The recently cloned rabbit cardiac calsequestrin gene is over 30 kb and
contains 11 exons. It also includes 1198 bp of 3'-untranslated, 85bp of 5'-untranslated,
and a single poly-A addition site (Arai et. al, 1991). The exon boundaries are located
at the predicted beginnings and ends of helixes and the sequences which encode ß
sheets are located within exons. All isoforms of the calsequestrin gene have similar
splicing patterns, so no alternative splicing pattern is apparent (Yano and Zarain
Calsequestrin is an anchored protein network within the lumen of the SR (Yano and Zarain-Hertzberg, 1994) that is retained within the lumen of the SR after transcription. The protein includes a signal sequence in its first 20 amino acids, that targets the protein to the SR, which is immediately cleaved off the functional protein after translation. The protein does not include KDEL, the amino acid sequence which has been shown to cause a protein to remain in the ER (Munro and Pellum, 1987). Since there is no apparent mechanism to keep the calsequestrin inside the SR, it must either use an unknown mechanism or bind to another SR protein.
The calcium buffer, calsequestrin, is a low-affinity (kD 100µM), high-capacity (up to 45 moles calcium/ mole of calsequestrin) calcium-binding glycoprotein. When there is an influx of calcium into the SR, calsequestrin can bind large amounts of calcium due to its high capacity. When the release channels are opened, the calcium can quickly dissociate from the calsequestrin, due to its low affinity, and move into the cytoplasm by diffusion. The function of the calcium buffer is also to reduce the calcium gradient across the membrane and to keep the calcium ions from precipitating within the lumen.
Calsequestrin is a highly conserved protein and shares up to 90% homology
in different species (fig 4). In the phylogenic tree in figure 4, rabbit cardiac
calsequestrin is indicated to share more homology to other cardiac calsequestrins
than the rabbit skeletal calsequestrin. This comparison shows that the cardiac
isoform is more similar to calsequestrins with similar function than to different
isoforms within the same species. Interestingly, chicken skeletal calsequestrin is
ranked as more homologous to cardiac calsequestrins than other skeletal
calsequestrins. This raises a question of the difference in avian evolution of calcium
proteins compared to other vertebrates.
As mentioned above, two calsequestrin isoforms have been detected in mammals. One is expressed in cardiac and slow-twitch skeletal muscle (cardiac isoform), while the other is expressed in fast-twitch and slow-twitch skeletal muscle (fast-twitch isoform). These two isoforms are encoded by separate genes, but have similar properties and 65% amino acid identity (Choi and Clegg, 1990). The primary structure for both proteins has been determined through cDNA cloning.
Each type of muscle uniquely expresses the two isoforms of calsequestrin during muscle development. Cardiac muscle expresses only the cardiac isoform and the mRNA increases with development. Fast-twitch skeletal muscle coexpresses the cardiac and fast-twitch isoform in fetal and neonatal stages. The cardiac isoform mRNA level gradually decreases during development and is undetectable in adult fast-twitch skeletal muscle. The fast-twitch isoform mRNA increases and eventually replaces the cardiac isoform in adult fast-twitch muscle, indicating a switch in isoforms during development. This occurrence suggests that the two genes may share some common regulatory mechanisms (Arai et. al, 1991). In adult slow-twitch skeletal muscle, the fast-twitch isoform mRNA is strongly expressed while the cardiac isoform mRNA is barely detectable (Arai et. al, 1991).
The heart is the first working organ in the chicken embryo. The initial heart beat occurs between Hamburger and Hamilton development stages 9 - 10 (1951), before the SR calcium pool exists. The first coordinated contractions are not localized in any area, but occur in an unpredictable sequence along the right ventricular wall (Patten and Kramer, 1949). The expression of the calcium pool components during development in relation to the initial cardiac contraction has not been determined.
Experimentation with early chicken embryos has determined that when
calcium channels in the plasma membrane are blocked, early heart contractions do
not occur (Renaud et. al, 1984; Hasin and Barry, 1987; Sakai et. al, 1983). Therefore,
initial contractions of cardiac myocytes rely on the influx of extracellular calcium.
There is a developmental switch from the use of extracellular calcium for muscle
contraction to the use of intracellular calcium regulated by the SR, but it has not
been determined when or how this switch occurs. To understand the mechanism
and timing of this switch, it is important to determine the developmental stage and
location of initial calsequestrin expression to know when the calcium is first
buffered in the lumen of the SR.
