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J Appl Physiol 101: 728-733, 2006. First published May 25, 2006; doi:10.1152/japplphysiol.00214.2006
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Contractile and Ca2+-handling properties of the right ventricular papillary muscle in the late-gestation sheep fetus

T. N. Spencer,1 K. J. Botting,1 J. L. Morrison,2,* and G. S. Posterino1,*

1Discipline of Physiology, School of Molecular and Biomedical Science, University of Adelaide, and 2Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia

Submitted 19 February 2006 ; accepted in final form 8 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The force-generating capacity of cardiomyocytes rapidly changes during gestation and early postnatal life coinciding with a transition in cardiomyocyte nucleation in both mice and rats. Changes in nucleation, in turn, appear to coincide with important changes in the excitation-contraction coupling architecture. However, it is not clear whether similar changes are observed in other mammals in which this transition occurs prenatally, such as sheep. Using small (70–300 µM diameter) chemically skinned cardiomyocyte bundles from the right ventricular papillary muscle of sheep fetuses at 126–132 and 137–140 days (d) gestational age (GA), we aimed to examine whether changes in cardiomyocyte nucleation during late gestation coincided with developmental changes in excitation-contraction coupling parameters (e.g., Ca2+ uptake, Ca2+ release, and force development). All experiments were conducted at room temperature (23 ± 1°C). We found that the proportion of mononucleate cardiomyocytes decreased significantly with GA (126–132d, 45.7 ± 4.7%, n = 7; 137–140d, 32.8 ± 1.6%, n = 6; P < 0.05). When we then examined force development between the two groups, there was no significant difference in either the maximal Ca2+-activated force (6.73 ± 1.54 mN/mm2, n = 14 vs. 6.55 ± 1.25 mN/mm2, n = 7, respectively) or the Ca2+ sensitivity of the contractile apparatus (pCa at 50% maximum Ca2+-activated force: 126–132d, 6.17 ± 0.06, n = 14; 137–140d, 6.24 ± 0.08, n = 7). However, sarcoplasmic reticulum (SR) Ca2+ uptake rates (but not Ca2+ release) increased with GA (P < 0.05). These data reveal that during late gestation in sheep when there is a major transition in cardiomyocyte nucleation, SR Ca2+ uptake rates increase, which would influence total SR Ca2+ content and force production.

cardiomyocytes; excitation-contraction coupling; Ca2+ sensitivity

THE FORCE-GENERATING CAPACITY of cardiomyocytes changes rapidly during gestation and early development in the mouse (20). This change in force-generating capacity is correlated to important changes in protein expression and architecture within the excitation-contraction (E-C) coupling system (20, 27). E-C coupling broadly describes the sequence of events from the spread of the action potential at the surface membrane to activation of calcium (Ca2+) release from the sarcoplasmic reticulum (SR), muscle contraction, and Ca2+ uptake by the SR (3). In mice, a significant decrease in the Ca2+ sensitivity and increase in maximum Ca2+-activated force production of cardiomyocytes (20) occurs at a time coinciding with a transition in the nucleation of cardiomyocytes after birth [3–7 days (d) postnatal (PN)] (22). Siedner et al. (20) reported in the mouse a twofold increase in the maximum Ca2+-activated force development and a significant decrease in the Ca2+ sensitivity of the contractile apparatus between 19d gestational age (GA), when cells are predominantly mononucleate, and 7d PN, when cells are predominantly binucleate (22). Although changes in contractile apparatus properties of cardiomyocytes in mice may be suggestive of developmental changes in the human, it is not clear how well these changes correlate, particularly in terms of function. In this respect, sheep are similar to humans such that before 110d GA, fetal sheep heart growth occurs by cell proliferation of mononucleate cardiomyocytes (5). There is a decline in the number of mononucleate cardiomyocytes and an increase in binucleate cardiomyocytes after 110d GA. Binucleate cardiomyocytes have an increased myofibril density and are unable to undergo cytokinesis (cell division), relying on hypertrophic growth (increasing size) to match the growth requirements of the heart (5, 21). In addition, the functional changes in contractile apparatus seen in mice and rats (12) around this transition to binucleate cardiomyocytes may differ from the human because it occurs postnatally rather than in the intrauterine environment where right ventricular dominance occurs and changes in load are known to alter cardiomyocyte development (2).

