Jeffery D. Cromartie ('97): Honors Thesis

Development of Calcium Pools in Early Embryonic Chicken Hearts

Jeffery Cromartie ('97)

Abstract

Calcium plays a critical role in cellular homeostasis, neurotransmitter release, and muscle contraction. Intracellular calcium pools are regulated by calcium ion pumps, channels, and buffers. Sarco/endoplasmic reticulum calcium ATPase (SERCA) is the transmembrane protein which actively transports calcium ions from the cytosol into the sarcoplasmic reticulum (SR) for storage. The ryanodine receptor and the inositol 1,4,5,-trisphosphate receptor (IP3R) are both calcium channels which allow calcium ions to be released from the SR. Calsequestrin (CS) is a lumenal SR protein that binds calcium with low affinity and high capacity and acts as a buffer for the mM levels of lumenal calcium. The initial expression of the cardiac SERCA isoform (SERCA2), and IP3R, was determined in cardiac myocytes of chicken embryos (embryonic day 1 through 4) using indirect immunofluorescence labeling. SERCA2 first appears as a punctate pattern along the right margin of the ventricular wall where myocytes exhibit the first visible contractions (Patten and Kramer Physiol. Rev. 29: 31-47, 1949). Our observations not only support an anterior to posterior gradient of SERCA2 (Jorgenson and BashirDev. Biol. 106: 156-165, 1984), but also suggest an additional lateral to medial gradiaent of SERCA2 expression in early chicken heart development. By embryonic day 3, the heart is beating regularly but IP3R was not detected until embryonic day 3.5 to 4. Therefore, calcium pool development in cardiac myocytes is a sequential process that begins with SERCA2 expression and is not completed until embryonic day 4. Perhaps this sequential expression of proteins allows the SR to accumulate calcium via SERCA2 in order to fill the pool for later use when the SR becomes the major site of calcium regulation for cardiac contractions.

Introduction

Calcium ions regulate a wide range of cellular functions including neurotransmitter secretion, T-cell activation, and muscle excitation-contraction. Calcium ions are also believed to play a critical role in cell division (Zimmerman, 1989). Recently discovered transmembrane nuclear calcium ion gradients and a variety of intra-nuclear calcium ion binding proteins has sparked interest in this ion's involvement in the cell-cycle timing and division, gene expression, and protein activation (Gilchrist et al., 1994).

Although calcium is needed for life functions, high concentrations of calcium ions within a cell are toxic. A cell must be able to tightly regulate calcium ion concentrations, or it will die. Excess calcium can activate enzyme systems that can break down parts of a cell (Zimmerman, 1989). In addition to digesting itself, a cell with toxic levels of calcium will lose access to its energy source, ATP. As calcium ions bind to free phosphorus ions, it becomes harder for the cell to find phosphorus which slows down ATP synthesis (Zimmerman,1989). So, it is to a cell's advantage to actively regulate calcium ion levels even if the process takes some energy. Calcium control can consume as much as 15% of a myocytes energy (Zimmerman, 1989). Cell function and survival depend critically upon the precise regulation of intracellular calcium concentrations, and rapid changes in calcium levels in myocytes are controlled by the sarcoplasmic reticulum (Putney, 1990).

Intracellular calcium ions are regulated by calcium pools in the sarco/endoplasmic reticulum (SER). Calcium pools are composed of three main groups of proteins: pumps, channels, and buffers. Calcium pumps use ATP to actively pump calcium ions from the cytosol into the lumen (figure 1). The calcium pool buffer, calsequestrin (Sacchetto et al., 1995), stores the intracellular calcium ion supply, while calcium channels release calcium ions back into the cytoplasm.

Figure 1. The three components necessary for a functional calcium pool. Calcium pumps actively transport calcium ions from the cytoplasm to the lumen. Calcium buffers store calcium ions, and calcium channels allow the release of sequestered calcium ions back into the cytoplasm.

