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Am J Physiol Heart Circ Physiol 288: H2431-H2438, 2005. First published December 22, 2004; doi:10.1152/ajpheart.01069.2004
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Decreased Ca2+ extrusion via Na+/Ca2+ exchange in epicardial left ventricular myocytes during compensated hypertrophy

Mark R. Fowler, James R. Naz, Mark D. Graham, Gilles Bru-Mercier, Simon M. Harrison, and Clive H. Orchard

School of Biomedical Sciences, University of Leeds, Leeds, West Yorkshire, United Kingdom

Submitted 19 October 2004 ; accepted in final form 20 December 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

cardiac myocytes; epicardium; endocardium; sarcoplasmic reticulum; t tubules; spontaneously hypertensive rats

SYSTEMIC HYPERTENSION IS ASSOCIATED with cardiac morbidity and death (7); the increased afterload imposed on the heart by hypertension results in cardiac hypertrophy, which predisposes to heart failure. Such pressure overload leads to concentric hypertrophy, characterized by an increase in cell size and ventricular wall thickness. Remodeling also results in altered excitation-contraction coupling. In the spontaneously hypertensive rat (SHR), a genetic model of hypertension and cardiac hypertrophy (11, 13), this is characterized by maintained or enhanced systolic Ca2+ transient and contraction amplitude and a slowed time course of contraction and relaxation (4, 5, 20, 30).

A number of changes in the excitation-contraction coupling pathway have been identified that may account for the altered Ca2+ transient and contraction, including action potential prolongation, increased sarcoplasmic reticulum (SR) Ca2+ content (5), and larger amplitude Ca2+ sparks (30). However, the mechanisms underlying the changes in Ca2+ transient configuration are still not well understood. In many studies, an increase in Na+/Ca2+ exchange (NCX) expression has been reported during compensated hypertrophy, although, paradoxically, function often appears downregulated or unaltered. For example in the mouse, after surgically induced pressure overload, NCX transcript and protein expression increase but caffeine-evoked inward current and Ni2+-sensitive current decrease (34). Similarly, in another mouse model of compensated hypertrophy, NCX, SR Ca2+-ATPase (SERCA), and phospholamban protein levels increase, but NCX current is not significantly altered (16); in addition, in the SHR during compensated hypertrophy, expression of phospholamban, SERCA, and ryanodine receptor are unchanged (30). In contrast, in hypertrophied canine myocytes, Ca2+ extrusion via NCX and Ni2+-sensitive current are increased (31). Thus there appear to be differences that may be due to the model and to the degree of progression of hypertrophy. In addition, the ventricular myocardium displays regional variations in its structural, mechanical, and electrical properties in normal and hypertrophied hearts (6, 20, 22, 23), probably in part as a result of regional differences in wall stress (25). Recent work in the SHR has shown that the amplitude and time course of the action potential, Ca2+ transient, and contraction are altered differentially in subepicardial (Epi) and subendocardial (Endo) myocytes during compensated hypertrophy (20). However, the mechanisms underlying these regional changes in the Ca2+ transient have not been investigated. The present study was designed, therefore, to investigate the changes in Ca2+ handling that underlie the altered Ca2+ transient observed previously, in cardiac myocytes isolated from the Epi and Endo of the left ventricle of SHR and normotensive controls.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of ventricular myocytes. Male 20-wk-old Wistar-Kyoto rats (WKY) and SHR (300–400 g; Harlan) were killed by stunning followed by cervical dislocation: a schedule 1 procedure under the UK Home Office Animals (Scientific Procedures) Act of 1986 and sanctioned by the local ethical review committee. The heart was removed and perfused via the aorta with "isolation solution" containing (in mmol/l) 130 NaCl, 5.4 KCl, 0.4 NaH2PO4, 1.4 MgCl2, 0.75 CaCl2, 10 HEPES, 10 glucose, 20 taurine, 10 creatine, pH 7.1, at 37°C. When the coronary circulation was clear of blood, perfusion was switched to a Ca2+-free solution (isolation solution containing 0.1 mmol/l EGTA and no added Ca2+) for 4–5 min after which perfusion was continued for 5–10 min with Ca2+-free solution containing collagenase (1 mg/ml, type 2; Worthington Biochemical, Lakewood, NJ) and protease (6 µg/ml, type XIV; Sigma, St. Louis, MO). The left ventricle was isolated, and strips of Epi and Endo were dissected free and dispersed separately by agitation at 37°C in recycled collagenase- and protease-containing solution supplemented with 1% BSA. Cells were resuspended in Ca2+-containing (0.75 mmol/l) isolation solution and stored at room temperature (22–24°C) until use.

