Contraction and intracellular Ca2+, Na+, and H+ during acidosis in rat ventricular myocytes SIMON M. HARRISON, JAMES E. FRAMPTON, EILEEN MCCALL, MARK R. BOYETT, AND CLIVE H. ORCHARD Department of Physiology, University of Leeds, Leeds LS2 9JT, United Kingdom Harrison, Simon M., James E. Frampton, Eileen McCall, Mark R. Boyett, and Clive H. Orchard. Contraction and intracellular Ca”, Na’, and H+ during acidosis in rat ventricular myocytes. Am. J. Physiol. 262 (Cell Physiol. 31): C348-C357, 1992.-We have investigated the effect of a CO,induced (respiratory) acidosis on contraction and on intracellular Ca”, Na+, and pH (measured using the fluorescent dyes fura-2, sodium-binding benzofuran isophthalate, and 2’,7’bis(carboxyethyl)-5,6-carboxyfluorescein, respectively) in ventricular myocytes isolated from rat hearts. Initial exposure to acidosis led to a rapid decrease in intracellular pH that was accompanied by an abrupt decline in contractility. There were no consistent changes of intracellular Ns’ or Ca” during this period. The rapid decline of contractility was followed by a slower partial recovery, which was accompanied by increases in intracellular Na+, systolic and diastolic Ca”, and an increase in the Ca”’ content of the sarcoplasmic reticulum (estimated using caffeine). Intracellular pH did not change during this slow recovery. The slow rise of intracellular Na’ and the recovery of the twitch were blocked by the Na’-H’ exchange inhibitor amiloride. The sarcoplasmic reticulum inhibitor ryanodine blocked the recovery of the twitch but had no effect on the rise of intracellular Na’ induced during acidosis. It is concluded that a major cause of the initial decline of the twitch during acidosis is a decrease in the response of the contractile proteins to Ca”’ due to the decrease of intracellular pH. The subsequent slow recovery of the twitch is due to the decrease of intracellular pH activating the Na’-H+ exchange mechanism. This elevates intracellular Na’ and presumably, via the Na’-Ca”’ exchange mechanism, intracellular Ca2’. This in turn may lead to increased Ca” loading of, and hence release from, the sarcoplasmic reticulum, and it is this that underlies the partial recovery of contraction during acidosis in this preparation.
question why Ca2+ release from the SR, and hence the size of the Ca2+transient, increases during acidosis when previous studies have shown that acidosis has an inhibitory effect on all Ca2+ release mechanisms (including the SR; Refs 5, 9, 10, 22, 31, 32). There are two possible answers. First, that an increase in cytoplasmic Ca2+ during acidosis leads to increased Ca”’ loading of the SR, which then has more Ca2+ available for release (12) I t has previously been suggested that sueh Ca2+ I.oadin,g may occur because a decrease of intracellular pH leads to activation of the Na+-H+ exchange mechanism (2). The consequent rise of intracellular Na+ then, via the Na’-Ca2’ exchange mechanism, increases cytoplasmic Ca2+ (2), and hence Ca2+ uptake and release by the SR. A second possibility is that intracellular Ca2+ increases by other means [e.g., as a result of the displacement of Ca2+ by H+ from intracellular Ca2’ buffers or due to the decreased efficacy of either SR Ca2+ uptake (22) or the Na’-Ca”’ exchange (32) at lower intracellular pH] and that this leads to a secondary elevation of intracellular Na’ via Na’-Ca2+ exchange. Another possibility is that the Ca”’ load of the SR does not change, but that the magnitude of the Ca2’ current (I& which is thought to act as a graded trigger for Ca2+ release from the SR (8), may change (5, 21); although acidosis decreases Ica, this may, under certain circumstances, increase Ca2+ release from the SR (9). Alternatively, the amount of Ca2+ entering the cell (via Ica) that actually binds to the site that triggers SR Ca2’ release may change if acidosis alters Ca2+binding to this site in the same way that it appears to alter Ca2’ binding to other proteins within the cardiac cell (29). cardiac muscle; pH; muscle contraction The present study was designed, therefore, to investiIT HAS BEEN KNOWN for over 100 years that acidosis gate how acidosis alters intracellular Na+, Ca2+, and the decreases the strength of contraction of cardiac muscle Ca2+ content of the SR to further elucidate the mecha(13); during a COz-induced (respiratory) acidosis, the nisms underlying the increase in the size of the Ca2+ strength of contraction of cardiac muscle initially de- transient during acidosis. Myocytes isolated from the creases rapidly, with no change in the size of the Ca2+ ventricles of the rat heart were used, since the partial transient that induces contraction (1, ‘28). A major cause recovery of contraction during acidosis in this species of the initial decrease of contractile strength during appears to be dependent on altered Ca”’ release from the acidosis must, therefore, be a decrease in the responsive- SR (28). These cells were loaded with one of three ness of the contractile proteins to Ca2’ (19,30). However, fluorescent indicators to monitor the intracellular activon continued exposure to a respiratory acidosis, the force ity of either Ca”+ (Cai), H+ (pHi), or Na+ (Na;). Exposure of contraction shows a slow partial recovery that is to respiratory acidosis led to a rapid decrease of pHi, which showed little recovery. This was followed by an associated with an increase in the size of the Ca”+ transient (1). The partial recovery of force and the increase increase of Na;, Cai, the size of the twitch, and the in the size of the Ca”+ transient are both suppressed by amount of Ca”’ that could be released from the SR using inhibitors of sarcoplasmic reticulum (SR) function (28). caffeine (35). The rise of Nai and recovery of the twitch Therefore, this suggeststhat during acidosis, Ca2’ release were blocked by amiloride. These results suggest that from the SR is progressively increased and that this under these experimen .tal conditions the decrease of pHi increased release leads to the partial recovery in the activates th .e Na+-H’ exchange mechanism, increasing strength of contraction. These observations raise the Na’ influx. The resulting increase of Na; presumably C348
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then increases cytoplasmic Ca2’ change mechanism, which leads to of the SR, and hence an increase transient and contraction. Preliminary accounts of some ready been published (17, 24).
IONS,
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via the Na’-Ca2’ exincreased Ca2’ loading in the size of the Ca”+ of these data have al-
METHODS
Isolation of ventricular myocytes and loading with fluorescent dyes. Ventricular myocytes were dissociated from whole hearts using an enzymatic dispersion technique that has been described previously (11, 12, 16). To load the myocytes with fluorescent dye, freshly isolated cells were agitated gently in physiological salt solution containing Ca*’ (0.5 mM) and the acetoxymethyl ester of either fura- (5 ,uM for 10 min), sodiumbinding benzofuran isophthalate (SBFI, 11 PM for 2 h), or 2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF, 9 PM for 10 min) at room temperature. The cells were then centrifuged, the supernatant removed, and the cells resuspended in physiological salt solution and kept at room temperature until used. Apparatus. Ventricular myocytes were allowed to settle on the glass bottom of a chamber mounted on the stage of a Nikon Diaphot inverted microscope that was enclosed in a darkened Faraday cage. Solutions were pumped to the chamber at -3 ml/min. Two input lines controlled by electrically operated solenoid valves enabled a rapid solution changeover. The solution level in the chamber was controlled by an electronic feedback system. Experiments were carried out at room temperature (24-26°C). The cells were field stimulated via two platinum electrodes on either side of the bath. Fura-2- or SBFI-loaded myocytes were alternately excited with light of 340- and 380-nm wavelengths by using a rotating filter wheel (Cairn Research). This excitation light was transmitted to the cell under study by a 430-nm dichroic mirror beneath the microscope nose piece and a x40 oil-immersion objective lens. The resulting fluorescence was collected by the objective lens and transmitted to the side port of the microscope where it passed through a variable rectangular diaphragm that was arranged so that only fluorescence from the cell under study was measured. The fluorescence was reflected by a 580nm dichroic mirror to a photomultiplier tube (Thorn EM1 9844B) via a 510-nm emission filter. The output of the photomultiplier tube passed to the Cairn spectrophotometer which correlated the fluorescence signal with the particular excitation filter in the light path. The three fluorescence signals in response to excitation light from the three 340-nm excitation filters in the filter wheel were averaged (340 signal), as were the signals in response to the excitation light from the three 380-nm filters (380 signal). The ratio (340 signal/380 signal, a function of Cai when fura- was being used and of Nai when SBFI was being used) was determined using a custom-built analog divide circuit, the output of which was low pass filtered (time constant of 1.6 ms for fura-2, and 4 s for SBFI), displayed on a chart recorder (Gould), and stored on magnetic tape (Racal 7DS FM recorder) for later analysis. Fluorescence from BCECF-loaded myocytes was triggered and collected in a similar manner to that described above, but the excitation wavelengths were 440 and 490 nm, and fluorescence was monitored at 540 nm. The ratio (fluorescence emitted at 540 nm during excitation at 440 rim/emission at 540 nm during excitation at 490 nm) was used to obtain a qualitative estimate of pHi. The BCECF fluorescence ratio was filtered using a low-pass filter (time constant, 2.7 s) before being recorded as described above. To measure cell length (and hence contraction), cells were
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illuminated from above with long wavelength (red, ~600 nm) light. The cell image formed by the red light was collected by the objective lens and transmitted to the side port of the microscope where it was separated from the dye fluorescence by a 580-nm dichroic mirror. The cell image was then focused onto a linear 1,024-element photodiode array. From the output of the array, the length of the cell was measured electronically (4) Records of fura- fluorescence and cell length were averaged (n = 16 sweeps) and analyzed using a Tandon computer fitted with a Data Translation DT2805 analog-to-digital (A/D) board and running “Vacuum” data acquisition software, sampling each channel at 1 kHz. To obtain plots of twitch shortening (i.e., the amplitude of the twitch) against fluorescence (e.g., Figs. 3B, 5, or 6) or systolic against diastolic fluorescence (Fig. 3A), a CED 1401 A/D interface was used, run from a Tandon 386 computer, and using software written in Turbo Pascal to sample cell length and fluorescence signals at appropriate times. Estimation of Caiusing fura-2. The problems associated with the use of furaas a quantitative indicator of Ca; in cardiac myocytes have been widely debated and so shall not be discussed here (e.g., Refs. 6, 12, 26). However, becausethe present study is concerned with the effects of acidosis, any direct effects of pH on fura- fluorescence or the dissociation constant (&) of fura- for Ca*’ should be considered. Figure 1A shows the effect of acidosis on the in vitro excitation spectrum of fura-2, with emission recorded at 505 nm using a Perkin-Elmer spectrofluorimeter. A decrease in pH of 0.9 units slightly decreased fluorescence during excitation at 340 nm and increased fluorescence during excitation at 380 nm. Thus acidosis would decrease the 340/380 ratio described above, and hence the estimated Cai. This would, therefore, tend to minimize the increases in Ca; observed during acidosis in the present study. However, this effect is probably negligible in the present study because 1) the effect is small and, for the change of pHi that we estimate would occur in the present study (-0.2 pH units), would be even smaller and 2) no artifactual decrease in the fura- fluorescence ratio is observed during the period in which pHi is decreasing (e.g., Fig. 2). These results are consistent with those of Grynkiewicz et al. (14) who reported that pH variations between 6.75 and 7.05 hardly modified either the Ca’+-free or Ca*‘-bound spectra or the in vitro & of furafor Ca? However, as suggested previously, changes in the & of furafor Ca*’ would be expected to affect quantitative rather than qualitative determinations of Ca*+. Estimation of Na; using SBFI. At the excitation wavelengths used in the present study (340 and 380 nm), increases in Nai resulted in a decrease in 51O-nm fluorescence during excitation at 380 nm, with little change in the fluorescence at 510 nm during excitation with 340-nm light. Thus the ratio used to monitor Nai (fluorescence at 510 nm during excitation at 340 rim/fluorescence at 510 nm during excitation at 380 nm) increased as Nai increased (11). In vivo calibration of SBFI fluorescence was carried out in solutions containing 10 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.1 mM strophanthidin, 1 mg/l gramicidin D, 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; pH 7.1), 150 mM NaCl plus KC1 (Na’ and K’ were varied reciprocally over a range of [Na’]). When Nai activity was between 0 and 16 mM (the physiological range), the relationship between the SBFI fluorescence ratio and Na’ activity was linear (r* = 0.998), with a slope of ~0.06 ratio units/l0 mM change in Nai (16). This differed markedly from in vitro calibration which was also linear over this range, but which showed a higher fluorescence ratio at each Na+ activity, and an increased sensitivity to Na’ (slope) when compared with the in vivo calibration (16). This
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A
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Fig. 1. Effect of acidosis on the fluorescence of furaand sodium-binding benzofuran isophthalate (SBFI) in vitro. A: excitation spectra of furaat pH 7.4 and 6.5. Solution contained 0.5 PM fura2 (free acid), 130 mM KCl, 10 mM HEPES, 1 mM MgCIZ, and 400 nM [ Ca”+]. ([Ca”‘] was buffered to this level at each pH using 1 mM EGTA). Fluorescence was monitored at 505 nm. B: SBFI fluorescence at 510 nm during excitation at 340 and 380 nm (top) and the calculated fluorescence ratio (bottom) as pH was varied between 7.2 and 4.5. Solution constituents: 5 PM SBFI (free acid), 140 mM KCl, 10 mM NaCl, and 5 mM HEPES. C, top: effect of a 10 mM change of [Na’] with that of a 10 mM change of [K’] on SBFI fluorescence (solution constituents as above except where stated on the figure; values are in mM). C, bottom: effect of varying pH between 7.1 and 6.7 (the range expected in the present study) on the SBFI fluorescence ratio. Solution constituents are as in B.
