Effects of changes of pH on the contractile of cardiac muscle C. H. ORCHARD
AND
function
J. C. KENTISH
Department of Physiology, University of Leeds, Leeds LS2 9JT; and Department University College London, London WClE 6BT, United Kingdom
of Physiology,
ORCHARD,C. H., AND J. C. KENTISH. Effects of changes of pH on the contractile function of cardiac muscle. Am. J. Physiol. 258 (Cell Physiol. 27): C967C981, 1990.-It has been known for over 100 years that acidosis decreases the contractility of
4 Hz decreases pHi by -0.06 pH units; Ref. 14), it is sufficient to modulate the contractile response of heart muscle to the rate change (14). 3) A profound decrease of intracellular pH is one of the most marked consecardiac muscle. However, the mechanisms underlying this de- quences of myocardial ischemia, with decreases of pHi of crease are complicated because acidosis affects every step in 0.5 units being commonly observed and values of pHi as the excitation-contraction coupling pathway, including both the delivery of Ca2’ to the myofilaments and the response of low as 6.2 being reported (56). 4) Clinical disturbances the myofilaments to Ca2+.Acidosis has diverse effects on Ca2+ of acid-base status, such as diabetic ketoacidosis or hycan cause substantial changes in pH, or delivery. Actions that may diminish Ca2+ delivery include 1) poventilation, pHi that could affect cardiac function. 5) Acidosis may, inhibition of the Ca2’ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action to some extent, protect against hypoxia (12), reperfusion potential, when such shortening occurs. Conversely, Ca2” deliv- injury (78), and, at least in hepatocytes, ATP depletion ery may be increased by the prolongation of the action potential (61, 62). that is sometimes observed and by the rise of diastolic Ca2+ Although it is well established that extracellular acithat occurs during acidosis. This rise, which will increase the dosis causes a fall of developed force in cardiac muscle, uptake and subsequent release of Ca2’ by the sarcoplasmic the magnitude and rate of the force response depend on reticulum, may be due to 1) stimulation of Na+ entry via Na+H’ exchange, which will increase intracellular Ca2+ via Na+- how the acidosis is produced. Smith (117) first pointed Ca2+ exchange; 2) direct inhibition of Na+-Ca2+ exchange; 3) out that the negative inotropic effect of an extracellular acidosis was greater and more rapid if the decrease in mitochondrial release of Ca2+;and 4) displacement of Ca2+from pH, was produced by an elevated [CO,] (respiratory cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the acidosis) than if it was produced by a reduction in [HCO:] (metabolic acidosis). From such evidence, he contractile proteins to Ca2+,probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, and others (26, 105, 133) concluded that the negative possibly by a direct action on the cross bridges. Thus the final inotropism is due mainly to intracellular, rather than amount of force developed by heart muscle during acidosis is extracellular, acidosis; the faster action of CO2 is then the complex sum of these changes. explained because it can readily diffuse across the cell membrane and lower pHi (by dissociation to H+ and heart muscle; acidosis HCO; ), whereas extracellular HCO; and H+ are relatively impermeant. The faster time course of the fall in pHi during respiratory acidosis has been confirmed by IT HAS BEEN RECOGNIZED for over 100 years that a microelectrode measurements (43). These considerations decrease in extracellular pH reduces the contractility of suggest that the major actions of pH, are via changes of the heart (57). An understanding of how changes of pHi. For this reason, this review is concerned chiefly with extracellular pH (pH,) or intracellular pH (pHi) affect the mechanisms by which changes in pHi can affect force. cardiac function is important for our comprehension of However, it should be noted that a small part of the cardiac physiology and pathophysiology for five main negative inotropic effects of acidosis may result from reasons. 1) The cellular handling of Na+ and Ca2’, which direct effects of pH, on the properties of the cell memis central to cell function, is intimately linked to cellular brane (see Electrophysiology: Channels and Currents and H+ metabolism by Na+-H+ and Na+-Ca2+ exchanges, and Na+-H+ exchange). possibly by sharing common buffer sites within the cell. Within the last few years it has become possible to 2) Inotropic maneuvers such as changes of heart rate monitor discrete steps in the excitation-contraction coumay change pHi from its normal value (-7.0). Although pling pathway, and it has become clear that changes of this change is relatively small (increasing rate from 0 to pH may influence all of the steps in the pathway leading 0363-6143/90
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Copyright 0 1990 the American Physiological
Society
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to contraction, from the action potential to the generation of force by the myosin cross bridges within the myofibrils. Thus the net effect of changes of pH on contractile activity is complex. In addition, in studies using intact tissues it can be difficult to determine whether changes of pH are acting directly or whether the observed changes are secondary to other changes of cell function. In this article we will review recent studies that help us to understand the effect of changes of pH on contractile function of the heart. We will not be concerned with the regulation of intracellular pH (which is reviewed elsewhere, Refs. 52, 111).We will concentrate on the effects of acidosis, rather than alkalosis, for two reasons. First, it has clinical relevance (acidosis is a major component of myocardial ischemia), and second, more studies have examined the effects of acidosis than of alkalosis. Where alkalosis has been studied, the effects are in general found to be opposite to those of acidosis. This indicates that protons have an influence on the various steps in excitation-contraction (E-C) coupling even at normal values of pH. We will first describe the effects of changes of pH on measurements of intracellular [Ca”‘] and force, before discussing how variations of pH might alter cellular processes to bring about these changes. CONTRACTILE TO ACIDOSIS
RESPONSE
OF HEART
MUSCLE
Figure 1 shows a record of how force and cytosolic Ca2+ concentration (CaJ change in a ferret papillary muscle during and after exposure to a hypercapnic acidosis (see legend to Fig. 1 for details). Acidosis leads to a rapid decline of developed force followed by a slower partial recovery. The negative inotropic response to acidosis is well known (e.g., 26, 90, 91, 100, 105, 117, 122, l33), and a similar biphasic force response to acidosis has been observed in papillary muscles from a variety of other species (e.g., guinea pig, Ref. 53; cat, Refs. 90, 116) and with a variety of preparations, such as Langendorffperfused hearts (e.g., 4) and isolated ventricular myocytes (17). The muscle in Fig. 1 had been injected with the photoprotein aequorin, which emits light when it binds Ca2+. The aequorin light emission (a function of Cai) showed that systolic Cai increases during acidosis (5, 6, 102), reaching a maximum after -3-min exposure to the acid solution. Figure 1B shows that acidosis alters the time course of both the twitch and the Cai transient that accompanies it; the onset of twitch relaxation is earlier, twitch relaxation is faster, and the Cai transient declines more slowly during acidosis. Although small, these effects are statistically significant (5, 102). Measurements of pHi during sustained hypercapnic acidosis have shown either a monophasic decline in Purkinje fibers and rat ventricular muscle (44) or a rapid decline followed by a small partial recovery in guinea pig papillary muscles (108) and Langendorff-perfused ferret heart (4). This partial recovery of pHi may explain, in part, the partial recovery of force. In Fig. 1, pH, and pHi were decreased; when the primary change is of pHi alone, force still decreases, the size of the calcium transient still increases, and the changes of time course of the twitch
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A 20 nA
10 mN/mm*
IO nA
1 1 1
10 mN /mm*]
FIG. 1. Effect of acidosis on intracellular [Ca”‘] (monitored using photoprotein aequorin) and force in an isolated papillary muscle from a ferret heart. A: aequorin light (a function of Cai; top) and developed force (bottom). Period of acidosis is shown above records. B: aequorin light transients (top) and force (bottom) averaged during control conditions (a) and after 1.5min exposure to acidosis (b). c: aequorin light and developed force from a and b normalized (uncalibrated) to the same peak to show changes in time course. Force and light during acidosis are denoted by black circles. Temperature was 3O”C, and stimulation frequency was 0.33 Hz. Acidosis was induced by increasing [CO*] of the gas with which the muscle superfusate was equilibrated from 5% (pH, 7.35) to 30% (pH, 6.7). [HCOJ was constant throughout. Effect of acidosis on aequorin is to cause a small decrease in aequorin light emission at pCa 6 (6), which would tend to minimize changes shown above. During a respiratory acidosis induced using 15% CO2 (pH, 6.85), a decrease of pHi of 0.3 units has been reported in Langendorff-perfused ferret hearts (4). Thus pHi decreased by 60% of pH,. In experiment shown above, it might be expected, therefore, that pHi will have decreased by -0.6 x (7.35 - 6.7) = 0.39 units. However, it has recently been reported (126) that decreasing pH, by 0.43 pH units decreased pHi by only 0.13 pH units (i.e., 30% of pH,) in guinea pig papillary muscles. However, whether this is due to a species difference is unclear.