Morphology. Body weight and heart weight were recorded, and the heart weight-to-body weight ratio was used as an index of cardiac hypertrophy. Cell length and width were measured, and volume was calculated as described in Ref. 28 and used to ascertain cellular hypertrophy. To investigate t tubule morphology, cells were incubated with the lipophilic dye 4–2-[6-(dioctylamino)-2-naphthalenyl]-ethenyl-1-(3-sulfopropyl)-pyridinium (di-8-ANNEPS, 5 µmol/l; Molecular Probes) for 2 min before resuspension in Tyrode solution. Di-8-ANNEPS was excited at 488 nm; fluorescence was imaged at >505 nm with the use of a confocal microscope (see below).

Experimental setup. Myocytes were continually superfused in the experimental chamber with a modified Tyrode solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 1.2 MgCl2, 1 CaCl2, 0.4 NaH2PO4, 5 HEPES, and 10 glucose, pH 7.4. The myocytes were stimulated to contract at 1 Hz by stimuli delivered via platinum electrodes on each side of the experimental chamber; all experiments were performed at room temperature. Unless otherwise stated, all chemicals were from Sigma. Dilution of drugs into the Tyrode solution is given in the relevant sections.

Intracellular Ca2+ imaging. One milliliter of cell suspension was incubated with 10 µmol/l fluo 3-AM and 0.1% Pluronic F-127 (diluted from a 20% stock solution in DMSO) or 3 µmol/l fura 2-AM (Molecular Probes) in isolation solution for 20–30 min (fluo 3) or 10 min (fura 2) at room temperature.

We performed confocal imaging of fluo 3 fluorescence using a Zeiss LSM5 Pascal confocal microscope. Incident and emitted light were focused and collected with a x63/1.2 numerical aperture water-immersion objective lens. The experimentally derived full-width-half-maximum of the two-dimensional point spread function at the point of focus (the Airy Disc) was 0.39 ± 0.05 µm, derived with fluorescent microspheres (0.175 µm diameter, p-speck beads; Molecular Probes). Optimum confocality was achieved by setting the confocal aperture to the size of the Airy disc (calculated to be 31.26 µm) to give a z-axis resolution of 0.9 µm. Fluo 3 was excited at 488 nm, and emitted fluorescence was collected above 505 nm. Eight-bit, 512-pixel transverse line scans were acquired every 2–4 ms, and fluorescence (F) was normalized to baseline fluorescence (F0).

For whole cell epifluorescence measurements, fura 2 was alternately excited at 340 and 380 nm every 2 ms with a spectrophotometer (Cairn, Faversham, Kent, UK), and emitted fluorescence (510 ± 40 nm) was collected via a x40/1.3 numerical aperture oil-immersion objective lens as described previously (12). The 340-nm, 380-nm, and ratio signals were sampled at 1 kHz (CED 1401 AD converter) and displayed, stored, and analyzed with Spike2 software (CED, Cambridge, UK). The 340-to-380 nm fluorescence ratio was used as an index of intracellular Ca2+ (Cai2+) because it has previously been shown that fura 2 behaves identically in cells isolated from WKY and SHR (5).