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difference has been noted previously for fura- and may be due to the higher viscosity of the intracellular environment (33). The values of Nai given in the present paper were, therefore, obtained using the in vivo calibration procedure described above. Although each cell used was subjected to the calibration procedure, the variation between cells was small. For example, the mean &SE) ratio in 14 cells at 5 mM Na’ was 0.316 * 0.003. Another consideration is compartmentation of the dye. Previous reports have shown that Na’ is distributed evenly throughout the cytoplasm and intracellular organelles (15) so that errors associated with compartmentation of SBFI into intracellular organelles should be small. SBFI fluorescence from organelles with acidic interiors is unlikely to change, since at low pH SBFI becomes unresponsive to Na’ (15). However, SBFI fluorescence from mitochondria, which have an alkaline interior, would be expected to increase as bulk Nai rises. This may tend to slightly overestimate Nai during all interventions but would not be expected to make any gross qualitative changes to the present results. In this study gramicidin D was used to permeabilize the outer cell membrane (34), but not intracellular organelles, allowing the free movement of Na+,
6.9
6.7
K’, and H’ into and out of the cell (18, 25) such that the resulting calibration curve should reflect changes in cytoplasmic Na’ activity. The final problem to be considered is the direct effect of pH on SBFI fluorescence since it is possible that changes in pHi will affect the & of SBFI for Na’. Figure 1B shows the effect of pH on SBFI fluorescence in vitro (see figure legend for details); it shows the effect of pH on fluorescence monitored at 510 nm during excitation at either 340 nm or 380 nm as well as the 340/380 ratio described above. A reduction in pH decreased the fluorescence in response to excitation at both 340 and 380 nm (Fig. lB, top). The effect on the 340/380 ratio (Fig. lB, bottom) was complex and was the result of the differential effect of pH on fluorescence in response to excitation at the two wavelengths. However, over the pH range of interest in the present study (we estimate that the maximum decrease of pHi on exposure to the acid solution would be from -7.1 to 6.9; see Refs. 7 and 31), acidosis caused a 2% decrease in the in vitro fluorescence ratio (see Fig. lC, bottom). With the assumption that the percentage change in the fluorescence ratio is similar in vivo as observed in vitro (i.e., a decrease of 2%), then the fall of pHi induced at the onset of acidosis would result in a
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ACIDOSIS,
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change in the in vivo fluorescence ratio equivalent to a decrease in Na’ activity of -1.0 mM. This direct action of pH on SBFI could therefore explain the apparent fall or rise of intracellular Na’ activity (of between 0.4 and 1.3 mM), shown in Figs. 6-8 when the cell was either first exposed to, or removed from, the acid solution. Note that SBFI was calibrated in vivo at a pH of 7.1 and therefore the SBFI fluorescence ratio whilst the cell under study was in acid solution cannot be used to obtain a quantitative measure of Nai. Physiological interventions will vary intracellular Na’ and K’ reciprocally (because of the action of the Na’ pump). Figure 1C (top) compares the effect of a 10 mM increase in Na’ and K’ concentration on the SBFI ratio, showing that the effect of K+ on SBFI is negligible. Sohtions. The composition of physiological salt solution used during the cell isolation procedure was (in mM) 130.4 Na”, 142.4 Cl-, 5.4 K’, 5 HEPES, 10 glucose, 0.4 H2P0;, 3.5 Mg’+, 20 taurine, 10 creatine, 0.75 Ca”, set to pH 7.2 with NaOH at 26°C. The solution used during the experiments contained (in mM) 135 Na’, 5 K’, 1 Mg2+, 20 HCOi-, 102 Cl-, 1 SO:-, 1 Ca’)+, 20 acetate, 10 glucose, and 5 U/l insulin. This solution was equilibrated with either 5% C02-95% O2 (pH 7.3) or 15% C02-85% O:, (pH 6.85). Caffeine was dissolved in this solution just before use to give a concentration of 10 mM. This concentration of caffeine had no significant effect on furafluorescence in vitro at the excitation and emission wavelengths used in the present study. Amiloride was dissolved just before use to give a concentration of 1 mM. Amiloride itself gave a large fluorescent signal, on top of which the signal from SBFI within the cell was superimposed (Fig. 8). Ryanodine was kept as a concentrated stock that was added to the superfusate to give a final concentration of 1 PM. Statistical analysis. All data are expressed as means t SE of n preparations. Statistical comparisons were made using either a paired t test or Student’s t test as appropriate. RESULTS
Effect of acidosison Ca; and contraction. Figure 2 shows a slow time base recording of cell length and furafluorescence (a function of Cai), evoked by l-Hz stimulation before, during, and after exposure to respiratory acidosis. Increasing the percentage of CO, equlibrated with the superfusate from 5 to 15% will reduce extracellular pH by 0.45 pH units (from pH 7.3 to 6.85) and pHi by -0.2 pH units (from pH 7.1 to 6.9). The changes in cell shortening induced by this relatively mild acidosis are similar to the changes of developed force described previously in multicellular preparations contracting isometrically: an initial rapid decrease of contractile activity followed by a slower increase. The fluorescence trace
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shows that during the initial decrease in twitch shortening there was very little change in Ca;. However, during the slower recovery of twitch shortening there was a slow increase in the size of the Ca2+transient that was accompanied by a slow increase in diastolic or resting Cai. If the increase in systolic Cai (i.e., the peak of the Ca2’ transient) is a consequence of the increase in diastolic Ca; (see the introduction), it might be expected that systolic and diastolic Cai would increase in parallel. It has been shown previously (12) that systolic and diastolic Ca2+ increase in parallel during changes of stimulation rate (which are thought to increase cytoplasmic Ca2+via Na’-Ca2’ exchange). Figure 3A shows the systolic fura2 ratio plotted against the diastolic ratio for a representative cell during exposure to acidosis. For comparison, points obtained from the same cell when stimulation rate was changed between 0.2 and 2 Hz are also shown. In this, and five other cells, there was a close relationship between the increase in systolic and diastolic Cai observed during acidosis. There was no apparent difference between the relationship obtained in acidosis and that obtained on increasing stimulation rate. The relationship between twitch shortening and systolic fura- fluorescence ratio is shown in Fig. 3B. It is clear that for a given fura- 2 fluorescence ratio ( and hence Cdi 9 the twitch is smaller during acidosis than at normal pHi (when Cai was altered by changing stimulation rate). It appears9 therefore, that in agreement with previous studies, the contractile proteins are less responsive to Ca2+ during acidosis than at control pH. In support of this idea the Ca”+ transient was prolonged during acidosis, and the twitch was abbreviated (not shown); this is consistent with the off-rate of Ca2+ from troponin being increased during acidosis (28). Effect of acidosis on the Ca2’ load of the SR. It has been shown previously that the SR inhibitor ryanodine blocks the recovery of the twitch in rat cardiac muscle during respiratory acidosis (Ref. 28, see also Fig. 7), suggesting that an increase in Ca”+ release from the SR underlies the increase in the size of the Ca”+ transient and hence the recovery of the twitch during acidosis. However, it has been reported that the ability of the SR to accumulate Ca2’ is decreased during intracellular acidosis induced by the washout of 10 mM NH&l (20). We have, therefore, used caffeine to release Ca2’ from the SR during acidosis in an attempt to determine whether the Ca2+load of the SR increases during respiratory acidosis.
’ c
Fig. 2. Effect of respiratory acidosis on cell length (top; contraction is indicated by a downward deflection, indicating a decrease in cell length) and Cai, monitored as furafluorescence (bottom). Dashed line indicates mean level of diastolic Cai before acidosis. Stimulation rate was 1 Hz.
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0.5
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Fig. 3. A: comparison of the effect of a 5min exposure to acidosis at 1 Hz on the relationship between systolic and diastolic furafluorescence ratio (filled triangles), with the effect of changing stimulation rate on this relationship (stimulus rate changed from 1 to 0.2 to 2 to 0.5 to 1 Hz, open circles). B: comparison of the effect of a 5min exposure to acidosis at 1 Hz on the relationship between Ca; (monitored as furafluorescence, filled triangles) and the twitch, with the effect of changing stimulation rate in a different cell from A (stimulus rate changed from 1 to 0.2 to 2 to 1 Hz, open circles).
