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1Institute of Comparative Medicine, University of Glasgow Veterinary School, University of Glasgow, Glasgow G61 1QH; and 2Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Submitted 22 January 2004 ; accepted in final form 23 April 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1.5 times greater in amplitude in the horse compared with the rabbit. Ca2+ wave velocity was comparable between the species. The reason for this disparity in Ca2+ wave characteristics is unknown. Separate measurements of oxalate-supported Ca2+ uptake into the SR suggest that both horse and rabbit cardiomyocytes have comparable levels SERCA activity. The possible reasons for the observed differences in SR Ca2+ release between the horse and rabbit are discussed.
cardiac; waves; equine
One important feature of EC coupling is the duration of the ventricular muscle action potential. The equine action potential duration (APD) anticipated from electrocardiographic measurements would suggest a value of 0.6–1.0 s (11, 44) an estimate which is similar to recent in vitro measurements made on equine cardiac tissue (16). This value is considerably higher than that from the human, pig, dog, rabbit, and guinea pig, which, despite the wide range of resting heart rates, display comparable APD (0.2–0.35 s) (5, 21, 35, 53, 54). In smaller mammals, maintained depolarization causes elevated intracellular [Ca2+] concentration ([Ca2+]i) and an increased incidence of spontaneous Ca2+ release (2). These events activate transient inward currents that increase the probability of triggered arrhythmias (25, 29) in particular, early afterdepolarizations, which occur during repolarization, and delayed afterdepolarizations. The latter phenomena are also observed in heart failure where APD prolongation is also a consistent electrophysiological observation (4, 22, 40, 42).
Another feature of the equine heart is the slow heart rate experienced at rest (30–40 beats/min). Studies on isolated myocardium/myocytes from mammals, including the human, rabbit, and hamster, have shown that a direct imposition of a slow heart rate by pacing at slower than normal (resting) heart rates results in a prolonged APD (21, 46, 53). With the exception of the rat and mouse, this pacing can lead to diminution of the intracellular Ca2+ transient and decreased fractional shortening (28, 36, 37, 45). These studies therefore indicate that a slowing in heart rate and accompanying prolongation of APD cannot be simply imposed on the basic EC coupling mechanism without consequences for the electrical and mechanical stability of the heart.
In this study, we have compared the behavior of the SR from the horse with that of the rabbit. The marked differences observed in dynamic SR function between the two species highlight a possible modification of equine SR Ca2+ release and uptake, which may decrease the potential for arrhythmogenic activity while ensuring that the rise of intracellular Ca2+ is adequate to activate contractile proteins at low resting heart rates.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Equine hearts were obtained from horses with life-threatening or career-ending disorders unrelated to the cardiovascular system. A 7 x 7-cm section of midmyocardium from the left ventricle was cut into 1–3 mm3 of tissue. These were washed and resuspended in the following solution (in mM): 120 NaCl, 20 HEPES, 5.4 KCl, 0.52 NaH2PO4, 3.5 MgCl2, 6 H2O, 10 2,3-butanedione monoxime (BDM), 1 EGTA, and 11.1 glucose, pH 7.25 (20–21°C). An aliquot of tissue was then gently broken up further with an Ultraturrax homogenizer for 2 s at minimal speed (5,000 rpm). The cellular suspension, containing single-cell equine cardiomyocytes, was then filtered (250 µm mesh), washed, and resuspended in 1 ml of the above solution. The process was repeated with further aliquots of tissue until an adequate number of single cells was achieved. The final cellular suspension obtained was washed and resuspended in Krebs-Henseleit solution without the addition of BDM until use.