Measurement of intracellular Na+. Cells were incubated with 11 µmol/l SBFI-AM (Molecular Probes) for 2 h in the dark at room temperature before being resuspended in Tyrode solution and stored in the dark until use (12). SBFI-loaded cells were excited at 340 and 380 nm every 20 ms, and emitted fluorescence was collected at 510 ± 40 nm, using the system described above for fura 2 measurements. The ratio signal was filtered with a time constant of 0.66 s before sampling at 5 Hz. Individual myocytes were calibrated at the end of each experiment by exposure to a solution containing (in mmol/l) 10 EGTA, 5 HEPES, 0.1 strophanthidin, 0.005 gramicidin D, 5 or 20 Na+, and 150 KCl, minus [NaCl]. The linearity between the SBFI ratio and physiological concentrations of Na+ was confirmed by including 0 mmol/l Na+ in the calibration protocol in some experiments.

Analysis of Ca2+ release using confocal line-scan images. Spatially nonsynchronous Ca2+ release was detected and quantified with Zeiss image analysis software and Sigma Plot. A 100-ms portion of the line-scan image at the beginning of the Ca2+ transient was chosen for analysis, and regions of early and delayed Ca2+ release were identified by eye and included for analysis only if present in the same region in all of the 5–10 Ca2+ transients recorded. An F50 value was calculated as 50% of the amplitude of the whole cell Ca2+ transient (15, 18); this represented the threshold below which areas of delayed release were identified, that is, areas with an intensity below F50 at times >12 ms after the start of the upstroke of the Ca2+ transient (0 ms).

Statistics. Origin software (OriginLab) was used to fit a single exponential function to the declining phase of Ca2+ transients, taken from the peak at systole to the diastolic level (24), to calculate their decay rate constant, k, and to determine the periodicity of t tubule staining using fast Fourier transforms (3). Data are shown as means ± SE of n cells. Statistical significance was determined with unpaired t-tests, {chi}-square tests, or two-way ANOVA (with interactions) as appropriate, using SigmaStat software (Jandel Scientific). Significance was assumed at the 5% level.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphology. Table 1 summarizes the morphological data from WKY and SHR. These data show that, in the SHR, heart weight-to-body weight ratio, cell width, and cell volume are significantly increased and the cell length-to-cell width ratio is significantly decreased, consistent with previous work showing significant organ and cellular hypertrophy in the 20-wk-old SHR, with equal cellular hypertrophy in Epi and Endo myocytes (20).


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  		Table 1 Morphological data from WKY and SHR

 
Cai2+. Figure 1 shows representative whole cell Ca2+ transients recorded from left ventricular myocytes from the Epi region of WKY (Fig. 1A) and SHR (Fig. 1B). In control rats (WKY), the amplitude of the Ca2+ transient was significantly smaller in Epi than Endo myocytes (Fig. 1C). This regional difference was absent in SHR, in which the Epi Ca2+ transient amplitude was significantly greater (P < 0.05) than that in WKY Epi cells but was not significantly different from the SHR Endo Ca2+ transient (Fig. 1C). Endo Ca2+ transient amplitude was not significantly different in WKY and SHR.



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  		Fig. 1. Representative intracellular Ca2+ (Cai2+) transients during stimulation at 1 Hz in left ventricular myocytes from the epicardium (Epi) of Wistar-Kyoto rat (WKY; A) and spontaneously hypertensive rat (SHR; B) hearts. C: mean ± SE Cai2+ transient amplitude in Epi and endocardium (Endo) left ventricular myocytes from WKY and SHR hearts (WKY, Epi/Endo: n = 12/11; SHR, Epi/Endo: n = 13/13). AU, arbitrary units. D: mean ± SE time to peak of the Ca2+ transient. E: mean ± SE rate constant of the decay of the Ca2+ transient. Statistical comparison was by 2-way ANOVA with post hoc Holm-Sidak interactive comparison. Lines above each graph indicate bars that are significantly different (*P < 0.05).