Figure 4A shows Ca” transients obtained from a representative myocyte before and during acidosis, showing the increase in systolic Ca; during acidosis (note also the increase in diastolic Cai during acidosis). Figure 4B shows the increase of Cai produced by the rapid application of caffeine to the cell at control pH and during acidosis. The increase of Cai produced by caffeine appears to be due to the rapid release of Ca2+ from the SR (27). Although the amplitude of the rise of Cai produced by caffeine was not always increased during acidosis, the integral increased significantly (to 126 k 6% of control, P c 0.001, n = 18; see Fig. 4C). The simplest explanation of this result is, therefore, that the increase in the size of the Ca2+ transient, and hence the recovery of the twitch observed during acidosis, may be due in part to increased release of Ca2+ from the SR, secondary to an increased Ca2’ load (but seeDISCUSSION). This increased load may be due to the increase in diastolic Cai described above. We have gone on to investigate to what extent the changes described above may be due to changes of IIH; and Nai.
xl b wa> -t Q) 0E 0 ii5
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Fig. 4. A: superimposed Ca”’ transients in control conditions and after 5min exposure to acidosis. B: superimposed records of the rise in Cai produced by rapid application of 10 mM caffeine to the same cell as in A, in control conditions, and after 5-min exposure to acidosis as indicated. C: integrals of the records shown in B (time bar is common between B and C).
Changes of pHi during respiratory acidosis. Figure 5A shows a slow time base chart recording of cell length and BCECF fluorescence ratio (a function of pHi) from a representative ventricular myocyte. On exposure to the acid solution there was a rapid decrease in pHi that had a time course similar to the initial decrease of contractile activity. However, there was very little subsequent change of pHi, although the twitch showed marked recovery. This is shown graphically in Fig. 5B, which shows a plot of twitch shortening against BCECF fluorescence. During the onset of acidosis (points a-b) and recovery from acidosis (points c-d), the changes in the twitch occurred with a similar time course to the changes in BCECF fluorescence. However, the recovery of the twitch during acidosis occurred with little change of BCECF fluorescence (points b-c). Similar results have been obtained in six other cells.
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-$127L F s z
a 103-
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65 BCECF
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Fig. 5. Relationship between twitch shortening and intracellular pH (pH,) during acidosis. A: chart recording of cell length (top) and 2’,7’bis(carboxyethyl)-5,6+arboxyfluorescein (BCECF) fluorescence (a function of pH,; bottom) before, during, and after exposure to acidosis, as indicated above the records. B: plot of twitch shortening against BCECF fluorescence throughout the record shown in A. Arrows indicate the sequence of the data, and the lower case letters correspond to the points indicated in A. Stimulation rate was 1 Hz.
Fig. 6. during before, records. shown letters HZ.
It is unlikely, therefore, that the recovery of the twitch during acidosis is a direct consequence of a change of pHi, but the decrease of pHi may be indirectly responsible for the recovery of the twitch by increasing Cai (see the introduction and below). It appears likely, however, that the initial rapid decline in the twitch (e.g., Figs. 2 and 5), which coincides with the decrease of pHi, appears to be due to a reduction in the Ca2+ sensitivity of the contractile proteins (10). Effect of acidosis on Noi. It is likely that an increase of Nai, induced as a result of activation of Na+-H+ exchange, may, via the Na+-Ca2+ exchange mechanism, lead to an increase of Ca;. In this series of experiments we monitored Nai using the fluorescent indicator SBFI to investigate whether a rise in Na could underlie the increase of Cai observed during acidosis and could, therefore, form the link between the decrease of pHi and the increase of Cai (see above). Figure 6A shows a slow time base chart recording of cell length and Na; from a ventricular myocyte before, during, and after exposure to the acid solution. On exposure to acidosis there is an initial rapid decrease in the
SBFI fluorescence ratio, associated with the decline of contractility. Following this, there is a slow increase of Nai, from a mean resting value of 6 + 1 to 9 rt 1 mM (n = 6) during lo-min exposures to acidosis. This increase of Na, coincides with the recovery of the twitch during acidosis. This is shown graphically in Fig. 6B, which shows twitch shortening plotted against Nai. Induction of acidosis causes an abrupt fall in contractility that is associated with a reduction of SBFI fluorescence caused by the direct effect of pH on SBFI which leads to artifactual changes in Na; (see METHODS). Therefore, the initial decrease in the size of the twitch occurs with little actual change of Na, (points a-b). There is then a parallel increase in both Nai and the twitch (points b-c). Because pHi remains constant during acidosis (see Fig. 5), then this increase in SBFI fluorescence represents a true increase in Nai. On returning to normal pH the twitch increases with no actual change in Nai (points c-d, see above), but then shows a slower decrease that occurs in parallel with a decrease of Na, (points d-e). These data suggest that the increase of Na, may, via Na+-Ca2+ exchange, lead to the increase of Ca,, and hence the twitch.
Relationship between cell length and intracellular Na (Na;) acidosis. A: chart recording of cell length (top) and Na; (bottom) during, and after exposure to acidosis, as indicated above the B: plot of twitch shortening against Na; throughout the record in A. Arrows show the sequence of the data, and the lower case correspond to the points indicated in A. Stimulation rate was 1
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IONS,
AND
Three observations support this idea: 1) in some cells (not shown) long exposures to acidosis led to a biphasic change of Na;, an initial increase followed by a decrease. In these cells, the recovery of force was also biphasic, with the slow recovery of the twitch being followed by a decline. 2) The dependence of the twitch on Na; during recovery from acidosis (points d-e; during which the twitch declined by 2.1 t 0.4 pm/mm01 decrease in Na+, n = 5) was similar to the relationship observed during washoff of the cardiac glycoside strophanthidin (2.1 t 0.6 pm/mm01 Na+, n = 5, not shown; Refs. 3, 16). The effect of a pH change on the relationship between Na; and the twitch was quantified during recovery from acidosis so that the problem of pH affecting SBFI fluorescence (see METHODS) could be ignored. However, Fig. 6B shows that the slope of this relationship was similar during recovery from acidosis as during acidosis. 3) The half time for the rise in systolic Cai (68 t 1‘2 s, n = 12) was not significantly different (P = 0.933, unpaired t test) from the time course of the rise of Na; (69 t 7s, n = 11). While these data support the proposal that an increase in Na; leads to an increase in Cai via Na’-Ca”’ exchange, it should be stated that the role of the Na+Ca2+ exchange was not tested directly. However, we have gone on to try and determine both the source of this increase in Na; and its relation to the recovery of the twitch. Effect of ryanodine on the twitch and Na; during acidosis. It appeared possible that acidosis could increase Cai by displacement from cellular Ca”+ buffers and that this could, via Na+-Ca2+ exchange, lead to a secondary increase in Nai. To investigate whether this was the case, we used the SR inhibitor ryanodine to abolish the recovery of contraction during acidosis (and hence, presumably, the increase of the Cai transient), and monitored Na; during a subsequent exposure to acidosis. Figure 7A shows a slow time base chart recording of cell length and Nai before, during, and after acidosis under control conditions. Figure 7B shows records of cell length and Na; from the same cell when it was exposed to acidosis in the presence of 1 ,uM ryanodine. Although the recovery of the twitch in acidosis was abolished by ryanodine, there was no significant difference in the increase of Na; (P = 0.95, n = 5, paired t test) compared with control. These data make it unlikely that the abolition of twitch recovery in ryanodine is because of a change in the response of Na; during acidosis. They also make it unlikely that the observed increase of Na; is secondary to the rise of Ca. hffect of amiloride on the twitch and Nai during acidosis. Another possibility is that the decrease of pHi leads to activation of the Na’-H+ exchange mechanism, leading to increased Na+ influx, which may increase Cai and the twitch via Na’-Ca’+ exchange. In support of this hypothesis, Kim and Smith (20) reported that blocking Na+-H+ exchange in cultured chick ventricular myocytes with ethylisopropylamiloride (EIPA) abolished the rise of Cai during acidosis induced by the washout of NH&l. However, in contrast, a rise of Cai during acidosis was still observed by Kohomoto et al. (22) under conditions where both the Na+-H+ and Na’-Ca2+ exchange were inhibited. We have, therefore, used the Na’-H+ exchange
CONTRACTION
A 15%
lz +
co2
Control
112
$7 --;i3 0
ZL 95
'I
7.4 .- \0 SE -A
41.
I
B 15%
co2
Ryanodine
I
I
1 min Fig. 7. Effect of ryanodine on the response of cell length and Na; to acidosis. In each panel, top record shows cell length, and bottom record shows Na;, monitored as SBFI fluorescence. The cell was exposed to acidosis for the period indicated above the records. Records in A were obtained under control conditions. In B, 1 PM ryanodine was present throughout. Stimulation rate was 1 Hz.
inhibitor amiloride (1 mM) to investigate this possibility. Figure 8 shows slow time base records of cell length and SBFI fluorescence from a representative myocyte exposed to acidosis in the absence (A) and presence (B) of amiloride. It is clear that amiloride completely inhibits the rise of Nai and the recovery of the twitch during acidosis. Similar results were observed in seven other cells. This experiment supports the suggestion, therefore, that a decrease of pHi leads to increased Na+ influx on the Na’-H’ exchange mechanism and that this rise of Na; underlies the recovery of twitch shortening, by increasing Cai presumably via the Na’-Ca2’ exchange mechanism. DISCUSSION
The main findings in the present paper are that 1) the initial decrease in the twitch during acidosis occurs in parallel with a decrease of pHi, but with no demonstrable change in Na; or Cai, and is not abolished by the SR inhibitor ryanodine; and 2) the recovery of the twitch on continued exposure to respiratory acidosis is associated with an increase of Nai, Cai, and the amount of Ca”+ that can be released from the SR using caffeine. It is, however, independent of any change of pHi. This recovery of the twitch is abolished by ryanodine, which does not alter the rise of Na;, and by amiloride, which also inhibits the rise of Nai during acidosis. These data support the scheme proposed by Bountra
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ACIDOSIS,
15%
co2
IONS,
AND
Control
r l6
B 15%
co2
Amiloride
cCI
150 O5 E -z z 133 0
.-0
c, 2 u. :
0.33 0.31
I---I1
1 min
Fig. 8. Effect of amiloride on the response of cell length and Nai to acidosis. In each panel, top record shows cell length, and bottom record shows Nai, monitored as SBFI fluorescence. The cell was exposed to acidosis for the period indicated above the records. Records in A were obtained under control conditions. In B, 1 mM amiloride was present throughout. Stimulation rate was 1 Hz.