Calcium pools are extremely important in the development of the heart because calcium ions play a critical role in the excitation-contraction process (Lytton and MacLennan, 1991). When calcium pools are developed in cardiomyocytes, intracellular calcium is pumped by SERCA2 from the cytosol into the SER where the ions are sequestered and later released through calcium channels. Troponin is located in the sarcoplasm and requires calcium ions to facilitate activation-contraction of actin and myosin (Lim, et al., 1983). In order for relaxation to occur, calcium ions must be actively removed from the sarcoplasm.

The sarco/endoplasmic reticulum calcium ATP-ase (SERCA) has three main isoforms. SERCA 1 is expressed in fast-twitch muscle cells (Brandl et al., 1986, 1987). SERCA2 has two different isoforms, SERCA2a and SERCA2b, that differ in the carboxyl terminal region due to alternative mRNA splicing (Campbell, et al 1991). SERCA2a is the calcium pump expressed in cardiac myocytes (Brandl et al., 1987). SERCA2b is known as a general maintenance isoform and is expressed in all cells with the exception of cardiac myocytes and fast-twitch muscle cells (Brandl et al., 1987). SERCA3 is less well documented, but it has been demonstrated to be expressed in non-muscle tissues (Burk et al., 1989; Wu et al. 1988). Related isoforms of calcium pumps are found in the plasma membrane (Kasai 1990). Since the majority of calcium ion regulation of myocytes is associated with the SER, the plasma membrane calcium pumps play a limited role in calcium regulation by actively transporting calcium ions from the cytosol into the extracelluar fluid.

Calcium channels in the SER include the ryanodine receptor and the inositol 1-4-5 triphosphate (IP3) receptor (Fleischer and Inui, 1989). When opened, both receptors allow the release of sequestered calcium pools. During embryonic chicken development, the ryanodine receptor has not been detected until embryonic day four (Dutro, et al. 1993). The ryanodine receptor has been shown to be the primary calcium channel in adult chicken cardiomyocyte SR (Junker, et al. 1994). In contrast, the IP3 receptor is expressed in the intercalated discs and only makes up 1% of the calcium channels in the SER (Kijima, et al. 1993).

Chicken heart development begins when cardiac primordia fuse, forming a straight anterior to posterior ventricular tube. As development progresses, the ventricle begins to fold. Figure 2 is a line drawing adapted from Patten and Krammer (1949), which depicts the early folding pattern of the ventricle. The first contractions of the chicken heart occur between Hamburger and Hamilton (1953) stage 9-10 (embryonic day 1.5/ 29-38 hours) which roughly corresponds to 10-11 somite pairs (Patten and Krammer, 1949).

Figure 2. These line drawings depict chicken embryos at stages 11 (panel A) and early13 (panel B). Adapted from Patten and Kramer (1949).

The purpose of this study was to determine when and in what pattern the cardiac calcium pump (SERAC2) and the cardiac calcium channels ( the IP3-R and the Ryanodine-R) are initially expressed in relation to the development of the heart and the initial cardiac contractions.

Methods

Whole Embryo labeling
Fertilized chicken eggs were obtained from Hubbard farms in Statesville, NC. The eggs were placed in a Petersime model #4 incubator for 24-48 hours at 37š C, then cracked into petri dishes. Watman #1 (42.5 mm circles) filter paper was cut so that the centrally located hole was larger than the embryo and the filter paper portion is 5-10 mm wide. The filter paper rings were placed on the embryos, and the embryos and the paper were removed from the yolk using scissors and forceps. The embryos were then washed in a PBS (Phosphate Buffered Saline) solution for one minute to remove yolk, fixed in a 4% paraformaldehyde PBS solution for 5 minutes, transferred into a PBS + 1% BSA + 5 mg/ml lysine for 5 minutes, and then transferred to microscope slides which had been previously treated with subbing solution (0.05% K[CR(SO6H4)2 (H2O)2] 6 H20 + 0.3% gelatin + 0.04% NaN3). The embryos were then separated from the Watman paper using razor blades, and excess membranes were also cut away from the embryos.

