This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moorman, A. F. M. Articles by Boheler, K. R. Search for Related Content PubMed PubMed Citation Articles by Moorman, A. F. M. Articles by Boheler, K. R.

(Circulation Research. 1995;76:616-625.)
© 1995 American Heart Association, Inc.

Articles

Patterns of Expression of Sarcoplasmic Reticulum Ca²⁺-ATPase and Phospholamban mRNAs During Rat Heart Development

Antoon F. M. Moorman, Jacqueline L. M. Vermeulen, Maren U. Koban, Ketty Schwartz, Wouter H. Lamers, Kenneth R. Boheler

From the Cardiovascular Research Institute Amsterdam (A.F.M.M., J.L.M.V., W.H.L.), University of Amsterdam (Netherlands); Institut National de la Sante et de la Recherche Medicale (K.S.), U153, Paris, France; and the National Heart and Lung Institute (M.U.K., K.R.B.), University of London (UK).

Correspondence to Dr Antoon F.M. Moorman, Department of Anatomy and Embryology, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, Netherlands.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.

Key Words: sarcoplasmic reticulum Ca²⁺-ATPase • phospholamban • cardiac development

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
During embryonic development, myocardial function changes continuously concomitant with a complex remodeling of the heart (see Moorman and Lamers¹ for a recent review). Heart development starts with the formation of a slowly conducting² peristaltoid-contracting³ single myocardial tube. Subsequently, fast-conducting rapidly contracting atrial and ventricular chambers start to develop within the confines of the (slowly conducting) primary myocardial heart tube.⁴ As a result, five segments can be distinguished in such hearts; each segment has a distinct molecular phenotype and, as a consequence, a separate function. They comprise a slowly conducting inflow tract, a fast-conducting atrium, a slowly conducting atrioventricular canal, a fast-conducting ventricle, and a slowly conducting outflow tract. Local differences in the conduction velocity over the consecutive segments of the embryonic heart tube explain to a large extent how the embryonic heart can function without valves and a conduction system.⁴ Eventually, a double-circuited heart develops, in which valves and a conduction system have become essential elements for cardiac function.

Regional differences in conduction velocities in the embryonic heart obviously cannot be the only parameter to account for the observed differences in the patterns of contraction. This is most clearly demonstrated by the striking differences in contraction pattern of the embryonic atrium and ventricle, which both conduct the impulse relatively fast. The atrium is characterized by rapid short-lasting contractions and is relaxed during most of the heart cycle, whereas the ventricle has rapid long-lasting contractions. A major factor in the contractile process is the intracellular free calcium concentration, which in turn is largely controlled by the sarcoplasmic reticulum^{5 6} and hence by the molecular properties and concentration of its constituting components, which are involved in the release, clearance, and sequestration of calcium. This raises the intriguing possibility that the differences in the contraction pattern in the distinct segments of the embryonic heart are the result of different patterns of expression of the genes encoding the multiple protein components of the sarcoplasmic reticulum. As a first attempt to test this possibility, we wished to analyze the spatiotemporal changes in the expression of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) and phospholamban (PLB), proteins that play a central role in the contraction-relaxation cycle of the adult heart. To this end, we have cloned and sequenced four rat cardiac PLB clones. The largest of these clones, pRCPLB1, and the previously published SERCA2-specific clone, pRH39,⁷ were used for in situ analysis of their pattern of expression during rat heart development.

The principal function of SERCA2 is to convey calcium from the sarcoplasm into the sarcoplasmic reticulum; hence, it is largely responsible for the diastolic relaxation of the heart. Ca²⁺-ATPase is a single polypeptide with an M_r of 110 000.⁸ It belongs to the SERCA gene family and comprises at least three genes encoding five isoforms.^{8 9 10} In cardiac and slow-twitch muscle, SERCA2a is the predominant isoform^{7 9 11 12} ; an alternatively spliced isoform (SERCA2b) is expressed in smooth-muscle and nonmuscle cells.¹³ The calcium pump activity is blocked by PLB.^{5 14} PLB is a pentameric protein complex, composed of identical subunits of M_r 6000,¹⁵ that in its nonphosphorylated form binds to the calcium-free form of the calcium pump to inhibit its action. Phosphorylation of PLB prevents binding and allows the pump to exhibit its full activity. PLB is expressed in cardiac, slow-twitch, and smooth-muscle cells but not in fast-twitch muscle tissue^{16 17} and is encoded by a single gene directing the synthesis of the same protein in cardiac and slow-twitch muscle.¹⁸

Relatively little is known about the developmentally regulated expression of both genes in the mammalian heart. SERCA2 expression (mRNA, protein, and activity) increases during development; isoform switches have not been reported.^{12 19 20 21 22 23} These studies were confined to analyses of ventricular homogenates; the embryonic period was included in the mRNA studies only.^{12 19} In a recent in situ hybridization study, SERCA3 mRNA expression was found to be present in early stages of heart development.²⁴ PLB mRNA expression has been detected very early in mouse²⁵ and chicken^{26 27} cardiac development, and its level was reported not to change significantly during development.²⁰ In fetal extracts, it is not phosphorylated in the presence of Ca²⁺ or calmodulin.²¹

In the present study, we describe the clonal analysis of cardiac PLB clones from rat and describe the temporal and spatial distribution of both PLB and SERCA2 mRNAs in rat heart during embryonic and fetal development. The results from the in situ hybridization analyses show that SERCA2, but not PLB, can be detected very early in the cardiogenic plate of the presomite rat embryo. SERCA2 and PLB mRNA display very distinct patterns of expression during further development of the heart, indicating substantial differences in the contractile performance of the various components of the embryonic heart, which are in support of our model of heart development.¹

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Adult Wistar rats were obtained from HSD Animal Farm, Zeist, Netherlands. Embryonic age was calculated from dated matings, and neonatal age was determined from the time of birth. Birth normally occurred between 21.5 and 22.5 days after conception. Hearts at 9, 10, 11, 12, 13, 16, 20, and 22 days of embryonic development (ED), 10- and 23-day-old postnatal hearts, and adult hearts were studied.

Isolation and Characterization of PLB cDNA Clones
Clones for PLB were isolated and characterized as previously described for SERCA2⁷ and SORO-1.²⁸ Briefly, a cDNA library was prepared from poly (A⁺) RNA isolated from Wistar rats by using a modification of the techniques of Huynh et al.²⁹ Approximately 1x10⁶ phages were plated and screened to isolate putative cDNA clones for PLB. The library was screened with a 60-bp oligonucleotide corresponding to the 5' coding region of the dog PLB sequence (generously provided by A.-M. Lompré, Paris, France). Hybridizations (5x SSPE [1x SSPE contains 180 mmol/L NaCl, 10 mmol/L sodium phosphate, and 1 mmol/L EDTA], 5x Denhardt's solution, 0.5% sodium dodecyl sulfate [SDS], and 0.02 mg/mL denatured herring sperm DNA) were carried out at 42°C, and the membranes were washed under low-stringency conditions, with the final wash being in 2x SSPE and 0.1% SDS at 55°C. After the initial screening, seven putative clones were further analyzed; this analysis yielded four independent clones of different sizes. These clones were sequenced on both strands by use of the Sequenase kit (Amersham) and the T7 and T3 primers and internal oligonucleotides constructed for this purpose.