 
Figure 1D illustrates that, in both WKY and SHR, the time to peak of the Ca2+ transient was significantly shorter in Epi than in Endo myocytes. In addition, there was a small, but statistically significant, increase in time to peak in Endo myocytes from SHR, compared with Endo myocytes from WKY. However, there were no significant difference in k of the Ca2+ transient between Epi and Endo cells or between WKY and SHR (Fig. 1E). These data show that the normal regional differences in the Ca2+ transient in WKY are altered in the SHR, as reported previously (20). Subsequent experiments were designed to extend these observations by investigating the mechanisms that underlie these changes.

Ca2+ transport pathways. The differences in Ca2+ transient amplitude may be the result of changes in SR Ca2+ content, which may, in turn, be due to changes in SR or sarcolemmal Ca2+ transport. Figure 2 shows the protocol used to investigate this possibility: fura 2-loaded ventricular myocytes were stimulated to steady state at 1 Hz; stimulation was then stopped for 5 s before rapid application of 20 mmol/l caffeine to discharge SR Ca2+. The amplitude of the resulting caffeine-evoked Ca2+ transient was taken as a measure of SR Ca2+ content and its rate of decay as a measure of trans-sarcolemmal Ca2+ extrusion (24). Stimulation was recommenced after washout of caffeine; when steady state was reattained, caffeine was applied again, this time in the presence of 10 mM Ni2+ to inhibit NCX activity (10, 17, 24). The k value of the caffeine-evoked Ca2+ transient, compared with the electrically evoked Ca2+ transient, was used to calculate the proportion of Ca2+ removed from the cytoplasm by the SR [(ktwitch – kcaffeine)/ktwitch]. Similarly, (kcaffeine – kcaffeine+Ni)/ktwitch was used to calculate the proportion of Ca2+ removed by NCX (10, 24), which also enabled calculation of the proportion removed by non-SR, non-NCX, pathways.



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  		Fig. 2. Protocol used to assess sarcoplasmic reticulum (SR) Ca2+ content and SR and sarcolemmal Ca2+ removal. See text for further details. Caff, caffeine.

 
Figure 3A shows that the amplitude of the caffeine-evoked Ca2+ transient was significantly smaller in WKY Epi myocytes than in WKY Endo myocytes. This regional difference in SR Ca2+ load was absent in SHR myocytes; the caffeine-evoked Ca2+ transient amplitude was significantly greater in SHR Epi myocytes than in WKY Epi myocytes (P < 0.05) but was not significantly different from SHR Endo. There was no significant difference in the amplitude of the caffeine-evoked Ca2+ transient in Endo myocytes. These differences mirror, and therefore probably underlie, the differences in Ca2+ transient amplitude observed during electrical stimulation (Fig. 1C)



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  		Fig. 3. Mean ± SE results of SR Ca2+ content and Ca2+ extrusion in Epi and Endo left ventricular myocytes from WKY and SHR hearts. A: SR Ca2+ content. B and C: change in rate constant due to caffeine (B) and due to Ni2+ (C). NCX, Na+/Ca2+ exchange. D: fractional contribution of Ca2+ removal pathways to cellular Ca2+ handling. Bars show fractional contribution of SR Ca2+-ATPase uptake (SERCA; left pair of bars for each cell type), NCX (middle pair of bars for each cell type), and other mechanisms (right pair of bars for each cell type) in Epi and Endo left ventricular myocytes from WKY and SHR hearts. See text for further details. WKY, Epi/Endo: n = 13/13; SHR, Epi/Endo: n = 8/8. Statistical comparison was by 2-way ANOVA with post hoc Holm-Sidak interactive comparison. Lines above bars show which are significantly different (*P < 0.05).