Rabbit Single Ventricular Cardiomyocyte Isolation
New Zealand White rabbits (2–2.5 kg) underwent humane euthanasia with an intravenous injection of 500 IU heparin and an overdose of sodium pentobarbitone (100 mg/kg). The animal care and handling protocols followed NIH and our institution’s animal care guidelines. The hearts were rapidly excised and cannulated onto a Langendorff perfusion column via the aorta. The hearts were perfused retrogradely at a perfusion rate of 25 ml/min (37°C), initially with 150 ml of Krebs-Henseleit solution. Thereafter, the hearts were perfused with 75 ml of Krebs-Henseleit solution supplemented with 0.66 mg/ml collagenase (type 1, Worthington) and 0.04 mg/ml protease (type XIV, Sigma) for 2 min. At 2 min, the enzyme solution was collected and 50 µM CaCl2 was added to the recirculating perfusate. Finally, at 6 min the heart was perfused with 50 ml of 0.1% Krebs-Henseleit solution containing 0.1% bovine serum albumin and 50 µM CaCl2. The left ventricle was dissected free from the heart on the column, cut into chunks, and incubated (37°C) for 1 h in Krebs-Henseleit solution (no added Ca2+). The cell suspensions obtained at the end of the incubation period were filtered (250 µm mesh) back into Krebs-Henseleit solution at a concentration of 1 x 105 cell/ml. Unlike the preparation of equine myocytes, BDM was not used in rabbit cell isolation.
Studying SR Function in Horse and Rabbit Cardiomyocytes
[Ca2+]i <200 nM generally leads to the production of spontaneous whole cell SR Ca2+ release-Ca2+ waves. These events initiate in one area of the cell and subsequently propagate to the remainder. Their production is a function of the 1) SR Ca2+ release channel/RyR2, 2) Ca2+ uptake into the SR via the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump, and 3) SR luminal control including intra-SR buffering. The cells were then permeabilized to characterize and compare these Ca2+ events in both species without the influence of sarcolemmal ionic transport mechanisms.
Cell Permeabilization for Confocal Measurements
Cardiomyocytes (horse and rabbit) were allowed to settle onto the coverslip at the base of a small (300-µl volume) bath. 
-Escin (Sigma) was acutely applied from a freshly prepared stock solution to the cell suspension within the 300-µl bath to give a final concentration of 0.1 mg/ml. Permeabilization was ascertained using confocal microscopy to monitor the entry of fluorophore into the cell (0.5–1 min).
Solutions for Production of Spontaneous Ca2+ Waves Within Permeabilized Cells
Permeabilized cells were perfused with a mock intracellular solution with the following composition (in mM): 100 KCl, 5 Na2ATP, 10 disodium creatine phosphate (Na2CrP), 5.5 MgCl2, 25 HEPES, and 0.05 K2EGTA, pH 7.0 (20–21°C). The equilibrium concentrations of metal ions in the calibration solutions were calculated using a computer program (React; G. L. Smith) with known affinity constants for H+, Ca2+, and Mg2+ for EGTA (47) and for ATP and CrP (13). Corrections for ionic strength, details of pH measurement, allowance for EGTA purity, and the principles of the calculations are detailed elsewhere (39). Free Mg2+ was calculated to be 0.9 mM (verified by MgFura measurements). The [Ca2+] in the perfusing solution was varied by the addition of known amounts of 1 M CaCl2 stock solution. The fluorescent Ca2+ indicator Fluo-3 (Molecular Probes) was added to the solution to give a nominal final concentration of 10 µM. All other chemicals were supplied by Sigma.
Laser Scanning Confocal Fluo-3 Fluorescence Measurements in Free Solution and Permeabilized Cardiomyocytes
The Ca2+ sensitivity of Fluo-3 in free solution was measured using a series of Ca2+-buffered solutions (10 mM EGTA) based on the mock intracellular solution described above. Fluo-3 inside the permeabilized cardiomyocyte has a Ca2+ affinity indistinguishable from that of free solution (558 nM) (31).