RNA Analyses
Total RNA was isolated from rat tissues (heart, brain, liver, spleen, and skeletal muscle) by using the techniques of Chomczynski and Sacchi³⁰ and stored as a precipitate as previously described.³¹ The RNAs were used subsequently for Northern blot hybridization for size determinations of the PLB clones^{32 33} or for slot-blot hybridization for semiquantitative analyses of expression levels.³⁴ Standard hybridization conditions with a random-primed (Megaprime kit, Amersham) EcoRI insert of either pRCPLB1 or pRCPLB3 were used as previously described^{31 34} in all of the analyses of the membrane-bound RNA. All data generated from the slot blots were normalized relative to 18S RNA expression as described previously^{28 33 35} to ensure against loading differences between samples.

Tissue Processing
Up to 16 days of development, whole embryos were fixed; after 16 days, the heart was isolated. Fixation was performed in freshly prepared phosphate-buffered 4% paraformaldehyde for 4 hours or overnight at room temperature.³⁶ After dehydration in graded ethanol series, the specimens were incubated overnight in 1-butanol and embedded in Paraplast Plus (Monoject).³⁶ Sections (7 µm thick) were made and mounted onto 3-aminopropyltriethoxysilane (Sigma)–coated slides.

In Situ Hybridization
³⁵S-labeled RNA probes were allowed to hybridize to the sections essentially as described in detail previously³⁶ ; some minor modifications were made because RNA probes were used rather than cDNA probes. In short, sections were pretreated in 0.2N HCl for 20 minutes, in 2x standard saline citrate (SSC) (1x SSC contains 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate [pH 7.2]) for 10 minutes at 70°C, and in 0.1% pepsin (Sigma P 7000) in 0.01N HCl at 37°C for 1.5 minutes (10 and 11 ED embryos), 3 minutes (12 and 13 ED embryos), 15 minutes (16 ED embryos), or 20 minutes (late fetal, postnatal, and adult specimens). Sections were subsequently postfixed in 4% paraformaldehyde for 20 minutes and incubated in 10 mmol/L dithiothreitol for 5 minutes to prevent nonspecific binding of ³⁵S-labeled probe due to oxidation of the tissue.

Hybridization was carried out overnight at 52°C in 50% (vol/vol) deionized formamide, 0.1% Triton X-100, 10% (wt/vol) dextran sulfate, 2x SSC, 0.04% (wt/vol) each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll, 10 mmol/L dithiothreitol, 200 ng herring sperm DNA per microliter, and labeled probe.

Probe concentration was 50 pg (specific activity, 1000 dpm/pg) per microliter hybridization solution, and 6 µL of hybridization mixture was applied per section. No coverslips were used to improve efficiency of hybridization and to allow easy comparison of serial sections incubated with different probes and controls. Generally 5 to 10 sections were mounted onto a single microscope slide.

After hybridization, the sections were rinsed with 1x SSC and washed at 52°C in 1x SSC/50% formamide for 15 minutes (twice), in 1x SSC for 10 minutes, in RNaseA (10 µg/mL of 10 mmol/L Tris-HCl [pH 8.0], 5 mmol/L EDTA, and 500 mmol/L NaCl) for 30 minutes at 37°C, in 1x SSC for 10 minutes (twice), and finally in 0.1x SSC for 10 minutes. Sections were then dehydrated, air-dried, covered with photographic emulsion (nuclear research emulsion G5, Ilford), and exposed for 5 days. After development, sections were slightly stained with nuclear fast red, dehydrated, sealed in mounting medium (Malinol, Schmid GMBH), and photographed using Agfa Pan APX 25 (15 DIN) film and bright-field microscopy.

Probe Specifications and Preparation
The probes used to detect SERCA2 and PLB were generated from pRH39, containing an EcoRI insert corresponding to the 3' end of the SERCA2a cDNA,⁷ and pRCPLB1, the longest cDNA clone (983 bp) that was isolated from the rat cardiac cDNA library, respectively. For controls of specificity, probes for -myosin heavy chain (-MHC) were generated from the clone -120,³⁷ and probes for ß-myosin heavy chain (ß-MHC) were from the plasmid PUC 4H (kindly provided by V. Mahdavi). The rat connexin43 sequence was generously provided by Dr D.L. Paul (Harvard Medical School, Boston, Mass). The probe comprises 1500 bp, which contain the complete coding region.³⁸

³⁵S-labeled transcripts were made by in vitro transcription of linearized pBluescript in which the appropriate restriction fragment was subcloned. To allow efficient hybridization, RNA transcripts longer than 200 nt were subjected to alkaline hydrolysis to yield fragments with a mean size of 100 to 150 nt, as assessed by polyacrylamide gel electrophoresis.

Statistics
A two-tailed paired t test was used for all statistical analyses of RNA isolated from the same heart. Significance was accepted for a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and Characterization of Rat PLB cDNAs
To determine the amino acid sequence of rat cardiac PLB, 1x10⁶ recombinant phages from a rat cardiac cDNA library were screened by using an end-labeled synthetic oligonucleotide corresponding to the 5' coding region of dog PLB. Of the multiple positive clones isolated after stringent washing, five plasmids were rescued from ZAPII, four of which were further characterized by restriction mapping and sequencing. The largest of the clones, pRCPLB1, was 1000 nt in length and contained all the sequences found in the other four clones (data not shown). A partial restriction map and the strategy for sequencing the four clones are shown in Fig 1A and 1B, respectively. The complete sequence is shown in Fig 1C. Two polyadenylation sites were seen at positions 565 and 691, based on the locations of the poly (A⁺) tail seen in clones pRCPLB7, pRCPLB3, and pRCPLB2 at sites 575, 584, and 710, respectively. Like other PLB clones, rat PLB encodes a 52–amino acid protein characterized by a hydrophilic amino terminus and a hydrophobic carboxy terminus. This amino acid sequence is identical to mouse and rabbit cardiac and slow-twitch skeletal muscle PLB and differs from dog cardiac muscle at position 2 and from human PLB at position 27.

View larger version (30K): [in this window] [in a new window]   Figure 1. Schematic representation (A) and sequencing strategy (B) of the rat heart phospholamban clone pRCPLB1, the largest of four fully sequenced clones. Open boxes represent coding regions; thin lines, the untranslated regions. Arrows indicate the direction and extent of sequencing. The locations of several convenient restriction sites are indicated (RI represents EcoRI). The entire sequence of rat pRCPLB1 is also shown (C). The 5' and 3' untranslated regions are denoted by small letters, and the coding sequence is indicated by capital letters. The amino acids coded for by this clone are written, using the single-letter code, under the corresponding nucleic acid sequence. The underlined sequences show the signal sequence for polyadenylation. *Location of the last nucleotide before the addition of a poly (A+) tail from clones pRCPLB7, pRCPLB3, and pRCPLB2, respectively.

Expression of PLB in Rats
PLB transcripts have been reported in cardiac, slow skeletal, and smooth muscle cells.^{16 17} To confirm that the distribution of PLB mRNAs in rats is similar to transcripts from other species, total RNA was prepared from atria, right and left ventricles, liver, soleus muscle, and brain. In Northern blot experiments, no signals were detected from liver or brain RNA (Fig 2A), and only very weak signals were detected from soleus muscle RNA (slow skeletal muscle). The strongest signals were detected in cardiac tissues, with the ventricles showing a stronger signal than the atria (data not shown). Three strong signals were detectable on Northern blots (Fig 2A) at 2.3 kb and 2.0 kb and between 0.6 and 1.0 kb. This smallest band, which is much (approximately three times) stronger in signal intensity than the higher molecular weight bands, consists of at least two transcripts of 0.6 and 0.7 kb (see Fig 1C) with variable lengths of polyadenylation and a potential third transcript of 0.95 kb. Two known polyadenylation signals are present in pRCPLB1 as described above and in Fig 1C, although a third potential signal is located at nt 944 to 950 (ataaaaa). Therefore, we cannot exclude the possibility of a fifth transcript.