The older chicken embryos (stages 19 - 21) were prepared by cryostat sectioning at 12µm thickness. The frozen sections were melted directly onto a slide and a circle of rubber cement was put around the section to limit reagent volumes. The tissues were hydrated with phosphate bovine solution (PBS - 1% NaCl, 0.025% KCl, 0.18% Na2HPO4, 0.03% KH2PO4). Next, the tissues were hydrated with 1% bovine serum albumin (BSA) PBS solution for 5 minutes. The sections were then fixed in PBS with 4% paraformaldehyde for 5 minutes and left in PBS, 1% BSA and 5 mg/ml lysine for 5 minutes. The primary antibody (polyclonal rabbit antisera to canine cardiac calsequestrin; Jorgensen and Campbell, 1984) was added in PBS, 1% BSA and 0.25% saponin and incubated for 45 minutes at room temperature. This antibody was generously provided by Dr. Kevin Campbell at the University of Iowa. The sections were washed 3 times with PBS and the secondary antibody (FITC conjugated goat anti-rabbit, purchased from KPL, Gaithersburg, MD) in PBS, 1% BSA and 0.25% saponin was added and incubated for 45 minutes at room temperature. The sections were washed three times again, covered in mounting medium (90% glycerol, 10% PBS, 0.1% para-phenylenediamine) and sealed with a cover slip and clear fingernail polish. The slide was then photographed to observe labeling.
The younger embryos (stages 12 - 19) were whole-mounted on slides. The eggs were cracked into petri dishes and the embryo was rolled to the top of the yolk. A donut-shaped ring of 42.5mm Whatman #3 filterpaper was placed on top of the embryo. While holding the ring with forceps, the membranes on the margin of the paper were cut away. The paper was carefully lifted, with the embryo, and put into PBS to remove excess yolk. The embryos were emersed into buffered 4% paraformaldehyde for 10 minutes at room temperature, then transferred into blocking solution (BSA and 5 mg/ml lysine) for 5 minutes at room temperature. The embryo was placed on the slide, yolk side down, and rubber cement was added around the edges. The slide was put into a humid chamber and the primary antibody in BSA and saponin was added to each slide. The embryos were incubated overnight at 4° C. The next day, the antibody was removed and the embryo was washed throughout the day with three changes of buffer while being maintained at 4° C. At the end of the day, the embryo was covered with secondary antibody and incubated overnight. The antibody was removed and the embryos were washed with three changes of buffer the next day, covered with mounting medium, sealed with a coverslip, and photographed the embryo.
All slides were viewed with an epifluorescence microscope. They were
photographed with T-max 400 35mm film (Kodak) and developed according to the
Calsequestrin may not be the only calcium binding protein in the lumen of the SR in muscle cells. Calreticulin, a functional analogue of calsequestrin, is a constituent calcium-binding protein in cardiac and skeletal muscle. Calreticulin is expressed in early fetal rat skeletal and cardiac muscle, but is down regulated during development. Calsequestrin appears later in development and becomes the major calcium binding protein and plays a major role in SR maturation (Imanaka-Yoshida et. al, 1996). In future experiments, chicken embryos should be labeled for calreticulin to determine if and when it appears in development of the heart.
Muscle development is thought to require a network of interdependent, temporally regulated gene activation. Protein expression during muscle differentiation is known to induce expression of later proteins. Little is known about the formation of SR, but calsequestrin immunoreactivity is detected in myoblasts before the appearance of recognizable SR (Choi and Clegg, 1990). After the formation of the SR, skeletal calsequestrin immunoreactivity takes on a fibrous appearance, similar to that of newly formed SR, suggesting that calsequestrin may initiate SR formation (Choi and Clegg, 1990). However, each muscle type expresses a unique program of SR proteins during muscle development. Cardiac SR development relies on the abundance of a single calsequestrin isoform, not two distinct isoforms as in skeletal muscle (Yano and Zarain-Hertzberg, 1994). SR gene transcripts are coordinately induced during muscle differentiation (Arai et. al, 1991). Contractile protein mRNA and SR gene transcripts appear simultaneously during skeletal muscle differentiation, indicating the coordination of their expression. The SR gene transcripts appear just before myotube formation and then simultaneously with contractile protein mRNA. These observations suggest that the activation of SR genes is under the control of muscle development differentiation regulators, such as the myoD gene family (Arai et. al, 1991).
However, in this research it was shown that SR transcripts are not
coordinately induced. Each SR calcium pool protein is initially expressed at a
different time and location, contradicting the idea that all SR proteins appear
simultaneously during development. In this study, calsequestrin has been found to
appear later in development than the calcium pump, but before the appearance of
IP3 receptors. From this information, we can hypothesize what event in SR
development turns on calsequestrin expression. Since the calcium pumps are
present in the early SR, perhaps the build up of calcium ions, or the flow of calcium
across the SR membrane, triggers calsequestrin transcription. Until the timing of
ryanodine receptor calcium channel expression is determined, we will not know if
the calcium is allowed to move out of the primitive SR when calsequestrin first
appears. Future research should include detecting for ryanodine receptors in early
embryos, as well as calreticulin, to further our knowledge of SR development.
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Tharin, S., E. Dziak, M. Michalak and M. Opas. Widespread tissue distribution of rabbit calreticulin, a non-muscle functional analogue of calsequestrin. Cell and Tissue Research. 269: 29-37, 1992.
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College, Davidson, NC 28036
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