Ca2+ Wave Measurements in Permeabilized Cardiomyocytes
Confocal line-scan images were recorded using a Bio-Rad Radiance 2000 confocal system. Fluo-3 in the perfusing solution was excited at 488 nm and measured >500 nm (HQ500LP emission filter) using epifluorescence optics of a Nikon Eclipse inverted microscope with a Fluor x60 water objective lens (1.2 numerical aperture; Plan Apochromat). The 3-mW Kr laser was set to 12% power, and the gain on the photomultiplier tube set to 25%. Iris diameter was set at 1.9 providing an axial (z) resolution of 0.9 µm and X-Y resolution of 0.5 µm based on full-width half-maximal amplitude measurements of images of 0.175 µm fluorescent beads (Molecular Probes). Data were acquired in a line-scan mode at 2 ms/line and the pixel dimension was 0.3 µm (512 pixels/scan; zoom = 1.4). The scanning laser line was oriented parallel with the long axis of the cell and placed approximately equidistant between the outer edge of the cell and the nucleus/nuclei, to ensure the nuclear area was not included in the scan line. The LaserScan (Bio-Rad) software saved the data as a series of image files each containing 30,000 line scans (i.e., 1 min of continuous recording). An experimental record typically comprised 4–5 line-scan image files; these were reviewed off-line and a single intracellular region (20 pixels wide) was selected based on the earliest events in the majority of Ca2+ waves. This ensured that any movement artifact after the increase in [Ca2+] did not affect the estimation of peak [Ca2+]. Simultaneous measurements were also taken from the extracellular solutions using the same method. Average background fluorescence values (Fbg) were subtracted from the fluorescence measurements to obtain accurate values of fluorophore-associated fluorescence. Permeabilized cells Fbg = 5.29 ± 0.43 (n = 6) and extracellular Fbg = 3.56 ± 0.21 (n = 6) (30).
Measurements of SR Ca2+ Uptake Rates in Cardiac Protein Homogenates
A sample of either horse or rabbit midmyocardium weighing 1 g (±1%) was placed into 5 ml of the following homogenization buffer and kept on ice: 100 mM KCl, 25 mM HEPES, 0.05 mM EGTA, 1 mM MgCl2, 1 mM DTT, 5 mg/ml leupeptin, and 10 mg/ml aprotonin. Protein concentration (total protein in mg/g of tissue) was assayed in triplicate using a standard Coomassie blue protein reagent and spectrophotometer (595 nm) absorption measurements. No differences in the yield of total protein were observed between rabbit myocardium (95.7 ± 2.1 mg protein/g wet wt; n = 10) and equine myocardium (89.6 ± 6.7; n = 6). The concentration of protein used for the functional measurements was 2 mg of protein/ml. This concentration of protein contributes a negligible amount of Ca2+ buffering to the solution (20). This was suspended in 2 ml of mock intracellular solution as for the permeabilized cells (see above). -Escin (0.1 mg/ml; Sigma) was added, and the homogenate was gently spun down with the use of a hand centrifuge (5 g, 1 min). The pellet was resuspended in the above mock intracellular solution. The free [Mg2+] was 0.9–1 mM in all solutions. This was calculated as for the above solutions with the use of a computer program (React, G. L. Smith). Fura-2 (10 µM; Molecular Probes) was used to monitor the [Ca2+] within the cuvette with the use of a dual-wavelength spectrophotometer (Cairn Research). Oxalate (10 mM; Sigma) was added to maintain low and constant levels of intra-SR [Ca2+]. Ruthenium Red (RuR) (3 µM) was used to block Ca2+-efflux via the ryanodine receptor. RuR can inhibit SERCA and quench the fluorescence signal from Fura-2 (24). However, at 3 µM RuR, the effects of RuR on SERCA are minimal and quench appeared to affect the fluorescence at both excitation wavelengths equally without affecting the Km of Fura for Ca2+. The time course of the decrease of [Ca2+] within the cuvette was monitored in response to the addition of an aliquot of CaCl2. All measurements were made at room temperature (20–22°C). At the end of a series of uptake measurements on an aliquot of cells, the Fura-2 signal was calibrated using two standard solutions: <10 nM [Ca2+], 5 mM EGTA, and 370 nM [Ca2+] (10 mM EGTA). In a separate set of measurements, a range of free [Ca2+]s was used to measure the affinity of Fura-2 for Ca2+ under our experimental conditions, a Kd value of 110 ± 20 nM was obtained, close to that measured in another study (18).