View larger version (42K): [in this window] [in a new window]   Figure 2. A, Northern blot analysis of total RNA hybridized with a random-labeled EcoRI insert of pRCPLB3 showing the existence of three strong signals in ventricle (V), which are absent in liver (L) and brain (B). The location of 18S RNA is as indicated. S indicates soleus muscle RNA. B, Ethidium bromide fluorescence of 28S ribosomal RNA, indicating the amount of RNA applied onto the gel.

Because the Northern blots seemed to show a difference in expression between the heart chambers, RNA was prepared from atria and the left and right ventricles of adult rats (250 g). These were analyzed on slot blots, and the values were normalized relative to 18S RNA (Fig 3). We were unable to demonstrate any difference in PLB expression between the left and right ventricles (n=4) (data not shown); however, we found a 2.7-fold increase in the relative levels of PLB mRNA expression in the ventricles (12.7 arbitrary units) relative to the atria (4.8 arbitrary units), which when analyzed by a two-tailed t test gave a value of P<.05 (Fig 3). It is unclear whether this difference in the accumulation of PLB transcripts in adult rat atria versus ventricle leads to a concomitant difference in PLB protein levels. As a summary for the clonal analysis of rat cardiac PLB, we conclude that rats have four (possibly five) distinct transcripts for PLB, that the amino acid sequence for PLB is very similar to that described previously for other mammalian species, and that PLB transcripts are expressed more abundantly in adult rat ventricle than atria.

View larger version (13K): [in this window] [in a new window]   Figure 3. A, Example of an autoradiogram of a slot blot where total RNA isolated from rat ventricle (V), atrium (A), and liver (L) was hybridized with either labeled pRCPLB1 (PLB) or an oligonucleotide complementary to 18S RNA (18S). Schematic representation of the relative expression of PLB in atria versus ventricle. Values obtained from slot-blot analyses are expressed in arbitrary units relative to 18S RNA, and the data are expressed as mean±SEM (n=7).

In Situ Hybridization Analysis
Hybridization to labeled probe has been visualized by bright-field microscopy, because this type of microscopy has the capability of showing the patterns and intensities of hybridization within the topographical context of the embryo with high sensitivity.³⁶ Data are interpreted as being semiquantitative within a particular developmental stage. Hybridizations were carried out by using cRNA probes to PLB, SERCA2, -MHC, ß-MHC, and connexin43. The distinct hybridization patterns obtained with the different probes constitute important tissue-intrinsic controls that are imperative for reliable mRNA localizations. In addition, use of ³⁵S-labeled sense RNA as a probe did not yield signals at any of the developmental stages, indicating that specific hybridization is being achieved and that anti-sense mRNA is not expressed (data not shown). The only exception to this pattern was the expression of anti-sense ß-MHC mRNA in the nuclei of postnatal atrial and ventricular myocardium (K.R. Boheler et al, unpublished data, 1994).

Developmental Appearance
In the presomite embryo of 9 days of development, no heart tube has been formed yet. The precardiac mesoderm is essentially a flattened cardiogenic plate that is part of the visceral wall of the coelom. In this youngest embryo tested, SERCA2 mRNA can already be detected unambiguously in the horseshoe-shaped cardiogenic plate (Fig 4). However, PLB mRNA cannot be detected.

View larger version (68K): [in this window] [in a new window]   Figure 4. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) mRNA expression in the cardiogenic plate of a 9-day-old rat embryo. Transverse sections are shown in the craniocaudal direction (a through d). At the cranial side (a), SERCA2 mRNA expression is present in the midline, rostral to the neural folds that are just sectioned. Expression continues caudally into the lateral rudiments of the cardiogenic plate (b, c, and d). Sections shown in panels b, c, and d are 75, 150, and 300 µm caudal to the section shown in panel a, respectively. nf indicates neural fold; ng, neural groove; ec, ectoderm; cp, cardiogenic plate (part of the visceral mesoderm); and en, endoderm. Bar=100 µm.

At ED 10, the almost straight heart tube has started to bulge toward the right. At both ends of the cardiac tube but predominantly at the venous pole, newly formed myocardium is still added to the heart. SERCA2 mRNA is present along the entire heart tube (Fig 5). A faint craniocaudal gradient of expression can now be recognized, increasing in concentration going from the arterial pole (outflow tract/cranial side) toward the venous pole (inflow tract/caudal side). PLB mRNA cannot be detected at this stage (data not shown).

View larger version (78K): [in this window] [in a new window]   Figure 5. Sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression in the looping cardiac tube of a 10-day-old rat embryo. Transverse sections are shown in the craniocaudal direction (a through d). SERCA2 expression can be detected along the whole heart tube. The intensity of hybridization is most strong in the caudal part. oft indicates outflow tract; ift, inflow tract; lht, looping heart tube between inflow and outflow tract, which will become largely ventricle; ng, neural groove; and fg, foregut. Bar=100 µm.

At ED 11, the heart is clearly looped. The plane of sectioning presented in Fig 6 shows the five segments of the heart with a clear gradient of expression of SERCA2 mRNA (Fig 6c). At the transition of the atrium to the atrioventricular canal, there seems to be a drop in intensity of hybridization. PLB mRNA cannot yet be detected at significant levels (Fig 6b). ß-MHC (Fig 6a), used as a control, is present in a gradient opposite that of SERCA2 mRNA, decreasing in concentration from outflow tract to inflow tract, similar to the protein pattern.^{1 39}

View larger version (64K): [in this window] [in a new window]   Figure 6. Patterns of gene expression in the rat embryo at day 11 of embryonic development. Serial transverse sections were allowed to hybridize to RNA probes complementary to ß-myosin heavy chain (a), phospholamban (PLB) (b), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) (c) mRNA. PLB mRNA cannot yet be detected. ift indicates inflow tract; A, atrium; avc, atrioventricular canal; V, ventricle; and oft, outflow tract. Bar=100 µm.

At EDs 12 to 13, the mRNA encoding PLB can first be detected. It is present in a highly distinct pattern (Fig 7a and 7c). The overall impression at ED 13 is that PLB mRNA expression is virtually opposite that of SERCA2 mRNA (Fig 7b and 7d); ie, PLB mRNA expression is highest in the outflow tract and decreases in concentration going toward the inflow tract, whereas the expression of SERCA2 mRNA is the inverse. Two other observations are noteworthy: (1) Initially, PLB mRNA is most strongly expressed in the most distal part of the outflow tract, in line with the statement above, but it is also present at a lower level in the dorsal wall of the atrium and inflow tract (arrows in 7a). This subtle expression of PLB mRNA in the venous pole of the heart seems to persist during further development (eg, see the expression around the superior caval vein at ED 16; Fig 8a). (2) Apart from the gradients of expression mentioned above, both PLB and SERCA2 mRNA tend to be present at slightly lower levels at the atrioventricular junction compared with the adjacent myocardium of atrium and ventricle.