and calcium transient are still observed (102). The different subcellular systems that might be affected by changes of pH to produce these effects are shown in Fig. 2. The remainder of this article will be concerned with how acidosis may affect these cellular processes to produce changes of Cai and force such as those shown in Fig. 1. EFFECT OF ACIDOSIS ON CONTRACTION COUPLING
Electrophysiology:
THE EXCITATIONPATHWAY
Channels and Currents
In early electrophysiological studies it was found that the cardiac action potential and, by implication, the ionic currents that caused it were much less sensitive than action force to changes in pH,. Indeed, near-normal potentials could be elicited under acid conditions (pH, 5.5-6.2) when force was zero (87, 131). Nevertheless, there were some effects on action potential configuration which indicated that pH did influence the underlying ionic currents. The predominant effect was on the height of the plateau, which was decreased by acidosis, suggesting a reduction of the slow inward current (Isi) carried chiefly by Ca2+ (27, 87). A fall of Isi could also explain the observed abbreviation of the action potential seen in
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Na Ca
3Na
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Na
2K
Cell membrane
Ca
H
3Na
Ca
Sarcoplasmic reticulum T-tubule Mitochondria
@
FIG. 2. Possible sites of action of H+ in the myocardial cell. The text refers to the following sites: 1, electrophysiology: channels and currents; 2a, sarcolemmal bound Ca2’; 2b, Na+-Ca2+ exchange; 2c, Na+-H+ exchange; 2d, Na+-K+-ATPase; 2e, Ca2’-ATPase; 3a, Ca2+ uptake by the SR; 3b, Ca2’ release from the SR; 4, intracellular (cytoplasmic) Ca’+ during acidosis; 5, contractile proteins; 6, cell metabolism and mitochondrial function (mitochondrial Ca2+ extrusion is shown as a single pump, although it is the result of several processes; see text).
Myofibrils
many studies (e.g., 27, 81, 87, 112). However, in other studies, the action potential was prolonged (17, 53, 64), indicating that other currents were affected (probably outward K+ currents). The direct effect of acidosis on Isi would affect force production because of the role of Isi in E-C coupling (see SZow inward current), but the effect on other currents that affect action potential duration could also be important: it is known that an increase in action potential duration leads to an increase of developed force (95), presumably by increasing Cai. The mechanism of this increase is probably that the prolonged action potential I) increases the time for which Isi flows and 2) increases Ca2+ influx (or decreases efflux) on the Na+-Ca2’ exchange mechanism (see Na’-Ca2+ exchange). Thus changes of action potential duration will alter the response to acidosis by altering Cai. The alterations of membrane currents underlying the changes in the action potential have been elucidated using voltage-clamp techniques. Slow inward current (IS;). Particular attention has been paid to the effects of pH on 1si. This reflects the importance of 1si in E-C coupling: the entry of Ca2+ during 1si via L-type Ca2+ channels not only loads the myocytes with Ca2+, but it may also be the trigger for Ca2+ release from the sarcoplasmic reticulum (SR) (47). The first direct evidence for the pH sensitivity of Isi was obtained with voltage clamp of multicellular preparations. These studies showed that external acidosis produced a large decrease of 1si in frog atrium (27, 132) and mammalian ventricle (53, 80, 131) and slowed the kinetics of inactivation and recovery of Isi (80). Because force development depends on the magnitude of Isi (9), some of the reduction in force could be explained by the fall in Isi.
However, the simultaneous recording of force and Isi showed that force was generally decreased relatively more than Isi (80, 131, but cf. 53), indicating additional actions of pH were involved. More recent studies on membrane currents have utilized voltage clamp of perfused (dialyzed) isolated myocytes. In the whole cell voltage-clamp configuration, the solution in the recording patch pipette is in contact with the cell cytoplasm and exchanges rapidly with it (in minutes). Thus the solution in the patch pipette can, in theory, control the composition and pH of the cytosol, so the pHi can be varied independently of pH,. This may also avoid any secondary changes in Cai or Nai, which complicated the interpretation of experiments on multicellular preparations. An additional advantage is that problems of K+ accumulation within extracellular clefts are avoided (see Na+ current). Such experiments with isolated myocytes have confirmed and extended the results from multicellular preparations. In all studies, the magnitude of 1si fell in acid solutions. This inhibition was seen whether pHi was reduced (by intracellular perfusion with acid solutions) at constant pH, (66, 68, 81, 112) or if pH, was reduced at constant pHi (66, 82), demonstrating that there are extracellular and intracellular sites of action of H+. Acidity was also found to slow the kinetics of Isi in some studies (137) but not others (66) Itappears likely, then, that there are both extracellular and intracellular sites of action of H+, predominantly on the conductance properties but also on the gating properties of Isi. The effects of extracellular acidosis on Isi could arise by two main mechanisms: by block by H+ at a specific site in the Ca2+ channel or by neutralization of
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some of the fixed negative charge associated with the membrane surface, thereby changing the transmembrane electric field in the vicinity of the Ca2+ channel. The second possibility can account for the finding that there is a positive shift of the current-voltage (I- V) relation by lo-15 mV when pH, is reduced (66, 82, 137). The increased extracellular [H+] would neutralize some of the negative charge on the outer surface of the membrane (although the overall membrane potential would be unaffected); this would make the membrane act as if the cell were hyperpolarized, and the I-V curves would shift in a positive direction. This shift would decrease the peak of Isi, since the peak current would occur at a more positive potential, where the driving force for entry of Ca2+ was reduced. However, this shift is not sufficient by itself to account for all of the drop in peak Isi during extracellular acidosis (82)) so an additional mechanism seems necessary, such as direct block of the channel by H+ The mechanism of Isi inhibition by intracellular acidosis (at constant pH,) has been investigated in detail by Kaibara and Kameyama (68), using open-cell-attached patch clamp, in which individual channel events could be analyzed in dialyzed cells. Most of the 60% fall of I$i when pHi was reduced from 7.2 to 6.2 was due to a reduction in the open probability of the channel. This resulted mainly from an increased lifetime of a state in which the channel was unavailable for opening. Kaibara and Kameyama (68) also found that 20% of the reduction of 1siwas due to a decrease in the unitary current through the Ca2+ channel. Although earlier studies had reported a positive shift (112) or no shift (81) in the I-V relationship, Kaibara and Kameyama found a negative shift of -10 mV in the activation and inactivation curves, which is consistent with an alteration of Ca2+ channel gating as a result of H+ neutralizing negative charges on the cytosolic surface of the sarcolemma. In intact cells it is possible that some of the effects of acidosis on Isi result from a secondary rise in Cai that reduces Isi amplitude and accelerates inactivation (18, 38). However, this is probably only a minor factor, since the effects of pHi on 1si amplitude are still present if the cell is perfused with ethylene glycol-bis(P-aminoethyl ether) -lV,AT,N’ ,N’ -tetraacetic acid (EGTA) (66,68). The relative roles of changes in pH, and pHi remain to be established conclusively. A problem with attempting to change pHi to known values in dialyzed cells is that the cell’s active H+ extrusion mechanism (Na+-H+ exchange) tends to oppose any applied changes in pHi produced by the solution in the patch pipette (66). However, if Na+-H+ exchange is prevented, by removal of external Na+ or by amiloride, 1si is affected much more by a given change in pHi than by an equivalent change in pH,. For example, for 50% inhibition of Isi, pHi needed only to be reduced from 7.2 to 6.6, compared with a change in pH, of 7.4 to 5.0 (with pHi well buffered; Ref. 66). Of course, in many situations there will be changes in both pH, and pHi so that the effects will combine. In summary, a decrease of pHi and, to a lesser extent, of pH, reduces the magnitude of Isi. This will decrease Ca2+ loading of the cell and decrease the trigger for Ca2+
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release from the SR (see pH dependence of Ca2+ release by the SR). Na+ current.