The SR is an important organelle in contractile function of the heart, as it controls intracellular Ca2+ concentration ([Ca2+]) by acting as a store of Ca2+. Ca2+-ATPases regulate the rate of Ca2+ sequestration and ultimately determine the Ca2+ content of the SR (25). Ca2+ uptake rates have only previously been examined in fetal and adult cardiac muscle from sheep by using isolated SR vesicle preparations (13). Mahony and Jones (13) showed that Ca2+ uptake in SR from 126–132d GA fetuses was reduced compared with adult. However, it is not clear from this study whether SR Ca2+ uptake reaches adult levels before or after birth because any changes that may occur over the late-gestation period have not been examined. The ryanodine receptors (RyRs) also play a major role in Ca2+ handling in terms of controlling the amount of Ca2+ released with every action potential. Currently, little is known about the function of the RyRs in developing sheep hearts. A previous study by Michalak (15) comparing isolated SR vesicle preparations from fetal (100–105 and 140–145d) and adult sheep hearts indicated functional immaturity in fetal SR, possibly arising from differences in the localization within the SR, function, and subtype of RyR present. However, it was not clear how these differences in SR properties equated to functional changes in Ca2+ release.

Therefore, this study aimed to investigate 1) the effect of GA on the maximum Ca2+-activated force and Ca2+ sensitivity of the contractile apparatus and 2) Ca2+ uptake and release by the SR, in developing sheep hearts, and 3) to identify whether these changes correlate with the transition from mononucleate to binucleate cardiomyocytes. We show that at a time when a significant decline in the proportion of mononucleate cardiomyocytes was found across late gestation, there is an increase in the rate of Ca2+ uptake by the SR but no other significant change in any other measured E-C coupling parameter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   All experiments were performed according to the guidelines and with approval of the University of Adelaide Animal Ethics Committee. Thirteen time-dated, pregnant Merino ewes underwent fetal and maternal vascular surgery at 110–120d GA under aseptic conditions. General anesthesia was induced by sodium thiopentone (1.25 g; Pentothal, Rhone Merieux, Pinkenba, Queensland, Australia) and maintained by inhalation of halothane (2.5–4%) in oxygen. Vascular catheters were inserted into the maternal jugular vein, fetal femoral and carotid arteries, fetal jugular vein, and amniotic cavity (6, 7, 17). Fetal catheters were exteriorized through a small incision in the ewe's flank. At surgery, antibiotics were administered to the ewe (3.5 ml, 150 mg/ml procaine penicillin; 112.5 mg/ml, benzathine penicillin; 2 ml, 250 g/ml dihydrostreptomycin, Lyppards, Adelaide, Australia) and fetus (1 ml, 150 mg/ml procaine penicillin; 112.5 mg/ml benzathine penicillin; 1 ml, 250 g/ml dihydrostreptomycin, Lyppards). Antibiotics were administered intramuscularly to the ewe for 3 days after surgery and intra-amniotically to the fetus (5 ml; 100 mg/ml ampicillin, Lyppards) for 4 days after surgery (6). Fetal blood-gas samples (1 ml) were collected daily for the measurement of PO2, PCO2, pH, and hemoglobin (ABL 520 analyzer, Radiometer, Copenhagen, Denmark).