Table 1

Antibodies Used in This Study

Antibody	Dilution	Source
Anti-SERCA2 (3H2) (Campbell, et al. 1991)	5 µg/ml	Dr. Doug Fambrough Johns Hopkins University
Anti-Ryanodine Receptor € (34C) € (110 E) (Dutro, et al. 1993)	1: 400 1: 100	Dr. John Sutko University of Nevada Los Vegas
Anti-IP₃ Receptor Rabbit Serum (Mignery, et al. 1989)	1:200	Dr. Pietro DeCamilli Yale University
Secondary Antibody Fluorescein-Labeled	0.05 mg/ ml	Kirkegaard and Perry Labs, MD

Antibody solutions were prepared using PBS +1% BSA+ 0.25% saponin with appropriate dilution of antibodies or anti-serum (table 1). Rubber cement was placed around the embryo, buffered antibody solution was added as soon as the cement dried, and embryos were placed in a humid chamber and incubated overnight at 4° C. The following day, the embryos were washed three times with PBS, then buffered (PBS + 1% BSA+ 0.25% saponin) secondary antibody solution (table 1) was added, and the embryos were again incubated overnight in a humid chamber at 4° C. On the third day, the embryos were washed with PBS three times during the day. At the end of the third day, each embryo was covered with 12 microliters of mounting media (10 ml PBS+ 90 ml glycerol [heat glycerol 30-60 seconds in a 600 watt Microwave] then added 0.1 g para-phenylenediamine and stored in alloquots at -20° C). Cover slips were placed on the embryos, and the cover slips were sealed with nail polish. Embryos were then photographed using a 35mm camera loaded with TMAX 400 black and white film. The camera was attached to a model BH-2 Olympus epi-fluorescent microscope with a model PM-10AD fluorescent lamp attachment.

Cryosections
Embryos stages 19 and higher were frozen on microscope slides with dry ice. The frozen embryos were then removed with razor blades, placed on cryosectioning chucks, and embedded with TBS tissue freezing media (Triangle Biomedical). The embryos were then sectioned using a Microm cryostat into 20 micron sections. The cardiac sections were placed on subbed slides (see whole embryo labeling section), fixed in 4% para-formaldehyde for 5 minutes, and transferred to a PBS + 1% BSA + 5 ug/ml of lysine for 5 minutes. The cardiac sections were then surrounded by a mote of Stanford rubber cement, and incubated with buffered antibody solution (PBS + 1% BSA+ 0.25% saponin) for 1 hour. The sections were washed three times in a PBS solution for 5 minutes, and incubated with buffered (PBS + 1% BSA+ 0.25% saponin) secondary antibody solution (table 1), followed by three final PBS washes for 5 minutes each. The treated sections were covered with mounting media and sealed with nail polish (see whole embryo labeling section). The sections were viewed and photographed as above. Also adult cardiac and sartorius sections were fixed, blocked (see above) and then treated to a 1% SDS (sodium dodecyl sulfate) wash for 5 minutes followed by a 5 minute PBS wash before antibody incubation and observation (see above).

Bodipy-Labeled Ryanodine Probing
Adult chicken sartorius muscle and adult chicken hearts were obtained from Harris Teeter Supermarket. The tissue was frozen at -25° C and mounted on cryosectioning chucks. Both tissue samples were cryosectioned into 20 micron sections, placed on subbed slides, fixed in 4% para-formaldehyde for 5 minutes, and transferred to a PBS + 1% BSA + 5 micrograms per milliliter of lysine solution for 5 minutes. The tissue sections were then treated with PBS + 1% BSA+ 1% triton X-100 + 0.25% saponin +1mM FL/X Bodipy Ryanodine (using ethanol or DMSO as a solvent) solution for 12 hours (see table 2) . The following day, the sections were washed in a 50 ml bath of PBS + 1 gram of activated charcoal for ten minutes followed by a 5 minute wash in 50 ml PBS. The sections were then observed as above.

Table 2

Bodipy-Ryanodine Incubation Solution

Buffered Antibody Solution
(PBS +1% BSA+ 0.25% Saponin)

Add 1% Triton

Bring entire Solution up to 1 mM Bodipy Ryanodine using EtOH or DMSO to initially solubilize the Bodipy Ryanodine.