The time course of Ca2+ uptake was used to assess SR Ca2+ uptake capacity by: 1) conversion of free [Ca2+] to total [Ca2+] assuming the major Ca2+ buffer in solution was EGTA, 2) differentiation of the total [Ca2+] with respect to time and correcting the Ca2+ uptake rate for the amount of homogenate protein. The value of instantaneous Ca2+ uptake rate was plotted against the corresponding free [Ca2+] to generate the sigmoid relationship shown in Fig. 6B. The resultant data were fitted to a logistic curve using Origin (OriginLab) to obtain a maximal rate of Ca2+ uptake (Vmax) and Km for each data set.
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Data were expressed as means ± SE. Comparisons were performed by using the unpaired Student's t-test, and differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Figure 1 shows photographs of typical adult hearts from the horse, rabbit, and mouse. As indicated by Table 1, the typical body weights, heart weights, and cardiac outputs are 200-fold larger in the horse compared with the rabbit. The heart rate is, however, almost five times lower in the horse compared with the rabbit and hence the scaled cardiac output (in ml·kg body wt–1·min–1) is comparable between the two species.
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Figure 2 shows typical transmission images of single isolated cardiomyocytes from horse, rabbit, and mouse hearts. Strikingly, whereas the intact whole horse heart, shown in Fig. 1, is almost five times larger in every aspect compared with the rabbit heart (and a similar relationship exists between the rabbit and mouse heart), the cardiomyocytes from each species are of similar dimensions and cell length was not significantly different (P > 0.05). Although cell width was greater in the horse (P < 0.05), the extent was small [cell length: 118.6 ± 4.1 vs. 116.0 ± 5.7 µm; cell width 25.1 ± 1.0 µm vs. 28.7 ± 0.6 µm, rabbit (n = 24) vs. horse (n = 82), respectively].
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Figure 3 shows the protocol used to initiate Ca2+ waves within both horse and rabbit cardiomyocytes and is described in detail in our previous study (30). Because of the presence of Fluo-3 (10 µM) in the perfusing solution, the fluorescence signal recorded from line-scan images of the cell contains signals from both intracellular and extracellular compartments. The extracellular signal (black trace; Fig. 3A) resembles therefore the changes in fluorescence due to solution changes, and the intracellular fluorescence signal (gray trace; Fig. 3A) relates to the cytoplasmic changes of [Ca2+], which occur in the cell as a result of these solution changes. Increasing [Ca2+]i from 40 to 565 nM within this equine cardiomyocyte (as indicated above the fluorescence trace in Fig. 3) initiated a series of Ca2+ waves recorded as transient increases in fluorescence at regular intervals at the intracellular site. The delay between the increased cytosolic [Ca2+] and the first Ca2+ wave is caused by a period of net SR Ca2+ uptake and diffusion of [Ca2+] into the cell. No obvious differences in this delay between the two species were apparent. The frequency of Ca2+ waves was constant after 15 s, suggesting a new steady SR Ca2+ content was achieved. At the point indicated, the perfusing solution was changed to one containing 375 nM free [Ca2+] (buffered by 10 mM EGTA). The high EGTA concentration effectively clamped the [Ca2+], buffering Ca2+ release, and uptake by the SR. The [Ca2+] was subsequently reduced to <1 nM [Ca2+] by perfusion with 10 mM EGTA solution (no added Ca2+) and then to 33 µM [Ca2+] by perfusion with 10 mM EGTA solution (not shown). The fluorescence signal in the last three solutions (10 mM EGTA) were used to convert the fluorescence signal into [Ca2+] as shown in Fig. 3B.
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150 nM. The highest Ca2+ concentration reached occurred at the peak of the Ca2+ wave and again using the average trace in Fig. 3C was 3.5 µM. A similar protocol was performed in both rabbit and equine cardiomyocytes across the range of [Ca2+] from 40 to 260 nM (n = 8 cells from 3 different rabbits and n = 8 cells from 4 different horses) and from 40 nM to 565 nM [Ca2+] (n = 8 cells from 3 different rabbits and n = 5 cells from 2 different horses). Comparison of Ca2+ Wave Characteristics in Horse and Rabbit
Ca2+ wave frequency and velocity.