Muscle collection.   Postmortem was performed between 126–140d GA. The ewe was killed with an overdose of sodium pentobarbitone (25 ml at 325 mg/ml, Lethobarb, Lyppards, Adelaide, Australia). The fetus was removed by hysterotomy, weighed, and killed by exsanguination. The heart was excised, weighed, perfused with heparin and KCl, and transported in ice-cold saline. The heart was hung on a Langendorff apparatus and reverse perfused through the aorta with a Ca2+-free Tyrode's solution at a rate of 10.6 ml/min (Minipulse 3 pump, Gilson, Villeirs, France), while the medial papillary muscle was removed from the right ventricle (4, 18, 26) and immediately placed in cool, well-oxygenated Tyrode's solution. The heart was then dissociated by perfusion with collagenase (120 units/ml, Worthington Biochemical, Lakewood, NJ) and protease (Sigma, Castle Hill, NSW, Australia) in Tyrode's solution to digest the extracellular matrix (23, 24). Approximately 300 ml of KB buffer were then perfused through the heart to flush out the enzymes. Dissociated cells were collected after trituration and then fixed in 1% paraformaldehyde for later analysis.

Bundle isolation.   Under a dissecting microscope, small bundles of cardiomyocytes of 70–300 µm diameter were isolated from the papillary muscle and then attached to a force transducer and stationary pin by use of fine suture silk. The bundle was then briefly immersed in a high-EGTA physiological solution (solution 1; see Table 1) to ensure that the bundle was fully relaxed and then stretched by 20% of its slack length before skinning (either saponin or Triton 2%X-100). Length and diameter of the bundle were then measured. Subsequent force responses were recorded by using a PowerLab/8Sp (ADInstruments, Castle Hill, NSW, Australia) data-acquisition system and recorded onto both a paper chart recorder (Ross Recorders) and PowerLab Chart v4.1 computer software (ADInstruments).


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  		Table 1. Physiological solutions

 
Ca2+ uptake and release by the sarcoplasmic reticulum.   In a number of bundles, measurement of Ca2+ uptake rates were examined before the specific contractile apparatus properties. Similarly, it was necessary to determine whether there was any change in Ca2+ uptake before determining any changes to Ca2+ release, as the former affects the latter. To ensure that optimal SR function was retained after skinning, we used the cardiomyocyte skinning procedure described previously in rat cardiac muscle (9, 16). Briefly, bundles were first exposed to a weakly buffered skinning solution (Table 1) containing 50 µg/ml of saponin for 25 min to preferentially permeabilize the surface membrane. We confirm that this skinning procedure similarly minimizes any damage to the SR Ca2+ storage capacity of sheep cardiomyocytes (data not shown). The bundle was then washed in solution 3 for 5 min to remove the saponin. The SR was completely depleted of its stored Ca2+ by exposing the bundle to the release solution, containing 30 mM caffeine. The cardiomyocyte bundle was then exposed to a load solution (pCa 6.0) for time increments between 2 and 10 min. The load solution was made by mixing proportions of solutions 1, 2, and 3 together (see Table 1) to give a free [Ca2+] of 1 µM (pCa 6.0; 0.5 mM total EGTA); the pCa was confirmed by use of an Orion Ca2+-sensitive electrode (Thermo Electron). During exposure to the load solution, a small, slowly increasing force response was observed, as the SR loaded with Ca2+, consistent with Ca2+ loading seen in rat trabeculae (10). Between each load period the bundle was briefly (30 s) equilibrated in solution 3 to stop SR loading before it was again exposed to the release solution. The area of the caffeine-induced force response was measured in this study as it has been rigorously shown to provide an accurate qualitative estimate of SR Ca2+ uptake and Ca2+ content (8–10). This is because in the continuous presence of caffeine, and providing there are no changes to Ca2+ sensitivity or maximum force of the contractile apparatus (neither occur in this study; see RESULTS), the duration of the waveform principally reflects the amount of Ca2+ released from the SR; the more Ca2+ released, the greater the duration of the waveform as the longer it takes for the force transient to decay as Ca2+ diffuses out of the fiber (8). To ascertain the rate of Ca2+ uptake, the area was simply normalized to the maximum load time in each bundle (units are expressed in %).