In general, the actions of pH changes on the fast inward Na+ current (1& are small and inconsistent. The maximum rate of depolarization of the action potential, which reflects the magnitude of &, has been reported to be little affected by acidosis (53, 67), or depressed by acidosis (27, 124, 129). The latter action is likely to be because of 1) a shift of lNa threshold in the depolarizing direction, which has been ascribed to protonation of fixed charge on the membrane surface (22), or 2) in Purkinje fibers at least, a small decrease in resting membrane potential due to accumulation of K+ in extracellular clefts (22). K+ currents. The outward currents carried chiefly by K+ also seem to be affected by changes of pH, although results are variable. In some studies on isolated myocytes, the time-dependent outward current (delayed rectifier, 1k) was little changed (81), although there was an increase in a time-independent outward current at positive potentials (112, but cf. 137). The latter was not due to a Ca2+activated K+ conductance (112). This increase in outward current would, like the decrease in 1si, tend to decrease action potential duration at low pH. In addition, acid solutions increased an outward current recorded at negative potentials (probably the inward rectifier, &I)3 which could explain the small hyperpolarization of the resting potential seen in some studies (112). In contrast to the above, in other studies it was found that the action potential was prolonged during acidosis; this has been seen in both multicellular preparations (53, 64) and in isolated ventricular myocytes (e.g., Ref. 17). This prolongation may be due to a decrease in 1k (27) or in lkl (63), which could also explain the small depolarization of the resting membrane potential sometimes observed during acidosis (17). Obviously more work is needed before we can explain these divergent results on action potential duration or on K+ currents. One possibility is that the direction of the response varies with the magnitude of the pH change, since Sato et al. (112) found a moderate fall of pHi increased outward current, whereas a large fall decreased it. Arrhythmogenesis. Under certain conditions, intracellular acidosis can lead to abnormal electrical rhythms. Coraboeuf et al. (30) found that a fall of pHi in Purkinje fibers provoked repolarization abnormalities and transient depolarizations, which induced fiber reexcitation. In what may be another manifestation of the same phenomenon, the injection of acid into myocytes has been shown to directly induce the transient inward current (&J (81). Iti can also be induced by excessive Ca2+ loading of cells (24, 70) and is thought to be due to spontaneous release of Ca2+ from the SR, causing activation of the electrogenic Na+-Ca2+ exchange (which carries net current into the cell). The acid-induced genesis of Iti is therefore probably due to a rise in Cai caused by the low pHi (see Diastolic [Ca2+]). Because 1ti is an inward (depolarizing) current that can occur during diastole, it can lead to the generation of arrhythmias (86). Thus acidosis may increase the likelihood of arrhythmogenesis in cardiac cells. In addition, a spontaneous release of Ca2+
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during diastole will reduce the Ca2+ transient, and therefore, the force, in the subsequent contraction of the cell (3); this could contribute to the negative inotropic effect of acidosis in cardiac muscle. It has also been found that a decrease of pHi reduces the coupling between cardiac cells by decreasing the conductance of the gap junctions (32, 109). For example, a fall of pHi from 7.2 to 6.8 in Purkinje fibers increased the junctional resistance by ~30% (109). This, in addition to any reduction of lNa (see above), would tend to slow propagation of the action potential, as discussed by Gettes (58), and could reduce the synchrony of contraction within the ventricle. A severe reduction of conduction velocity in part of the ventricle would also increase the likelihood of arrhythmogenesis by reentrant excitation and cause a decrease in the efficiency of pumping. Ion Regulation by the Sarcolemma: Pumps, and Exchangers
Cu2+ Binding
Sites,
The cell membrane may alter contractile function during acidosis not only by altering events during the action potential (action potential duration and the magnitude of Isi; see Electrophysiology: Channels and Currents) but also by altering ion flux through pumps and exchangers in the cell membrane that are active in both systole and diastole, hence altering Cai (Fig. 2). Sarcolemmal bound Ca2+. It has been suggested (84) that some of the Ca2+ that enters the cell cytoplasm to initiate contraction comes from a pool that is bound to sites on the sarcolemmal membrane, although this view is still controversial. In “gas-dissected” membranes and sarcolemmal vesicles from the canine heart (83), Ca2+ binding to sarcolemmal sites was decreased as pH was lowered from 8.5 to 5.5, with an apparent pK for inhibition of 6.6-7.15. If Ca2” is bound to the external side of the cell membrane, then flux of Ca2’ from these sites into the cell will be via Isi and Na+-Ca2+ exchange, and a decrease in Ca2+ bi .nding to these Sl tes could contribute to decrea .sed influx into the cell v ia these processes (see Slow irward current and Na+-Ca2+ exchange). If Ca2+ is bound to the inside of the membrane, its displacement by H+ may contribute to the increase in resting Ca2+ observed during acidosis (see Diastolic [Ca2+J). Na+-Ca2+ exchange. In the resting myocyte Na+-Ca2” exchange is an important route for Ca2+ extrusion. This exchanger uses the electrochemical gradient for Na+ entry into the cell to extrude Ca2+ from the cell, exchanging three Na+ for each Ca2+ (for review, see Ref. 40). However, it has been suggested that during the action potential the exchanger may reverse and cause Ca2+ entry into the cell. The exchanger may, therefore, be a route for both Ca2+ influx and efflux from the cell, the direction depending on the transmembrane electrochemical gradients for Na+ and Ca2+. Changes in the activity of this exchange mechanism may, therefore, have profound effects on cell function. The effect of pH on the Na+Ca2+ exchange mechanism has been studied in both canine sarcolemmal vesicles (106) and isolated guinea pig myocytes (37). In isolated vesicles the initial rate of Nai-dependent Ca2’ uptake into the vesicles showed a sigmoidal dependence on pH
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over the range pH 5-9, with acidosis inhibiting uptake. N&-dependent Ca2+ efflux also showed pH dependence, with alkalosis (pH 9) stimulating efflux and acidosis (pH 6) inhibiting efflux, in agreement with work on squid axons (35). Thus the exchanger appears to be inhibited by acidosis, regardless of the direction in which it is working. In single cell studies (37) this inhibition appears to occur whether the pH change is applied at the inner or outer cell surface. The cause of this inhibition is less clear, but it appears to be a direct action on the exchanger and may be, in part, due to competition between Ca2+ and H+. Thus, if Na+-Ca2’ exchange extrudes Ca2+ from the cell during diastole (there must be a net efflux to balance Ca2+ influx during the action potential), inhibition of the exchanger will lead to less Ca2+ extrusion, and hence an increase of Cai. Conversely, if the exchanger normally reverses during the action potential, and brings Ca2+ into the cell, inhibition of the exchanger will lead to less Ca2’ influx via the exchanger during the action potential. Na+-H+ exchange. This exchange mechanism is thought to be electroneutral, exchanging one Na+ for one H+, and using the concentration gradient for Na+ entry to extrude H+ from the cell. Thus changes in the transsarcolemmal Na+ or H+ gradient will result in altered fluxes through the exchanger. However, unlike many of the systems discussed above, the Na+-Hf exchange mechanism does not appear to be very active when pHi is close to, or above, normal pH (42, 69). It appears, rather, to be activated by a decrease in pHi and thus acts to help recovery of pHi from acid loads (lO7), but appears to play little role in recovery from alkali loads. In addition, unlike the Na+-Ca2+ exchange mechanism, it is unlikely that the Naf-H+ exchange mechanism will reverse to produce H” influx, since under normal conditions (or even when Na; is elevated), the exchanger is far from equilibrium, with the gradient for Na+ influx being far greater than that for H+ influx. This assumes, however, that the exchange is electroneutral and requires no additional energy input. However, the exchanger may have a requirement for ATP (128). An intracellular acidosis at constant pH, will stimulate H+ efflux and hence Na+ influx on the exchanger. In the presence of an active Na+-K+-ATPase, the Na+ will be removed rapidly from the cell, and only a small increase in Na; is observed. However, if this ATPase is inhibited by strophanthidin, a larger increase of Nai is observed (69). Presumably the inhibition of the Na+-K+-ATPase by acidosis (see Na+-K+-A TPase) means that the increase of Nai observed during a decrease of pHi is greater than it would be in the absence of such inhibition. Bountra and Vaughan-Jones (16) have recently reported that the decrease of pHi that occurs during an “ammonium rebound” results in an increase of intracellular Na+, which can be blocked by amiloride (a blocker of Na+-H+ exchange). This increase of Na+ was associated with an increase of developed force. This suggests that the following chain of events can occur when pHi is decreased (16) stimulation of Na+-H’ exchange f?H+li