Whereas Ca2+ waves were initiated in rabbit cardiomyocytes at a [Ca2+] of 250–270 nM with a frequency of 0.16 ± 0.02 waves/s (n = 9), no Ca2+ waves were evident at this [Ca2+]i in the horse (n = 9) (Fig. 4D,a). However, if the [Ca2+] was subsequently increased to 540–560 nM, Ca2+ waves were produced from cardiomyocytes from both species (Fig. 4A). Figure 4A,b and c, shows single Ca2+ waves from both horse and rabbit at this higher [Ca2+]. Although in 80% of cells, the point of initiation of the Ca2+ wave was in the same region of the cell during a 120-s line-scan image, 20% of cells showed variation in the region of cell in which the Ca2+ wave began (Fig. 4A,a). This feature of Ca2+ waves was similar between the two species. Ca2+ waves appear similar at the point of initiation; however, the duration of release was markedly elevated and prolonged in the horse relative to the rabbit. The [Ca2+] signal trace of a series of Ca2+ waves from the horse and rabbit in Fig. 4C, a and b, respectively, emphasize this observation. It can also be seen that at these higher [Ca2+]s (550 nM) Ca2+ waves in the horse occurred with a significantly lower frequency (0.12 ± 0.01 waves/s; n = 25) compared with the rabbit (0.48 ± 0.10 waves/s; n = 6), as shown in Fig. 4D,b (P < 0.05).
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Ca2+ wave maximum and minimum values. Figure 5A shows that characteristic profile of spontaneous SR Ca2+ release taken from an average of a series of Ca2+ waves from a single cardiomyocyte in both the horse (black trace) and rabbit (gray trace). The average maximum and minimum [Ca2+] values from a number of cells is presented in Fig. 5B. It can be seen that the maximum peak of Ca2+ release is significantly greater in the horse (3.28 ± 0.26 µM, n = 5) compared with the rabbit (2.04 ± 0.19 µM, n = 6, P < 0.05). The peak [Ca2+] in equine cells, represent fluorescence values close to Fmax for Fluo-3. Therefore, to verify the peak [Ca2+] observed in equine cells, a limited set of measurements were made using Fluo-5F, a dye with a lower Ca2+ sensitivity (30). In two experiments, the peak [Ca2+] was 3.25 and 3.75 µM. These values were in close agreement with the mean obtained using Fluo-3 and confirm the differences in peak [Ca2+] observed with Fluo-3 between the two species.
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SR Ca2+ wave release characteristics.
To characterize Ca2+ release from the SR, the rate of rise and fall of the Ca2+ wave was assessed at two set [Ca2+]s (1.0 and 1.5 µM). These measurements were performed on the average Ca2+ wave of each cell as shown in Fig. 3C. The rate of rise of Ca2+ during the wave in the rabbit was significantly greater than the horse at both [Ca2+] as shown in Table 2 and Fig. 5A. A similar observation was made during the rate of fall. The duration of the rise phase between 600 nM and peak [Ca2+] was also assessed. The duration of rise was nearly fourfold greater in the horse compared with the rabbit (Table 2) (P < 0.05). A value of 600 nM was used as the starting point for the measurement because the mean [Ca2+]o was 560 nM.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The Use of Ca2+ Waves to Study SR Function
This study has focused on SR Ca2+ release via RyR2 and SR Ca2+ uptake via SERCA. Many authors (7, 10, 43, 52, 55) have used Ca2+ wave characteristics to assess SR function. The data obtained from these studies have been fundamental to our understanding of EC coupling within ventricular cardiomyocytes. The importance of studying Ca2+ waves becomes even more pertinent in a species that shows a prolonged APD and low resting heart rate, i.e., the horse. The greater period of time held at more positive potentials during the prolonged action potential would be anticipated to lead to a greater influx Ca2+ via the Na+/Ca2+ exchanger (outward mode). This would lead to an elevation of [Ca2+]i, a higher SR Ca2+ content, and the production of spontaneous Ca2+ waves. These events increase the probability of arrhythmogenic transient inward currents, which are incompatible with normal heart function.
