Sarcolemmal distribution of ICa and INCX and Ca autoregulation in mouse ventricular myocytes. Heart

Sarcolemmal distribution of I Ca and I NCX and Ca 2 (cid:2) autoregulation in mouse ventricular myocytes. Am Physiol ajpheart.00117.2017.—The balance of Ca 2 (cid:2) inﬂux and efﬂux regulates the Ca 2 (cid:2) load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca 2 (cid:2) inﬂux, via L-type Ca 2 (cid:2) current ( I Ca ), and efﬂux, via the Na (cid:2) /Ca 2 (cid:2) exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of I Ca and NCX current ( I NCX ), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca 2 (cid:2) measurements in intact and detubulated (DT) cells. In contrast to the rat, I NCX was located predominantly at the surface membrane, and the hysteresis between I NCX and Ca 2 (cid:2) observed in intact myocytes was preserved after detubulation. Immuno-staining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both

T-TUBULES are invaginations of the surface membrane of cardiac ventricular myocytes that play a central role in excitationcontraction coupling. Contraction is initiated by Ca 2ϩ influx [Ca 2ϩ current (I Ca )] through L-type Ca 2ϩ channels (LTCCs); this activates ryanodine receptors (RyRs) in the adjacent sarcoplasmic reticulum (SR) membrane to cause Ca 2ϩ release from the SR [Ca 2ϩ -induced Ca 2ϩ release (CICR)]. I Ca , RyRs, and thus CICR occur predominantly at t-tubules (6,8,14), which results in a near-synchronous rise in cytosolic Ca 2ϩ throughout the cell to levels sufficient to activate the contractile proteins.
For myocytes to relax, Ca 2ϩ must be removed from the cytosol. This is achieved by Ca 2ϩ reuptake into the SR and Ca 2ϩ efflux from the cell. Although SR Ca 2ϩ uptake is the main route of Ca 2ϩ removal from the cytosol, sarcolemmal Ca 2ϩ efflux pathways, the Na ϩ /Ca 2ϩ exchanger (NCX) and sarcolemmal Ca 2ϩ ATPase, also play an important role (1,25). Evidence largely from rat cardiac myocytes suggests that, like influx, Ca 2ϩ efflux also occurs predominantly at the t-tubules (11,14,37) where, it has been proposed, NCX has privileged access to Ca 2ϩ released from the SR (2,20,41).
The balance between sarcolemmal Ca 2ϩ influx and efflux determines the Ca 2ϩ load of the cell and thus the amplitude of the Ca 2ϩ transient and is maintained by a process called "autoregulation," which involves regulation of both Ca 2ϩ influx and efflux by cytoplasmic Ca 2ϩ . For example, sensitizing CICR has only a short-lived effect on the Ca 2ϩ transient amplitude (17,39,42) because the resulting increase in SR Ca 2ϩ release decreases I Ca by Ca 2ϩ -dependent inactivation of I Ca and increases Ca 2ϩ efflux by stimulating NCX (17,36). These changes reduce the Ca 2ϩ transient amplitude back to baseline levels with an accompanying decrease in SR Ca 2ϩ content (17,39,42). The role of t-tubules in autoregulation is unknown; however, because I Ca and its inactivation by Ca 2ϩ as well as NCX current (I NCX ) and its stimulation by Ca 2ϩ released from the SR have been reported to occur predominantly at the t-tubules (6,14,30), it seems likely that they play an important role in autoregulation. Therefore, this study was designed to determine the sarcolemmal distribution of I Ca and I NCX and the consequences for the role of the t-tubules in autoregulation in mice.

MATERIALS AND METHODS
Myocyte isolation and detubulation. Ventricular myocytes were isolated from the hearts of male C57BL/6 mice aged between 11 and 13 wk. All procedures were performed in accordance with United Kingdom legislation and approved by the University of Bristol Ethics Committee. Mice were injected with heparin (500 IU by intraperitoneal injection) and killed by cervical dislocation. The heart was excised and washed in isolation solution supplemented with 0.1 mM CaCl 2 and 10 U/ml heparin. The heart was then Langendorff perfused with isolation solution for 4 min followed by enzyme solution (isolation solution plus 0.1 mM CaCl 2, 265 U/ml collagenase, and 0.3 U/ml protease) for~15 min. The ventricles were then removed and shaken in enzyme solution for 4 -6 min before being filtered and centrifuged. Cells were resuspended in isolation solution (pH 7.4) plus 0.1 mM CaCl2 and stored for 2-8 h before use on the day of isolation. Detubulation (DT), the physical and functional uncoupling of the t-tubules from the surface membrane, was achieved using formamideinduced osmotic shock, as previously described, by incubating cells with 1.5 M formamide for 2 min before centrifugation and resuspending the cells in Tyrode solution (5). Data from intact and DT myocytes were obtained from separate groups of cells.