 
Figure 3 also shows the change in k (see above) due to caffeine (ktwitch – kcaffeine; Fig. 3B) and to Ni2+ (kcaffeine – kcaffeine+Ni; Fig. 3C), representing the effect of inhibiting SR Ca2+ uptake and NCX, respectively, showing that there were no significant differences between Epi and Endo cells or between WKY and SHR. These data suggest that there is no significant effect of region or hypertrophy on absolute SR Ca2+ uptake (Fig. 3B) or NCX activity (Fig. 3C). However, these data were used to calculate the proportion of Ca2+ removed from the cytoplasm by SR, NCX, and the slow (sarcolemmal Ca2+-ATPase and mitochondria) extrusion pathways, as described above. Figure 3D shows that the calculated proportion of Ca2+ extruded by each pathway is similar to that reported previously for the rat (19). These data also show that there was no significant effect of animal strain or region on the contribution made by SERCA or the slow mechanisms. However, the proportion of Ca2+ extruded by NCX in Epi cells was significantly lower in SHR than in WKY (1.7 ± 0.3%, n = 8 vs. 4.0 ± 0.6%, n = 13; P < 0.05). Thus, in Epi cells, the ratio of NCX to SR Ca2+ removal was lower in SHR than in WKY (0.018 ± 0.005, n = 8 vs. 0.044 ± 0.007, n = 13; P < 0.05), whereas this ratio was not significantly different in WKY and SHR Endo myocytes. These data suggest that the fraction of Ca2+ extruded by NCX is lower and that the fraction taken up by the SR is higher in Epi SHR cells. This may explain the greater SR Ca2+ content and hence Ca2+ transient amplitude observed in these cells (above).

Intracellular Na+. Intracellular Na+ (Nai+) is an important regulator of NCX (and of Na+/H+ exchange) and hence of cell function. We therefore measured Nai+ to investigate whether it could contribute to the changes reported above. However, there was no apparent effect of animal strain or region on Nai+ [WKY Epi: 11.7 mmol/l (n = 2), WKY Endo: 10.2 ± 3.1 mmol/l (n = 3); SHR Epi: 10.1 ± 1.2 mmol/l (n = 8), SHR Endo: 7.2 ± 3.3 mmol/l (n = 6)]. Thus it appears unlikely that changes in Nai+ underlie the observed changes in Ca2+ handling.

t Tubule spacing. The t tubules are an important site for excitation-contraction coupling, but t tubule density is labile (1, 14, 18). There is, however, little available information regarding t tubule structure or function during hypertrophy, although such changes could decrease the synchronization of Ca2+ release and thus slow the time to peak of the Ca2+ transient (18). We have therefore investigated whether there are regional or strain differences in t tubule spacing that might account for the observed differences in time to peak of the Ca2+ transient (Fig. 1D). Di-8-ANNEPS was used in conjunction with confocal microscopy to visualize the cell membrane, and fast Fourier transforms of longitudinal intensity profiles were used to determine the periodicity of t tubule staining. Figure 4 shows representative Epi and Endo myocytes from WKY and SHR hearts, stained with di-8-ANNEPS (top) and intensity profiles of this staining from a 70-µm longitudinal section of each cell, avoiding the nucleus (middle). Fourier transforms generated from these profiles are shown in Fig. 4, bottom. The t tubule periodicity was not significantly different between regions or between WKY and SHR (Epi vs. Endo; WKY: 1.75 ± 0.03 µm vs. 1.84 ± 0.02 µm, n = 20; SHR: 1.81 ± 0.02 µm vs. 1.84 ± 0.02 µm, n = 11 and 13, respectively; all P > 0.05). In addition, visual inspection of images of cells stained with di-8-ANNEPS showed little apparent difference in t tubule distribution. Therefore it is unlikely that changes in t tubule spacing underlie the observed changes in the time course of the Ca2+ transient. However, other factors may alter the synchrony of Ca2+ release and hence the time course of the Ca2+ transient; this was therefore investigated in the next series of experiments.