t[Na”]i
via Na+-Ca2+ exchange -
T[Ca2+]i
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To account for the observed results, this scheme must that it overcomes the decrease in the responsiveness of the contractile proteins to Ca2+ (see Contrczctile Proteins) and 2) assume that, despite being inhibited (see Na’-Ca2+ exchange), Na+-Ca2+ exchange does continue to function during acidosis. Under most conditions intracellular acidosis produces a net decrease of force, but the above mechanism may contribute to the partial recovery of force seen during maintained acidosis (e.g., Fig. 1A). It might be expected that an extracellular acidosis would lead to changes in the opposite direction, that is, decreased H+ efflux and decreased Na+ influx on the exchanger so that Nai would decrease, thus enhancing Ca2+ removal on the Na+-Ca2+ exchanger. Bountra and Vaughan-Jones (16) have reported that an extracellular acidosis does produce small changes of Na; (a decrease in Purkinje fibers, an increase in papillary muscles). The small response may be due to inhibition of the Na’-H’ exchanger by extracellular acidosis (125). Thus changes of pH will alter the activity of the Na’H+ exchanger by altering the transsarcolemmal H+ gradient or by direct inhibition. This will alter Na+ flux, and hence the activity of Na+-Ca2+ exchanger. The sarcolemma also possesses a Cl--HCO; exchange (127). Although this is activated under alkaline conditions, it seems to have little activity at or below normal values of pH (127) and so is unlikely to play a part in the responses to acidosis considered in this review. Na+-K+-ATPase. It is well recognized that changes in the activity of the Na+-K+-ATPase can cause changes of Nai and, via the Na+-Ca2+ exchanger (see Na+-Ca2+ exchange), to changes of Cai. For example, inhibition of the ATPase with cardiotonic steroids can lead to an increase in Nai from its normal value of -5 mmol/l to -20 mmol/ 1 (33), thereby producing a decrease in the electrochemical gradient for Na+, a decrease in Ca2+ extrusion by the exchanger, and hence an increase in cytosolic Ca2+, thus having a positive inotropic effect. The effect of pH on this pump has not been widely studied in heart muscle. However, the effect of pH on the activity of the Na+-K+-dependent ATPase has been studied in isolated ATPase from embryonic chick hearts (12l), cultured chick heart cells (121), and rat hearts (8). All three preparations showed a maximum ATPase activity at about pH 7.5, with the activity decreasing at pH values either above or below pH 7.5. However, the magnitude of the effect of acidosis differed between the two preparations. At pH 6, activity fell to -40% of that at pH 7.5 in the preparations from chick hearts but fell to ~10% at pH 6 in the preparation from rat heart. However, whether this represents a species difference or a difference in the isolation procedure employed is unclear. The consequence of this inhibition is that Nai would be expected to rise in acidosis and may lead to an increase of Cai via Na+-Ca2+ exchange. Thus stimulation of the Na+-H+ exchanger or inhibition of the Na+-K+ pump by acidosis would lead to a similar effect, a rise of Cai. Ca2+-ATPase. We are not aware of any work on the pH sensitivity of the sarcolemmal Ca2+-ATPase in heart muscle, but by analogy with the SR Ca2+-ATPase (see 1) elevate Cai sufficiently
REVIEW
pH dependence
of Ca2+ uptake by the SR), it might
expected that acidosis would inhibit
be
this pump.
Sarcoplasmic Reticulum
The relative contributions of Ca2+ influx across the cell membrane, and Ca2+ release from the SR, to the size of the Ca2+ transient that initiates contraction remains unknown. It currently appears likely that the amount of Ca2+ contributed by each of these sources to activation of the myofilaments varies between species (lo), although the SR seems to be a major source of activator Ca2’ in most mammals. Effects of pHi on SR function will therefore influence the amount of Ca2+ released in response to an action potential, and hence affect force production. The effect of changes of pH on the role of the SR as a source of Ca2+ in heart muscle will depend on the pH dependence of both Ca2+ uptake by the SR and Ca2+ release from the SR. pH dependence of Ca2+ uptake by the SR. Ca2+ is pumped into the SR by a Ca2+-ATPase, which is thought to pump 2 mol Ca2+ for each mole of ATP split. The translocation of Ca2+ from the cell cytoplasm to the lumen of the SR can be characterized by four steps: 1) Ca2+ binding to the ATPase; 2) ATP binding to the ATPase; 3) splitting of ATP, phosphorylation of the pump protein, and the shedding of ADP; and 4) the release of Ca2+ and inorganic phosphate from the ATPase. Two types of studies have provided evidence that this pump is affected by changes of pH: experiments on isolated SR vesicles and experiments on either intact or skinned cardiac muscle. The first experiments on the effect of changes of pH on the SR were undertaken to test the suggestion that physiological Ca2+ release from the SR was due to a depolarization-induced change in pHi (96,97). Although the hypothesis that a change of pHi is the trigger for Ca2’ release from the SR now seems unlikely in cardiac muscle (48), the results of these experiments remain of interest. Nakamaru and Schwartz (96, 97) showed that increasing pH decreased Ca2+ binding to isolated SR. This change of Ca2+ binding required the presence of ATP but could not be ascribed to changes in the amount of ATP bound to the SR. These results are consistent with the idea that the Ca2+-ATPase of the SR membrane is pH dependent. More recently, Schwartz and his colleagues (88) have investigated the pH dependence of the Ca2+-ATPase of the SR in cardiac muscle. Using a quench-flow technique, they examined the effect of changes of pH on the rate of formation and disappearance of the phosphorylated intermediate in the Ca2+ translocation sequence described above. The rate of formation of this intermediate appeared to be independent of pH in the absence of Ca2+ but showed a marked pH dependence in the presence of Ca2+, the rate of formation becoming slower as pH was decreased from 7.6 to 6.0. This suggests that the Ca2+ binding step of the ATPase may be sensitive to changes of pH, possibly because of a decrease in the affinity of the Ca2+ binding site (92,130). Because the on-rate for Ca2+ binding is normally diffusion limited, Mandel et al. (88) suggested that acidosis slowed a conformational change that occurs in the en-
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zyme after the binding of Ca2+ and before MgATP is bound. However, the steps that involve the binding of ATP and the splitting of ATP appear to be pH independent. These conclusions agree with those of Scofano et al. (115) in skeletal muscle. The slower rate of disappearance of the phosphorylated intermediate, in acid conditions, observed by Mandel et al. (88) may be due to a decrease in the rate of Ca2+ release into the lumen of the SR. The results from these experiments appear to indicate, therefore, that the rate of Ca2+ uptake into the SR falls if pH is reduced. Fabiato and Fabiato (51) used a technique modified from that of Endo (45, 46) to examine the effect of pH on Ca2+ loading of the SR in mechanically skinned cardiac muscle cells. This technique was to expose the cell to a solution of known [Ca”‘] and pH (7.4-6.2)for a fixed period before releasing the Ca2+ stored in the SR by using caffeine at pH 7.0 and measuring the amplitude of the ensuing tension transient. The tension transient became smaller as the pH during the loading period was decreased, suggesting that Ca2+ loading of the SR is pH dependent, with a decrease of pH inhibiting the rate of Ca2+ uptake by the SR. However, the use of caffeine to assess the role of the SR may be inappropriate because it is not clear that the Ca2+ that can be released by caffeine is the same as that released in response to a more physiological stimulus. In addition, the use of peak force during the caffeine contracture (rather than the force-time integral) may be inaccurate if there are changes in the time course of release. In view of these problems, Fabiato (48) reexamined this problem using both force and the photoprotein aequorin to monitor Ca2+ in skinned cardiac cells. In this study, instead of assaying SR Ca2+ content using caffeine, he examined the effect of changes of pH on Ca2+ release induced by a rise in “trigger” Ca2+ (“calcium-induced calcium release,” see below). If pH was decreased 10 s (or more; Fig. 3) before such a release was induced, the subsequent release was smaller than control. However, if the pH change was induced
AND
function
J. C. KENTISH
Department of Physiology, University of Leeds, Leeds LS2 9JT; and Department University College London, London WClE 6BT, United Kingdom
of Physiology,
ORCHARD,C. H., AND J. C. KENTISH. Effects of changes of pH on the contractile function of cardiac muscle. Am. J. Physiol. 258 (Cell Physiol. 27): C967C981, 1990.-It has been known for over 100 years that acidosis decreases the contractility of
