Muscle-specific transcriptional regulation of the cardiac/slow-twitch SERCA2 gene (original) (raw)

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Abstract

1 The abbreviations used are: bp, base pair(s); tss, transcription start site; DLM, dorsal longitudinal muscle; DVM, dorso-ventral muscle; dcm, direct control muscles, specifically the basalare and pterale I and II; TT, tergotrochanter muscle; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indolyl ␤-D-galactopyranoside.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes

Molecular and Cellular Biochemistry, 2017

The cardiac sarco/endoplasmic reticulum Ca 2+-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position −78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing −254 and −2579 bp of 5′-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes

Cardiovascular Research, 2004

Objective: Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. Methods: SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca 2 +-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. Results: Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (À 40%) and KN-93 (À 20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were cotransfected. Conclusion: Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways.

SERCA is critical to control the Bowditch effect in the heart

Scientific reports, 2018

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCA induced positive staircase and arrhythmia, while SERCA accentuated the negative staircase of wild type. At the stimulation frequencies tested, no sig...

Transcriptional changes following restoration of SERCA2a levels in failing rat hearts

The FASEB Journal, 2004

Heart failure is characterized at the cellular level by impaired contractility and abnormal Ca 2+ homeostasis. We have previously shown that restoration of a key enzyme that controls intracellular Ca 2+ handling, the sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a), induces functional improvement in heart failure. We used high-density oligonucleotide arrays to explore the effects of gene transfer of SERCA2a on genetic reprogramming in a model of heart failure. A total of 1,300 transcripts were identified to be unmodified by the effect of virus alone. Of those, 251 transcripts were found to be up-or down-regulated upon failure. A total of 51 transcripts which were either up-(27) or down-(24) regulated in heart failure were normalized to the nonfailing levels by the restoration of SERCA2a by gene transfer. The microarray analysis identified new genes following SERCA2a restoration in heart failure, which will give us insights into their role in the normalization of multiple pathways within the failing cell.

Replacement of the Muscle-Specific Sarcoplasmic Reticulum Ca21ATPase Isoform SERCA2a by the Nonmuscle SERCA2b Homologue Causes Mild Concentric Hypertrophy and Impairs Contraction-Relaxation of the Heart

2000

The cardiac sarco(endo)plasmic reticulum Ca 2ϩ -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 Ϫ/Ϫ 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 Ϫ/Ϫ mice corresponded to only 50% of wild-type SERCA2 levels. Cardiac phospholamban mRNA levels were unaltered in SERCA2a Ϫ/Ϫ mice, but total phospholamban protein levels increased 2-fold. The transgenic phenotype was characterized by a Ϸ20% increase in embryonic and neonatal mortality (early phenotype), with histopathologic evidence of major cardiac malformations. Adult SERCA2a Ϫ/Ϫ 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 ␤-adrenergic response. Ca 2ϩ uptake levels in SERCA2a Ϫ/Ϫ cardiac homogenates were reduced by Ϸ50%. In isolated cells, relaxation and Ca 2ϩ 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. (Circ Res. 2001;89:838-846.)

Modulation of SERCA: implications for the failing human heart

Basic Research in Cardiology, 2002

Human heart failure is characterized by distinct alterations in the intracellular homeostasis and key regulators of the sarcoplasmic reticulum Ca 2+ sequestration mechanisms. Systolic peak Ca 2+ is reduced, diastolic Ca 2+ levels are increased and diastolic Ca 2+ decay is prolonged. Recently specific changes in the expression, function and modulation of SR Ca 2+ -ATPase (SERCA) have been elucidated. As such, in a variety of studies SERCA expression appeared to be decreased in the failing human heart, although these findings have been discussed controversially depending on the studied tissue, especially with respect to the non-failing samples and regional variation in the obtained samples. However, consistent findings of a diminished Ca 2+ dependent SERCA activation were found. Increasing evidence has been provided that one of the underlying mechanisms for a decreased activation of SERCA is its altered regulation. With respect to this, the modulations through phospholamban and Ca 2+ -dependent protein kinase II (CaMK II) play a detrimental role in regulating SERCA function. Phospholamban phosphorylation of SERCA at the serine-16 and threonine-17 site is diminished in human heart failure resulting in decreases in the apparent affinity for Ca 2+ of the SR Ca 2+ uptake rates. In contrast, activation of CaMK II leads to an increased maximal velocity of SR Ca 2+ sequestration that may enhance SR Ca 2+ -load. Additional regulation has been recently elucidated by changes in the apparent coupling ratio of Ca 2+ transported per ATP hydrolysed. This review summarizes recent advances in the understanding how SERCA is modulated under physiological and pathophysiological conditions.