and Vaughan-Jones (2) in guinea pig papillary muscles; a decrease of pHi leads, via the Na+-H’ exchanger, to an increase of Na; that, via the Na’-Ca2’ exchanger, increases Ca;, and hence the strength of contraction. The present data elucidate the role of the SR in this scheme and provide more evidence that the increase of Ca; is indeed secondary to the increase of Na;. Before considering this scheme in more detail, the methods used in the present paper should be discussed. Use of single cells and fluorescent indicators. The use of single cells for studies of cardiac function is now well establ ished. Using si.ngle cells in the present study enabled us to correlate changes of contractile act .ivity with changes - in the activity of intracellular ions in the same cell, unlike studies using multicellular preparations, in which the mean contractile activity of several thousand cells is compared with the activity of intracellular ions in one, or a few, superficial cells of the preparation that, one hopes, are representative of all the cells in the preparation. It should also be noted that we measured contractile activity by monitoring changes in cell length during isotonic contractions. This is different from studies in multicellular preparations in which isometric force is a more common measure of contractile activity. However, the response to acidosis observed in the present study
CONTRACTION
c355
was qualitatively similar to the response of papillary muscles from rat hearts in previous studies. It is likely, however, that there are quantitative differences in the response to acidosis because acidosis appears to have less effect on isotonic contractions than on isometric contractions in intact ferret ventricular muscle (23). This may be because of the different trajectory taken on the length-tension relationship by isometric and isotonic contractions (23); isotonic contractions will contract to a point at the bottom of the length-tension relation where changes in inotropic state have less effect on the position of the curve than at its peak, where isometric contractions will occur. Role of Cai and the SR in the response to acidosis. It has previously been shown in multicellular preparations that the partial recovery of contractile activity during acidosis is associated with an increase in systolic Cai (1). It has also been shown that in unstimulated preparations, acidosis leads to an increase in resting Cai (28). This led to the suggestion that an increase of resting Ca2’ during acidosis led to an increase in the Ca2’ load of the SR, and hence an increase in release in response to the action potential, and hence to recovery of the twitch (28). The present study extends these observations in two ways. First, it demonstrates that diastolic Cai increases in stimulated preparations during acidosis, and that this increase in diastolic Ca2’ occurs over a similar time course as the increase in systolic Ca2+, and that there is a close relationship between systolic and diastolic Ca;. This is consistent with the idea (see the introduction) that an increase in diastolic Cai leads to an increased Ca”+ load in the SR, and hence an increased release and Ca2’ transient. In this case the SR acts as an “amplifier” of diastolic Cai; it is, therefore, surprising that the relationship between systolic and diastolic Cai during acidosis is the same as that observed when stimulation rate was changed, since acidosis is known to have inhibitory effects on both the uptake and release of Ca2’ by the SR (22, 31). The reason for this common relationship is obscure. Second, the increase of Cai induced by the application of caffeine is increased during acidosis. However, this increase of Cai may not accurately represent the amount of Ca”’ available for release from the SR during acidosis for a number of reasons. 1) Because resting Ca2+ and intracellular [H+] increase during acidosis, it is possible that the ability of the cytoplasmic Ca2’ buffers to buffer Ca”’ is decreased so that for a given Ca2’ release into the cell cytoplasm, more Ca2’ remains free. This would be expected, however, to alter the amplitude of the caffeine-induced increase of Cai, which was not always observed. 2) It is possible that the inhibition of Ca”’ extrusion mechanisms, such as the Na+-Ca2+ exchanger (32) and the sarcolemmal Ca2+ATPase by acidosis, leads to less Ca2+ extrusion so that Cai increases more than if it was being extruded rapidly from the cell cytoplasm. It seemsunlikely that this is the case, however, because the Ca2’ efflux pathways are capable of lowering Cai more rapidly during the stimulus-induced Ca2+transient in the presence of caffeine than is observed during the caffeineinduced increase of Cai (not shown). 3) It has been suggested that caffeine acts on the SR via the normal
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C356
ACIDOSIS,
IONS,
AND
Ca*+-induced Ca”’ release mechanism (27). In this case, acidosis may inhibit the response to caffeine as it does to a Ca*’ trigger (9). This would, however, tend to minimize the rise of Ca; produced by caffeine. In conclusion, it is not possible to state unequivocally that the Ca*’ load of the SR increases during acidosis, although it seems likely. The data with ryanodine (Fig. 7), however, suggest that a functional SR is necessary for the recovery of the twitch during acidosis. Kim and Smith (20) reported that Cai still increased during acidosis in the presence of ryanodine; however, this increase was presumably insufficient to increase the size of the twitch during acidosis due to the inhibition of SR Ca*’ uptake by ryanodine. Role of pHi in the response to acidosis. pHi decreased rapidly at the beginning of the exposure to acidosis but then recovered very little during the first lo-min exposure to acidosis, in agreement with previous studies (e.g., Ref. 7). Because the initial decrease in the twitch occurred in parallel with the decrease of pHi, but with no consistent change of Nai or Cai, and was unaffected by ryanodine or amiloride, it seems most likely that this initial decrease in the twitch is due predominantly to the decrease in the sensitivity of the contractile proteins to Ca*’ that occurs in acidosis (Fig. 3B; see Refs. 10,19,30) although it appears likely that there is also a decrease in Ca*+ release from the SR during this period (28). However, the slower recovery of the twitch occurred with no change of pHi and was unlikely, therefore, to be due directly to a change of pHi. Role of Na; in the response to acidosis: relationship between Nai and the twitch. It is probable, however, that the decrease of pHi will activate the Na+-H’ exchange mechanism, which appears to be responsible for extruding acid loads, and hence increase Na; (2). This is supported by two observations. First, the increase of Na; is abolished by the Na+-H+ exchange blocker amiloride. Second, the rise of Nai still occurs when the rise of the twitch is inhibited by the application of ryanodine. However, it is possible that there is still some rise in diastolic Ca*’ during acidosis in the presence of ryanodine (20), for example, if part of the rise of diastolic Ca*+ were due to displacement of Ca*’ from cytoplasmic buffers (22). This may influence Na’ flux on the Na+-Ca*’ exchange mechanism. If Nai were being altered by the changes of Cai, then abolition of the Ca*+ transient with ryanodine would be expected to have some effect on Na;; however, ryanodine had no effect (P = 0.95) on the changes in Na; induced during acidosis, making it unlikely that Nai was being determined by Cai. It seemsmore likely, therefore, that the rise of Na; is induced by the decrease of pHi and that the rise of Na; leads to the increase of Cai via the Na’-Ca*’ exchange mechanism. These results agree with those of Bountra and Vaughan-Jones (2) who suggested that such a mechanism could increase Cai. However, although these workers predicted a recovery of force during acidosis on the basis of their measurements of pHi and Nai, such a recovery was not observed in their experiments on guinea pig papillary muscles during respiratory acidosis. The reasons for this are unclear but may be due to the difficulty of correlating the contractile activity of many
CONTRACTION
cells with ion measurements from one cell. This will be especially true in papillary muscles that are relatively thick, and which will therefore have diffusion gradients from the surface to the core of the muscle. In single cells, however, in which such gradients will be nonexistent and ion concentrations and contraction are monit*ored from the same cell, there is a clear correlation between Na;, Cai, and contraction during the recovery of twitch shortening during acidosis. The results observed using ryanodine suggest that the effect of increased Nai on twitch amplitude requires a functional SR. We thank Dr. Margaret Orchard for the use of the spectrofluorimeter and Gillian Harrison for expert technical assistance. We thank the British Heart Foundation, the Medical Research Council, and the Wellcome Trust for financial support. Address reprint requests to S. M. Harrison. Received
8 July
1991; accepted
in final
form
25 September
1991.
REFERENCES 1. Allen, D. G., and C. H. Orchard. The effects of changes of pH on intracellular calcium transients in mammalian cardiac muscle. J. Physiol. Lond. 335: 555-567, 1983. 2. Bountra, C., and R. D. Vaughan-Jones. Effect of intracellular and extracellular pH on contraction in isolated, mammalian cardiac muscle. J. Physiol. Lond. 418: 163-187, 1989. 3. Boyett, M. R., and S. M. Harrison. The relationship between the intracellular sodium activity, measured with SBFI, and the strength of contraction of ventricular myocytes isolated from the guinea-pig (Abstract). J. Physiol. Lond. 423: 6OP, 1990. 4. Boyett, M. R., M. Moore, B. R. Jewell, R. A. P. Montgomery, M. S. Kirby, and C. H. Orchard. An improved apparatus for the optical recording of contraction of single heart cells. Pfluegers Arch. 413: 197-205,1988. 5. Chesnais, J. M., E. Coraboeuf, M. P. Sauviat, and J. M. Vassas. Sensitivity to H’, Li’, and Mg”+ of the slow inward sodium current in frog atria1 fibres. J. Mol. Cell. Cardiol. 7: 627-642, 1975. 6. Cleeman, L., and M. Morad. Role of Ca” channel in cardiac excitation-contraction coupling in the rat: evidence from Ca”’ transients and contraction. J. Physiol. Lond. 432: 283-312, 1991. 7. Ellis, D., and R. C. Thomas. Microelectrode measurement of the intracellular pH of mammalian heart cells. Nature Lond. 262: 224-225,1976. 8. Fabiato, A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol. 245 (Cell Physiol. 14): Clc14, 1983. 9. Fabiato, A. Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells. Cell Calcium 6: 95-108, 1985. 10. Fabiato, A., and F. Fabiato. Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J. Physiol. Lond. 276: 233-255, 1978. 11. Frampton, J. E., S. M. Harrison, M. R. Boyett, and C. H. Orchard. Ca2’ and Na’ in rat ventricular myocytes showing different force-frequency relationships. Am. J. Physiol. 261 (Cell Physiol. 30): C739-C750, 1991. 12. Frampton, J. E., C. H. Orchard, and M. R. Boyett. Diastolic, systolic and sarcoplasmic reticulum [Ca”] during inotropic interventions in isolated rat myocytes. J. Physiol. Lond. 437: 351-375, 1991. 13. Gaskell, W. H. On the tonicity of the heart and blood vessels. J. Physiol. Lond. 3: 48-75, 1880. 14. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca”’ indicators with greatly improved fluorescent properties. J. Biol. Chem. 260: 3440-3450, 1985. 15. Harootunian, A. T., J. P. Y. Kao, B. K. Eckert, and R. Y. Tsien. Fluorescence ratio imaging of cytosolic free Na’ in individual fibroblasts and lymphocytes. J. Biol. Chem. 264: 19458-19467, 1989. 16. Harrison, S. M., E. McCall, and M. R. Boyett. The relation-
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ACIDOSIS,
17.
22.
23.
24.
25.
26.