To ascertain whether there was any effect of GA on Ca2+ release rates, the rate of rise (in s) of the caffeine-induced force transient (estimated in the most linear phase of the force transient between 20 and 80% of the peak force) was also measured.

Force-calcium relationship.   All bundles were also chemically skinned in solution 1 containing 2% Triton-X 100 for 30 min. This procedure destroys all membranes, leaving only the contractile apparatus intact. The bundle was washed in fresh solution 1 for 5 min and then equilibrated in a weakly buffered (2 mM) EGTA solution by combining proportions of solutions 1 and 3. The force-pCa relationship was then determined by activating the bundle in solutions of increasing free [Ca2+], created by combining solutions 1 and 2 in ratios from 1:1 to 1:10 (pCa 6.7 to 5.5; confirmed with a Ca2+ electrode). The precise pCa in each activation ratio was subsequently measured by using an Orion Ca2+-sensitive electrode. Bundles were maximally activated by exposure to solution 2 (pCa 4.1). The maximum Ca2+-activated force responses in bundles were normalized to the cross-sectional area of the bundle (mN/mm2) for comparison. Cross-sectional area was determined by the equation area = {pi}r2, assuming the muscle bundle had a cylindrical form and taking the average diameter at three locations across the fiber bundle. Submaximal force relative to the maximum Ca2+-activated force was used in determination of the force-pCa relationship.

For each fiber bundle, the relative force produced for each free [Ca2+] was plotted by use of GraphPad Prism v4.01 (GraphPad Software, San Diego, CA) and a sigmoidal dose-response curve (Hill equation) fitted. Parameters Max (pCa 4.5) and Min (pCa7.0) of the fitted curve were set to 100 and 0%, respectively. The average R2 value for fitted curves was 0.97 ± 0.01 (n = 21). From each resulting curve the pCa required to produce 20% (EC20), 50% (EC50), and 80% (EC80) of maximum Ca2+-activated force and the Hill slope were measured and averaged as reported in previous studies (10, 19).

Proportion of mononucleate cardiomyocytes.   Dissociated cardiomyocytes were pipetted onto a slide (1 x 106) and stained with methylene blue. The proportion of mononucleate cardiomyocytes in isolated right ventricular cardiomyocytes was determined by counting the number of mononucleate cardiomyocytes in 200 cardiomyocytes by use of an Olympus VANOX-T microscope (Olympus Optical, Tokyo, Japan).

Data analysis.   Comparisons between groups were analyzed for statistical significance with either a Student's unpaired t-test or a one- or two-way analysis of variance, where appropriate.

Included in the study were fetal sheep in which, because of technical difficulties, blood-gas data were not obtained (n = 2); however, their weight was within two standard deviations of the mean of the fetuses at that GA (7, 14).

All results were considered to be significantly different if P < 0.05. Data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Fetal characteristics.   The animals were divided into two groups: 126–132d GA (n = 7) and 137–140d GA (n = 6). The groups consisted of both twins (126–132d, n = 4; 137–140d, n = 4) and singletons (126–132d, n = 3; 137–140d, n = 2), males (126–132d, n = 2; 137–140d, n = 2) and females (126–132d, n = 5; 137–140d, n = 4).

Fetal blood-gas data are shown in Table 2. There was a difference between the two groups in mean gestational PO2 (126–132d, 20.5 ± 0.6 mmHg; 137–140d, 19.1 ± 0.4 mmHg; P < 0.05) and mean gestational PCO2 (126–132d, 41.9 ± 0.9 mmHg; 137–140d, 48.8 ± 1.6 mmHg; P < 0.05), but both groups were within accepted "normoxic" ranges. There was also a difference in total hemoglobin (126–132d, 8.5 ± 0.4 g/dl; 137–140d, 10.9 ± 0.7 g/dl; P < 0.05). Blood pH levels were measured to monitor the acidosis of the fetus. There was no significant difference in the mean gestational pH (126–132d, 7.365 ± 0.009; 137–140d, 7.365 ± 0.008).