Impaired Cardiac Performance in Heterozygous Mice with a Null Mutation in the Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2 (SERCA2) Gene

Journal of Biological Chemistry, 1999

The sarco(endo)plasmic reticulum Ca 2؉-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca 2؉ 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 ϳ45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca 2؉ uptake into the sarcoplasmic reticulum were reduced by ϳ35%. 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 Ca 2؉ sequestering activity, and that the deficit in Ca 2؉ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Slowed relaxation and preserved maximal force in soleus muscles of mice with targeted disruption of the Serca2 gene in skeletal muscle

The Journal of physiology, 2011

Sarcoplasmic reticulum Ca2+ ATPases (SERCAs) play a major role in muscle contractility by pumping Ca2+ from the cytosol into the sarcoplasmic reticulum(SR) Ca2+ store, allowing muscle relaxation and refilling of the SRwith releasableCa2+.Decreased SERCAfunction has been shown to result in impaired muscle function and disease in human and animal models. In this study,we present a newmouse modelwith targeted disruption of the Serca2 gene in skeletal muscle (skKO) to investigate the functional consequences of reduced SERCA2 expression in skeletal muscle. SkKO mice were viable and basic muscle structure was intact. SERCA2 abundance was reduced inmultiplemuscles, and by asmuch as 95% in soleusmuscle, having the highest content of slow-twitch fibres (40%). The Ca2+ uptake rate was significantly reduced in SR vesicles in total homogenates. We did not find any compensatory increase in SERCA1 or SERCA3 abundance, or altered expression of several other Ca2+-handling proteins. Ultrastructural analysis revealed generally well-preserved muscle morphology, but a reduced volume of the longitudinal SR. In contracting soleus muscle in vitro preparations, skKO muscles were able to fully relax, but with a significantly slowed relaxation time compared to controls. Surprisingly, the maximal force and contraction rate were preserved, suggesting that skKO slow-twitch fibres may be able to contribute to the total muscle force despite loss of SERCA2 protein. Thus it is possible that SERCA-independent mechanisms can contribute to muscle contractile function.

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Myocardial SERCA2a transcription regulation : a balance between contraction- and calcium-dependent signaling

2006

and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Table of Contents Chapter 1 General Introduction Chapter 2 Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes Chapter 3 GATA4-dependent control of SERCA2a transcription: Implications for contractile arrest-induced SERCA2a expression 57 Chapter 4 Contractile activity of cardiomyocytes induces opposite changes in SERCA2a and ANF transcription through stimulation of RhoA-SRF signaling Chapter 5 Measuring the activity of SERCA2a promoter constructs in left 103 and right ventricles of the rat heart. A method for the in vivo analysis of load-dependent transcriptional regulation Chapter 6 Conclusions and General Discussion Chapter 7

Multiple Sp1 Binding Sites in the Cardiac/Slow Twitch Muscle Sarcoplamsic Reticulum Ca[IMAGE]-ATPase Gene Promoter Are Required for Expression in Sol8 Muscle Cells

Journal of Biological Chemistry, 1996

The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA2) gene encodes a Ca 2؉ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.

Sp1 and Sp3 transcription factors are required for trans-activation of the human SERCA2 promoter in cardiomyocytes