IONS,
ship between contraction and intracellular sodium in rat and guinea-pig ventricular myocytes. J. Physiol. Lond. In press. Harrison, S. M., E. McCall, M. R. Boyett, and C. H. Orchard. Changes in intracellular sodium and pH during respiratory acidosis in rat ventricular myocytes (Abstract). Biophys. J. 59: 434a, 1991. Henderson, P. J. F., J. D. McGivan, and J. B. Chappell. The action of certain antibiotics on mitochondrial, erythrocyte and artificial phospholipid membranes. Biochem. J. 111: 521~535,1969. Kentish, J. C., and R. J. Solaro. Differential pH sensitivity of Ca2+ activated force production in myofibrils from adult and neonatal rat hearts (Abstract). J. Physiol. Lond. 394: 39P, 1987. Kim, D., and T. W. Smith. Cellular mechanisms underlying calcium-proton interactions in cultured chick ventricular cells. J. Physiol. Lond. 398: 391-410, 1988. Kohlhardt, M., K. Haap, and M. R. Figulla. Influence of low extracellular pH upon the calcium inward current and isometric contractile force in mammalian ventricular myocardium. Pfluegers Arch. 394: 264-270,1976. Kohomoto, O., K. W. Spitzer, M. A. Movsesian, and W. H. Barry. Effects of intracellular acidosis on [Ca”]i transients, transsarcolemmal Ca2’ fluxes, and contraction in ventricular myocytes. Circ. Res. 66: 622-632, 1990. Lee, J. A., and D. G. Allen. Comparison of the effect of inotropic interventions on isometric tension and shortening in isolated ferret ventricular muscle. Cardiovasc. Res. 23: 748-755, 1989. McCall, E., S. M. Harrison, M. R. Boyett, and C. H. Orchard. Intracellular sodium activity, intracellular pH and contractility in isolated rat ventricular myocytes during respiratory acidosis (Abstract). J. Physiol. Lond. 429: 17P, 1990. Myers, V. B., and D. A. Haydon. Ion transfer across lipid membranes in the presence of gramicidin A. Biochim. Biophys. Acta 274: 313-322, 1972. Noble. D.. and T. Powell. The attenuation and slowing of
AND
CONTRACTION
27.
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30.
31.
32.
33.
34.
35.
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calcium signals in cardiac muscle by fluorescent indicators (Abstract). J. Physiol. Lond. 425: 54P, 1990. O’Neill, S. C., and D. A. Eisner. A mechanism for the effects of caffeine on Ca*’ release during diastole and systole in isolated rat ventricular myocytes. J. Physiol. Lond. 430: 519-536, 1990. Orchard, C. H. The role of the sarcoplasmic reticulum in the response of ferret and rat heart muscle to acidosis. J. Physiol. Lond. 384: 431-449,1987. Orchard, C. H., D. G. Allen, M. R. Boyett, A. C. Elliott, and M. S. Kirby. The effects of acidosis on intact cardiac muscle. In: Modulation of Cardiac Calcium Sensitivity, edited by D. G. Allen and J. A. Lee. London: Martin Dunitz, chapt. 7. In press. Orchard, C. H., D. L. Hamilton, P. Astles, E. McCall, and B. R. Jewell. The effect of acidosis on the relationship between calcium and force in isolated ferret cardiac muscle. J. Physiol. Lond. 436: 559-578,199l. Orchard, C. H., and J. C. Kentish. Effects of changes of pH on the contractile function of cardiac muscle. Am. J. Physiol. 258 (CeZl Physiol. 27): C967-C981, 1990. Philipson, K. D., M. M. Bersohn, and A. Y. Nishimoto. Effects of pH on Na’-Ca*’ exchange in canine cardiac sarcolemmal vesicles. Circ. Res. 50: 287-293, 1982. Poenie, M., J. Alderton, R. Steinhardt, and R. Y. Tsien. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science Wash. DC 233: 886-889, 1986. Rink, T. J., C. Montecucco, T. R. Hesketh, and R. Y. Tsien. Lymphocyte membrane potential assesed with fluorescent probes. Biochim. Biophys. Acta 595: 15-30, 1980. Smith, G. L., M. Valdeomillos, D. A. Eisner, and D. G. Allen. Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles. J. Gen. Physiol. 92: 35l-368,1988.
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question why Ca2+ release from the SR, and hence the size of the Ca2+transient, increases during acidosis when previous studies have shown that acidosis has an inhibitory effect on all Ca2+ release mechanisms (including the SR; Refs 5, 9, 10, 22, 31, 32). There are two possible answers. First, that an increase in cytoplasmic Ca2+ during acidosis leads to increased Ca”’ loading of the SR, which then has more Ca2+ available for release (12) I t has previously been suggested that sueh Ca2+ I.oadin,g may occur because a decrease of intracellular pH leads to activation of the Na+-H+ exchange mechanism (2). The consequent rise of intracellular Na+ then, via the Na’-Ca2’ exchange mechanism, increases cytoplasmic Ca2+ (2), and hence Ca2+ uptake and release by the SR. A second possibility is that intracellular Ca2+ increases by other means [e.g., as a result of the displacement of Ca2+ by H+ from intracellular Ca2’ buffers or due to the decreased efficacy of either SR Ca2+ uptake (22) or the Na’-Ca”’ exchange (32) at lower intracellular pH] and that this leads to a secondary elevation of intracellular Na’ via Na’-Ca2+ exchange. Another possibility is that the Ca”’ load of the SR does not change, but that the magnitude of the Ca2’ current (I& which is thought to act as a graded trigger for Ca2+ release from the SR (8), may change (5, 21); although acidosis decreases Ica, this may, under certain circumstances, increase Ca2+ release from the SR (9). Alternatively, the amount of Ca2+ entering the cell (via Ica) that actually binds to the site that triggers SR Ca2’ release may change if acidosis alters Ca2+binding to this site in the same way that it appears to alter Ca2’ binding to other proteins within the cardiac cell (29). cardiac muscle; pH; muscle contraction The present study was designed, therefore, to investiIT HAS BEEN KNOWN for over 100 years that acidosis gate how acidosis alters intracellular Na+, Ca2+, and the decreases the strength of contraction of cardiac muscle Ca2+ content of the SR to further elucidate the mecha(13); during a COz-induced (respiratory) acidosis, the nisms underlying the increase in the size of the Ca2+ strength of contraction of cardiac muscle initially de- transient during acidosis. Myocytes isolated from the creases rapidly, with no change in the size of the Ca2+ ventricles of the rat heart were used, since the partial transient that induces contraction (1, ‘28). A major cause recovery of contraction during acidosis in this species of the initial decrease of contractile strength during appears to be dependent on altered Ca”’ release from the acidosis must, therefore, be a decrease in the responsive- SR (28). These cells were loaded with one of three ness of the contractile proteins to Ca2’ (19,30). However, fluorescent indicators to monitor the intracellular activon continued exposure to a respiratory acidosis, the force ity of either Ca”+ (Cai), H+ (pHi), or Na+ (Na;). Exposure of contraction shows a slow partial recovery that is to respiratory acidosis led to a rapid decrease of pHi, which showed little recovery. This was followed by an associated with an increase in the size of the Ca”+ transient (1). The partial recovery of force and the increase increase of Na;, Cai, the size of the twitch, and the in the size of the Ca”+ transient are both suppressed by amount of Ca”’ that could be released from the SR using inhibitors of sarcoplasmic reticulum (SR) function (28). caffeine (35). The rise of Nai and recovery of the twitch Therefore, this suggeststhat during acidosis, Ca2’ release were blocked by amiloride. These results suggest that from the SR is progressively increased and that this under these experimen .tal conditions the decrease of pHi increased release leads to the partial recovery in the activates th .e Na+-H’ exchange mechanism, increasing strength of contraction. These observations raise the Na’ influx. The resulting increase of Na; presumably C348
0363-6143/92
$2.00 Copyright
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
ACIDOSIS,
then increases cytoplasmic Ca2’ change mechanism, which leads to of the SR, and hence an increase transient and contraction. Preliminary accounts of some ready been published (17, 24).