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  		Fig. 4. t Tubule distribution. Top: representative Epi and Endo myocytes from WKY and SHR hearts, stained with di-8-ANNEPS. Scale bar = 20 µm. Middle: intensity profiles of a 70-µm line drawn through the cell outside the nucleus. Bottom: corresponding fast-Fourier transforms showing frequencies (and corresponding periodicities) of WKY Epi [0.549 (1.821 µm)], WKY Endo [0.538 (1.857 µm)], SHR Epi [0.546 (1.831 µm)], and SHR Endo [0.538 (1.858 µm)]. Statistical comparison of group data was by 2-way ANOVA with post hoc Holm-Sidak interactive comparison (see text for further details).

 
Synchronization of Cai2+ release. Fluo 3-loaded ventricular myocytes were electrically stimulated at 0.5 Hz. Line-scan images of Cai2+ were obtained by confocal microscopy and analyzed as described in METHODS. Figure 5 shows line-scan images from cells showing synchronous (A, top) and nonsynchronous (B, top) Ca2+ release. Figure 5, bottom, shows the intensity profiles across the line scans at the times indicated. The cell shown in Fig. 5A was classified as synchronous because F/F0 > F50 (dotted line) across the width of the cell at 13 ms (see METHODS). In contrast, the nonsynchronized release shown in Fig. 5B shows regions of the cell in which F/F0 < F50 at times after 12 ms: regions of delayed release are clearly evident in the line-scan image at 15 and 31 ms. However, the occurrence of synchronous and nonsynchronous Ca2+ release thus defined was not significantly different in Epi and Endo cells from WKY or SHR hearts (%Epi vs. Endo cells showing nonsynchronous Ca2+ release were as follows: 38 vs. 33% in WKY, n = 8 and 15, respectively; and 38 vs. 50% in SHR, n = 13 and 6, respectively; all P > 0.05; {chi}-squared test).



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  		Fig. 5. Synchronization of Ca2+ release. Top: line-scan images of Cai2+ from representative cells showing synchronous (A) and nonsynchronous (B) Ca2+ release. Bottom: intensity profiles across the line scans at the times indicated. Cells showing nonsynchronous Ca2+ release were defined as those having regions in which fluorescence/baseline fluorescence < F50 (50% of the amplitude of the whole cell Ca2+ transient) at times >12 ms. See METHODS for more details.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cardiac and cellular morphology in SHR. The SHR has been widely used to study cardiac remodeling in response to hypertension (4,5, 20,30). This model allows investigation of a cardiac pathology that is genetic and develops slowly, more closely mimicking the human condition, compared with models in which hypertrophy is induced rapidly. The present study showed significant organ and cell hypertrophy at 20 wk in the SHR; heart weight-to-body weight ratio was significantly increased, and individual myocytes had significantly larger cell widths and estimated volumes compared with the normotensive controls. The cell length-to-width ratio was also significantly decreased, suggesting concentric hypertrophy. These changes are similar to those described previously, and are consistent with compensated hypertrophy in the SHR at 20 wk (20). The changes in morphology were accompanied by regional changes in Ca2+ transient amplitude similar to those described previously (4, 20).

SR Ca2+ content and SR Ca2+ handling. The present data show that Ca2+ transient amplitude and SR Ca2+ content are greater in Epi SHR myocytes than in Epi WKY myocytes; however, Endo Ca2+ transient amplitude and SR Ca2+ content are unaltered in SHR compared with WKY. This increase in SR content is similar to the enhanced SR content of SHR myocytes described previously (5), although SR content has also been reported to be unaltered in the SHR (30); the present work suggests that such variation may reflect regional differences in the origin of the myocytes used. Increased SR Ca2+ content has also been reported in a canine model of compensated hypertrophy, induced by chronic AV block (31). SR Ca2+ release, and hence Ca2+ transient amplitude, increase markedly with SR Ca2+ content (32), suggesting that the increased SR Ca2+ content may underlie the larger Ca2+ transient amplitude in Epi SHR myocytes. The reason that this change occurs only in Epi cells is unclear; it has previously been shown that cellular hypertrophy occurs equally in Epi and Endo myocytes from the 20-wk-old SHR (20) and that the increased wall stress would be expected to occur predominantly at the endocardium. Therefore, this may be a response to more subtle regional differences (25). The resulting abolition of the normal regional difference in Ca2+ transient amplitude will result in more homogeneous contraction across the ventricular wall.