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  		Table 2. Measurements of fetal growth and health

 
The mean fetal weight increased with GA from 126–132d to 137–140d fetuses; however, there was no significant difference in fetal heart weight between the two groups (126–132d, 27.3 ± 2.5 g; 137–140d, 31.0 ± 2.3 g). There was a significant difference in the mean relative fetal heart weight (126–132d, 8.1 ± 0.6 g/kg; 137–140d, 6.6 ± 0.2 g/kg), indicating that the heart size relative to body weight was smaller in the 137–140d group.

Proportion of mononucleate cardiomyocytes.   The mean proportion of mononucleate cardiomyocytes in the right ventricle decreased significantly with GA (126–132d, 45.7 ± 4.7%, n = 7; 137–140d, 32.8 ± 1.6%, n = 6; P < 0.05; see Fig. 1).


Figure 1
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  		Fig. 1. Proportion of mononucleate cardiomyocytes in the right ventricle. Mean percentage of mononucleate cardiomyocytes by age, 126–132 (n = 7) and 137–140 days (n = 6). Mean ± SE; *significance (P < 0.05). A significant decrease in percentage mononucleate cardiomyocyte was observed with the increase in gestational age (GA).

 
Calcium uptake and Ca2+ release by the sarcoplasmic reticulum.   Figure 2 shows raw data from a single cardiomyocyte bundle in which caffeine-induced force responses were elicited after a given load period. The relative area of caffeine-induced force responses can be seen to increase with increasing Ca2+ loads. The mean data are presented in Fig. 3. By 10 min of loading the peak of the caffeine-induced force as a percentage of the maximum Ca2+-activated force was 14 ± 7% (n = 7) and 23 ± 10% (n = 4) for the 126–132 and 137–140d groups, respectively (indicating that the force responses were not saturated), which were not significantly different (P > 0.05). The normalized area of the caffeine-induced force response was found to be significantly different between groups (P < 0.05). This indicated that Ca2+ uptake rates increase with increasing GA.


Figure 2
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  		Fig. 2. Caffeine-induced force responses in a single cardiomyocyte bundle. A cardiomyocyte bundle from a 138d GA sheep was subjected to a load-release cycle (described in MATERIALS AND METHODS). Specific solutions are described in detail in Table 1. After a load period (sequentially between 2 and 10 min) in the load solution (0.25 mM total EGTA, pCa 6.0), the fiber bundle was equilibrated in a weakly buffered (50 µM total EGTA solution, pCa7.0; solution 3) before the sarcoplasmic reticulum (SR) was emptied of all its Ca2+ by use of 30 mM caffeine (C) solution (50 µM total EGTA, pCa 7.0; period of exposure indicated by the solid bar under the force response). It can be seen (from left to right, top to bottom) that the area of the caffeine-induced force response increases with increasing load time. Maximum Ca2+-activated force (see Fig. 4) was 0.625 mN.

 

Figure 3
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  		Fig. 3. Calcium uptake by fetal cardiomyocyte bundles of the 126–132d (bullet) and 137–140d ({blacksquare}) GA groups. Mean data (±SE) of the Ca2+ uptake experiments described in the MATERIALS AND METHODS and illustrated in Fig. 2. The normalized area of caffeine-induced force responses after a period of SR Ca2+ loading is shown. The normalized area significantly increased with time and between GA groups (*P < 0.001 and #P < 0.03, respectively). Sample size: n = 7 and n = 4 for each time point for 126–132 and 137–140d GA groups, respectively. All time points were ascertained in each bundle. Lines were drawn by using a variable-slope sigmoidal dose-response (Hill equation) by use of Graphpad software and are for illustrative purposes.

 
We also measured the rate of rise of the caffeine-induced force response elicited after a 10-min load period. The rate of rise between 20 and 80% of the peak force response was not significantly different between groups (126–132d, 3.89 ± 0.38 s, n = 7; 137–140d, 3.78 ± 0.54 s, n = 4; P > 0.05), indicating no difference in Ca2+ release rates.