IONS,
AND
via the Na’-Ca2’ exincreased Ca2’ loading in the size of the Ca”+ of these data have al-
METHODS
Isolation of ventricular myocytes and loading with fluorescent dyes. Ventricular myocytes were dissociated from whole hearts using an enzymatic dispersion technique that has been described previously (11, 12, 16). To load the myocytes with fluorescent dye, freshly isolated cells were agitated gently in physiological salt solution containing Ca*’ (0.5 mM) and the acetoxymethyl ester of either fura- (5 ,uM for 10 min), sodiumbinding benzofuran isophthalate (SBFI, 11 PM for 2 h), or 2’,7’-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF, 9 PM for 10 min) at room temperature. The cells were then centrifuged, the supernatant removed, and the cells resuspended in physiological salt solution and kept at room temperature until used. Apparatus. Ventricular myocytes were allowed to settle on the glass bottom of a chamber mounted on the stage of a Nikon Diaphot inverted microscope that was enclosed in a darkened Faraday cage. Solutions were pumped to the chamber at -3 ml/min. Two input lines controlled by electrically operated solenoid valves enabled a rapid solution changeover. The solution level in the chamber was controlled by an electronic feedback system. Experiments were carried out at room temperature (24-26°C). The cells were field stimulated via two platinum electrodes on either side of the bath. Fura-2- or SBFI-loaded myocytes were alternately excited with light of 340- and 380-nm wavelengths by using a rotating filter wheel (Cairn Research). This excitation light was transmitted to the cell under study by a 430-nm dichroic mirror beneath the microscope nose piece and a x40 oil-immersion objective lens. The resulting fluorescence was collected by the objective lens and transmitted to the side port of the microscope where it passed through a variable rectangular diaphragm that was arranged so that only fluorescence from the cell under study was measured. The fluorescence was reflected by a 580nm dichroic mirror to a photomultiplier tube (Thorn EM1 9844B) via a 510-nm emission filter. The output of the photomultiplier tube passed to the Cairn spectrophotometer which correlated the fluorescence signal with the particular excitation filter in the light path. The three fluorescence signals in response to excitation light from the three 340-nm excitation filters in the filter wheel were averaged (340 signal), as were the signals in response to the excitation light from the three 380-nm filters (380 signal). The ratio (340 signal/380 signal, a function of Cai when fura- was being used and of Nai when SBFI was being used) was determined using a custom-built analog divide circuit, the output of which was low pass filtered (time constant of 1.6 ms for fura-2, and 4 s for SBFI), displayed on a chart recorder (Gould), and stored on magnetic tape (Racal 7DS FM recorder) for later analysis. Fluorescence from BCECF-loaded myocytes was triggered and collected in a similar manner to that described above, but the excitation wavelengths were 440 and 490 nm, and fluorescence was monitored at 540 nm. The ratio (fluorescence emitted at 540 nm during excitation at 440 rim/emission at 540 nm during excitation at 490 nm) was used to obtain a qualitative estimate of pHi. The BCECF fluorescence ratio was filtered using a low-pass filter (time constant, 2.7 s) before being recorded as described above. To measure cell length (and hence contraction), cells were
CONTRACTION
c349
illuminated from above with long wavelength (red, ~600 nm) light. The cell image formed by the red light was collected by the objective lens and transmitted to the side port of the microscope where it was separated from the dye fluorescence by a 580-nm dichroic mirror. The cell image was then focused onto a linear 1,024-element photodiode array. From the output of the array, the length of the cell was measured electronically (4) Records of fura- fluorescence and cell length were averaged (n = 16 sweeps) and analyzed using a Tandon computer fitted with a Data Translation DT2805 analog-to-digital (A/D) board and running “Vacuum” data acquisition software, sampling each channel at 1 kHz. To obtain plots of twitch shortening (i.e., the amplitude of the twitch) against fluorescence (e.g., Figs. 3B, 5, or 6) or systolic against diastolic fluorescence (Fig. 3A), a CED 1401 A/D interface was used, run from a Tandon 386 computer, and using software written in Turbo Pascal to sample cell length and fluorescence signals at appropriate times. Estimation of Caiusing fura-2. The problems associated with the use of furaas a quantitative indicator of Ca; in cardiac myocytes have been widely debated and so shall not be discussed here (e.g., Refs. 6, 12, 26). However, becausethe present study is concerned with the effects of acidosis, any direct effects of pH on fura- fluorescence or the dissociation constant (&) of fura- for Ca*’ should be considered. Figure 1A shows the effect of acidosis on the in vitro excitation spectrum of fura-2, with emission recorded at 505 nm using a Perkin-Elmer spectrofluorimeter. A decrease in pH of 0.9 units slightly decreased fluorescence during excitation at 340 nm and increased fluorescence during excitation at 380 nm. Thus acidosis would decrease the 340/380 ratio described above, and hence the estimated Cai. This would, therefore, tend to minimize the increases in Ca; observed during acidosis in the present study. However, this effect is probably negligible in the present study because 1) the effect is small and, for the change of pHi that we estimate would occur in the present study (-0.2 pH units), would be even smaller and 2) no artifactual decrease in the fura- fluorescence ratio is observed during the period in which pHi is decreasing (e.g., Fig. 2). These results are consistent with those of Grynkiewicz et al. (14) who reported that pH variations between 6.75 and 7.05 hardly modified either the Ca’+-free or Ca*‘-bound spectra or the in vitro & of furafor Ca? However, as suggested previously, changes in the & of furafor Ca*’ would be expected to affect quantitative rather than qualitative determinations of Ca*+. Estimation of Na; using SBFI. At the excitation wavelengths used in the present study (340 and 380 nm), increases in Nai resulted in a decrease in 51O-nm fluorescence during excitation at 380 nm, with little change in the fluorescence at 510 nm during excitation with 340-nm light. Thus the ratio used to monitor Nai (fluorescence at 510 nm during excitation at 340 rim/fluorescence at 510 nm during excitation at 380 nm) increased as Nai increased (11). In vivo calibration of SBFI fluorescence was carried out in solutions containing 10 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.1 mM strophanthidin, 1 mg/l gramicidin D, 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES; pH 7.1), 150 mM NaCl plus KC1 (Na’ and K’ were varied reciprocally over a range of [Na’]). When Nai activity was between 0 and 16 mM (the physiological range), the relationship between the SBFI fluorescence ratio and Na’ activity was linear (r* = 0.998), with a slope of ~0.06 ratio units/l0 mM change in Nai (16). This differed markedly from in vitro calibration which was also linear over this range, but which showed a higher fluorescence ratio at each Na+ activity, and an increased sensitivity to Na’ (slope) when compared with the in vivo calibration (16). This
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c350
A
ACIDOSIS,
IONS,
AND
CONTRACTION
60
I
260
300
340
Excitation
380
wavelength
340
nm
380
nm
Fig. 1. Effect of acidosis on the fluorescence of furaand sodium-binding benzofuran isophthalate (SBFI) in vitro. A: excitation spectra of furaat pH 7.4 and 6.5. Solution contained 0.5 PM fura2 (free acid), 130 mM KCl, 10 mM HEPES, 1 mM MgCIZ, and 400 nM [ Ca”+]. ([Ca”‘] was buffered to this level at each pH using 1 mM EGTA). Fluorescence was monitored at 505 nm. B: SBFI fluorescence at 510 nm during excitation at 340 and 380 nm (top) and the calculated fluorescence ratio (bottom) as pH was varied between 7.2 and 4.5. Solution constituents: 5 PM SBFI (free acid), 140 mM KCl, 10 mM NaCl, and 5 mM HEPES. C, top: effect of a 10 mM change of [Na’] with that of a 10 mM change of [K’] on SBFI fluorescence (solution constituents as above except where stated on the figure; values are in mM). C, bottom: effect of varying pH between 7.1 and 6.7 (the range expected in the present study) on the SBFI fluorescence ratio. Solution constituents are as in B.
I
(nm)
pH
7.1
i."
_'-:--: 4.5
5.0
5.5
6.0
6.5
7.0
[K+]
130
140
140
[Nat1
10
10
20
130
J
4.5
5.0
Kt,
10
Nat
1 1 1 1
5.5
6.0
PH
6.5
7.0
pH
7.1
difference has been noted previously for fura- and may be due to the higher viscosity of the intracellular environment (33). The values of Nai given in the present paper were, therefore, obtained using the in vivo calibration procedure described above. Although each cell used was subjected to the calibration procedure, the variation between cells was small. For example, the mean &SE) ratio in 14 cells at 5 mM Na’ was 0.316 * 0.003. Another consideration is compartmentation of the dye. Previous reports have shown that Na’ is distributed evenly throughout the cytoplasm and intracellular organelles (15) so that errors associated with compartmentation of SBFI into intracellular organelles should be small. SBFI fluorescence from organelles with acidic interiors is unlikely to change, since at low pH SBFI becomes unresponsive to Na’ (15). However, SBFI fluorescence from mitochondria, which have an alkaline interior, would be expected to increase as bulk Nai rises. This may tend to slightly overestimate Nai during all interventions but would not be expected to make any gross qualitative changes to the present results. In this study gramicidin D was used to permeabilize the outer cell membrane (34), but not intracellular organelles, allowing the free movement of Na+,
6.9
6.7
K’, and H’ into and out of the cell (18, 25) such that the resulting calibration curve should reflect changes in cytoplasmic Na’ activity. The final problem to be considered is the direct effect of pH on SBFI fluorescence since it is possible that changes in pHi will affect the & of SBFI for Na’. Figure 1B shows the effect of pH on SBFI fluorescence in vitro (see figure legend for details); it shows the effect of pH on fluorescence monitored at 510 nm during excitation at either 340 nm or 380 nm as well as the 340/380 ratio described above. A reduction in pH decreased the fluorescence in response to excitation at both 340 and 380 nm (Fig. lB, top). The effect on the 340/380 ratio (Fig. lB, bottom) was complex and was the result of the differential effect of pH on fluorescence in response to excitation at the two wavelengths. However, over the pH range of interest in the present study (we estimate that the maximum decrease of pHi on exposure to the acid solution would be from -7.1 to 6.9; see Refs. 7 and 31), acidosis caused a 2% decrease in the in vitro fluorescence ratio (see Fig. lC, bottom). With the assumption that the percentage change in the fluorescence ratio is similar in vivo as observed in vitro (i.e., a decrease of 2%), then the fall of pHi induced at the onset of acidosis would result in a
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ACIDOSIS,
IONS,
AND
change in the in vivo fluorescence ratio equivalent to a decrease in Na’ activity of -1.0 mM. This direct action of pH on SBFI could therefore explain the apparent fall or rise of intracellular Na’ activity (of between 0.4 and 1.3 mM), shown in Figs. 6-8 when the cell was either first exposed to, or removed from, the acid solution. Note that SBFI was calibrated in vivo at a pH of 7.1 and therefore the SBFI fluorescence ratio whilst the cell under study was in acid solution cannot be used to obtain a quantitative measure of Nai. Physiological interventions will vary intracellular Na’ and K’ reciprocally (because of the action of the Na’ pump). Figure 1C (top) compares the effect of a 10 mM increase in Na’ and K’ concentration on the SBFI ratio, showing that the effect of K+ on SBFI is negligible. Sohtions. The composition of physiological salt solution used during the cell isolation procedure was (in mM) 130.4 Na”, 142.4 Cl-, 5.4 K’, 5 HEPES, 10 glucose, 0.4 H2P0;, 3.5 Mg’+, 20 taurine, 10 creatine, 0.75 Ca”, set to pH 7.2 with NaOH at 26°C. The solution used during the experiments contained (in mM) 135 Na’, 5 K’, 1 Mg2+, 20 HCOi-, 102 Cl-, 1 SO:-, 1 Ca’)+, 20 acetate, 10 glucose, and 5 U/l insulin. This solution was equilibrated with either 5% C02-95% O2 (pH 7.3) or 15% C02-85% O:, (pH 6.85). Caffeine was dissolved in this solution just before use to give a concentration of 10 mM. This concentration of caffeine had no significant effect on furafluorescence in vitro at the excitation and emission wavelengths used in the present study. Amiloride was dissolved just before use to give a concentration of 1 mM. Amiloride itself gave a large fluorescent signal, on top of which the signal from SBFI within the cell was superimposed (Fig. 8). Ryanodine was kept as a concentrated stock that was added to the superfusate to give a final concentration of 1 PM. Statistical analysis. All data are expressed as means t SE of n preparations. Statistical comparisons were made using either a paired t test or Student’s t test as appropriate. RESULTS
Effect of acidosison Ca; and contraction. Figure 2 shows a slow time base recording of cell length and furafluorescence (a function of Cai), evoked by l-Hz stimulation before, during, and after exposure to respiratory acidosis. Increasing the percentage of CO, equlibrated with the superfusate from 5 to 15% will reduce extracellular pH by 0.45 pH units (from pH 7.3 to 6.85) and pHi by -0.2 pH units (from pH 7.1 to 6.9). The changes in cell shortening induced by this relatively mild acidosis are similar to the changes of developed force described previously in multicellular preparations contracting isometrically: an initial rapid decrease of contractile activity followed by a slower increase. The fluorescence trace
c351
CONTRACTION
shows that during the initial decrease in twitch shortening there was very little change in Ca;. However, during the slower recovery of twitch shortening there was a slow increase in the size of the Ca2+transient that was accompanied by a slow increase in diastolic or resting Cai. If the increase in systolic Cai (i.e., the peak of the Ca2’ transient) is a consequence of the increase in diastolic Ca; (see the introduction), it might be expected that systolic and diastolic Cai would increase in parallel. It has been shown previously (12) that systolic and diastolic Ca2+ increase in parallel during changes of stimulation rate (which are thought to increase cytoplasmic Ca2+via Na’-Ca2’ exchange). Figure 3A shows the systolic fura2 ratio plotted against the diastolic ratio for a representative cell during exposure to acidosis. For comparison, points obtained from the same cell when stimulation rate was changed between 0.2 and 2 Hz are also shown. In this, and five other cells, there was a close relationship between the increase in systolic and diastolic Cai observed during acidosis. There was no apparent difference between the relationship obtained in acidosis and that obtained on increasing stimulation rate. The relationship between twitch shortening and systolic fura- fluorescence ratio is shown in Fig. 3B. It is clear that for a given fura- 2 fluorescence ratio ( and hence Cdi 9 the twitch is smaller during acidosis than at normal pHi (when Cai was altered by changing stimulation rate). It appears9 therefore, that in agreement with previous studies, the contractile proteins are less responsive to Ca2+ during acidosis than at control pH. In support of this idea the Ca”+ transient was prolonged during acidosis, and the twitch was abbreviated (not shown); this is consistent with the off-rate of Ca2+ from troponin being increased during acidosis (28). Effect of acidosis on the Ca2’ load of the SR. It has been shown previously that the SR inhibitor ryanodine blocks the recovery of the twitch in rat cardiac muscle during respiratory acidosis (Ref. 28, see also Fig. 7), suggesting that an increase in Ca”+ release from the SR underlies the increase in the size of the Ca”+ transient and hence the recovery of the twitch during acidosis. However, it has been reported that the ability of the SR to accumulate Ca2’ is decreased during intracellular acidosis induced by the washout of 10 mM NH&l (20). We have, therefore, used caffeine to release Ca2’ from the SR during acidosis in an attempt to determine whether the Ca2+load of the SR increases during respiratory acidosis.