View larger version (104K): [in this window] [in a new window]   Figure 7. Patterns of expression of phospholamban (PLB) mRNA (a and c) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) mRNA (b and d) in sagittal sections of the rat heart at embryonic days 12 (a and b) and 13 (c and d). PLB mRNA can now be detected for the first time (arrows in inflow tract [ift] and outflow tract [oft] of the rat heart at embryonic day 12 [a]). A indicates atrium; avc, atrioventricular canal; and V, ventricle. Bar=100 µm.

View larger version (76K): [in this window] [in a new window]   Figure 8. Four-chamber view of a fetal rat heart at embryonic day 16. Serial sections were allowed to hybridize to cRNA probes for the mRNAs encoding phospholamban (PLB) (a), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2) (b), and connexin43 (c). LV indicates left ventricle; LA, left atrium; and oft, outflow tract. Bar=100 µm.

Development of the "Adult" Pattern of Expression
At ED 16, the four-chambered heart can be clearly recognized, although ventricular septation has not been entirely completed. In essence, the adult pattern of expression has become established. SERCA2 mRNA (Fig 8b) is still most abundantly expressed in the atria but is also present in substantial levels in the ventricles. The ventricular trabeculations display a lower intensity of hybridization than the septum and the ventricular free wall. PLB mRNA (Fig 8a) is now clearly expressed in the ventricles, but its expression in the atria remains relatively low. This overall pattern of expression does not change significantly during further fetal development (see Fig 9) and is similar in the postnatal and adult heart (not shown). PLB mRNA remains less abundant in the atria (Figs 7 and 9), whereas the difference between the atrial and ventricular levels of SERCA2 mRNA becomes less pronounced because of the relative increase of SERCA2 mRNA in the ventricles.

View larger version (124K): [in this window] [in a new window]   Figure 9. Four-chamber view of a fetal rat heart at embryonic day 20. Overview demonstrates the myocardium surrounding the pulmonary (arrows) and caval veins (cv). Serial sections were allowed to hybridize to cRNA probes for the mRNAs encoding -myosin heavy chain (a), ß-myosin heavy chain (b), phospholamban (c), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (d). e indicates esophagus; lb, left principal bronchus; cv, caval vein; and LV, left ventricle. Bar=1 mm.

At ED 16, the embryonic outflow tract is still present. Its myocardium forms a myocardial cuff around the outflow of the ventricles. Fig 8 clearly shows that it still has the same molecular phenotype as in the previous stages, ie, low levels of SERCA2 mRNA and high expression of PLB mRNA. Note that the mRNA encoding the gap junctional protein connexin43 is virtually absent from the outflow tract (Fig 8c) (see "Discussion").

Pattern of Expression in the Conduction System
The conduction system in the formed heart constitutes the sinoatrial node, the atrioventricular node, the atrioventricular bundle, and bundle branches. The sinoatrial and atrioventricular nodes develop in the inflow tract and atrioventricular canal, respectively. Both segments become incorporated in the atria during further development. The expression of SERCA2 and PLB mRNA in the nodal areas did not reveal a pattern that was significantly different from the surrounding working myocardium. In contrast, the ventricular conduction system, comprising bundle and bundle branches, displayed a distinct molecular phenotype because of the low abundance or absence of SERCA2 mRNA (Figs 9 and 10). The expression of PLB mRNA was not found to be significantly different from the surrounding ventricular working myocardium. The low levels of SERCA2 mRNA are in line with the poorly developed sarcoplasmic reticulum that is present in these structures.⁴⁰

View larger version (72K): [in this window] [in a new window]   Figure 10. Details of the sections shown in Fig 9 of the rat heart at embryonic day 20, highlighting the pattern of expression of the atrioventricular bundle (arrowheads) at the top of the interventricular septum and the myocardium of the trabeculae (small arrows). Serial sections were allowed to hybridize to cRNA probes for the mRNAs encoding -myosin heavy chain (a), ß-myosin heavy chain (b), phospholamban (c), and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (d). RA indicates right atrium; LV, left ventricle; ivs, interventricular septum; and v, atrioventricular valves. Bar=200 µm.

Caval and Pulmonary Myocardium
In rodents the caval and pulmonary veins are surrounded by myocardium.^{41 42} The overviews shown in Fig 9 clearly demonstrate that this myocardium displays the atrial phenotype, as assessed by the expression of the mRNAs encoding -MHC, ß-MHC, PLB, and SERCA2. Similar findings have been reported previously.^{39 43 44}

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sarcoplasmic reticulum is the key regulator of the free sarcoplasmic calcium concentration, with calcium mobilization being largely responsible for sarcomeric contraction and calcium uptake responsible for diastolic relaxation.⁴⁵ Calcium uptake into the sarcoplasmic reticulum of the cardiac muscle is accomplished by the SERCA2 calcium pump,^{7 9 11 12} the activity of which is regulated by PLB.¹⁴ The present study was initiated to explore the possibility of whether local differences in SERCA2 and PLB mRNA accumulation exist in the developing heart. Such differences could eventually contribute to our understanding of the embryonic contraction pattern. We now have described that SERCA2 mRNA can be detected in the cardiogenic plate of the presomite embryo and that SERCA2 and PLB mRNA display unique spatial patterns of expression during cardiac development.

The strength of the hybridization signal in the cardiogenic plate makes it likely that the embryo starts well before 9 days with the accumulation of SERCA2 mRNA in the precardiac mesoderm. PLB mRNA can first be detected at 12 days of development, indicating that the PLB-mediated regulatory machinery of the SERCA2 calcium pump develops far beyond the time that the primary function, ie, calcium pump activity, has been established. This early outbreak of SERCA2 transcription suggests that the encoded protein plays an indispensable role in the contraction-relaxation cycle of the early myocardium, as the first contractions in embryonic rat hearts appear in embryos with three somites, which are 9 days and 14 hours old.⁴⁶ They appear in the lateral rudiments that are still separated from each other by the developing foregut and appear first at the left side and 2 hours later at the right side. At a comparable stage of cardiac development, desmin and myosin protein have been detected in the cardiogenic plate of the rat and chicken embryo.^{47 48} The very early onset of cardiac myogenesis may explain why myogenic basic helix-loop-helix proteins, which would activate cardiac-specific transcription, have not been identified so far.⁴⁹ The very early appearance of SERCA2 mRNA makes it an excellent marker for the study of early heart development.

The crucial feature of our observations is that the mRNAs encoding SERCA2 and PLB, respectively, are expressed along the cardiac tube in virtually opposite patterns, SERCA2 mRNA being expressed at highest levels in the upstream part of the cardiac tube and PLB mRNA prevailing in the downstream compartments. These observations are potentially of great significance for our insight into the (regulation of the) embryonic cardiac contraction patterns, provided the mRNA patterns reflect protein levels. The validity of the latter assumption is supported, at least in part, by the observed increase of SERCA2 mRNA and protein during rat heart development.^{12 19 22} In further support of this conclusion are immunohistochemical studies performed in developing chicken hearts that show a distribution of SERCA2 protein that is comparable to the mRNA distribution described in the present study.⁵⁰

The potential implication of the observed molecular patterns is that the upstream part of the heart would have a greater capacity to clear calcium and, hence, would be relaxed for a greater part of the heart cycle than the downstream compartments. This is precisely what can be noticed by direct observation of the contracting embryonic heart and matches the differences in the atrial and ventricular action potential duration.⁵¹ This effect may even be enhanced by the occurrence of an additional calcium-mobilizing muscarinic system in embryonic chicken hearts.⁵² Acetylcholinesterase has been supposed to be a marker for this system.⁵³ It decreases in concentration along a craniocaudal gradient and is absent from the atrium and inflow tract.⁵⁴ Taken together, these data provide a plausible explanation for the differing contraction times in the distinct segments of the embryonic heart: short contraction times in the inflow tract and atrium and increasing times going from the atrioventricular canal and ventricle toward the outflow tract. The low levels of PLB mRNA and high concentrations of SERCA2 mRNA explain why the atria are relaxed during most of the heart cycle, allowing them to function as the drainage pool of the body. Recently, the PLB mRNA distribution in developing chicken heart was described.²⁷ We think that this study shows in essence a similar distribution pattern, but this pattern was not recognized as such, possibly because of the use of less sensitive ³²P-labeled probes and/or of dark-field illumination.