Measurement of membrane currents. Myocytes were placed in a chamber mounted on a Diaphot inverted microscope (Nikon UK, Kingston-upon-Thames, UK). Membrane currents and cell capacitance were recorded using the whole cell patch-clamp technique using an Axopatch 200B patch-clamp amplifier, a Digidata 1440A analogto-digital converter, and pClamp 10 software (Molecular Devices, Reading, UK), which was also used for data acquisition (at 2 kHz) and analysis. Pipette tip resistances were typically 1.2-2.0 M⍀ when filled with pipette solution. All experiments were performed at room temperature.
To monitor Ca 2ϩ influx and efflux, and thus Ca 2ϩ balance, during a Ca 2ϩ transient, holding potential was set to Ϫ80 mV; a 500-ms ramp to Ϫ40 mV was used to inactivate Na ϩ current followed by step depolarization to 0 mV for 100 ms to activate I Ca at a frequency of 1 Hz. I Ca was measured as the difference between peak inward current and current at the end of the pulse to 0 mV, and the integral was taken as a measure of Ca 2ϩ influx. Inactivation of ICa was quantified by measuring the time to 50% inactivation (T 50%). The current representing Ca 2ϩ removed by NCX after the step depolarization (INCX,tail) was measured by fitting a single-exponential function to 350 ms of the current trace starting 20 ms after repolarization from 0 to Ϫ80 mV and extrapolating back to when the membrane was repolarized. The integral of the exponential was taken as a measure of Ca 2ϩ efflux during the Ca 2ϩ transient (13,15). This analysis was performed using MATLAB R2015a (Mathworks, Natick, MA).
To determine the distribution of I NCX between the surface and t-tubule membranes, I NCX was measured in intact and DT myocytes during the application of 10 mM caffeine to cause spatially and temporally uniform release of SR Ca 2ϩ (4); the resulting inward current due to Ca 2ϩ extrusion via NCX was recorded at Ϫ80 mV, and I NCX was taken as the difference between the peak current and the current after caffeine washout.
The distribution of ICa, INCX, and membrane capacitance (a function of membrane area), and, thus, current density, between the surface and t-tubular membranes was calculated from measurements in intact (whole cell) and DT (surface membrane only) myocytes, as previously described (7,8). In brief, the currents and capacitance of the surface sarcolemma were calculated from those measured in DT myocytes, corrected for incomplete DT assessed using confocal imaging of Di-8-ANEPPS-stained cells as 12.7%; t-tubular currents and capacitance were calculated from the difference between those in intact cells and those in the surface sarcolemma.
Measurement of intracellular Ca 2ϩ . Fluo-4 fluorescence was excited at 450 -488 nm, and emitted fluorescence was collected at wavelengths Ͻ560 nm. Fluorescence was normalized to fluorescence just before application of caffeine (F/F0). The rate of decay of Ca 2ϩ transients was obtained by fitting single exponential functions to the declining phase of the ICa-and caffeine-induced Ca 2ϩ transients.
Analysis of NCX hysteresis loops. NCX hysteresis loops were produced by plotting I NCX against F/F0 during application of 10 mM caffeine to release SR Ca 2ϩ , as previously described (14,41). Loops were quantified by calculating the area within the loop and normalizing to the rectangle defined by maximum and minimum INCX and F/F0 (41).
Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 10 min before being permeabilized with 0.1% Triton X-100 and stained with primary antibodies for RyR (MA3-916, Thermo Fisher) or NCX (R3F1, Swant) overnight. Cells were then incubated in Alexa fluor 488-conjugated anti-mouse secondary antibody before being mounted with ProLong Gold. Cells were imaged on an LSM 880 confocal microscope (Zeiss) in Airyscan "super-resolution" mode, with a 1.4 numerical aperture ϫ63 oil-immersion objective.