4 Hz decreases pHi by -0.06 pH units; Ref. 14), it is sufficient to modulate the contractile response of heart muscle to the rate change (14). 3) A profound decrease of intracellular pH is one of the most marked consecardiac muscle. However, the mechanisms underlying this de- quences of myocardial ischemia, with decreases of pHi of crease are complicated because acidosis affects every step in 0.5 units being commonly observed and values of pHi as the excitation-contraction coupling pathway, including both the delivery of Ca2’ to the myofilaments and the response of low as 6.2 being reported (56). 4) Clinical disturbances the myofilaments to Ca2+.Acidosis has diverse effects on Ca2+ of acid-base status, such as diabetic ketoacidosis or hycan cause substantial changes in pH, or delivery. Actions that may diminish Ca2+ delivery include 1) poventilation, pHi that could affect cardiac function. 5) Acidosis may, inhibition of the Ca2’ current, 2) reduction of Ca2+ release from the sarcoplasmic reticulum, and 3) shortening of the action to some extent, protect against hypoxia (12), reperfusion potential, when such shortening occurs. Conversely, Ca2” deliv- injury (78), and, at least in hepatocytes, ATP depletion ery may be increased by the prolongation of the action potential (61, 62). that is sometimes observed and by the rise of diastolic Ca2+ Although it is well established that extracellular acithat occurs during acidosis. This rise, which will increase the dosis causes a fall of developed force in cardiac muscle, uptake and subsequent release of Ca2’ by the sarcoplasmic the magnitude and rate of the force response depend on reticulum, may be due to 1) stimulation of Na+ entry via Na+H’ exchange, which will increase intracellular Ca2+ via Na+- how the acidosis is produced. Smith (117) first pointed Ca2+ exchange; 2) direct inhibition of Na+-Ca2+ exchange; 3) out that the negative inotropic effect of an extracellular acidosis was greater and more rapid if the decrease in mitochondrial release of Ca2+;and 4) displacement of Ca2+from pH, was produced by an elevated [CO,] (respiratory cytoplasmic buffer sites by H+. Acidosis inhibits myofibrillar responsiveness to Ca2+ by decreasing the sensitivity of the acidosis) than if it was produced by a reduction in [HCO:] (metabolic acidosis). From such evidence, he contractile proteins to Ca2+,probably by decreasing the binding of Ca2+ to troponin C, and by decreasing maximum force, and others (26, 105, 133) concluded that the negative possibly by a direct action on the cross bridges. Thus the final inotropism is due mainly to intracellular, rather than amount of force developed by heart muscle during acidosis is extracellular, acidosis; the faster action of CO2 is then the complex sum of these changes. explained because it can readily diffuse across the cell membrane and lower pHi (by dissociation to H+ and heart muscle; acidosis HCO; ), whereas extracellular HCO; and H+ are relatively impermeant. The faster time course of the fall in pHi during respiratory acidosis has been confirmed by IT HAS BEEN RECOGNIZED for over 100 years that a microelectrode measurements (43). These considerations decrease in extracellular pH reduces the contractility of suggest that the major actions of pH, are via changes of the heart (57). An understanding of how changes of pHi. For this reason, this review is concerned chiefly with extracellular pH (pH,) or intracellular pH (pHi) affect the mechanisms by which changes in pHi can affect force. cardiac function is important for our comprehension of However, it should be noted that a small part of the cardiac physiology and pathophysiology for five main negative inotropic effects of acidosis may result from reasons. 1) The cellular handling of Na+ and Ca2’, which direct effects of pH, on the properties of the cell memis central to cell function, is intimately linked to cellular brane (see Electrophysiology: Channels and Currents and H+ metabolism by Na+-H+ and Na+-Ca2+ exchanges, and Na+-H+ exchange). possibly by sharing common buffer sites within the cell. Within the last few years it has become possible to 2) Inotropic maneuvers such as changes of heart rate monitor discrete steps in the excitation-contraction coumay change pHi from its normal value (-7.0). Although pling pathway, and it has become clear that changes of this change is relatively small (increasing rate from 0 to pH may influence all of the steps in the pathway leading 0363-6143/90
$1.50
Copyright 0 1990 the American Physiological
Society
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to contraction, from the action potential to the generation of force by the myosin cross bridges within the myofibrils. Thus the net effect of changes of pH on contractile activity is complex. In addition, in studies using intact tissues it can be difficult to determine whether changes of pH are acting directly or whether the observed changes are secondary to other changes of cell function. In this article we will review recent studies that help us to understand the effect of changes of pH on contractile function of the heart. We will not be concerned with the regulation of intracellular pH (which is reviewed elsewhere, Refs. 52, 111).We will concentrate on the effects of acidosis, rather than alkalosis, for two reasons. First, it has clinical relevance (acidosis is a major component of myocardial ischemia), and second, more studies have examined the effects of acidosis than of alkalosis. Where alkalosis has been studied, the effects are in general found to be opposite to those of acidosis. This indicates that protons have an influence on the various steps in excitation-contraction (E-C) coupling even at normal values of pH. We will first describe the effects of changes of pH on measurements of intracellular [Ca”‘] and force, before discussing how variations of pH might alter cellular processes to bring about these changes. CONTRACTILE TO ACIDOSIS
RESPONSE
OF HEART
MUSCLE
Figure 1 shows a record of how force and cytosolic Ca2+ concentration (CaJ change in a ferret papillary muscle during and after exposure to a hypercapnic acidosis (see legend to Fig. 1 for details). Acidosis leads to a rapid decline of developed force followed by a slower partial recovery. The negative inotropic response to acidosis is well known (e.g., 26, 90, 91, 100, 105, 117, 122, l33), and a similar biphasic force response to acidosis has been observed in papillary muscles from a variety of other species (e.g., guinea pig, Ref. 53; cat, Refs. 90, 116) and with a variety of preparations, such as Langendorffperfused hearts (e.g., 4) and isolated ventricular myocytes (17). The muscle in Fig. 1 had been injected with the photoprotein aequorin, which emits light when it binds Ca2+. The aequorin light emission (a function of Cai) showed that systolic Cai increases during acidosis (5, 6, 102), reaching a maximum after -3-min exposure to the acid solution. Figure 1B shows that acidosis alters the time course of both the twitch and the Cai transient that accompanies it; the onset of twitch relaxation is earlier, twitch relaxation is faster, and the Cai transient declines more slowly during acidosis. Although small, these effects are statistically significant (5, 102). Measurements of pHi during sustained hypercapnic acidosis have shown either a monophasic decline in Purkinje fibers and rat ventricular muscle (44) or a rapid decline followed by a small partial recovery in guinea pig papillary muscles (108) and Langendorff-perfused ferret heart (4). This partial recovery of pHi may explain, in part, the partial recovery of force. In Fig. 1, pH, and pHi were decreased; when the primary change is of pHi alone, force still decreases, the size of the calcium transient still increases, and the changes of time course of the twitch
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A 20 nA
10 mN/mm*
IO nA
1 1 1
10 mN /mm*]
FIG. 1. Effect of acidosis on intracellular [Ca”‘] (monitored using photoprotein aequorin) and force in an isolated papillary muscle from a ferret heart. A: aequorin light (a function of Cai; top) and developed force (bottom). Period of acidosis is shown above records. B: aequorin light transients (top) and force (bottom) averaged during control conditions (a) and after 1.5min exposure to acidosis (b). c: aequorin light and developed force from a and b normalized (uncalibrated) to the same peak to show changes in time course. Force and light during acidosis are denoted by black circles. Temperature was 3O”C, and stimulation frequency was 0.33 Hz. Acidosis was induced by increasing [CO*] of the gas with which the muscle superfusate was equilibrated from 5% (pH, 7.35) to 30% (pH, 6.7). [HCOJ was constant throughout. Effect of acidosis on aequorin is to cause a small decrease in aequorin light emission at pCa 6 (6), which would tend to minimize changes shown above. During a respiratory acidosis induced using 15% CO2 (pH, 6.85), a decrease of pHi of 0.3 units has been reported in Langendorff-perfused ferret hearts (4). Thus pHi decreased by 60% of pH,. In experiment shown above, it might be expected, therefore, that pHi will have decreased by -0.6 x (7.35 - 6.7) = 0.39 units. However, it has recently been reported (126) that decreasing pH, by 0.43 pH units decreased pHi by only 0.13 pH units (i.e., 30% of pH,) in guinea pig papillary muscles. However, whether this is due to a species difference is unclear.