The opposite patterns of expression of SERCA2 and PLB mRNA along the cardiac tube are reminiscent of the opposite patterns of expression of - and ß-MHC mRNA and protein in rat, mouse,^{39 43} and chicken,⁵⁵ pointing to the presence of region-specific programs of gene expression. Thyroid hormone and/or its receptor distribution may play a crucial role in this process. The expression of both -MHC^{56 57} and SERCA2^{58 59} is stimulated by thyroid hormone, whereas the expression of ß-MHC^{57 60} and PLB^{58 59} are downregulated. The thyroid hormone receptor has recently been shown to modulate the expression of the SERCA2 gene.⁶¹ The functional consequence of this pattern of expression is that the upstream compartments of the heart (inflow tract and atrium) contract rapidly (short lasting, as discussed above), owing to the expression of the fast myosin isoform, whereas the downstream compartments (atrioventricular canal, ventricle, and outflow tract) contract slowly (long lasting, as discussed above), owing to the expression of the slow myosin isoform.

The outflow tract has a very special function in the embryonic heart, which has no valves. Because of its length, its very slow propagation of the impulse (overlapping the next cycle), and the long duration of its action potentials, the outflow tract has a peristaltoid contraction form and functions as a sphincter that can substitute for the semilunar valves to prevent regurgitation of the blood.⁴ The high levels of PLB mRNA and low levels of SERCA2 mRNA (see Figs 6, 7, and 8) match the required slow relaxation times. The low conduction velocities in this cardiac segment are in accord with the very low abundance of connexin43 mRNA (Fig 8 and M.J.A. van Kempen et al, unpublished data, 1994) and protein.⁵³ Connexin43 is the major component of cardiac gap junctions,⁶² which mediate the electrotonic coupling of the cardiocytes.⁶³

The very distinct patterns of expression of PLB mRNA and SERCA2 mRNA are suggestive of an adequately functioning sarcoplasmic reticulum before birth, at least regarding the PLB-mediated calcium uptake. However, it often has been assumed that the prenatal sarcoplasmic reticulum would be morphologically and biochemically poorly developed.^{21 22 23} The conclusion emerged that it would be rather deficient in calcium regulation; hence, a sarcolemmal Na⁺-Ca²⁺ exchanger has been proposed to provide an alternative mechanism for the regulation of calcium.⁶⁴ At first glance, these data seem to conflict with the present study. However, one should realize that the atrium has almost never been included in studies involving the development of the sarcoplasmic reticulum. Also, in rat only a 10% increase of SERCA2 protein content and activity (calcium uptake) has been described to occur between 15-day-old fetuses and adult controls.¹²

Obviously, the functional significance of our observations can be fully exploited only if the local differences in calcium movements can be established, which is hard to accomplish because of the size of the compartments of the embryonic heart.

	Acknowledgments

 
This study was supported by an INSERM/MW-NWO grant to Drs Moorman and Schwartz, a concerted action grant (BMH1-CT92-1171) between the European Economic Community and INSERM to Drs Schwartz and Boheler, and a British Heart Foundation Project Grant (PG/93148) to Dr Boheler. Dr Koban was supported by Bundesministerium für Forschung und Technik (01ZZ9105-1.23). The authors express their appreciation to Rob Charles and Frits de Jong for many stimulating discussions, to Cars Gravemeijer for excellent photography, and to Remco ten Hagen for dedicated secretarial support.

	Footnotes

 
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received June 9, 1994; accepted December 15, 1994.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Moorman AFM, Lamers WH. Molecular anatomy of the developing heart. Trends Cardiovasc Med. 1994;4:257-264.

2. Kamino K. Optical approaches to ontogeny of electrical activity and related functional organization during early heart development. Physiol Rev. 1991;71:53-91. [Abstract/Free Full Text]

3. Patten BM. Initiation and early changes in the character of the heartbeat in vertebrate embryos. Physiol Rev. 1949;29:31-47. [Free Full Text]

4. de Jong F, Opthof T, Wilde AAM, Janse MJ, Charles R, Lamers WH, Moorman AFM. Persisting zones of slow impulse conduction in developing chicken hearts. Circ Res. 1992;71:240-250. [Abstract/Free Full Text]

5. Colyer J. Control of the calcium pump of cardiac sarcoplasmic reticulum: a specific role for the pentameric structure of phospholamban? Cardiovasc Res. 1993;27:1766-1771. [Free Full Text]

6. Lompré AM, de la Bastie D, Schwartz K. The sarcoplasmic reticulum: physiology, biochemistry and molecular biology. In: Swynghedauw B, ed. Research in Cardiac Hypertrophy and Failure. London, England: John Libbey, Eurotext; 1990:213-224.

7. Lompré AM, de la Bastie D, Boheler KR, Schwartz K. Characterization and expression of the rat heart sarcoplasmic reticulum Ca²⁺-ATPase mRNA. FEBS Lett. 1989;249:35-41. [Medline] [Order article via Infotrieve]

8. MacLennan DH, Brandl CJ, Korczak B, Green NM. Amino-acid sequence of a Ca²⁺ + Mg²⁺-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature. 1985;316:696-700. [Medline] [Order article via Infotrieve]

9. Brandl CJ, Green NM, Korczak B, MacLennon DH. Two Ca²⁺ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986;44:597-607. [Medline] [Order article via Infotrieve]

10. Burk SE, Lytton J, MacLennan DH, Shull GE. cDNA cloning, functional expression, and mRNA tissue distribution of a third organellar Ca²⁺ pump. J Biol Chem. 1989;264:18561-18568. [Abstract/Free Full Text]

11. Brandl CJ, deLeon S, Martin DR, MacLennan DH. Adult forms of the Ca²⁺ ATPase of sarcoplasmic reticulum. J Biol Chem. 1987;262:3768-3774. [Abstract/Free Full Text]

12. Komuro I, Kurabayashi M, Shibazaki Y, Takaku F, Yazaki Y. Molecular cloning and characterization of a Ca²⁺ + Mg²⁺-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. J Clin Invest. 1989;83:1102-1108.

13. Lytton J, MacLennan DH. Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca²⁺-ATPase gene. J Biol Chem. 1988;263:15024-15031. [Abstract/Free Full Text]

14. James P, Inui M, Tada M, Chiesi M, Carafoli E. Nature and site of phospholamban regulation of the Ca²⁺ pump of sarcoplasmic reticulum. Nature. 1989;342:90-92. [Medline] [Order article via Infotrieve]

15. Fujii J, Ueno A, Kitano K, Tanaka S, Kadoma M, Tada M. Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban. J Clin Invest. 1987;79:301-304.