Force production and the force-calcium relationship.   Figure 4 shows the force-pCa relationship after Triton treatment of the same cardiomyocyte bundle in which load experiments were initially determined (see Fig. 2). The mean maximal Ca2+-activated force between groups was not significantly different (126–132d, 4.05 ± 0.54 mN/mm2, n = 14; 137–140d, 4.97 ± 0.71 mN/mm2, n = 7).


Figure 4
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  		Fig. 4. Ca2+-activated force responses in a single cardiomyocyte bundle. The force-pCa relationship was subsequently determined in the same single cardiomyocyte bundle (138d GA sheep) in which Ca2+ uptake was previously determined (see Fig. 2). The bundle was first Triton X-100 treated to destroy all membrane structures (see MATERIALS AND METHODS) before being exposed to a series of Ca2+-EGTA solutions (mixtures of solutions 1 and 2; see Table 1) with increasing free Ca2+ concentration. Numbers 1–11 in the trace represent the following pCa values determined by use of a Ca2+-sensitive electrode (pCa): 6.70, 6.61, 6.30, 6.2, 6.04, 5.89, 5.81, 5.78, 5.65, 5.79, 4.1. Maximum force was 0.633 mN. Bundle cross-sectional area was 0.0745 mm2.

 
Similarly, the Ca2+ sensitivity of cardiomyocyte bundles was not related to the increase in GA (see Table 3). The mean EC20, EC50, and EC80 showed no significant difference between groups. No significant difference in the slope of the linear rise in the force-calcium relationship (Hill coefficient) between 20 and 80% was found.


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  		Table 3. Force-pCa relationship across gestational age

 
These data indicate that there were no changes in Ca2+-activated force production by contractile apparatus between the two age groups and indicate no change in contractile function over this gestational period.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The purpose of this study was to determine whether changes in several E-C coupling parameters (contractile force production, Ca2+ uptake, and Ca2+ release by the SR) coincided with that of the transition in the heart from predominantly mononucleate cardiomyocytes and growth through cell proliferation to predominantly binucleate cardiomyocytes and growth through hypertrophy. A significant decline in the proportion of mononucleate cardiomyocytes was found between the 126–132 and 137–140d groups, and this coincided with a significant increase in the rate of Ca2+ uptake by the SR with increasing GA. However, there was no significant change in Ca2+-activated force development.

Force production and the force-calcium relationship.   In an early sheep study by Anderson et al. (1), increases in contractility with development from 90d gestation to 70d after birth were described; the force of contraction of electrically stimulated isolated trabeculae carneae and moderator muscle from left and right ventricles, respectively, increased with development saturating at around 30d after birth. At the time the authors were not able to ascertain what steps in E-C coupling were involved in this process. More recently in mice, the transition in nucleation of cardiomyocytes was found to coincide with an increase in maximum Ca2+-activated force and a decrease in the Ca2+ sensitivity of the contractile apparatus between 19d GA and 7d PN (20). Western blot analysis has shown that the adult cardiac isoform of troponin-I is first present in the mouse heart at 19d GA and progressively becomes the dominant isoform expressed (20). A transition from beta- to {alpha}-myosin heavy chain isoform is observed across late gestation in the mouse heart and is believed to contribute to the increase in contractile force (20). However, we find that across late gestation in fetal sheep (126–140d), there was no significant change in maximum Ca2+-activated force and Ca2+ sensitivity of the contractile apparatus occurred (see Table 3). Recent SDS-PAGE Western blot analysis in the ~140d GA fetal sheep heart found that single adult isoforms of both myosin and troponin-T are expressed (11) with predominantly (>90%) cardiac (adult) troponin-I. Coupled with the result in our study, indicating no significant change in the contractile force production, these data indicate that near-complete maturation of the fetal sheep heart contractile apparatus must occur before 126d GA. As a consequence, there is no association with the late-gestational transition in nucleation and changes in force observed by Anderson et al. (1) during the same gestational period. Furthermore, these data demonstrate an important temporal difference in the maturation of the contractile apparatus between species (rats vs. sheep).