Measuring Ca2+ Wave Characteristics in Permeabilized Cells
Ca2+ wave characteristics are sensitive to changes in [Ca2+]i (9, 30). For this reason, equine and rabbit Ca2+ wave characteristics were measured and compared in permeabilized cardiomyocytes. The permeabilized cell has the advantage that [Ca2+]i can be accurately calibrated and standardized. Previous studies (31, 32) have established that SR Ca2+ release events are indistinguishable from those observed in intact cells and are regulated by known modulators of RyR2 activity (Ca2+-calmodulin and cADP ribose).
Ca2+ Wave Characteristics at 260 nM [Ca2+]i
Ca2+ waves in rabbit cardiomyocytes were apparent at 260 nM [Ca2+]i (frequency 0.16 waves/s). However, even during prolonged test periods (3–4 min), Ca2+ waves in isolated equine cardiomyocytes were not observed. It is conceivable that these events occur in horse cardiomyocytes at frequencies <0.005 waves/s. However, linear extrapolation of the relationship between [Ca2+]i and wave frequency in the rabbit suggest that the free [Ca2+]i would need to be as low as 20 nM before waves occurred at such low frequencies (30). It is unlikely that Ca2+ overload occurs at such [Ca2+]i. The lowest frequency Ca2+ waves reported have been observed in a study of intact rat myocardium (23). These events were termed "sporadic" Ca2+ waves and occurred at a frequency of 0.02–0.2 waves/s. These waves would have been observed during the long test period used in this study. Therefore, it would seem valid to conclude that the threshold for spontaneous Ca2+ waves in horse ventricular cardiomyocytes is higher than that for rabbit.
Localized SR Ca2+ release originating from a single cluster of RyR2s is called a Ca2+ spark (8). The spontaneous production of these events is a function of both cytoplasmic and luminal [Ca2+] (7, 33). Ca2+ wave initiation is thought to be the result of both the "temporal and spatial summation of Ca2+ sparks" in a discreet area of the cell (7). This process leads to recruitment of Ca2+ release from sufficiently sensitized adjacent clusters and subsequent wave propagation. Ca2+ sparks were observed at 250 nM [Ca2+]i in both rabbit and horse cardiomyocytes (results not shown). Therefore, SR Ca2+ release events with the potential to initiate Ca2+ waves were present in both species, but while conditions were sufficient for wave propagation in the rabbit, they were not sufficient in the horse. Therefore, to produce propagating Ca2+ waves in equine cells, the [Ca2+]i was raised.
Ca2+ Wave Characteristics at 560 nM [Ca2+]i
The main characteristic of equine Ca2+ waves at 560 nM [Ca2+]i was their low frequency and large amplitude (relative to rabbit waves at the same free [Ca2+]i). The factors determining the frequency of spontaneous SR Ca2+ release are complex. One view is that the system can be described in terms of having a simple threshold [Ca2+] value with which the SR triggers Ca2+ release (43). Another view, which has been examined in detail, involves luminal control (34). This group suggests that high luminal [Ca2+] increases the open probability of RyR2. Thus both approaches suggest that SR luminal [Ca2+] is the main determinant of Ca2+ wave frequency. A longer time between Ca2+ waves could be attributed to a slower rise of luminal Ca2+ or a relative insensitivity of RyR2 to luminal Ca2+. Whereas this investigation does not come to any definitive conclusions as to why equine SR Ca2+ release occurs with such low frequency when exposed to similar [Ca2+]s as the rabbit, it is useful to consider the various possibilities.