16. Jorgensen AO, Jones LR. Localization of phospholamban in slow but not fast canine skeletal muscle fibers: an immunocytochemical and biochemical study. J Biol Chem. 1986;261:3775-3781. [Abstract/Free Full Text]

17. Eggermont JA, Wuytack F, Verbist J, Casteels R. Expression of endoplasmic-reticulum Ca²⁺-pump isoforms and of phospholamban in pig smooth-muscle tissues. Biochem J. 1990;271:649-653. [Medline] [Order article via Infotrieve]

18. Fujii J, Lytton J, Tada M, MacLennan DH. Rabbit cardiac and slow-twitch muscle express the same phospholamban gene. FEBS Lett. 1988;227:51-55. [Medline] [Order article via Infotrieve]

19. Lompré AM, Lambert F, Lakatta EG, Schwarz K. Expression of sarcoplasmic reticulum Ca²⁺-ATPase and calsequestrin genes in rat heart during ontogenic development and aging. Circ Res. 1991;69:1380-1388. [Abstract/Free Full Text]

20. Arai M, Otsu K, MacLennan DH, Periasamy M. Regulation of sarcoplasmic reticulum gene expression during cardiac and skeletal muscle development. Am J Physiol. 1992;262:C614-C620. [Abstract/Free Full Text]

21. Pegg W, Michalak M. Differentiation of sarcoplasmic reticulum during cardiac myogenesis. Am J Physiol. 1987;252:H22-H31. [Abstract/Free Full Text]

22. Nakanishi T, Jarmakani JM. Developmental changes in myocardial mechanical function and subcellular organelles. Am J Physiol. 1984;246:H615-H625.

23. Nayler WG, Fassold E. Calcium accumulating and ATPase activity of cardiac sarcoplasmic reticulum before and after birth. Cardiovasc Res. 1977;11:231-237. [Medline] [Order article via Infotrieve]

24. Anger M, Samuel JL, Marotte F, Wuytack F, Rappaport L, Lompré AM. In situ mRNA distribution of sarco(endo)plasmic reticulum Ca²⁺-ATPase isoforms during ontogeny in the rat. J Mol Cell Cardiol. 1994;26:539-550. [Medline] [Order article via Infotrieve]

25. Ganim JR, Luo W, Ponniah S, Grupp I, Kim HW, Ferguson DG, Kadambi V, Neumann JC, Doetschman T, Kranias EG. Mouse phospholamban gene expression during development in vivo and in vitro. Circ Res. 1992;71:1021-1030. [Abstract/Free Full Text]

26. Will H, Küttner I, Vetter R, Will-Shahab L, Kemsies C. Early presence of phospholamban in developing chick heart. FEBS Lett. 1983;155:326-330. [Medline] [Order article via Infotrieve]

27. Toyofuku T, Doyle DD, Zak R, Kordylewski L. Expression of phospholamban mRNA during early avian muscle morphogenesis is distinct from that of -actin. Dev Dyn. 1993;196:103-113. [Medline] [Order article via Infotrieve]

28. Martin XJ, Schwartz K, Boheler KR. Characterization of a novel transcript of retroviral origin expressed in rat heart and liver. J Mol Cell Cardiol. 1995;27:589-597. [Medline] [Order article via Infotrieve]

29. Huynh TV, Young RA, Davis RW. Constructing and screening cDNA libraries in Igt10 and Igt11. In: Glover DM, ed. DNA Cloning: A Practical Approach. Oxford, England/Washington, DC: IRL Press; 1985:49-78.

30. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

31. Boheler KR, Carrier L, de la Bastie D, Alien PD, Komajda M, Mercadier JJ, Schwartz K. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest. 1991;88:323-330.

32. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory; 1989.

33. Mercadier JJ, Lompré AM, Duc P, Boheler KR, Fraysse JB, Wisnewsky C, Allen PD, Komajda M, Schwartz K. Altered sarcoplasmic reticulum Ca²⁺-ATPase gene expression in the human ventricle during end-stage heart failure. J Clin Invest. 1990;85:305-309.

34. Carrier L, Boheler KR, Chassagne C, de la Bastie D, Wisnewsky C, Lakatta EG, Schwartz K. Expression of the sarcomeric actin isogenes in the rat heart with development and senescence. Circ Res. 1992;70:999-1005. [Abstract/Free Full Text]

35. Mendez RE, Pfeffer JM, Ortola FV, Bloch KD, Anderson S, Seidman JG, Brenner BM. Atrial natriuretic peptide transcription, storage and release in rats with myocardial infarction. Am J Physiol. 1987;253:H1449-H1455. [Abstract/Free Full Text]

36. Moorman AFM, de Boer PAJ, Vermeulen JLM, Lamers WH. Practical aspects of radio-isotopic in situ hybridization on RNA. Histochem J. 1993;25:251-260. [Medline] [Order article via Infotrieve]

37. Schiaffino S, Samuel JL, Sassoon D. Nonsynchronous accumulation of -skeletal actin and ß-myosin heavy chain mRNAs during early stages of pressure-overload–induced cardiac hypertrophy demonstrated by in situ hybridization. Circ Res. 1989;64:937-948. [Abstract/Free Full Text]

38. Beyer EC, Paul DL, Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987;105:2621-2629. [Abstract/Free Full Text]

39. de Groot IJM, Lamers WH, Moorman AFM. Isomyosin expression pattern during rat heart morphogenesis: an immunohistochemical study. Anat Rec. 1989;224:365-373. [Medline] [Order article via Infotrieve]

40. Virágh S, Challice CE. The development of the conduction system in the mouse embryo heart, IV: differentiation of the atrioventricular conduction system. Dev Biol. 1982;89:25-40. [Medline] [Order article via Infotrieve]

41. Kramer AW Jr, Marks LS. The occurrence of cardiac muscle in the pulmonary veins of rodentia. J Morphol. 1965;117:135-150. [Medline] [Order article via Infotrieve]

42. Nathan H, Gloobe H. Myocardial atrio-venous junctions and extensions (sleeves) over the pulmonary and caval veins. Thorax. 1970;25:317-324. [Abstract/Free Full Text]

43. Lyons GE, Schiaffino S, Sassoon D, Barton P, Buckingham ME. Developmental regulation of myosin expression in mouse cardiac muscle. J Cell Biol. 1990;111:2427-2437. [Abstract/Free Full Text]

44. Jones WK, Sánchez A, Robbins J. Murine pulmonary myocardium: developmental analysis of cardiac gene expression. Dev Biol. 1994;200:117-128.

45. Fabiato A, Fabiato F. Calcium and cardiac excitation-contraction coupling. Annu Rev Physiol. 1979;41:473-484. [Medline] [Order article via Infotrieve]

46. Goss CM. The first contractions of the heart in rat embryos. Anat Rec. 1938;70:505-524.

47. Baldwin HS, Jensen KL, Solursh M. Myogenic differentiation of the precardiac mesoderm in the rat. Differentiation. 1991;47:163-172. [Medline] [Order article via Infotrieve]

48. Han Y, Dennis JE, Cohen-Gould L, Bader DM, Fischman DA. Expression of sarcomeric myosin in the presumptive myocardium of chicken embryos occurs within six hours of myocyte commitment. Dev Dyn. 1992;193:257-265. [Medline] [Order article via Infotrieve]

49. Olson EN. Regulatory mechanisms for skeletal muscle differentiation and their relevance to gene expression in the heart. Trends Cardiovasc Med. 1992;2:163-170.