Staining at the cell surface and in the center of the cell was determined from a binary cell image obtained using Otsu's method (27). The perimeter of the cell was outlined manually, and staining within a band that extended 2 m inside this outline was taken as the cell edge; staining from the image inside this band, excluding the nuclei, was taken as the cell center.
Staining was quantified as follows: Normalized staining density ϭ %bright pixels %total pixels (1) where %bright pixels is the percentage of bright pixels in a given area relative to the total number of bright pixels in the cell and %total pixels is the percentage of pixels in a given area relative to the total number of pixels in the cell. Statistical analysis. Data are expressed as means Ϯ SE. Errors of derived variables (e.g., t-tubule ICa and INCX densities) and the subsequent statistical analysis were calculated using propagation of errors from the constituent measurements (8). Student's t-tests and two-way ANOVA with the Bonferroni post hoc test were used as appropriate and performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA). The limit of statistical confidence was P Ͻ 0.05. All statistical tests were performed on the number of cells. Sample sizes (n numbers) are given as c/h, where c is the number of cells used from h number of hearts.
Autoregulation model. The mathematical model of autoregulation described by Eisner et al. (12) was used to simulate the data obtained in intact and DT myocytes. Baseline values for intact cells were those used by Eisner et al. (12) except that the transsarcolemmal Ca 2ϩ efflux fraction (r) was decreased from 10% to 8% (the value measured in the current experiments). The relative changes obtained experimentally in DT cells were used to model the data in these cells: I Ca was decreased from 10 to 4.1, fractional SR Ca 2ϩ release (f) was decreased from 0.7 to 0.287, and r was maintained at 8%, the value measured in DT cells. Changes in I NCX relative to steady state obtained from the experimental data were incorporated for intact and DT myocytes.
I NCX was measured in intact and DT myocytes during application of 10 mM caffeine to determine its distribution. Figure 1 shows representative records of intracellular Ca 2ϩ , monitored as fluo-4 fluorescence, and the associated I NCX in intact (Fig. 1A) and DT (Fig. 1B) myocytes during application of caffeine. DT had no significant effect on either the ampli-tude or rate of decay (k Caff ) of the caffeine-induced Ca 2ϩ transient ( Fig. 1, C and D), suggesting that SR Ca 2ϩ content and sarcolemmal Ca 2ϩ efflux are unchanged by DT of mouse cells. Figure 1E shows whole cell I NCX density and the calculated density of I NCX at the surface and t-tubular membranes, showing that the density of I NCX in the t-tubules is about half that in the surface membrane (P Ͻ 0.01). Thus,~25% of total I NCX appears to occur in the t-tubules, consistent with the small, although statistically nonsignificant, decrease in mean k Caff on DT (Fig. 1D).
Previous work in rat ventricular myocytes has shown that when caffeine is applied to release Ca 2ϩ from the SR, I NCX is greater for a given cytoplasmic Ca 2ϩ concentration as Ca 2ϩ increases than during the subsequent decrease. It has been suggested that this hysteresis arises because Ca 2ϩ released from the SR has "privileged" access to NCX due to the proximity of NCX to RyRs. Thus, during Ca 2ϩ release, NCX is responding to a higher local Ca 2ϩ concentration than that reported by a Ca 2ϩ indicator in the cytoplasm, while during the falling phase, the local Ca 2ϩ concentration surrounding NCX is closer to bulk cytoplasmic Ca 2ϩ concentration (41). Hysteresis is lost after DT in rat ventricular myocytes, consistent with the hysteresis arising at the t-tubules as a result of localization of I NCX to the t-tubules, close to the site of Ca 2ϩ release, in these cells (14,41). Because the majority of I NCX appears to be located at the surface sarcolemma in mouse ventricular myocytes, we investigated whether the hysteresis and its response to DT were different in these cells by plotting I NCX against F/F 0 . Figure 2A shows that intact cells showed a marked hysteresis that was not abolished by DT: I NCX density for a given Ca 2ϩ was greater during the rising phase than the declining phase of the caffeine-induced transient in both cell types, with no significant difference in the area ratio of the loop (see Fig. 2B and MATERIALS AND METHODS). However, the loop was shifted in DT cells, with a greater I NCX density for a given Ca 2ϩ , consistent with a similar Ca 2ϩ release but greater I NCX density at the cell surface. These data are consistent with the majority of NCX being located at the surface sarcolemma in the mouse and the location of I NCX determining the hysteresis, which arises at the site of highest I NCX density: the t-tubules in the rat and the surface sarcolemma in the mouse.