and calcium transient are still observed (102). The different subcellular systems that might be affected by changes of pH to produce these effects are shown in Fig. 2. The remainder of this article will be concerned with how acidosis may affect these cellular processes to produce changes of Cai and force such as those shown in Fig. 1. EFFECT OF ACIDOSIS ON CONTRACTION COUPLING
Electrophysiology:
THE EXCITATIONPATHWAY
Channels and Currents
In early electrophysiological studies it was found that the cardiac action potential and, by implication, the ionic currents that caused it were much less sensitive than action force to changes in pH,. Indeed, near-normal potentials could be elicited under acid conditions (pH, 5.5-6.2) when force was zero (87, 131). Nevertheless, there were some effects on action potential configuration which indicated that pH did influence the underlying ionic currents. The predominant effect was on the height of the plateau, which was decreased by acidosis, suggesting a reduction of the slow inward current (Isi) carried chiefly by Ca2+ (27, 87). A fall of Isi could also explain the observed abbreviation of the action potential seen in
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Na Ca
3Na
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Na
2K
Cell membrane
Ca
H
3Na
Ca
Sarcoplasmic reticulum T-tubule Mitochondria
@
FIG. 2. Possible sites of action of H+ in the myocardial cell. The text refers to the following sites: 1, electrophysiology: channels and currents; 2a, sarcolemmal bound Ca2’; 2b, Na+-Ca2+ exchange; 2c, Na+-H+ exchange; 2d, Na+-K+-ATPase; 2e, Ca2’-ATPase; 3a, Ca2+ uptake by the SR; 3b, Ca2’ release from the SR; 4, intracellular (cytoplasmic) Ca’+ during acidosis; 5, contractile proteins; 6, cell metabolism and mitochondrial function (mitochondrial Ca2+ extrusion is shown as a single pump, although it is the result of several processes; see text).
Myofibrils
many studies (e.g., 27, 81, 87, 112). However, in other studies, the action potential was prolonged (17, 53, 64), indicating that other currents were affected (probably outward K+ currents). The direct effect of acidosis on Isi would affect force production because of the role of Isi in E-C coupling (see SZow inward current), but the effect on other currents that affect action potential duration could also be important: it is known that an increase in action potential duration leads to an increase of developed force (95), presumably by increasing Cai. The mechanism of this increase is probably that the prolonged action potential I) increases the time for which Isi flows and 2) increases Ca2+ influx (or decreases efflux) on the Na+-Ca2’ exchange mechanism (see Na’-Ca2+ exchange). Thus changes of action potential duration will alter the response to acidosis by altering Cai. The alterations of membrane currents underlying the changes in the action potential have been elucidated using voltage-clamp techniques. Slow inward current (IS;). Particular attention has been paid to the effects of pH on 1si. This reflects the importance of 1si in E-C coupling: the entry of Ca2+ during 1si via L-type Ca2+ channels not only loads the myocytes with Ca2+, but it may also be the trigger for Ca2+ release from the sarcoplasmic reticulum (SR) (47). The first direct evidence for the pH sensitivity of Isi was obtained with voltage clamp of multicellular preparations. These studies showed that external acidosis produced a large decrease of 1si in frog atrium (27, 132) and mammalian ventricle (53, 80, 131) and slowed the kinetics of inactivation and recovery of Isi (80). Because force development depends on the magnitude of Isi (9), some of the reduction in force could be explained by the fall in Isi.
However, the simultaneous recording of force and Isi showed that force was generally decreased relatively more than Isi (80, 131, but cf. 53), indicating additional actions of pH were involved. More recent studies on membrane currents have utilized voltage clamp of perfused (dialyzed) isolated myocytes. In the whole cell voltage-clamp configuration, the solution in the recording patch pipette is in contact with the cell cytoplasm and exchanges rapidly with it (in minutes). Thus the solution in the patch pipette can, in theory, control the composition and pH of the cytosol, so the pHi can be varied independently of pH,. This may also avoid any secondary changes in Cai or Nai, which complicated the interpretation of experiments on multicellular preparations. An additional advantage is that problems of K+ accumulation within extracellular clefts are avoided (see Na+ current). Such experiments with isolated myocytes have confirmed and extended the results from multicellular preparations. In all studies, the magnitude of 1si fell in acid solutions. This inhibition was seen whether pHi was reduced (by intracellular perfusion with acid solutions) at constant pH, (66, 68, 81, 112) or if pH, was reduced at constant pHi (66, 82), demonstrating that there are extracellular and intracellular sites of action of H+. Acidity was also found to slow the kinetics of Isi in some studies (137) but not others (66) Itappears likely, then, that there are both extracellular and intracellular sites of action of H+, predominantly on the conductance properties but also on the gating properties of Isi. The effects of extracellular acidosis on Isi could arise by two main mechanisms: by block by H+ at a specific site in the Ca2+ channel or by neutralization of
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some of the fixed negative charge associated with the membrane surface, thereby changing the transmembrane electric field in the vicinity of the Ca2+ channel. The second possibility can account for the finding that there is a positive shift of the current-voltage (I- V) relation by lo-15 mV when pH, is reduced (66, 82, 137). The increased extracellular [H+] would neutralize some of the negative charge on the outer surface of the membrane (although the overall membrane potential would be unaffected); this would make the membrane act as if the cell were hyperpolarized, and the I-V curves would shift in a positive direction. This shift would decrease the peak of Isi, since the peak current would occur at a more positive potential, where the driving force for entry of Ca2+ was reduced. However, this shift is not sufficient by itself to account for all of the drop in peak Isi during extracellular acidosis (82)) so an additional mechanism seems necessary, such as direct block of the channel by H+ The mechanism of Isi inhibition by intracellular acidosis (at constant pH,) has been investigated in detail by Kaibara and Kameyama (68), using open-cell-attached patch clamp, in which individual channel events could be analyzed in dialyzed cells. Most of the 60% fall of I$i when pHi was reduced from 7.2 to 6.2 was due to a reduction in the open probability of the channel. This resulted mainly from an increased lifetime of a state in which the channel was unavailable for opening. Kaibara and Kameyama (68) also found that 20% of the reduction of 1siwas due to a decrease in the unitary current through the Ca2+ channel. Although earlier studies had reported a positive shift (112) or no shift (81) in the I-V relationship, Kaibara and Kameyama found a negative shift of -10 mV in the activation and inactivation curves, which is consistent with an alteration of Ca2+ channel gating as a result of H+ neutralizing negative charges on the cytosolic surface of the sarcolemma. In intact cells it is possible that some of the effects of acidosis on Isi result from a secondary rise in Cai that reduces Isi amplitude and accelerates inactivation (18, 38). However, this is probably only a minor factor, since the effects of pHi on 1si amplitude are still present if the cell is perfused with ethylene glycol-bis(P-aminoethyl ether) -lV,AT,N’ ,N’ -tetraacetic acid (EGTA) (66,68). The relative roles of changes in pH, and pHi remain to be established conclusively. A problem with attempting to change pHi to known values in dialyzed cells is that the cell’s active H+ extrusion mechanism (Na+-H+ exchange) tends to oppose any applied changes in pHi produced by the solution in the patch pipette (66). However, if Na+-H+ exchange is prevented, by removal of external Na+ or by amiloride, 1si is affected much more by a given change in pHi than by an equivalent change in pH,. For example, for 50% inhibition of Isi, pHi needed only to be reduced from 7.2 to 6.6, compared with a change in pH, of 7.4 to 5.0 (with pHi well buffered; Ref. 66). Of course, in many situations there will be changes in both pH, and pHi so that the effects will combine. In summary, a decrease of pHi and, to a lesser extent, of pH, reduces the magnitude of Isi. This will decrease Ca2+ loading of the cell and decrease the trigger for Ca2+
REVIEW
release from the SR (see pH dependence of Ca2+ release by the SR). Na+ current.