50. Jorgensen AO, Bashir R. Temporal appearance and distribution of the Ca²⁺ + Mg²⁺ ATPase of the sarcoplasmic reticulum in developing chick myocardium as determined by immunofluorescence labeling. Dev Biol. 1984;106:156-165.[Medline] [Order article via Infotrieve]

51. Couch JR, West TC, Hoff HE. Development of the action potential of the prenatal rat heart. Circ Res. 1969;24:19-31. [Abstract/Free Full Text]

52. Oettling G, Schmidt E, Drews U. An embryonic Ca⁺⁺ mobilizing muscarinic system in the chick embryo heart. J Dev Physiol. 1989;12:85-94. [Medline] [Order article via Infotrieve]

53. van Kempen MJA, Fromaget C, Gros D, Moorman AFM, Lamers WH. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res. 1991;68:1638-1651. [Abstract/Free Full Text]

54. Lamers WH, te Kortschot A, Moorman AFM, Los JA. Acetylcholinesterase in prenatal rat heart: a marker for the early development of the cardiac conductive tissue? Anat Rec. 1987;217:361-370. [Medline] [Order article via Infotrieve]

55. de Jong F, Geerts WJC, Lamers WH, Los JA, Moorman AFM. Isomyosin expression pattern during formation of the tubular chicken heart: a 3D immunohistochemical analysis. Anat Rec. 1990;226:213-227. [Medline] [Order article via Infotrieve]

56. Chizzonite RA, Zak R. Regulation of myosin isoenzyme composition in fetal and neonatal rat ventricle by endogenous thyroid hormones. J Biol Chem. 1984;259:12628-12632. [Abstract/Free Full Text]

57. Lompré AM, Nadal-Ginard B, Mahdavi V. Expression of the cardiac ventricular - and ß-myosin heavy chain genes is developmentally and hormonally regulated. J Biol Chem. 1984;259:6437-6446. [Abstract/Free Full Text]

58. Nagai R, Zarain-Herzberg A, Brandl CJ, Fujii J, Tada M, MacLennan DH, Alpert NR, Periasamy M. Regulation of myocardial Ca²⁺-ATPase and phospholamban mRNA expression in response to pressure overload and thyroid hormone. Proc Natl Acad Sci U S A. 1989;86:2966-2970. [Abstract/Free Full Text]

59. Kiss E, Jakab G, Kranias EG, Edes I. Thyroid hormone–induced alterations in phospholamban protein expression: regulatory effects of sarcoplasmic reticulum Ca²⁺ transport and myocardial relaxation. Circ Res. 1994;75:245-251. [Abstract/Free Full Text]

60. Everett AW, Clark WA, Chizzonite RA, Zak R. Change in synthesis rates of - and ß-myosin synthesis by thyroid hormone. J Biol Chem. 1983;258:2421-2425. [Abstract/Free Full Text]

61. Zarain-Herzberg A, Marques J, Sukovich D. Thyroid hormone receptor modulates the expression of the rabbit cardiac sarco(endo)plasmic reticulum Ca²⁺-ATPase gene. J Biol Chem. 1994;269:1460-1467. [Abstract/Free Full Text]

62. Beyer EC, Kistler J, Pane DL, Goodenough DA. Antisera directed against connexin 43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989;108:595-605. [Abstract/Free Full Text]

63. Spray DC, Burt JM. Structure-activity relations of the cardiac gap junction channel. Am J Physiol. 1990;258:C195-C205. [Abstract/Free Full Text]

64. Boerth SR, Zimmer DB, Artman M. Steady-state mRNA levels of the sarcolemmal Na⁺-Ca²⁺ exchanger peak near birth in developing rabbit and rat hearts. Circ Res. 1994;74:354-359.[Abstract/Free Full Text]

This article has been cited by other articles:

				A. Guo and H.-T. Yang Ca2+ removal mechanisms in mouse embryonic stem cell-derived cardiomyocytes Am J Physiol Cell Physiol, January 1, 2009; 297(3): C732 - C741. [Abstract] [Full Text] [PDF]

				I Stoykov, H C van Beeren, A F M Moorman, V M Christoffels, W M Wiersinga, and O Bakker Effect of amiodarone and dronedarone administration in rats on thyroid hormone-dependent gene expression in different cardiac components Eur. J. Endocrinol., June 1, 2007; 156(6): 695 - 702. [Abstract] [Full Text] [PDF]

				I Stoykov, B Zandieh-Doulabi, A F M Moorman, V Christoffels, W M Wiersinga, and O Bakker Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart. J. Endocrinol., May 1, 2006; 189(2): 231 - 245. [Abstract] [Full Text] [PDF]

				A. C. Fijnvandraat, R. H. Lekanne Deprez, and A. F.M. Moorman Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes Cardiovasc Res, May 1, 2003; 58(2): 303 - 312. [Abstract] [Full Text] [PDF]

				A. C. Fijnvandraat, A. C.G. van Ginneken, P. A.J. de Boer, J. M. Ruijter, V. M. Christoffels, A. F.M. Moorman, and R. H. Lekanne Deprez Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube Cardiovasc Res, May 1, 2003; 58(2): 399 - 409. [Abstract] [Full Text] [PDF]

				S. Minamisawa, Y. Wang, J. Chen, Y. Ishikawa, K. R. Chien, and R. Matsuoka Atrial Chamber-specific Expression of Sarcolipin Is Regulated during Development and Hypertrophic Remodeling J. Biol. Chem., March 7, 2003; 278(11): 9570 - 9575. [Abstract] [Full Text] [PDF]

				D. Franco, S. Demolombe, S. Kupershmidt, R. Dumaine, J. N Dominguez, D. Roden, C. Antzelevitch, D. Escande, and A. F.M Moorman Divergent expression of delayed rectifier K+ channel subunits during mouse heart development Cardiovasc Res, October 1, 2001; 52(1): 65 - 75. [Abstract] [Full Text] [PDF]

				S. Demolombe, D. Franco, P. de Boer, S. Kuperschmidt, D. Roden, Y. Pereon, A. Jarry, A. F. M. Moorman, and D. Escande Differential expression of KvLQT1 and its regulator IsK in mouse epithelia Am J Physiol Cell Physiol, February 1, 2001; 280(2): C359 - C372. [Abstract] [Full Text] [PDF]

				A. F. M. Moorman, A. C. Houweling, P. A. J. de Boer, and V. M. Christoffels Sensitive Nonradioactive Detection of mRNA in Tissue Sections: Novel Application of the Whole-mount In Situ Hybridization Protocol J. Histochem. Cytochem., January 1, 2001; 49(1): 1 - 8. [Abstract] [Full Text]

				E. Mirit, C. Gross, Y. Hasin, A. Palmon, and M. Horowitz Changes in cardiac mechanics with heat acclimation: adrenergic signaling and SR-Ca regulatory proteins Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2000; 279(1): R77 - R85. [Abstract] [Full Text] [PDF]

				W. M Franz, O. J Mueller, M. Fleischmann, P. Babij, N. Frey, M. Mueller, U. Besenfelder, A. F.M Moorman, G. Brem, and H. A Katus The 2.3 kb smooth muscle myosin heavy chain promoter directs gene expression into the vascular system of transgenic mice and rabbits Cardiovasc Res, September 1, 1999; 43(4): 1040 - 1048. [Abstract] [Full Text] [PDF]