Results

Whole Embryo Labeling
Immunofluorescent labeling of SERCA2 was observed from H/H stage 10-13. At H/H stage 10 (figure 3), the heart is the tube like structure in the center of the photo. The bright areas are cells which contain the calcium pump SERCA2, and the dark circular structures within the labeled cells are nuclei. Notice that the SERCA2 expression pattern is scattered throughout the developing ventricle in a punctate fashion. Some of the labeled cells seem brighter than others, but the positioning of the brighter cells seems random. The cardiomyocyte nuclei appear to be free of labeling. Also, note that the ventricular wall (on the right side of the photo) is not intensely labeled. The round spot in the lower left hand quadrant is an air bubble in the mounting media. Note that there are many unlabeled cells which are not visible.

Figure 3. A stage 10 (12 somites pairs) embryonic heart. Anti-calcium pump (SERCA2) antibodies (3H2) were used to label the heart followed by secondary fluorescein-labeled antibodies. (The anterior is to the top.) The objective lens is 10x.

As development continues, the expression of SERCA2 becomes much more dense and centers in the middle of the ventricle. By early stage 11 ( 13 somites pairs) figure 4, SERCA2 expression is now densely centered in a patch formation, and all labeled cells within the patch appear bright. The patch appears to be located in the center of the ventricle leaving the right ventricular margin relatively unlabeled. A punctate pattern continues along the perimeter of the central patch. Cells scattered around the patch appear to vary in brightness much more so than the central patch.

Figure 4. An early stage 11 (13 somite pairs/40 hours) embryonic heart labeled using the same methods as above. (The anterior is to the top.)

At H/H mid-stage 11 (14 somite pairs; figure5), the patch-like pattern of cells expressing SERCA2 appears to migrate and fold along the right ventricular wall, centering the expression of SERCA2 along the lateral edge of the ventricle. The punctate expression is visible toward the medial portion of the ventricle as SERCA2 expression spreads. The lateral edge of the ventricle appears extremely bright. The dark areas within the ventricle are cells which are not expressing SERCA2.

Figure 5. A mid-stage 11 (14 somite pairs/ 45 hours) embryonic heart labeled using the same methods as above. (The anterior is to the top.)

By stage 14, (figure 6; 19 somite pairs; 48-50 hours), labeling of SERCA2 occurs uniformly throughout the embryonic heart. Notice that the atrium is starting to fold into position above the ventricle. The labeling on the upper left edge of the photo is the cranial region. The punctate patterns of SERCA2 are no longer apparent, and the intensity of labeling seems to be uniform throughout the heart.

Figure 6. A stage 14 (19 somite pairs/50 hours) embryonic heart labeled using the same methods as above. The anterior is to the top. The objective lens is 3.2X.

Whole embryo immunofluorescent labeling of the ryanodine receptor and the IP3 receptor were both negative through H/H stage 17 (data not presented). Since embryos past stage 17 are too think to be used in whole embryo mounting, so past this stage embryonic hearts were cryosectioned as the search continued.

Cryosectioning Labeling Results
Embryonic cardiac cryosections labeled with anti-IP3 receptor polyclonal antibodies were negative until stage 21 (H/H) (figure 7), and by stage 23 cardiac cryosections revealed more intense labeling (data not shown). All attempts to label embryonic and adult cardiac sections with anti-ryanodine antibodies (34C and 110E) showed no detectable labeling (data not shown), although 110E and 34C both labeled adult chicken sartorius tissue very clearly (data not shown). Since antibody labeling did not work, the newly released bodipy-labeled ryanodine was employed to probe for the ryanodine receptor. There is no published protocol for this type of labeling.

Figure 7. A stage 21 (3.5 days) cardiac cryosection which has been labeled with polyclonal rabbit anti-chicken IP3-receptor followed by secondary anti-rabbit fluorescein-labeled antibodies (bottom panel). The photo on the top is the negative control which received no anti-IP3-receptor antibodies, but did receive secondary fluorescein antibodies. The objective lens is is 10X.