NCX and RyR distribution. Immunohistochemistry was used to investigate the distribution of NCX and RyR. Figure 2C shows confocal images of a representative mouse myocyte stained for NCX, showing marked striations within the cell and continuous staining at the cell surface, consistent with NCX being present at the t-tubular and surface membranes and thus with the measured distribution of I NCX . Figure 2D shows a representative cell stained for RyRs, which showed marked striations, consistent with t-tubular localization, with less staining at the cell surface, although two or sometimes three distinct areas of RyRs were observed coinciding with the mouth of t-tubules (Fig. 2D,ii). Staining of NCX and RyRs at the cell edge and cell center, quantified using Eq. 1, are shown in Fig.  2E; the apparent density of NCX staining was higher than that of RyR at the cell surface (P Ͻ 0.0001, Bonferroni post hoc test) but similar in the cell center, although both proteins had a greater density at the cell edge than at the cell center (P Ͻ 0.0001, two-way ANOVA). However, although the images were obtained using Airyscan "super-resolution" to minimize the contribution of out of focus light, there may be more surface membrane than t-tubular membrane in the optical field, since the former is likely to be present in the full depth of the field. This could lead to a higher apparent NCX density at the cell surface, although its effect on measured RyR density is unclear. However, although quantification is difficult, these data show that Ca 2ϩ release sites and NCX are present at the I Ca distribution in mouse ventricular cells. Since I NCX distribution appears to be different in mouse and rat myocytes, we also investigated the distribution of I Ca in these cells. Figure 3 shows representative I Ca and the elicited Ca 2ϩ transients in intact (Fig. 3A) and DT (Fig. 3B) myocytes. Consistent with previous reports in rat myocytes, the Ca 2ϩ transient amplitude (Fig. 3C) and rate of decay (Fig. 3D) were significantly decreased by DT (5,6,21).
I Ca in intact and DT cells was used to calculate the distribution of I Ca between the surface sarcolemma and t-tubule membranes. Figure 3E shows that I Ca density was about four times greater in the t-tubule membrane compared with the surface sarcolemma (P Ͻ 0.0001), as in the rat (6,8,14). However, in contrast to the rat, there was no significant change in the rate of inactivation of I Ca after DT (Fig. 3F), suggesting that inactivation was similar at the surface and t-tubular membranes.
Effect of DT on Ca 2ϩ autoregulation. Since the distribution of Ca 2ϩ -handling proteins determines the role of the t-tubules in Ca 2ϩ handling (4,6,14,38), we investigated the effect of detubulation on the recovery of systolic Ca 2ϩ transient amplitude and Ca 2ϩ flux via I Ca and I NCX after depletion of SR Ca 2ϩ by a high concentration (10 mM) of caffeine, and during and after application of a low concentration (200 M) of caffeine to sensitize CICR (26,42), both of which elicit autoregulation. Figure 4 shows that after application of 10 mM caffeine, Ca 2ϩ transient amplitude was initially small and gradually recovered to steady state with successive beats in both intact (Fig. 4A,i) and DT (Fig. 4B,i) myocytes. Recovery was accompanied by a decrease in the amplitude and integral of I Ca , and an increase in the amplitude and integral of I NCX in both intact (Fig. 4A,ii) and DT (Fig. 4B,ii) myocytes. However, steadystate Ca 2ϩ transient amplitude was significantly smaller in DT cells (P Ͻ 0.0001), consistent with reduced I Ca and loss of t-tubules, and the half-time (t 1/2 ) to reach steady state was significantly longer (8.7 Ϯ 1.0 s for intact cells vs. 12.6 Ϯ 0.4 s for DT cells, P Ͻ 0.01); thus, the rate of recovery due to SR refilling was slower in DT myocytes (Fig. 4C). Figure 4D shows that recovery of Ca 2ϩ transient amplitude was accompanied by a small reduction in Ca 2ϩ influx via I Ca in both intact and DT cells, although Ca 2ϩ influx, and thus the rate of Ca 2ϩ accumulation, was significantly smaller in DT myocytes (Fig.  4, D and E). In contrast, Ca 2ϩ efflux via I NCX gradually increased with continued stimulation in both cell types (Fig.  4F), reflecting an increase in SR Ca 2ϩ content and release, although Ca 2ϩ efflux was significantly smaller in DT cells (Fig. 4, F and G), which is likely to reflect the decrease in Ca 2ϩ transient amplitude due to reduced I Ca rather than loss of NCX (above).
