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
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).
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.
(Campbell, et al. 1991)
|5 µg/ml||Dr. Doug Fambrough|
Johns Hopkins University
(Dutro, et al. 1993)
| Dr. John Sutko|
University of Nevada
(Mignery, et al. 1989)
|1:200||Dr. Pietro DeCamilli|
|Secondary Antibody Fluorescein-Labeled||0.05 mg/ ml||Kirkegaard and Perry|
|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.|
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.
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.
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.
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.
Summary of Calcium Pool Development
(Dutro et al., 1993)
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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College, Davidson, NC 28036
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