Bodipy- labeled ryanodine techniques were beginning to show striations on adult sartorius muscle (data not shown). The technique has not been refined enough to effectively label embryonic cardiac tissue.

Discussion

Our results confirm the observations of Jorgensen and Bashir (1984) who noticed both the initial SERCA2 expression in the bulbus ventricular region of the single tubular heart at stage 9-10 and the initial anterior to posterior SERCA2 expression gradient within the developing ventricle. Also, Patten and Krammer (1949), using moving picture records, first observed the initial contractions along the lateral margin of the ventricle at stage 10. According to their observations, the first contractions appeared to involve only a few cells at a time, and the impulse did not seem to spread by conduction. These observations match the expression of SERCA2 which at stage 10 is only expressed in a few cardiomyocytes in a punctate pattern throughout the ventricle (figure 3). Patten and Krammer also observed that about 7-10 hours after the initial punctate contractions the entire right side on the ventricle began to contract, soon followed by the entire ventricle contracting. This initial contraction pattern correlates directly with the expression of SERCA2 (figure 5 and 6). Since SERCA2 facilitates muscular relaxation by removing calcium ions from the cytosol, and SERCA2 expression occurs at the same time as the initial contractions (stages 9-10), it would appear that SERCA2 is one of the last, if not the last, protein necessary for the initial cardiac contraction-relaxation. According to the observations of Lim et al. (1983), sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combinations with densitometry and electron micrographs reveal detectable quantities of myosin heavy chain, actinin, actin, and tropomyosin accumulating in pre-contractile cardiomyocytes. These data further support the hypothesis that SERCA2 is the last in a series of proteins necessary for cardiac contraction-relaxation.

Whole embryo labeling allowed us to observe the intense patch of labeling located in the center of the ventricle (early stage 11). By late stage 11, SERCA2 expression is highest along the lateral wall of the ventricle while expression is not as intense toward central area of the ventricle. This suggest that either the ventricle twists during stage 11, making the central patch appear to have moved the lateral margin of the ventricle or the cells in the central patch area stop expressing SERCA2 by late stage 11, and the cells on the lateral margin start to express SERCA2. The lateral movement of the ventricle seems to be more likely. By stage 13, SERCA2 is expressed evenly throughout the ventricle. This suggests a lateral to medial ventricular expression gradient of SERCA2 from late stage 11 to stage 13 in addition to the anterior to posterior gradient witnessed by Jorgensen and Bashir.

Antibodies 110E and 34C have been successfully used in western blot analysis to detect cardiac ryanodine Dutro et al (1993), but, in this study, no immunofluorescent labeling even in adult cardiac cyrosections was detected. The anti-ryanodine antibodies, 110E and 34C, may not recognize native cardiac ryanodine receptor epitopes. Clearly labeled striations were apparent when 110E and 34C were used to immunoflourescently label adult sartorius skeletal muscle. Since 34C and 110E are functional as demonstrated by the labeled sartorius striations, this suggest that the cardiac ryanodine receptor epitope might be somehow sheltered or contorted such that 110E and 34C can not recognize it unless the ryanodine receptor has by exposed to denaturing agents such as SDS (sodium dodecyl sulfate). Preliminary trails with SDS did not show labeling.

Without calcium channels, calcium pools are incomplete and unable to release intracellular calcium stores, which is one mechanism to facilitate contraction. Dutro et al. (1993), using both anti-ryanodine receptor antibodies (110E and 34C) and ryanodine binding, observed that the ryanodine receptor is present from embryonic day 4 through day 20. They also observed that ryanodine, which can block the ryanodine receptor, causes a slower contraction rate in embryonic hearts. Since the ryanodine receptor is the primary calcium channel in the SER of cardiomyocytes in adults (Junker, 1994), it appears the contraction rate of day 4 embryonic heart and older are dependent on intracellular calcium stores. The IP3 receptor is expressed at stage 21 (day 3.5), but the IP3 receptor is expressed in very limited quantities even in adult cardiac tissue (Kijima, et al.1993). It is doubtful that the IP3 receptor plays a major role in the release of sequestered intracellular calcium stores.