The ratio between Ca 2ϩ influx and efflux was calculated to compare Ca 2ϩ balance in intact and DT cells (Fig. 4H). In both cell types, the ratio was initially greater than 1 after caffeine, reflecting net Ca 2ϩ influx, and gradually decreased toward 1, which represents the steady-state balance of influx and efflux. However, the initial ratio was slightly lower in DT than intact myocytes. These data suggest that reduced Ca 2ϩ influx via I Ca , and therefore slower Ca 2ϩ accumulation, underlies the slower recovery of Ca 2ϩ transient amplitude in DT cells and thus that the t-tubules play an important role in autoregulation.
To test this idea further, Ca 2ϩ autoregulation was investigated during application and washout of 200 M caffeine to intact (Fig. 5A) and DT (Fig. 5B) myocytes. Application of caffeine to intact myocytes caused a transient increase in Ca 2ϩ transient amplitude, which recovered to steady state, while washout of caffeine caused a transient decrease in Ca 2ϩ transient amplitude, which also recovered to steady state, consistent with previous work (e.g., Ref. 39). DT cells showed a similar response, although the Ca 2ϩ transient amplitude was smaller; mean data are shown in Fig. 5C. Interestingly, the half-time to recover to steady state during application of caffeine was not changed by DT (5.7 Ϯ 0.7 s in intact cells vs.  washout caused only a small increase in both cell types (Fig.  5E). Ca 2ϩ efflux increased on application of 200 M caffeine, while on washout efflux was reduced (Fig. 5F). Although both influx and efflux were significantly (P Ͻ 0.001) reduced in DT cells, the overall balance was not significantly different (Fig.  5G), whether during application 200 M caffeine, where the balance favored Ca 2ϩ efflux, or whether during washout of caffeine, where the balance favored net Ca 2ϩ influx.
Taken together, these data suggest that t-tubules play an important role in recovery from a decrease in SR Ca 2ϩ load, since DT cells recover more slowly, but not in recovery from an increased SR Ca 2ϩ release, since intact and DT cells recover at similar rates. To test this idea further, we incorporated the data from intact and DT myocytes into the model of Eisner et al. (12), as described in MATERIALS AND METHODS. The results of these simulations are shown in Fig. 6, which shows that after SR Ca 2ϩ depletion (Fig. 6A), DT cells recovered much more slowly than intact cells (t 1/2 : 10.7 stimuli in intact cells vs. 26.8 stimuli in DT cells), as observed experimentally (cf. Fig. 4C). In contrast, the rate of recovery during sensitization of CICR (Fig. 6B) was similar in intact and DT myocytes (t 1/2 : 8.8 stimuli in intact cells vs. 12.8 stimuli in DT cells), consistent with the data from the experiments (cf. Fig. 5C).

DISCUSSION
The present study was designed to investigate the role of the t-tubules in Ca 2ϩ autoregulation in mouse ventricular myocytes. The data show that the majority of I Ca occurs in the t-tubules, whereas, in contrast to rat myocytes, I NCX occurs mainly at the surface sarcolemma and the hysteresis between Ca 2ϩ and I NCX persists after DT. Although autoregulation to steady state occurred after DT, the time course of recovery was slower during recovery from decreased SR Ca 2ϩ release but, interestingly, not from increased SR Ca 2ϩ release. This suggests that t-tubules play a role in recovery from decreased, but not increased, SR Ca 2ϩ release, which may reflect the localization of I Ca and I NCX .
Distribution of I Ca and I NCX in the mouse sarcolemma. Previous work has shown that I Ca and I NCX occur predomi-nantly in the t-tubules in rat ventricular myocytes (6,8,11,14,21,37,44). The present study demonstrated a slightly higher t-tubular membrane fraction (41%) than that previously reported for rat myocytes (6,8,30), consistent with the higher t-tubule density reported previously for the mouse (18,29); it also showed that I Ca is located predominantly in the t-tubules of mouse myocytes, consistent with a previous report using DT in mouse myocytes (19). The presence of I Ca at the t-tubules in both species, in close proximity to the majority of RyRs, allows the tight coupling between Ca 2ϩ influx and SR Ca 2ϩ release that underlies CICR, while the higher t-tubule density in the mouse may reflect its high heart rate and the consequent need for rapid activation.
