was delimited to the -1467 to -1105 bp fragment. We identified the E box/AT-rich element located at -1115 bp as critical for maximal enhancer activity. Gel mobility shift studies revealed that this E box/AT-rich element specifically binds a protein which is induced during Sol8 myogenesis. This region includes two other cis -acting elements, CArG and MCAT, which also bind specific nuclear protein complexes from Sol8 myotubes. Mutagenesis of each of these sites resulted in decreased SERCA/TK-CAT promoter activity. Based on these data, we propose that the E box/AT-rich element may contribute along with CArG and MCAT elements to the overall activation and regulation of the SERCA2 gene promoter.
3T3 Cells↗
MEF2B is a component of a smooth muscle-specific complex that binds an A/T-rich element important for smooth muscle myosin heavy chain gene expression.
To understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a smooth muscle-specific marker. In this study, we demonstrate that the SMHC promoter region (-1594 to -1462 base pairs) containing the A/T-rich element can activate the heterologous thymidine kinase promoter in smooth muscle cells, but not in fibroblasts. Mutations of this A/T-rich element decreased SMHC promoter activity significantly. Both gel mobility shift assays and DNase I footprinting revealed that this region binds to specific protein complexes from smooth muscle nuclear extracts, whereas nuclear extracts from skeletal muscle and fibroblasts produced a different binding pattern. We also demonstrate that the protein complex obtained from smooth muscle nuclear extract reacts with MEF2B-specific antibody, but not with antibodies specific to MEF2A, MEF2C, or MEF2D, suggesting that only MEF2B protein binds to the A/T-rich element. Furthermore, MEF2B overexpression in smooth muscle cells up-regulated the SMHC promoter, suggesting that MEF2B is important for SMHC gene regulation. This is the first report demonstrating a role for MEF2 factors in smooth muscle-specific gene expression.
3T3 Cells↗
Myosin heavy chain isoform expression in rat smooth muscle development.
Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.
Aging↗
Myosin heavy chain isoforms in smooth muscle.
In recent years, significant progress has been made toward understanding smooth muscle myosin heavy chain (SMHC) structure. Molecular cloning analysis has identified four different MHC isoforms. They are products of a single gene and result from alternative mRNA splicing. In addition, two non-muscle MHC isoforms are also expressed in smooth muscle cells. Studies show that SMHC expression is highly tissue specific and does not appear in cardiac or skeletal muscle cells. Each smooth muscle tissue is characterized by a specific pattern of MHC isoform expression that changes during development and disease. This review essentially focuses on SMHC isoforms and their expression in mammals.
Alternative Splicing↗
Cloning DNA fragments between two adjacent/overlapping restriction sites using a "positive stuffer".
Here we describe a solution to a common problem encountered in recombinant DNA cloning when directional cloning of a DNA fragment into a predetermined plasmid requires the use of restriction enzymes with adjacent or overlapping recognition sites. In preparing the double-digested plasmid, only one enzyme will often cut, whereas the second will not because of the lack of a sufficiently long stretch of double-stranded DNA at its recognition site. The problem can be solved by construction of a "user-friendly" intermediary plasmid in which the desired restriction sites are separated by a positively selectable stuffer with resistance to neomycin. This approach is particularly useful in cases where the choices of restriction sites are severely limited, for example, when it is necessary to clone an additional piece of DNA into a complex vector already containing multiple gene cassettes.
Binding Sites↗
Efficient cloning method that selects the recombinant clones.
Directional cloning using cohesive ends is the most efficient cloning method. However, sometimes it is necessary to use blunt ends to clone a DNA fragment into the plasmid vector. Compared with that of cohesive ends, efficiency of blunt-end ligation is low. Compared with the native blunt ends (e.g., SmaI or EcoRV), blunt-end ligation is particularly difficult when blunt ends are derived from overhangs. This results in low efficiency of insertion and high background from self-ligation of the vector. To remedy the problem, we developed a "positive selector" cloning strategy that provides positive selection for the recombinant clones. It is particularly useful when making complex recombinant constructs and the choice of restriction sites is limited.
Cloning, Molecular↗
Identification of targeted embryonic stem cells using long-distance PCR.
Explore the source record for details and available documents.
Animals↗