Cardiovascular Research, 2003

Objectives: The sarcoplasmic reticulum (SR) Ca 2 + ATPase (SERCA) is essential to the removal of cytosolic calcium following cardiac contraction, and its abundance and activity are significantly altered during perinatal development and in failing myocardium. The objective of the current study was to identify cis regulatory elements and nuclear transcription factors responsible for transactivating SERCA2 gene expression in cardiomyocytes. Methods: Primary cultures of neonatal rat ventricular myocytes were transiently transfected with luciferase (LUX) reporter gene constructs containing deletions of the SERCA2 promoter or which harbored mutations in consensus Sp1 transcription factor binding sites. Cotransfection assays, electrophoretic mobility shift, and supershift assays were also performed to delineate the regulatory role of specific transcription factors. Results: We identified a putative AP-1-like element and a consensus Egr-1 binding site, but neither Egr-1 nor 12-O-tetradecanoylphorbol 13-acetate (TPA) significantly modified human SERCA2 promoter activity in vitro. Maximal activity of the SERCA2 promoter required the proximal 177 bp, and strong activation was observed with a 125-bp construct, within which an Sp1 site and a CAAT box were important. Mutation analysis also revealed the importance of two Sp1 sites between À 125 and À 200. Sp1 and Sp3 transcription factors were subsequently identified to bind to oligonucleotide probes corresponding to only the two most proximal Sp1 sites. Conclusions: These studies provide direct evidence that regulation of human SERCA2 gene expression in cardiomyocytes depends on transactivation events elicited by Sp1 and Sp3 transcription factors.

Multiple Sp1 Binding Sites in the Cardiac/Slow Twitch Muscle Sarcoplamsic Reticulum Ca2⁺-ATPase Gene Promoter Are Required for Expression in Sol8 Muscle Cells

Journal of Biological Chemistry, 1996

The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca 2؉-ATPase (SERCA2) gene encodes a Ca 2؉ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (؊1810 base pair (bp) to ؊1110 bp) and one proximal (؊284 bp to ؊72 bp), within the SERCA2 promoter. The proximal promoter region from ؊284 bp to ؊80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.

Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle

Biochemical Journal, 1999

Cardiac muscle expresses sarcoplasmic\endoplasmic-reticulum Ca# + pump isoform SERCA2a ; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100-200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (meanpS.E.M.) were (29p4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation Abbreviations used : DTT, dithiothreitol ; PNS, postnuclear supernatant ; RT-PCR, reverse-transcriptase-mediated PCR ; SERCA, sarcoplasmic/ endoplasmic-reticulum Ca 2 + ATPase ; UTR, untranslated region. 1 To whom correspondence should be addressed (e-mail groverak!fhs.csu.mcmaster.ca). velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is posttranscriptional and pretranslational.

Regulation oftheHumanCardiac/Slow-Twitch Troponin C GenebyMultiple, Cooperative, Cell-Type-Specific, and MyoD-Responsive Elements

1993

expression ofthehumancTnC(HcTnC) gene.Atleast fourseparate elements cooperate to confer tissue-specific expression ofthis geneindifferentiated myotubes; a basal promoter(between -61and -13)augmentstranscription 9-fold, upstreammajorregulatory sequences(between -68and-142and between -1319and-4500) augment transcription asmuchas39-fold, andatleast twoenhancer-like elements inthefirst intron (between +58and+1028andbetween +1029and+1523)independently augment transcription 4-to5-fold. Theseenhancers inthefirst intron increase myotube-specific chloramphenicol acetyltransferase activity whenlinked totheir own promoter elements ortotheheterologous simian virus 40 promoter, andtheeffects aremultiplicative rather thanadditive. Eachofthemajormyotube regulatory regions iscapable ofresponding directly orindirectly tothemyogenic determination factor, MyoD.AMyoDexpression vector in1OT1/2 cells induced constructs carrying either theupstream HcTnCpromoter elements orthefirst intron ofthegene300-to50...

SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice

2021

SummaryThe DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice.

SERCA2a: its role in the development of heart failure and as a potential therapeutic target

Research Reports in Clinical Cardiology, 2014

The complexity of heart physiology has delayed the implementation of efficient, feasible, and safe therapies to fight against heart diseases for many years. As knowledge of the precise mechanisms governing cardiac hypertrophy and heart failure development increases, the availability of new therapeutic alternatives also grows. Since the cardiomyocyte physiology deeply depends on the correct calcium handling, many efforts to describe accurately the excitation-contraction coupling process in the heart and the proteins involved have been made. Among the proteins participating in calcium handling, sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase-2a (SERCA2a), whose expression and function is decreased in heart failure, stands out because of its critical role regulating Ca 2+ concentration in the cardiomyocyte. The importance of SERCA2a has been reflected in numerous studies aimed to describe its expression and function. Recently, gene therapy to deliver SERCA2a has shown promising results in human clinical trials. This paper reviews the current literature knowledge exploring diverse approaches to rescue SERCA2a expression in heart failure. It also discusses some data suggesting other possible therapies that could improve SERCA2a expression and function in cardiac diseases.