The caffeine-induced decrease in the rate constant of decline of Cai2+ was not significantly different in Epi and Endo cells or in WKY or SHR, in agreement with previous work that used other rat models of hypertrophy (19); there was, however, a small, although statistically insignificant, increase in the proportion of Ca2+ removed by SERCA in Epi SHR myocytes and no change in Endo myocytes, in agreement with previous work in aortic-banded rats showing unaltered SR contribution to Ca2+ handling (26).

This analysis is similar to that described previously (10) and is based on comparison of k during electrically and caffeine-evoked Ca2+ transients. However, this analysis does have limitations; for example, after electrical stimulation, depolarization of the cell membrane will tend to inhibit Ca2+ extrusion via NCX, whereas this will occur to a lesser extent after application of caffeine (where a smaller membrane depolarization secondary to inward NCX current is observed). Therefore, this analysis may underestimate the role of the SR and may overestimate that of NCX. In addition, the rate of decline of the electrically evoked Cai2+ transient depends on its amplitude (2) (and this study, not shown), which may decrease k derived from WKY Epi cells compared with other cell types. However, correction for this did not change the trend or the statistical significance of our data. It is also interesting to note that there was no Cai2+ dependence of the rate of decline of caffeine-evoked Cai2+ transients (either with or without Ni2+), which confirms that the Cai2+ dependence of decay of the electrically evoked Ca2+ transient is a characteristic of SR Ca2+ uptake (2).

The present data suggest, however, that SERCA activity is not altered significantly in SHR myocytes. The larger SR Ca2+ content may, however, be explained by changes in sarcolemmal Ca2+ extrusion pathways, which are in competition with SERCA for cytosolic Ca2+, so that changes in sarcolemmal Ca2+ extrusion may alter SR Ca2+ content. The contribution of the "slow" Ca2+ removal pathways (mitochondria and sarcolemmal Ca2+ ATPase) to the decline of Cai2+ does not appear to be altered in SHR myocytes (Fig. 3D); thus it appears more likely that changes in NCX activity may underlie the observed change in SR Ca2+content.

NCX activity. The Ni2+-induced decrease in k was not significantly different in either Epi or Endo cells or WKY and SHR, although there was a tendency toward reduced Ca2+ extrusion activity via NCX in SHR Epi cells (Fig. 3C). Figure 3D shows that the proportion of Ca2+ extruded via NCX was significantly lower (P < 0.05) in Epi SHR cells. Decreased Ca2+ extrusion via NCX would allow more Ca2+ uptake by SERCA and could, therefore, account for the greater SR Ca2+ content observed in SHR Epi cells (see above). A similar decrease in NCX activity has been observed in a rabbit model of hypertrophy (27), despite an increase in NCX mRNA and protein expression. A decrease in NCX activity has also been reported in a guinea pig model of hypertrophy (21) and in hypertrophied mouse heart, in which NCX current was decreased despite enhanced protein expression (34).

Thus these data suggest a significant reduction in NCX-mediated Ca2+ extrusion in SHR Epi myocytes. The cause of the reduced contribution of NCX to Ca2+ removal is, however, unclear. As cell volume increases, the surface area-to-volume ratio decreases, which may reduce the rate of Ca2+ extrusion. However, cell volume and the increase in cell volume in SHR are the same in Epi and Endo myocytes (20); it has also been reported that surface area-to-volume ratio is unchanged during hypertrophy in the rat (8), making this unlikely. An increase in Nai+ has been reported in hypertrophied and failing dog and rabbit hearts (9,33). In the absence of changes of NCX expression, this would reduce Ca2+ efflux via NCX, thus increasing cellular Ca2+ content. However, in the present study, Nai+ was unaltered, making this explanation unlikely. NCX activity is also modulated by membrane potential, and action potential prolongation has been reported during compensated hypertrophy in the SHR (4), which would tend to decrease Ca2+ efflux on the exchanger and could, therefore, help explain the present data. Thus the larger SR Ca2+ content in Epi SHR myocytes appears to be due to a change in the balance between Ca2+ sequestration by the SR and extrusion from the cell due to a 57.5% decrease in NCX-mediated Ca2+ extrusion and a small (albeit nonsignificant) fractional increase in SR Ca2+ uptake (Fig. 3D).