However, the present work also demonstrated that, in contrast to rat ventricular myocytes, I NCX is located predominantly at the cell surface in mouse myocytes. Although this might explain some of the discrepancies in the literature regarding the location of NCX determined using immunological techniques (35,37), it is unclear why the distribution of I NCX should be different in the two species, which have similar Ca 2ϩ -handling properties, with similar action potential configurations, fractions of SR and trans-sarcolemmal Ca 2ϩ flux (1,10,22,25), and kinetics of contraction and relaxation (23), although computer modeling suggests that NCX distribution has relatively little effect on whole cell Ca 2ϩ handling (31). One possibility is that location of NCX at the cell surface is energetically favorable, which might be important at the higher heart rates in the mouse, because it will avoid the futile Ca 2ϩ cycling that results from NCX being in close proximity to the main site of SR Ca 2ϩ release, by decreasing the amount of released Ca 2ϩ that is immediately removed via NCX, enabling more of the released Ca 2ϩ to activate the contractile proteins. The small t-tubular I NCX is, however, associated with a small (nonsignificant) decrease in the rate of decay of the caffeine-induced Ca 2ϩ transient after DT in the mouse, in contrast to the marked decrease observed in the rat (14).
This species difference in the sarcolemmal distribution of Ca 2ϩ -handling protein function raises questions about the distribution in other species. Although, as far as we are aware, corresponding data are not available for large mammals, previous work in cultured guinea pig myocytes (28) showed no significant change in I NCX density with time in culture, whereas cell capacitance decreased (correlating with loss of t-tubules), suggesting a similar I NCX density in the surface sarcolemma and t-tubules of guinea pig myocytes (28). Nevertheless, the maintained I NCX density might have been due to other mechanisms upregulating I NCX in culture.
It has been suggested that the hysteresis between I NCX and bulk cytoplasmic Ca 2ϩ is due to the proximity of NCX to the site of Ca 2ϩ release, so that the exchanger responds to a higher Ca 2ϩ concentration than monitored in the bulk cytosol during Ca 2ϩ release. The hysteresis is abolished after DT of rat myocytes (14), suggesting that this occurs at the t-tubules, where the majority of NCX is located close to the site of SR Ca 2ϩ release, and that surface sarcolemmal I NCX responds mainly to changes in global Ca 2ϩ concentration. The present observation that the hysteresis is present in both intact and DT mouse myocytes is consistent with the observed distribution of I NCX in these cells and suggests that I NCX at the surface sarcolemma is located close to sites of Ca 2ϩ release and responding to local changes of Ca 2ϩ concentration during Ca 2ϩ release (and to changes in global Ca 2ϩ concentration during the descending phase). The staining data support this idea: RyRs were observed not only in striations in the cell interior, consistent with localization at the t-tubules, but also at the surface membrane in clusters at the mouth of t-tubules, close to NCX, which, in agreement with the I NCX measurements, was also observed at both the t-tubules and cell surface. These data suggest that RyRs, and thus Ca 2ϩ release, occur at the t-tubules and cell surface and thus that it is the distribution of I NCX that determines the site of the hysteresis in these cells. These data also suggest that the site of origin of arrhythmogenic delayed afterdepolarizations, which are caused by acti- DT was significantly different from intact from the 8th stimulus (P Ͻ 0.05) to the 20th stimulus (P Ͻ 0.001). P values here and subsequently represent the results of a Bonferroni post hoc test. D: mean Ca 2ϩ influx. DT was significantly different from intact from the 1st stimulus (P Ͻ 0.0001) to 20th stimulus (P Ͻ 0.01). E: mean cumulative Ca 2ϩ influx. DT was significantly different from intact from the 9th stimulus (P Ͻ 0.05) to 20th stimulus (P Ͻ 0.0001). F: mean Ca 2ϩ efflux. DT was significantly different from intact from the 10th stimulus (P Ͻ 0.05) to 20th stimulus (P Ͻ 0.0001). G: mean cumulative Ca 2ϩ efflux. DT was significantly different from intact from the 10th stimulus (P Ͻ 0.05) to 20th stimulus (P Ͻ 0.0001). H: mean Ca 2ϩ influx-to-efflux ratio. C-H: all during recovery after caffeine. Intact: n ϭ 14/7; DT: n ϭ 10/7. vation of I NCX by SR Ca 2ϩ release, will depend on the location of I NCX and Ca 2ϩ release and may thus be different in different species.