This raises a question: where is the calcium coming from to facilitate contraction between day 1.5, when the contractions begin, and day 4, the earliest the ryanodine receptor is detected? There are two likely answers. First, there could be an undiscovered calcium channel in the SER which is expressed before day 4 that could allow possible intracellular stores to be released. Second, calcium channels on the plasma membrane could allow extracellular calcium to flood in and facilitate contraction. However, one of the most common plasma membrane calcium channels, the nitrendipine receptor, is not expressed until embryonic day 3 (Renaud, et. al., 1984). Also, calcium pumps on the plasma membrane, which pump calcium from the cytosol into the extracellular fluid, could be aiding in early cardiac relaxation. The sodium-calcium exchanger, could also aid in extracting calcium from the sarcoplasma while SERCA2 expression is low in stage 9. However since the ryanodine receptor, which constitutes the vast majority of SER calcium channels, is not detected until embryonic day 4, it seems as though there might be a functional calcium channel on the plasma membrane which allows calcium influxes necessary for contraction.

Recent immunofluorescent studies using anti-calsequestrin antibodies have show that calsequestrin is initially expressed in cardiac myocytes at stage 14 (embryonic day 2; Permar, personal communication). This suggests that intracellular calcium storage is capable well before embryonic day 4. Calcium ions are pumped into the lumen and bind to calsequestrin. If there is no SER calcium channel, then calcium ion levels in the lumen should rise. Perhaps the intracellular calcium pools need time to fill their stores with the aid of SERCA2 before the expression of SER calcium channels.

Table 3

Summary of Calcium Pool Development

Stagees: Hamburger and Hamilton	Stage 10 (Day 1.5) (33-38h)	Stage 11 (Day 1.75) (40-45h)	Stage 14 (Day 2.25) (50-52h)	Stage 17 (Day 2.5) (52-64h)	Stage 21 (Day 3.5) (84h)	Stage 24 (Day 4) (96h)
Protein Expression	Calcium Pump Begins	Calcium Pump Spreads	Calcium Pump Spreads Throughout Entire Heart	Calcium Pump Continues	Calcium Pump Continues	Calcium Pump Continues
-	-	-	Calsequestr in Begins	Calsequestrin Throughout Heart	Calsequestrin Continues	Calsequestrin Continues
-	-	-	-	-	IP₃ Receptor Begins	IP₃ Receptor Present
-	?	?	?	?	?	Ryanodine Receptor Present (Dutro et al., 1993)

In summary, it appears the development of the SER in cardiac myocytes is a sequential process (table 3). SERCA2 is expressed before both calsequestrin and the IP3-R, and SERCA2 closely coincides with the initial cardiac contractions/ relaxations. The calcium buffer calsequestrin is expressed after SERCA2, and calsequestrin could possibly allow intracellular calcium storage to build up if the ryanodine receptor is not already present. If the ryanodine receptor is not expressed until day 4, then it would seem likely that plasma membrane calcium exchangers or channels are responsible for the calcium ion supply necessary to keep the embryonic heart contracting between day 1.5 and day 4. However, since it is possible that the ryanodine receptor is present at low levels earlier in development, the exact sequence of SER development remains a mystery.

Future analysis will attempt to achieve immunofluorescent labeling of the ryanodine receptor before embryonic day 4 (stage 24). Since immunofluorescence shows more data than western blot analysis, it is possible that the Ryanodine receptor is present early than stage 24. Efforts will center around using brief SDS washes to expose the epitope of the cardiac ryanodine receptor (Brown et al, 1996), so that hopefully 110E and 34C will show labeling. Also, future research will seek to optimize bodipy-ryanodine labeling methods, thus providing researchers with a potentially useful tool for observing the ryanodine receptor.

Learning more about calcium pool components and interactions could greatly enhance our understanding of cardiac development, SER development, and ion regulation. Calcium regulates such a broad variety of critical life functions and interacts with so many different proteins that is of considerable importance to understand role of calcium ions in this complex and wonderful experience we call life.

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