’ c
Fig. 2. Effect of respiratory acidosis on cell length (top; contraction is indicated by a downward deflection, indicating a decrease in cell length) and Cai, monitored as furafluorescence (bottom). Dashed line indicates mean level of diastolic Cai before acidosis. Stimulation rate was 1 Hz.
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ACIDOSIS,
IONS,
AND
CONTRACTION
0.5
Acidosis
(15% co2) 0.4
Control 0
0.3
0.2 0
I
I
I
200
400
600
Time
0.35 Diastolic
Fluorescence
0 8@
Control (0.2-2Hz)
O O. 0 -,80
Ratio
0
0 8
0
O”
0.5
I
80
0
(ms)
B
.2 0.4 Q) 0 5 0.3 0 iii g 0.2 3 LL 0
0.1 Acidosis
0.34
0.58 Systolic
Fluorescence
Ratio
Fig. 3. A: comparison of the effect of a 5min exposure to acidosis at 1 Hz on the relationship between systolic and diastolic furafluorescence ratio (filled triangles), with the effect of changing stimulation rate on this relationship (stimulus rate changed from 1 to 0.2 to 2 to 0.5 to 1 Hz, open circles). B: comparison of the effect of a 5min exposure to acidosis at 1 Hz on the relationship between Ca; (monitored as furafluorescence, filled triangles) and the twitch, with the effect of changing stimulation rate in a different cell from A (stimulus rate changed from 1 to 0.2 to 2 to 1 Hz, open circles).
Figure 4A shows Ca” transients obtained from a representative myocyte before and during acidosis, showing the increase in systolic Ca; during acidosis (note also the increase in diastolic Cai during acidosis). Figure 4B shows the increase of Cai produced by the rapid application of caffeine to the cell at control pH and during acidosis. The increase of Cai produced by caffeine appears to be due to the rapid release of Ca2+ from the SR (27). Although the amplitude of the rise of Cai produced by caffeine was not always increased during acidosis, the integral increased significantly (to 126 k 6% of control, P c 0.001, n = 18; see Fig. 4C). The simplest explanation of this result is, therefore, that the increase in the size of the Ca2+ transient, and hence the recovery of the twitch observed during acidosis, may be due in part to increased release of Ca2+ from the SR, secondary to an increased Ca2’ load (but seeDISCUSSION). This increased load may be due to the increase in diastolic Cai described above. We have gone on to investigate to what extent the changes described above may be due to changes of IIH; and Nai.
xl b wa> -t Q) 0E 0 ii5
40
C Acid
30 20 10
3 LL 0 I
I
I
I
I
1
0
1
2
3
4
5
Time
(s)
Fig. 4. A: superimposed Ca”’ transients in control conditions and after 5min exposure to acidosis. B: superimposed records of the rise in Cai produced by rapid application of 10 mM caffeine to the same cell as in A, in control conditions, and after 5-min exposure to acidosis as indicated. C: integrals of the records shown in B (time bar is common between B and C).
Changes of pHi during respiratory acidosis. Figure 5A shows a slow time base chart recording of cell length and BCECF fluorescence ratio (a function of pHi) from a representative ventricular myocyte. On exposure to the acid solution there was a rapid decrease in pHi that had a time course similar to the initial decrease of contractile activity. However, there was very little subsequent change of pHi, although the twitch showed marked recovery. This is shown graphically in Fig. 5B, which shows a plot of twitch shortening against BCECF fluorescence. During the onset of acidosis (points a-b) and recovery from acidosis (points c-d), the changes in the twitch occurred with a similar time course to the changes in BCECF fluorescence. However, the recovery of the twitch during acidosis occurred with little change of BCECF fluorescence (points b-c). Similar results have been obtained in six other cells.
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ACIDOSIS,
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AND
CONTRACTION
-$127L F s z
a 103-
e
d
9-l
-
1 min
a
b 46
65 BCECF
Fluorescence
10
Ratio
12
14
16
Nai (mmolh)
Fig. 5. Relationship between twitch shortening and intracellular pH (pH,) during acidosis. A: chart recording of cell length (top) and 2’,7’bis(carboxyethyl)-5,6+arboxyfluorescein (BCECF) fluorescence (a function of pH,; bottom) before, during, and after exposure to acidosis, as indicated above the records. B: plot of twitch shortening against BCECF fluorescence throughout the record shown in A. Arrows indicate the sequence of the data, and the lower case letters correspond to the points indicated in A. Stimulation rate was 1 Hz.
Fig. 6. during before, records. shown letters HZ.
It is unlikely, therefore, that the recovery of the twitch during acidosis is a direct consequence of a change of pHi, but the decrease of pHi may be indirectly responsible for the recovery of the twitch by increasing Cai (see the introduction and below). It appears likely, however, that the initial rapid decline in the twitch (e.g., Figs. 2 and 5), which coincides with the decrease of pHi, appears to be due to a reduction in the Ca2+ sensitivity of the contractile proteins (10). Effect of acidosis on Noi. It is likely that an increase of Nai, induced as a result of activation of Na+-H+ exchange, may, via the Na+-Ca2+ exchange mechanism, lead to an increase of Ca;. In this series of experiments we monitored Nai using the fluorescent indicator SBFI to investigate whether a rise in Na could underlie the increase of Cai observed during acidosis and could, therefore, form the link between the decrease of pHi and the increase of Cai (see above). Figure 6A shows a slow time base chart recording of cell length and Na; from a ventricular myocyte before, during, and after exposure to the acid solution. On exposure to acidosis there is an initial rapid decrease in the
SBFI fluorescence ratio, associated with the decline of contractility. Following this, there is a slow increase of Nai, from a mean resting value of 6 + 1 to 9 rt 1 mM (n = 6) during lo-min exposures to acidosis. This increase of Na, coincides with the recovery of the twitch during acidosis. This is shown graphically in Fig. 6B, which shows twitch shortening plotted against Nai. Induction of acidosis causes an abrupt fall in contractility that is associated with a reduction of SBFI fluorescence caused by the direct effect of pH on SBFI which leads to artifactual changes in Na; (see METHODS). Therefore, the initial decrease in the size of the twitch occurs with little actual change of Na, (points a-b). There is then a parallel increase in both Nai and the twitch (points b-c). Because pHi remains constant during acidosis (see Fig. 5), then this increase in SBFI fluorescence represents a true increase in Nai. On returning to normal pH the twitch increases with no actual change in Nai (points c-d, see above), but then shows a slower decrease that occurs in parallel with a decrease of Na, (points d-e). These data suggest that the increase of Na, may, via Na+-Ca2+ exchange, lead to the increase of Ca,, and hence the twitch.
Relationship between cell length and intracellular Na (Na;) acidosis. A: chart recording of cell length (top) and Na; (bottom) during, and after exposure to acidosis, as indicated above the B: plot of twitch shortening against Na; throughout the record in A. Arrows show the sequence of the data, and the lower case correspond to the points indicated in A. Stimulation rate was 1
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c354
ACIDOSIS,
IONS,
AND
Three observations support this idea: 1) in some cells (not shown) long exposures to acidosis led to a biphasic change of Na;, an initial increase followed by a decrease. In these cells, the recovery of force was also biphasic, with the slow recovery of the twitch being followed by a decline. 2) The dependence of the twitch on Na; during recovery from acidosis (points d-e; during which the twitch declined by 2.1 t 0.4 pm/mm01 decrease in Na+, n = 5) was similar to the relationship observed during washoff of the cardiac glycoside strophanthidin (2.1 t 0.6 pm/mm01 Na+, n = 5, not shown; Refs. 3, 16). The effect of a pH change on the relationship between Na; and the twitch was quantified during recovery from acidosis so that the problem of pH affecting SBFI fluorescence (see METHODS) could be ignored. However, Fig. 6B shows that the slope of this relationship was similar during recovery from acidosis as during acidosis. 3) The half time for the rise in systolic Cai (68 t 1‘2 s, n = 12) was not significantly different (P = 0.933, unpaired t test) from the time course of the rise of Na; (69 t 7s, n = 11). While these data support the proposal that an increase in Na; leads to an increase in Cai via Na’-Ca”’ exchange, it should be stated that the role of the Na+Ca2+ exchange was not tested directly. However, we have gone on to try and determine both the source of this increase in Na; and its relation to the recovery of the twitch. Effect of ryanodine on the twitch and Na; during acidosis. It appeared possible that acidosis could increase Cai by displacement from cellular Ca”+ buffers and that this could, via Na+-Ca2+ exchange, lead to a secondary increase in Nai. To investigate whether this was the case, we used the SR inhibitor ryanodine to abolish the recovery of contraction during acidosis (and hence, presumably, the increase of the Cai transient), and monitored Na; during a subsequent exposure to acidosis. Figure 7A shows a slow time base chart recording of cell length and Nai before, during, and after acidosis under control conditions. Figure 7B shows records of cell length and Na; from the same cell when it was exposed to acidosis in the presence of 1 ,uM ryanodine. Although the recovery of the twitch in acidosis was abolished by ryanodine, there was no significant difference in the increase of Na; (P = 0.95, n = 5, paired t test) compared with control. These data make it unlikely that the abolition of twitch recovery in ryanodine is because of a change in the response of Na; during acidosis. They also make it unlikely that the observed increase of Na; is secondary to the rise of Ca. hffect of amiloride on the twitch and Nai during acidosis. Another possibility is that the decrease of pHi leads to activation of the Na’-H+ exchange mechanism, leading to increased Na+ influx, which may increase Cai and the twitch via Na’-Ca’+ exchange. In support of this hypothesis, Kim and Smith (20) reported that blocking Na+-H+ exchange in cultured chick ventricular myocytes with ethylisopropylamiloride (EIPA) abolished the rise of Cai during acidosis induced by the washout of NH&l. However, in contrast, a rise of Cai during acidosis was still observed by Kohomoto et al. (22) under conditions where both the Na+-H+ and Na’-Ca2+ exchange were inhibited. We have, therefore, used the Na’-H+ exchange
CONTRACTION
A 15%
lz +
co2
Control
112
$7 --;i3 0
ZL 95
'I
7.4 .- \0 SE -A
41.