Effects of DT on autoregulatory mechanisms. The present data show that, in agreement with previous studies in other species (17,39), recovery of Ca 2ϩ transient amplitude following altered SR Ca 2ϩ release is associated with reciprocal changes in Ca 2ϩ influx via I Ca and Ca 2ϩ efflux via NCX.
Changes in Ca 2ϩ transient amplitude result in changes in Ca 2ϩ influx through Ca 2ϩ -dependent inactivation of I Ca . In rabbit cells, Ca 2ϩ influx can increase by 50 -60% in the absence of SR Ca 2ϩ release (16,33); in the rat, Ca 2ϩ influx can double after SR Ca 2ϩ depletion (40). Although Ca 2ϩ -dependent inactivation also occurs in mouse myocytes, as seen by the faster inactivation during application of 200 M caffeine and slower inactivation on wash off in the present study, only small differences in the rate of inactivation between intact and DT myocytes were observed. This suggests, in contrast to rat myocytes in which Ca 2ϩ -dependent inactivation occurs predominantly at the t-tubules (6), that inactivation is similar at the surface and t-tubular membranes. The reason for this species difference is unknown but may reflect differences in local Ca 2ϩ and/or local LTCC regulation at the two sites. The lack of change in the rate of inactivation of I Ca after DT, despite the smaller Ca 2ϩ transient, could be explained by the absence of basal Ca 2ϩ -dependent inactivation of I Ca or by rapid inactivation, which is manifested as a change in peak I Ca rather than its duration. However, studies using intracellular Ca 2ϩ chelators or barium as the charge carrier for I Ca to inhibit Ca 2ϩ -induced inactivation have suggested that there is substantial basal I Ca inactivation, which mainly affects the rate of inactivation rather than peak I Ca (32,34,43), making these explanations unlikely. Thus, the most likely cause for I Ca inactivation being unaffected by DT appears to be that local Ca 2ϩ release, and thus inactivation, at the cell surface is similar to that at the t-tubules. In this case, the smaller, slower Ca 2ϩ transient in DT myocytes is due to loss of the quantitatively more important t-tubular CICR and loss of synchronization of Ca 2ϩ release. A similar local Ca 2ϩ release at the cell surface and t-tubules can explain why the hysteresis occurs in DT myocytes given the observed distribution of I NCX . Thus, it appears that, in contrast to the rat, both I NCX density and DT was significantly different from intact on the 1st stimulus (P Ͻ 0.01), from the 4th to 10th stimulus (P Ͻ 0.05), 12th to 35th stimulus (P Ͻ 0.05), 37th (P Ͻ 0.05), 50th to 53rd stimulus (P Ͻ 0.05), and 55th and 56th stimulus (P Ͻ 0.05). P values here and subsequently represent the results of a Bonferroni post hoc test. D: mean T50% of inactivation of ICa. E: mean Ca 2ϩ influx. DT was significantly different from intact for all stimuli (P Ͻ 0.0001). F: mean Ca 2ϩ efflux. DT was significantly different from intact for all stimuli (ranging from P Ͻ 0.01 to P Ͻ 0.0001) except the 2nd stimulus (not significant). G: mean Ca 2ϩ influx-to-efflux ratio. DT was significantly different from intact for the 2nd stimulus (P Ͻ 0.01) and 52nd stimulus (P Ͻ 0.001). C-G: all showing before, during, and after application of 200 M caffeine (indicated by solid bar). Intact: n ϭ 13/6; DT: n ϭ 10/4. Ca 2ϩ -dependent inactivation of I Ca are similar at the t-tubular and surface membranes and that the decrease in Ca 2ϩ influx after DT is predominantly due to loss of t-tubular I Ca rather than an altered rate of inactivation.