Despite these changes, the k for the decline of the electrically evoked Ca2+ transient is not significantly different in Epi and Endo myocytes from WKY and SHR (Fig. 1E), suggesting that the overall rate of Ca2+ removal from the cytoplasm is unaltered and thus that the decrease in NCX activity is compensated by an increase in the activity of other Ca2+ removal pathways, such as the SR (Fig. 3D). In addition, SR Ca2+ uptake is such a dominant Ca2+ removal mechanism that changes in NCX activity may have little effect on the rate of decline of the Ca2+ transient. The absence of any change in the time course of decline of the Ca2+ transient in SHR is in contrast to previous work showing slowing of the Ca2+ transient and accompanying contraction (4,5, 20,30), although the reason for this difference is unknown.

Spatial distribution of Cai2+ release. In addition to regional differences in Ca2+ transient amplitude, there were regional and hypertrophy-induced changes in its time to peak (Fig. 1D). In normal ventricular myocytes, Ca2+ release is temporally and spatially synchronized because it occurs at t tubules throughout the myocyte. Acute loss of t tubules results in nonsynchronous Ca2+ release and hence slower time to peak of the Ca2+ transient (3). Decreased t tubule density has been reported in cell cultures(18) and during pathological conditions (1, 14). Thus it seemed possible that changes in mechanical stress might alter t tubule morphology and hence the kinetics of Ca2+ release in compensated hypertrophy. However, analysis of t tubule spacing with Fourier transforms of di-8-ANNEPS staining profiles showed no significant differences between Epi or Endo WKY or SHR myocytes. Similarly, there were no regional or strain differences in the number of cells showing nonhomogeneous Ca2+ release. Thus it appears unlikely that differences in t tubule distribution and synchronization of Ca2+ release in the different cell types caused the differences in the time to peak of the Ca2+ transient. The differences in time to peak may, therefore, reflect local ultrastructural differences that affect the efficiency of coupling of Ca2+ handling proteins.

However, a significant fraction of each group of cells did display nonsynchronous Ca2+ release. Similar regions of "late" Ca2+ release have also been described in ventricular myocytes from Wistar rats (18), although the mechanism is unclear; it is unlikely to be due to t tubule structure because the t tubule system in Wistar myocytes is extensive (18), and in the present study we did not observe groups of cells with and without a dense t tubule structure that might account for these differences. Thus it appears more likely that inhomogeneities within the t tubules, in either the distribution(29) or regulation of key excitation-contraction coupling proteins, underlie such inhomogeneous Ca2+ release.

In summary, there are regional changes in Ca2+ handling that accompany compensated cellular hypertrophy in SHR. In particular, a marked decrease in NCX activity in Epi myocytes causes an increase in SR Ca2+ content and Ca2+ transient amplitude. This results in loss of the normal inhomogeneity in SR Ca2+ load and Ca2+ transient amplitude between the epi- and endocardium but will help increase strength of contraction in the face of an increased afterload.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge financial support from the British Heart Foundation and Wellcome Trust and thank Dr. D. Steele for the use of p-speck beads.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. H. Orchard, Dept. of Physiology, Univ. of Bristol, Bristol BS8 1TD, UK (E-mail: clive.orchard{at}bristol.ac.uk)

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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 RESULTS
 DISCUSSION
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