In general, the actions of pH changes on the fast inward Na+ current (1& are small and inconsistent. The maximum rate of depolarization of the action potential, which reflects the magnitude of &, has been reported to be little affected by acidosis (53, 67), or depressed by acidosis (27, 124, 129). The latter action is likely to be because of 1) a shift of lNa threshold in the depolarizing direction, which has been ascribed to protonation of fixed charge on the membrane surface (22), or 2) in Purkinje fibers at least, a small decrease in resting membrane potential due to accumulation of K+ in extracellular clefts (22). K+ currents. The outward currents carried chiefly by K+ also seem to be affected by changes of pH, although results are variable. In some studies on isolated myocytes, the time-dependent outward current (delayed rectifier, 1k) was little changed (81), although there was an increase in a time-independent outward current at positive potentials (112, but cf. 137). The latter was not due to a Ca2+activated K+ conductance (112). This increase in outward current would, like the decrease in 1si, tend to decrease action potential duration at low pH. In addition, acid solutions increased an outward current recorded at negative potentials (probably the inward rectifier, &I)3 which could explain the small hyperpolarization of the resting potential seen in some studies (112). In contrast to the above, in other studies it was found that the action potential was prolonged during acidosis; this has been seen in both multicellular preparations (53, 64) and in isolated ventricular myocytes (e.g., Ref. 17). This prolongation may be due to a decrease in 1k (27) or in lkl (63), which could also explain the small depolarization of the resting membrane potential sometimes observed during acidosis (17). Obviously more work is needed before we can explain these divergent results on action potential duration or on K+ currents. One possibility is that the direction of the response varies with the magnitude of the pH change, since Sato et al. (112) found a moderate fall of pHi increased outward current, whereas a large fall decreased it. Arrhythmogenesis. Under certain conditions, intracellular acidosis can lead to abnormal electrical rhythms. Coraboeuf et al. (30) found that a fall of pHi in Purkinje fibers provoked repolarization abnormalities and transient depolarizations, which induced fiber reexcitation. In what may be another manifestation of the same phenomenon, the injection of acid into myocytes has been shown to directly induce the transient inward current (&J (81). Iti can also be induced by excessive Ca2+ loading of cells (24, 70) and is thought to be due to spontaneous release of Ca2+ from the SR, causing activation of the electrogenic Na+-Ca2+ exchange (which carries net current into the cell). The acid-induced genesis of Iti is therefore probably due to a rise in Cai caused by the low pHi (see Diastolic [Ca2+]). Because 1ti is an inward (depolarizing) current that can occur during diastole, it can lead to the generation of arrhythmias (86). Thus acidosis may increase the likelihood of arrhythmogenesis in cardiac cells. In addition, a spontaneous release of Ca2+
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INVITED
during diastole will reduce the Ca2+ transient, and therefore, the force, in the subsequent contraction of the cell (3); this could contribute to the negative inotropic effect of acidosis in cardiac muscle. It has also been found that a decrease of pHi reduces the coupling between cardiac cells by decreasing the conductance of the gap junctions (32, 109). For example, a fall of pHi from 7.2 to 6.8 in Purkinje fibers increased the junctional resistance by ~30% (109). This, in addition to any reduction of lNa (see above), would tend to slow propagation of the action potential, as discussed by Gettes (58), and could reduce the synchrony of contraction within the ventricle. A severe reduction of conduction velocity in part of the ventricle would also increase the likelihood of arrhythmogenesis by reentrant excitation and cause a decrease in the efficiency of pumping. Ion Regulation by the Sarcolemma: Pumps, and Exchangers
Cu2+ Binding
Sites,
The cell membrane may alter contractile function during acidosis not only by altering events during the action potential (action potential duration and the magnitude of Isi; see Electrophysiology: Channels and Currents) but also by altering ion flux through pumps and exchangers in the cell membrane that are active in both systole and diastole, hence altering Cai (Fig. 2). Sarcolemmal bound Ca2+. It has been suggested (84) that some of the Ca2+ that enters the cell cytoplasm to initiate contraction comes from a pool that is bound to sites on the sarcolemmal membrane, although this view is still controversial. In “gas-dissected” membranes and sarcolemmal vesicles from the canine heart (83), Ca2+ binding to sarcolemmal sites was decreased as pH was lowered from 8.5 to 5.5, with an apparent pK for inhibition of 6.6-7.15. If Ca2” is bound to the external side of the cell membrane, then flux of Ca2’ from these sites into the cell will be via Isi and Na+-Ca2+ exchange, and a decrease in Ca2+ bi .nding to these Sl tes could contribute to decrea .sed influx into the cell v ia these processes (see Slow irward current and Na+-Ca2+ exchange). If Ca2+ is bound to the inside of the membrane, its displacement by H+ may contribute to the increase in resting Ca2+ observed during acidosis (see Diastolic [Ca2+J). Na+-Ca2+ exchange. In the resting myocyte Na+-Ca2” exchange is an important route for Ca2+ extrusion. This exchanger uses the electrochemical gradient for Na+ entry into the cell to extrude Ca2+ from the cell, exchanging three Na+ for each Ca2+ (for review, see Ref. 40). However, it has been suggested that during the action potential the exchanger may reverse and cause Ca2+ entry into the cell. The exchanger may, therefore, be a route for both Ca2+ influx and efflux from the cell, the direction depending on the transmembrane electrochemical gradients for Na+ and Ca2+. Changes in the activity of this exchange mechanism may, therefore, have profound effects on cell function. The effect of pH on the Na+Ca2+ exchange mechanism has been studied in both canine sarcolemmal vesicles (106) and isolated guinea pig myocytes (37). In isolated vesicles the initial rate of Nai-dependent Ca2’ uptake into the vesicles showed a sigmoidal dependence on pH
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over the range pH 5-9, with acidosis inhibiting uptake. N&-dependent Ca2+ efflux also showed pH dependence, with alkalosis (pH 9) stimulating efflux and acidosis (pH 6) inhibiting efflux, in agreement with work on squid axons (35). Thus the exchanger appears to be inhibited by acidosis, regardless of the direction in which it is working. In single cell studies (37) this inhibition appears to occur whether the pH change is applied at the inner or outer cell surface. The cause of this inhibition is less clear, but it appears to be a direct action on the exchanger and may be, in part, due to competition between Ca2+ and H+. Thus, if Na+-Ca2’ exchange extrudes Ca2+ from the cell during diastole (there must be a net efflux to balance Ca2+ influx during the action potential), inhibition of the exchanger will lead to less Ca2+ extrusion, and hence an increase of Cai. Conversely, if the exchanger normally reverses during the action potential, and brings Ca2+ into the cell, inhibition of the exchanger will lead to less Ca2’ influx via the exchanger during the action potential. Na+-H+ exchange. This exchange mechanism is thought to be electroneutral, exchanging one Na+ for one H+, and using the concentration gradient for Na+ entry to extrude H+ from the cell. Thus changes in the transsarcolemmal Na+ or H+ gradient will result in altered fluxes through the exchanger. However, unlike many of the systems discussed above, the Na+-Hf exchange mechanism does not appear to be very active when pHi is close to, or above, normal pH (42, 69). It appears, rather, to be activated by a decrease in pHi and thus acts to help recovery of pHi from acid loads (lO7), but appears to play little role in recovery from alkali loads. In addition, unlike the Na+-Ca2+ exchange mechanism, it is unlikely that the Naf-H+ exchange mechanism will reverse to produce H” influx, since under normal conditions (or even when Na; is elevated), the exchanger is far from equilibrium, with the gradient for Na+ influx being far greater than that for H+ influx. This assumes, however, that the exchange is electroneutral and requires no additional energy input. However, the exchanger may have a requirement for ATP (128). An intracellular acidosis at constant pH, will stimulate H+ efflux and hence Na+ influx on the exchanger. In the presence of an active Na+-K+-ATPase, the Na+ will be removed rapidly from the cell, and only a small increase in Na; is observed. However, if this ATPase is inhibited by strophanthidin, a larger increase of Nai is observed (69). Presumably the inhibition of the Na+-K+-ATPase by acidosis (see Na+-K+-A TPase) means that the increase of Nai observed during a decrease of pHi is greater than it would be in the absence of such inhibition. Bountra and Vaughan-Jones (16) have recently reported that the decrease of pHi that occurs during an “ammonium rebound” results in an increase of intracellular Na+, which can be blocked by amiloride (a blocker of Na+-H+ exchange). This increase of Na+ was associated with an increase of developed force. This suggests that the following chain of events can occur when pHi is decreased (16) stimulation of Na+-H’ exchange f?H+li