I
B 15%
co2
Ryanodine
I
I
1 min Fig. 7. Effect of ryanodine on the response of cell length and Na; to acidosis. In each panel, top record shows cell length, and bottom record shows Na;, monitored as SBFI fluorescence. The cell was exposed to acidosis for the period indicated above the records. Records in A were obtained under control conditions. In B, 1 PM ryanodine was present throughout. Stimulation rate was 1 Hz.
inhibitor amiloride (1 mM) to investigate this possibility. Figure 8 shows slow time base records of cell length and SBFI fluorescence from a representative myocyte exposed to acidosis in the absence (A) and presence (B) of amiloride. It is clear that amiloride completely inhibits the rise of Nai and the recovery of the twitch during acidosis. Similar results were observed in seven other cells. This experiment supports the suggestion, therefore, that a decrease of pHi leads to increased Na+ influx on the Na’-H’ exchange mechanism and that this rise of Na; underlies the recovery of twitch shortening, by increasing Cai presumably via the Na’-Ca2’ exchange mechanism. DISCUSSION
The main findings in the present paper are that 1) the initial decrease in the twitch during acidosis occurs in parallel with a decrease of pHi, but with no demonstrable change in Na; or Cai, and is not abolished by the SR inhibitor ryanodine; and 2) the recovery of the twitch on continued exposure to respiratory acidosis is associated with an increase of Nai, Cai, and the amount of Ca”+ that can be released from the SR using caffeine. It is, however, independent of any change of pHi. This recovery of the twitch is abolished by ryanodine, which does not alter the rise of Na;, and by amiloride, which also inhibits the rise of Nai during acidosis. These data support the scheme proposed by Bountra
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ACIDOSIS,
15%
co2
IONS,
AND
Control
r l6
B 15%
co2
Amiloride
cCI
150 O5 E -z z 133 0
.-0
c, 2 u. :
0.33 0.31
I---I1
1 min
Fig. 8. Effect of amiloride on the response of cell length and Nai to acidosis. In each panel, top record shows cell length, and bottom record shows Nai, monitored as SBFI fluorescence. The cell was exposed to acidosis for the period indicated above the records. Records in A were obtained under control conditions. In B, 1 mM amiloride was present throughout. Stimulation rate was 1 Hz.
and Vaughan-Jones (2) in guinea pig papillary muscles; a decrease of pHi leads, via the Na+-H’ exchanger, to an increase of Na; that, via the Na’-Ca2’ exchanger, increases Ca;, and hence the strength of contraction. The present data elucidate the role of the SR in this scheme and provide more evidence that the increase of Ca; is indeed secondary to the increase of Na;. Before considering this scheme in more detail, the methods used in the present paper should be discussed. Use of single cells and fluorescent indicators. The use of single cells for studies of cardiac function is now well establ ished. Using si.ngle cells in the present study enabled us to correlate changes of contractile act .ivity with changes - in the activity of intracellular ions in the same cell, unlike studies using multicellular preparations, in which the mean contractile activity of several thousand cells is compared with the activity of intracellular ions in one, or a few, superficial cells of the preparation that, one hopes, are representative of all the cells in the preparation. It should also be noted that we measured contractile activity by monitoring changes in cell length during isotonic contractions. This is different from studies in multicellular preparations in which isometric force is a more common measure of contractile activity. However, the response to acidosis observed in the present study
CONTRACTION
c355
was qualitatively similar to the response of papillary muscles from rat hearts in previous studies. It is likely, however, that there are quantitative differences in the response to acidosis because acidosis appears to have less effect on isotonic contractions than on isometric contractions in intact ferret ventricular muscle (23). This may be because of the different trajectory taken on the length-tension relationship by isometric and isotonic contractions (23); isotonic contractions will contract to a point at the bottom of the length-tension relation where changes in inotropic state have less effect on the position of the curve than at its peak, where isometric contractions will occur. Role of Cai and the SR in the response to acidosis. It has previously been shown in multicellular preparations that the partial recovery of contractile activity during acidosis is associated with an increase in systolic Cai (1). It has also been shown that in unstimulated preparations, acidosis leads to an increase in resting Cai (28). This led to the suggestion that an increase of resting Ca2’ during acidosis led to an increase in the Ca2’ load of the SR, and hence an increase in release in response to the action potential, and hence to recovery of the twitch (28). The present study extends these observations in two ways. First, it demonstrates that diastolic Cai increases in stimulated preparations during acidosis, and that this increase in diastolic Ca2’ occurs over a similar time course as the increase in systolic Ca2+, and that there is a close relationship between systolic and diastolic Ca;. This is consistent with the idea (see the introduction) that an increase in diastolic Cai leads to an increased Ca”+ load in the SR, and hence an increased release and Ca2’ transient. In this case the SR acts as an “amplifier” of diastolic Cai; it is, therefore, surprising that the relationship between systolic and diastolic Cai during acidosis is the same as that observed when stimulation rate was changed, since acidosis is known to have inhibitory effects on both the uptake and release of Ca2’ by the SR (22, 31). The reason for this common relationship is obscure. Second, the increase of Cai induced by the application of caffeine is increased during acidosis. However, this increase of Cai may not accurately represent the amount of Ca”’ available for release from the SR during acidosis for a number of reasons. 1) Because resting Ca2+ and intracellular [H+] increase during acidosis, it is possible that the ability of the cytoplasmic Ca2’ buffers to buffer Ca”’ is decreased so that for a given Ca2’ release into the cell cytoplasm, more Ca2’ remains free. This would be expected, however, to alter the amplitude of the caffeine-induced increase of Cai, which was not always observed. 2) It is possible that the inhibition of Ca”’ extrusion mechanisms, such as the Na+-Ca2+ exchanger (32) and the sarcolemmal Ca2+ATPase by acidosis, leads to less Ca2+ extrusion so that Cai increases more than if it was being extruded rapidly from the cell cytoplasm. It seemsunlikely that this is the case, however, because the Ca2’ efflux pathways are capable of lowering Cai more rapidly during the stimulus-induced Ca2+transient in the presence of caffeine than is observed during the caffeineinduced increase of Cai (not shown). 3) It has been suggested that caffeine acts on the SR via the normal
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C356
ACIDOSIS,
IONS,
AND
Ca*+-induced Ca”’ release mechanism (27). In this case, acidosis may inhibit the response to caffeine as it does to a Ca*’ trigger (9). This would, however, tend to minimize the rise of Ca; produced by caffeine. In conclusion, it is not possible to state unequivocally that the Ca*’ load of the SR increases during acidosis, although it seems likely. The data with ryanodine (Fig. 7), however, suggest that a functional SR is necessary for the recovery of the twitch during acidosis. Kim and Smith (20) reported that Cai still increased during acidosis in the presence of ryanodine; however, this increase was presumably insufficient to increase the size of the twitch during acidosis due to the inhibition of SR Ca*’ uptake by ryanodine. Role of pHi in the response to acidosis. pHi decreased rapidly at the beginning of the exposure to acidosis but then recovered very little during the first lo-min exposure to acidosis, in agreement with previous studies (e.g., Ref. 7). Because the initial decrease in the twitch occurred in parallel with the decrease of pHi, but with no consistent change of Nai or Cai, and was unaffected by ryanodine or amiloride, it seems most likely that this initial decrease in the twitch is due predominantly to the decrease in the sensitivity of the contractile proteins to Ca*’ that occurs in acidosis (Fig. 3B; see Refs. 10,19,30) although it appears likely that there is also a decrease in Ca*+ release from the SR during this period (28). However, the slower recovery of the twitch occurred with no change of pHi and was unlikely, therefore, to be due directly to a change of pHi. Role of Na; in the response to acidosis: relationship between Nai and the twitch. It is probable, however, that the decrease of pHi will activate the Na+-H’ exchange mechanism, which appears to be responsible for extruding acid loads, and hence increase Na; (2). This is supported by two observations. First, the increase of Na; is abolished by the Na+-H+ exchange blocker amiloride. Second, the rise of Nai still occurs when the rise of the twitch is inhibited by the application of ryanodine. However, it is possible that there is still some rise in diastolic Ca*’ during acidosis in the presence of ryanodine (20), for example, if part of the rise of diastolic Ca*+ were due to displacement of Ca*’ from cytoplasmic buffers (22). This may influence Na’ flux on the Na+-Ca*’ exchange mechanism. If Nai were being altered by the changes of Cai, then abolition of the Ca*+ transient with ryanodine would be expected to have some effect on Na;; however, ryanodine had no effect (P = 0.95) on the changes in Na; induced during acidosis, making it unlikely that Nai was being determined by Cai. It seemsmore likely, therefore, that the rise of Na; is induced by the decrease of pHi and that the rise of Na; leads to the increase of Cai via the Na’-Ca*’ exchange mechanism. These results agree with those of Bountra and Vaughan-Jones (2) who suggested that such a mechanism could increase Cai. However, although these workers predicted a recovery of force during acidosis on the basis of their measurements of pHi and Nai, such a recovery was not observed in their experiments on guinea pig papillary muscles during respiratory acidosis. The reasons for this are unclear but may be due to the difficulty of correlating the contractile activity of many
CONTRACTION
cells with ion measurements from one cell. This will be especially true in papillary muscles that are relatively thick, and which will therefore have diffusion gradients from the surface to the core of the muscle. In single cells, however, in which such gradients will be nonexistent and ion concentrations and contraction are monit*ored from the same cell, there is a clear correlation between Na;, Cai, and contraction during the recovery of twitch shortening during acidosis. The results observed using ryanodine suggest that the effect of increased Nai on twitch amplitude requires a functional SR. We thank Dr. Margaret Orchard for the use of the spectrofluorimeter and Gillian Harrison for expert technical assistance. We thank the British Heart Foundation, the Medical Research Council, and the Wellcome Trust for financial support. Address reprint requests to S. M. Harrison. Received
8 July
1991; accepted
in final
form
25 September
1991.
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