				A. Ribadeau-Dumas, M. Brady, S. Y Boateng, K. Schwartz, and K. R Boheler Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene products are regulated post-transcriptionally during rat cardiac development Cardiovasc Res, August 1, 1999; 43(2): 426 - 436. [Abstract] [Full Text] [PDF]

				P. Boknik, C. Unkel, U. Kirchhefer, U. Kleideiter, O. Klein-Wiele, J. Knapp, B. Linck, H. Luss, F. Ulrich Muller, W. Schmitz, et al. Regional expression of phospholamban in the human heart Cardiovasc Res, July 1, 1999; 43(1): 67 - 76. [Abstract] [Full Text] [PDF]

				S. Alcolea, M. Theveniau-Ruissy, T. Jarry-Guichard, I. Marics, E. Tzouanacou, J.-P. Chauvin, J.-P. Briand, A. F. M. Moorman, W. H. Lamers, and D. B. Gros Downregulation of Connexin 45 Gene Products During Mouse Heart Development Circ. Res., June 25, 1999; 84(12): 1365 - 1379. [Abstract] [Full Text] [PDF]

				E. Mirit, A. Palmon, Y. Hasin, and M. Horowitz Heat acclimation induces changes in cardiac mechanical performance: the role of thyroid hormone Am J Physiol Regulatory Integrative Comp Physiol, February 1, 1999; 276(2): R550 - R558. [Abstract] [Full Text] [PDF]

				E. Kolossov, B.K. Fleischmann, Q. Liu, W. Bloch, S. Viatchenko-Karpinski, O. Manzke, G.J. Ji, H. Bohlen, K. Addicks, and J. Hescheler Functional Characteristics of ES Cell-derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein J. Cell Biol., December 28, 1998; 143(7): 2045 - 2056. [Abstract] [Full Text] [PDF]

				J. Ya, M. W. Schilham, P. A. J. de Boer, A. F. M. Moorman, H. Clevers, and W. H. Lamers Sox4-Deficiency Syndrome in Mice Is an Animal Model for Common Trunk Circ. Res., November 16, 1998; 83(10): 986 - 994. [Abstract] [Full Text] [PDF]

				H. K. B. SIMMERMAN and L. R. JONES Phospholamban: Protein Structure, Mechanism of Action, and Role in Cardiac Function Physiol Rev, October 1, 1998; 78(4): 921 - 947. [Abstract] [Full Text] [PDF]

				B. S. Cain, D. R. Meldrum, K. S. Joo, J.-F. Wang, X. Meng, J. C. Cleveland Jr., A. Banerjee, and A. H. Harken Human SERCA2a levels correlate inversely with age in senescent human myocardium J. Am. Coll. Cardiol., August 1, 1998; 32(2): 458 - 467. [Abstract] [Full Text] [PDF]

				J. Weil, T. Eschenhagen, G. Fleige, C. Mittmann, E. Orthey, and H. Scholz Localization of preproenkephalin mRNA in rat heart: selective gene expression in left ventricular myocardium Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H378 - H384. [Abstract] [Full Text] [PDF]

				J. Cernohorsky, V. Pelouch, B. Korecky, and R. Vetter Thyroid control of sarcolemmal Na+/Ca2+ exchanger and SR Ca2+-ATPase in developing rat heart Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H264 - H273. [Abstract] [Full Text] [PDF]

				I. Gombosova, P. Boknik, U. Kirchhefer, J. Knapp, H. Luss, F. U. Muller, T. Muller, U. Vahlensieck, W. Schmitz, G. S. Bodor, et al. Postnatal changes in contractile time parameters, calcium regulatory proteins, and phosphatases Am J Physiol Heart Circ Physiol, June 1, 1998; 274(6): H2123 - H2132. [Abstract] [Full Text] [PDF]

				A. F.M. Moorman, F. de Jong, M. M.F.J. Denyn, and W. H. Lamers Development of the Cardiac Conduction System Circ. Res., April 6, 1998; 82(6): 629 - 644. [Full Text] [PDF]

				D. Franco, W. H Lamers, and A. F.M Moorman Patterns of expression in the developing myocardium: towards a morphologically integrated transcriptional model Cardiovasc Res, April 1, 1998; 38(1): 25 - 53. [Full Text] [PDF]

				J. Ya, M. J. B. van den Hoff, P. A. J. de Boer, S. Tesink-Taekema, D. Franco, A. F. M. Moorman, and W. H. Lamers Normal Development of the Outflow Tract in the Rat Circ. Res., March 9, 1998; 82(4): 464 - 472. [Abstract] [Full Text] [PDF]

				J. Ya, E. B. H. W. Erdtsieck-Ernste, P. A. J. de Boer, M. J. A. van Kempen, H. Jongsma, D. Gros, A. F. M. Moorman, and W. H. Lamers Heart Defects in Connexin43-Deficient Mice Circ. Res., February 23, 1998; 82(3): 360 - 366. [Abstract] [Full Text] [PDF]

				M. U Koban, A. F.M Moorman, J. Holtz, M. H Yacoub, and K. R Boheler Expressional analysis of the cardiac Na-Ca exchanger in rat development and senescence Cardiovasc Res, February 1, 1998; 37(2): 405 - 423. [Abstract] [Full Text] [PDF]

				K. Wong, K. R Boheler, J. Bishop, M. Petrou, and M. H Yacoub Clenbuterol induces cardiac hypertrophy with normal functional, morphological and molecular features Cardiovasc Res, January 1, 1998; 37(1): 115 - 122. [Abstract] [Full Text] [PDF]

				K. Wong, K. R. Boheler, M. Petrou, and M. H. Yacoub Pharmacological Modulation of Pressure-Overload Cardiac Hypertrophy : Changes in Ventricular Function, Extracellular Matrix, and Gene Expression Circulation, October 7, 1997; 96(7): 2239 - 2246. [Abstract] [Full Text]

				D. Yabe, T. Nakamura, N. Kanazawa, K. Tashiro, and T. Honjo Calumenin, a Ca2+-binding Protein Retained in the Endoplasmic Reticulum with a Novel Carboxyl-terminal Sequence, HDEF J. Biol. Chem., July 18, 1997; 272(29): 18232 - 18239. [Abstract] [Full Text] [PDF]

				D. R. Meldrum, J. C. Cleveland Jr, B. C. Sheridan, R. T. Rowland, A. Banerjee, and A. H. Harken Cardiac Surgical Implications of Calcium Dyshomeostasis in the Heart Ann. Thorac. Surg., April 1, 1996; 61(4): 1273 - 1280. [Abstract] [Full Text]

				D. L. Baker, V. Dave, T. Reed, and M. Periasamy 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 J. Biol. Chem., March 8, 1996; 271(10): 5921 - 5928. [Abstract] [Full Text] [PDF]

				M. Ver Heyen, S. Heymans, G. Antoons, T. Reed, M. Periasamy, B. Awede, J. Lebacq, P. Vangheluwe, M. Dewerchin, D. Collen, et al. Replacement of the Muscle-Specific Sarcoplasmic Reticulum Ca2+-ATPase Isoform SERCA2a by the Nonmuscle SERCA2b Homologue Causes Mild Concentric Hypertrophy and Impairs Contraction-Relaxation of the Heart Circ. Res., October 26, 2001; 89(9): 838 - 846. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moorman, A. F. M. Articles by Boheler, K. R. Search for Related Content PubMed PubMed Citation Articles by Moorman, A. F. M. Articles by Boheler, K. R.