Ca2+ uptake and Ca2+ release by the sarcoplasmic reticulum.   We extended the work of Mahony and Jones (13) by examining fetuses in late gestation (126–132 and 137–140d GA) using a more functionally intact preparation. We found a significant increase in Ca2+ loading by the SR across these two gestational groups when we examined the area of the caffeine-induced force response as an estimate of Ca2+ uptake (see Fig. 3). These data are consistent with the previous findings of Mahony and Jones (13) and may contribute to the increased force of contraction observed in the Anderson study (1). Although, we did not examine adult hearts, our data suggest that SR Ca2+ loading capacity may continue after birth, and further examination of these properties in sheep at different ages after birth is required. Nevertheless, these data indicate that the transition in nucleation of cardiomyocytes coincides with changes in Ca2+ uptake properties.

We also report that there is no significant change in the rate of Ca2+ release from the SR (as indicated by the absence of any significant change in the rise times of the caffeine-induced Ca2+ response after 10-min loading) between GA groups. Recently, Michalak (15), using isolated SR vesicle preparations from sheep heart, reported differences between fetal and adult SR properties. Michalak noted that calcium-induced calcium release from passively loaded preparations was not different between age groups examined (e.g., 100–105d, 10–45d, and adult). However, it was noted that, in fetal preparations, Ca2+ release was not isolated to the heavy SR fraction but diffuse throughout the SR (heavy and light), suggesting a functional difference between fetal and adult SR. It is not clear how these data equate to functional changes in vitro. We show no significant change in the rate of Ca2+ release in the two late-GA groups examined. This is likely consistent with the finding of Michalak. Recently, Perez et al. (19a) comparing Ca2+ release parameters between newborn and adult rat cardiomyocytes reported that the RyRs are fully functional and not different in inherent properties between the age groups examined. However, the contribution of RyRs to the total Ca2+ release transient mediated by an action potential was smaller in the newborn rats. The results of the Perez et al. study indicate a temporal difference in RyR function with development, which may not be apparent when using caffeine as a stimulus of RyR activity. However, given the differences in contractile apparatus development between rats and sheep (described above), it is not clear whether the same mechanisms prevail in sheep. It will be important to further investigate action potential-mediated Ca2+ release in sheep cardiomyocytes with development.

In conclusion, during late gestation the fetal sheep heart undergoes a transition in nucleation of cardiomyocytes with a decrease in the proportion of mononucleate cardiomyocytes. Although in the mouse and rat this transition is temporally related to changes in the contractile force production, this is not the case for sheep, which are a better model to reflect the prenatal developmental changes in the human. Similarly, we report a temporal relationship with Ca2+ uptake but not Ca2+ release from the SR. To our knowledge, this is the first time Ca2+ handling has been examined in skinned fetal sheep cardiomyocyte bundles. Together, these data indicate that by ~126d GA, many E-C coupling parameters have matured, but further work is required.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was funded by the National Health and Medical Research Council of Australia. J. L. Morrison was supported by a Postdoctoral Fellowship (PF 03A 1283) from the National Heart Foundation, the Heart and Stroke Foundation of Canada, and a Maternal Fetal and Newborn/Canadian Institutes of Health Research top up award.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Laura O'Carroll and Jayne Skinner for technical support with animal preparation and care and Stacey Dunn for technical assistance with muscle fiber studies. We thank Prof. Caroline McMillen for interest in and support of this project.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. S. Posterino, Discipline of Physiology, School of Molecular and Biomedical Science, Adelaide Univ., Adelaide, South Australia, Australia 5005 (e-mail: giuseppe.posterino{at}adelaide.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Co-senior authors. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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