In contrast to I Ca , regulation of I NCX appears similar to that previously reported, with an increase in I NCX associated with an increase in Ca 2ϩ transient amplitude in intact and DT myocytes. DT resulted in changes in k Caff and I NCX consistent with loss of the t-tubular fraction of I NCX (25%), while the hysteresis between I NCX and cytosolic Ca 2ϩ showed a larger current for a given Ca 2ϩ in DT myocytes, with no loss of hysteresis, consistent with the greater density of I NCX in the surface membrane and similar Ca 2ϩ release at the surface and t-tubular membranes. Thus, the observed changes are consistent with the observed distribution of I NCX , with no evidence for altered regulation.
Effects of DT on autoregulation. As in previous studies (4,14), DT had no effect on SR Ca 2ϩ content, as assessed by releasing SR Ca 2ϩ using 10 mM caffeine, but did result in a smaller and slower voltage-stimulated Ca 2ϩ transient, due to decreased I Ca and thus CICR and loss of synchronization of SR Ca 2ϩ release. The smaller Ca 2ϩ transient, in turn, decreased I NCX , even though it was present at the surface sarcolemma.
Although DT had no effect on the rate of recovery of Ca 2ϩ transient amplitude to steady state when SR Ca 2ϩ release was increased using low-dose caffeine, recovery was slower from a decreased SR Ca 2ϩ content after caffeine-induced SR Ca 2ϩ depletion or on washout of 200 M caffeine. This asymmetric response to changes in SR Ca 2ϩ release in DT cells was not due to the nonlinear response of fluo-4 to Ca 2ϩ because it was still present after the conversion of fluorescence to Ca 2ϩ (not shown), an idea supported by the model. It may, however, be explained by the differential distribution of I Ca and I NCX . In this case, when an increase in SR Ca 2ϩ release occurs, Ca 2ϩ efflux via NCX is the main mechanism to decrease Ca 2ϩ transient amplitude and return the cell to steady state; thus, in DT mouse cells, in which the majority of I NCX occurs at the surface, recovery is similar in intact and DT myocytes and is not affected by the loss of I Ca . However, when SR Ca 2ϩ release is decreased, Ca 2ϩ influx via I Ca is necessary to refill the SR and increase Ca 2ϩ transient amplitude to steady state and is thus essential for recovery. Since steady-state SR Ca 2ϩ content is similar in intact and DT myocytes but Ca 2ϩ influx via I Ca is smaller in DT cells, more stimuli are required to refill the SR to steady state, so that recovery is slower in DT than in intact cells, even though the majority of NCX is present and quantitatively shows greater changes over time than I Ca . Slowing of recovery from caffeine has also been reported after DT of rat myocytes (4), consistent with a key role for I Ca , which, like I NCX , occurs predominantly in the t-tubules in this species.
Although t-tubules uncoupled from the surface membrane by DT may reseal within the cell and, in principle, sequester and release Ca 2ϩ (5,24), it seems unlikely that they can explain the current data, since they are electrically uncoupled from the surface membrane. Therefore, Ca 2ϩ channels in such resealed tubules will not undergo activation (and thus inactivation), an idea supported by the lack of Ca 2ϩ release in the center of DT myocytes (5). Because they do not release Ca 2ϩ and have a small volume, they are also unlikely to take up Ca, and previous work inhibiting surface NCX in DT rat myocytes during application of caffeine showed that only~2% of Ca 2ϩ removal occurs into non-SR sinks within the cell (9), which includes mitochondria, and this is likely to be even less in mouse myocytes, which have a smaller fraction of NCX within the t-tubules. Thus, resealed t-tubules are unlikely to play an important role in normal Ca 2ϩ cycling or autoregulation, an idea supported by the effects of DT on the Ca 2ϩ transient and autoregulation.
Thus, it appears that the t-tubules play an important role in autoregulation in mouse myocytes during recovery from decreased SR Ca 2ϩ release, because of the high t-tubular I Ca density, but play little role in the recovery from increased Ca 2ϩ release. These data also suggest that the response of a particular species to such changes will depend on the distribution of I Ca and I NCX between the t-tubules and surface sarcolemma. Although the distribution of Ca 2ϩ fluxes in human myocytes is unknown, the current work shows that the distribution of I Ca and I NCX determines cell function and enhances our understanding of how this occurs. Thus, when human data become available, it should be possible to better predict the consequent changes in cell function in both physiological conditions and after t-tubule disruption and changes in the distribution of I Ca and I NCX , which have been reported in heart failure (14) and which may also affect autoregulation.