SR Ca2+ Uptake
If the rate of Ca2+ uptake is slowed then the time taken to reach threshold for Ca2+ release will increase, and the frequency will subsequently decrease. The comparative investigation of SERCA Ca2+ uptake rates (per milligram of cell protein) in rabbit and equine myocardial homogenates found no significant difference between the two species. One possible artifact in these measurements is the differential retrieval of cell protein from rabbit and equine myocardium. However, based on the similar cellular dimensions and comparable protein retrieval values (see MATERIALS AND METHODS), there is no evidence that retrieval of cell protein from rabbit and equine myocardium is markedly different. It is therefore unlikely an artifact is obscuring a differential in SERCA expression. Furthermore, it has been demonstrated that a reduction of SERCA-mediated Ca2+ uptake rate by application of 2,5-di-tert-butyl hydroquinone, while reducing the frequency of Ca2+ waves does not affect the peak [Ca2+] (19). Hence, whereas this parameter may have been a plausible explanation for a decrease in wave frequency it would not have explained the concurrent increase in Ca2+ wave amplitude.
SR Ca2+ Capacity and Release
If the capacity of the SR for Ca2+ in horse cardiac cells is greater than rabbit, a longer time will be taken for the SR to fill before the threshold for spontaneous Ca2+ release is reached and next wave produced. A main determinant of the functional size of the SR for Ca2+ is calsequestrin: an intra-SR Ca2+ binding protein (Kd 0.6 mM) (6, 51). A recent study using adenoviral-mediated overexpression (4-fold) of calsequestrin in rat ventricular cardiomyocytes showed that functional recharging of SR Ca2+ stores is slowed due to the increase in intra-SR buffering (51). This resulted in a decrease in the frequency of repetitive spontaneous Ca2+ sparks. The increased SR Ca2+ content in these cells also led to an increase in size of spontaneous (Ca2+ sparks) and stimulated SR Ca2+ release. This finding is similar to the situation found in equine Ca2+ waves, which are less frequent but larger in amplitude. However, no data have been published specifically relating to the relationship between SR buffering capacity and Ca2+ waves.
Overexpression of calsequestrin acts to stabilize the free intra-SR Ca2+ concentration adjacent to the luminal side of RyR and limits luminal Ca2+-dependent deactivation of RyR (51). In doing so, a prolonged rise time of Ca2+ release is observed (51). This is also the case in the equine Ca2+ wave (relative to the rabbit) and could be explained by an increased concentration of horse intra-SR buffer. This is further supported by the striking difference observed between the Ca2+ wave profile in the rabbit and horse (Fig. 5C). As soon as the rabbit peak [Ca2+] is reached, Ca2+ declines. The horse wave duration, however, remains elevated for a prolonged time (Fig. 5, A and C). Ca2+ wave decline is a function of Ca2+ uptake via SERCA, Ca2+ release via RyR2, and diffusion away from the cell. This suggests a larger Ca2+ release from equine SR during the wave. The larger Ca2+ wave amplitude in the horse relative to the rabbit supports this hypothesis (Fig. 5B). A very similar profile of SR Ca2+ release is also observed in stimulated transients and spontaneous Ca2+ sparks when intra-SR Ca2+ buffering is increased by overexpression of calsequestrin (51). Attempts to quantify the amount of calsequestrin in equine SR relative to the rabbit were unsuccessful due to the lack of a specific anti-equine antibody.
Whereas the duration of the rising phase of SR Ca2+ release is related to the inactivation of the release process, the rate of rise is related to the activation of RyR2 (51). An unusual finding is the decreased rate of Ca2+ release (at 1 and 1.5 µM) in the horse relative to the rabbit. A greater flux of Ca2+ through the RyR2 would expect to be achieved at a faster rate (51). Terentyev et al. (51) found no significant difference between the maximal rates of rise of Ca2+ release during stimulated transients overexpressing calsequestrin compared with control. The authors suggested that this may be due to slowed diffusion of Ca2+ through the luminal space and hence slowed transit of Ca2+ through open channels in cells overexpressing calsequestrin (51). If the horse has an increase in intra-SR buffering, then this may also be the case.