t[Na”]i
via Na+-Ca2+ exchange -
T[Ca2+]i
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To account for the observed results, this scheme must that it overcomes the decrease in the responsiveness of the contractile proteins to Ca2+ (see Contrczctile Proteins) and 2) assume that, despite being inhibited (see Na’-Ca2+ exchange), Na+-Ca2+ exchange does continue to function during acidosis. Under most conditions intracellular acidosis produces a net decrease of force, but the above mechanism may contribute to the partial recovery of force seen during maintained acidosis (e.g., Fig. 1A). It might be expected that an extracellular acidosis would lead to changes in the opposite direction, that is, decreased H+ efflux and decreased Na+ influx on the exchanger so that Nai would decrease, thus enhancing Ca2+ removal on the Na+-Ca2+ exchanger. Bountra and Vaughan-Jones (16) have reported that an extracellular acidosis does produce small changes of Na; (a decrease in Purkinje fibers, an increase in papillary muscles). The small response may be due to inhibition of the Na’-H’ exchanger by extracellular acidosis (125). Thus changes of pH will alter the activity of the Na’H+ exchanger by altering the transsarcolemmal H+ gradient or by direct inhibition. This will alter Na+ flux, and hence the activity of Na+-Ca2+ exchanger. The sarcolemma also possesses a Cl--HCO; exchange (127). Although this is activated under alkaline conditions, it seems to have little activity at or below normal values of pH (127) and so is unlikely to play a part in the responses to acidosis considered in this review. Na+-K+-ATPase. It is well recognized that changes in the activity of the Na+-K+-ATPase can cause changes of Nai and, via the Na+-Ca2+ exchanger (see Na+-Ca2+ exchange), to changes of Cai. For example, inhibition of the ATPase with cardiotonic steroids can lead to an increase in Nai from its normal value of -5 mmol/l to -20 mmol/ 1 (33), thereby producing a decrease in the electrochemical gradient for Na+, a decrease in Ca2+ extrusion by the exchanger, and hence an increase in cytosolic Ca2+, thus having a positive inotropic effect. The effect of pH on this pump has not been widely studied in heart muscle. However, the effect of pH on the activity of the Na+-K+-dependent ATPase has been studied in isolated ATPase from embryonic chick hearts (12l), cultured chick heart cells (121), and rat hearts (8). All three preparations showed a maximum ATPase activity at about pH 7.5, with the activity decreasing at pH values either above or below pH 7.5. However, the magnitude of the effect of acidosis differed between the two preparations. At pH 6, activity fell to -40% of that at pH 7.5 in the preparations from chick hearts but fell to ~10% at pH 6 in the preparation from rat heart. However, whether this represents a species difference or a difference in the isolation procedure employed is unclear. The consequence of this inhibition is that Nai would be expected to rise in acidosis and may lead to an increase of Cai via Na+-Ca2+ exchange. Thus stimulation of the Na+-H+ exchanger or inhibition of the Na+-K+ pump by acidosis would lead to a similar effect, a rise of Cai. Ca2+-ATPase. We are not aware of any work on the pH sensitivity of the sarcolemmal Ca2+-ATPase in heart muscle, but by analogy with the SR Ca2+-ATPase (see 1) elevate Cai sufficiently
REVIEW
pH dependence
of Ca2+ uptake by the SR), it might
expected that acidosis would inhibit
be
this pump.
Sarcoplasmic Reticulum
The relative contributions of Ca2+ influx across the cell membrane, and Ca2+ release from the SR, to the size of the Ca2+ transient that initiates contraction remains unknown. It currently appears likely that the amount of Ca2+ contributed by each of these sources to activation of the myofilaments varies between species (lo), although the SR seems to be a major source of activator Ca2’ in most mammals. Effects of pHi on SR function will therefore influence the amount of Ca2+ released in response to an action potential, and hence affect force production. The effect of changes of pH on the role of the SR as a source of Ca2+ in heart muscle will depend on the pH dependence of both Ca2+ uptake by the SR and Ca2+ release from the SR. pH dependence of Ca2+ uptake by the SR. Ca2+ is pumped into the SR by a Ca2+-ATPase, which is thought to pump 2 mol Ca2+ for each mole of ATP split. The translocation of Ca2+ from the cell cytoplasm to the lumen of the SR can be characterized by four steps: 1) Ca2+ binding to the ATPase; 2) ATP binding to the ATPase; 3) splitting of ATP, phosphorylation of the pump protein, and the shedding of ADP; and 4) the release of Ca2+ and inorganic phosphate from the ATPase. Two types of studies have provided evidence that this pump is affected by changes of pH: experiments on isolated SR vesicles and experiments on either intact or skinned cardiac muscle. The first experiments on the effect of changes of pH on the SR were undertaken to test the suggestion that physiological Ca2+ release from the SR was due to a depolarization-induced change in pHi (96,97). Although the hypothesis that a change of pHi is the trigger for Ca2’ release from the SR now seems unlikely in cardiac muscle (48), the results of these experiments remain of interest. Nakamaru and Schwartz (96, 97) showed that increasing pH decreased Ca2+ binding to isolated SR. This change of Ca2+ binding required the presence of ATP but could not be ascribed to changes in the amount of ATP bound to the SR. These results are consistent with the idea that the Ca2+-ATPase of the SR membrane is pH dependent. More recently, Schwartz and his colleagues (88) have investigated the pH dependence of the Ca2+-ATPase of the SR in cardiac muscle. Using a quench-flow technique, they examined the effect of changes of pH on the rate of formation and disappearance of the phosphorylated intermediate in the Ca2+ translocation sequence described above. The rate of formation of this intermediate appeared to be independent of pH in the absence of Ca2+ but showed a marked pH dependence in the presence of Ca2+, the rate of formation becoming slower as pH was decreased from 7.6 to 6.0. This suggests that the Ca2+ binding step of the ATPase may be sensitive to changes of pH, possibly because of a decrease in the affinity of the Ca2+ binding site (92,130). Because the on-rate for Ca2+ binding is normally diffusion limited, Mandel et al. (88) suggested that acidosis slowed a conformational change that occurs in the en-
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INVITED
zyme after the binding of Ca2+ and before MgATP is bound. However, the steps that involve the binding of ATP and the splitting of ATP appear to be pH independent. These conclusions agree with those of Scofano et al. (115) in skeletal muscle. The slower rate of disappearance of the phosphorylated intermediate, in acid conditions, observed by Mandel et al. (88) may be due to a decrease in the rate of Ca2+ release into the lumen of the SR. The results from these experiments appear to indicate, therefore, that the rate of Ca2+ uptake into the SR falls if pH is reduced. Fabiato and Fabiato (51) used a technique modified from that of Endo (45, 46) to examine the effect of pH on Ca2+ loading of the SR in mechanically skinned cardiac muscle cells. This technique was to expose the cell to a solution of known [Ca”‘] and pH (7.4-6.2)for a fixed period before releasing the Ca2+ stored in the SR by using caffeine at pH 7.0 and measuring the amplitude of the ensuing tension transient. The tension transient became smaller as the pH during the loading period was decreased, suggesting that Ca2+ loading of the SR is pH dependent, with a decrease of pH inhibiting the rate of Ca2+ uptake by the SR. However, the use of caffeine to assess the role of the SR may be inappropriate because it is not clear that the Ca2+ that can be released by caffeine is the same as that released in response to a more physiological stimulus. In addition, the use of peak force during the caffeine contracture (rather than the force-time integral) may be inaccurate if there are changes in the time course of release. In view of these problems, Fabiato (48) reexamined this problem using both force and the photoprotein aequorin to monitor Ca2+ in skinned cardiac cells. In this study, instead of assaying SR Ca2+ content using caffeine, he examined the effect of changes of pH on Ca2+ release induced by a rise in “trigger” Ca2+ (“calcium-induced calcium release,” see below). If pH was decreased 10 s (or more; Fig. 3) before such a release was induced, the subsequent release was smaller than control. However, if the pH change was induced