RyR Sensitivity
A decrease in sensitivity of RyR2 to cytosolic Ca2+ could explain a lower frequency Ca2+ wave, because the threshold for SR Ca2+ release would increase and therefore the time taken to reach it. This conclusion is supported by previous work (43) using tetracaine to reduce the Ca2+ sensitivity of RyR2. Tetracaine caused lower frequency but larger SR Ca2+ waves that propagate at comparable velocities to Ca2+ waves under control conditions. The propagation of Ca2+ waves within cardiomyocytes occurs by saltatory propagation between clusters of RyR2 (26). Ca2+ wave velocity is proportional to the amplitude of SR Ca2+ release but inversely related to the distance between firing sites and the threshold for SR Ca release (26). The peak amplitude of the Ca2+ wave in the horse relative to the rabbit is larger and should therefore result in a faster wave. However, no significant difference in wave velocity between the two species was observed. The mean value of 114 ± 6 µm/s (n = 21) for horse Ca2+ waves is similar to the values reported by other studies in rat and rabbit (23, 27, 30, 50, 52) and faster than others (7, 48). This lack of difference in Ca2+ wave velocity may be due to differences in distance between SR Ca2+ release sites. Assuming release sites are clustered at the T-tubule junction, preliminary measurements suggest that inter-T-tubular distance is similar between the two species. Alternatively, a decrease in equine RyR sensitivity may be responsible. This would increase the threshold for SR Ca2+ release and as explained above, lower Ca2+ wave frequency, yet produce larger Ca2+ waves, such as those observed in equine cardiomyocytes. Further measurements are required to confirm this conclusion.
Implications for Intact Cell and Whole Heart of Equine Species
To initiate Ca2+ waves in horse cardiomyocytes, an approximately threefold increase in resting [Ca2+]i (assuming a resting Ca2+ of 150 nM) is required, compared with only a 0.3-fold increase in the rabbit. Furthermore, when Ca2+ waves in the horse do occur, they are more than four times less frequent than rabbit at the same [Ca2+]i (0.117 vs. 0.484 waves/s in horse and rabbit, respectively). The long diastolic periods without potentially arrhythmogenic spontaneous SR Ca2+ release allows at least 4–5 Ca2+ transients to be stimulated at an HR of 30 beats/min (0.5 Hz) without the potential for a Ca2+ wave to be produced, the SR presumably being partially depleted at each transient. This decreased propensity for spontaneous SR Ca2+ release at a resting [Ca2+] is beneficial in a species with a prolonged APD and an increased propensity for SR Ca2+ overload.
The horse has the ability to increase HR almost 10-fold (from 24 to 240 beats/min) during strenuous exercise (38). This is compared with humans, who may only achieve an approximately threefold rise of resting heart rate. This degree of tachycardia in the horse is likely to bring more Ca2+ into the SR via voltage-dependent L-type Ca2+ channels and would increase the propensity for Ca2+ overload if efflux from the cells were not sufficient to match Ca2+ influx. The adaptations required for this balance of sarcolemmal fluxes in the horse can only be examined using electrophysiological measurements within intact cells and are highly desirable experiments that need to be performed in light of the results of this investigation. A previous study (16) has assessed the characteristics of the various potassium channels required for cellular repolarization within equine cardiomyocytes. The conclusion reached in this study was that cardiac repolarization in the horse resembles that in humans (16). Therefore, changes in repolarization characteristics are unlikely to be the major adaptive change necessary to reduce the incidence of triggered arrhythmias that may occur during prolonged APD. This further emphasizes the significance of SR adaptation that may occur in the horse compared with other species when faced with such a prolonged APD.
Future Directions
Although the majority of animal models of heart failure result in a variation of function/expression of proteins that regulate cellular Ca2+ homeostasis, a commonality among most models is SR dysfunction (12). This ultimately leads to decreased contractility. The ultimate goal of cardiac therapeutic investigations has been to find agents that increase the inotropy of the heart by increasing SR Ca2+ content and Ca2+ release for a given trigger. However, this has often been impeded by the problematic side effect of SR Ca2+ overload and subsequent arrhythmias. The equine species apparently has the capacity for a high SR Ca2+ content and release but at the same time a reduced incidence of potentially arrhythmogenic Ca2+ waves in conditions of Ca2+ overload. Molecular cardiology studies within the equine species may therefore provide us with invaluable insights into potential therapeutic strategies for both human and animal heart failure.
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ACKNOWLEDGMENTS |
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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.
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