J
Mol Cell Cardiol
23, 1077-1086 (1991)
EDITORIAL
Protons
in Ischemia:
Where
Do They
S. C. Dennis’,
Come
W. Gevers*
From;
Where
Do They
Go To?
and L. H. Opie’
1Bioenergetics of Exercise, 2Cell Biology ofAtherosclerosis and 31schemic Heart Disease Research Units of the Medical Research Counciland the University of Cape Town, Cape Town, South Africa (Received 22 June 1990, accepted in revisedform 20 March 1991)
It is commonly thought that the accumulation of lactic acid from anaerobic glycolysis is the cause of an undesirable acidosis during myocardial ischemia. In 1977, Gevers [13] suggested that the major source of protons during anaerobic metabolism was not so much the formation of lactic acid as the turnover of glycolytically produced ATP. The purpose of this Editorial is to re-evaluate this proposal and expand it, taking into account how ischemia and reperfusion might influence proton transport across cell membranes. To understand the formation of protons in the under-perfused myocardium requires emphasis that a total cessation of coronary blood flow is not a prerequisite for myocardial ischemia. A complete absence of blood flow rarely occurs clinically and, even under experimental conditions with multiple coronary artery ligations, there is still some collateral circulation to the ischemic zone [31]. Myocardial ischemia more commonly arises from a partial restriction of coronary blood flow and, this has two immediate consequences. One is that the supply of O2 for energy production is decreased. The other is that the removal of metabolites such as lactate, CO, and protons is impaired. Depending on the degree of 0, demand and on the severity of the reduction in coronary blood flow, intracellular pH declines from the normal values of around 7.2 [39] to values as Please address all correspondence Observatory 7925, South Africa. 0022-2828/91/091077
+ 06 $03.00/O
to: L. H.
Opie,
Heart
low as 6.3 [31] or even 5.7 [16]. From the reported physiochemical and metabolic buffering capacity of mammalian cardiac muscle [39], this fall of around 1 pH unit corresponds to an acid-load of 51-77 mmol H + per liter of tissue H,O.
Protons: Where Do They Come From? In considering the production of protons in the ischemic myocardium, it is important to appreciate that there is a relatively high (0.4-S. 5 mM) free cytoplasmic Mg* + concentration in cardiac tissue [3, 151. This, together with the fact that intracellular [Mg2 + ] rises when ATP levels fall 1421, strongly suggests that all of the ATP4- and ADP3 - is bound to Mg2 + and is therefore fully ionized at cytosolic pH [13, II]. Another point to note is that the pKa’ of inorganic phosphate is 6.9 and the normal intracellular pH is around 7.2 [39]. From these values, it can be calculated that, before acidosis, 72 % of the cytosolic inorganic phosphate is in the HP04’ - form [II, 46).
Not from lactic acid When intracellular [Mg2 + ] and pH are taken into account and the likely charges on ATP, ADP, inorganic phosphate and lactate are Research
Unit,
University
of Cape
Town,
0 1991 Academic
Medical
Press
School,
Limited
1078
S. C. Dennis
summed, it is found that the glycolytic pathway does not produce a large net gain of protons [13]. Anaerobic glycolysis has as its
(1)
end-product mainly lactic acid molecules
lactate anions rather than (Equation 1):
- + 2 Pi2- + 2 lactate - + 2 MgATP2
glucose + 2 MgADP
The metabolism of a hexose unit of glycogen to 2lactate molecules and 3 MgATP molecules also does not produce a net gain of protons. In
Nat from continued gbco&tic formation of NADH+H’ Contrary to assumptions often made, a continued conversion of glucose or glycogen to pyruvate- and NADH + H + is also unlikely to contribute to ischemic acidosis. Mitochondrial pyruvate metabolism and cytoplasmic NADH + H+ re-oxidation are linked. For the tricarboxylate cycle to proceed, the 2-0x0glutarate ( = ketoglutarate) leaving the mitochondria in exchange for malate has to be regenerated by the malate/aspartate shuttle.
-
fact, when 3 MgATPs are generated by the glycolytic pathway, a proton is consumed (Equation 2):
+ 3 P:- 3 2 lactate-
(2)
(3)
et al.
+ 3 MgATPs-
NADH and protons formed from glycolysis are therefore either taken into the mitochondria together with pyruvate or are used in the reduction of pyruvate to lactate. The only possible contribution to acidosis from continued glycolytic NADH + H + production is when glutamate is oxidized [43]. In the conversion of glutamate to succinate, pyruvate is diverted to alanine and, under circumstances, some glycolytic these NADH+H+ formation could persist (Equation 3):
Glycolysis
P;- + MgADPMgATP* F pyruvate
+ NAD’
- + NADH
+
q
glutamate2-oxoglutarate*-7
F alanine Cytoplasm Mitochondrion NADH+
+ MgGDPNADH
+ P:- -
+ MgGTP2
- +--’ ” succinate2 -
EditOrid Increases however, protons. NADH NADH cytosolic reaches
in NADH and H+ concentrations, provide only a transient source of Without a re-oxidation of + H+ to NAD+ , further glycolytic + H+ formation is prevented and the [NADH + H + ]/[NAD+ ] ratio simply a new steady-state.
From decreased mitochondn’al ATP Instead,
(4)
the metabolic T MgATP2
n-synthesis?
generation
‘M”Tg’-‘i”i+
-
phosphorylation
In very early ischemia, the rapid transfer of phosphate from creatine phosphate to ADP MgATP2 T
)Mgr-
Mg~glycolysis
arising from (Equation6):
its hydrolysis
+Mg-r-
Thus, the metabolic generation of protons during ischemia is primarily a result of the increased reliance on glycolysis for ATP resynthesis. From carbon dioxide retention? In the early stages of regional myocardial ischemia, another likely cause of acidification is the accumulation of CO2 from at least some mitochondrial respiration residual (Equation 7): Mitochondrial
respiration
-
generated
by ATP
‘i++P:-
hydrolysis
However, when ATP is formed by glycolysis rather than by mitochondrial metabolism or creatine phosphate breakdown, the protons
(7)
also takes up the protons breakdown (Equation 5):
-
creatine-P2-
(6)
during ischemia is more a consequence of the decrease in mitochondrial ATP production in relation to glycolytic ATP formation. During oxidative phosphorylation, the influx of protons into the mitochondria exceeds the outward “pumping” of protons by the electron transport chain [44] and most of the protons produced by ATP hydrolysis are probably utilized in its re-synthesis (Equation 4):
of protons
oxidative
(5)
1079
are not consumed
+r-
+m
Khuri et al. [18] showed that the increase in tissue pC02 from around 50 to 190mmHg during the first 30min of coronary artery occlusion is not entirely due to the 46 to 200 nmol/l rise in [H + ] displacing the HCOs-/CO2 equilibrium towards CO2 formation. As ischemia continued, there was a gradual decline in tissue pCOz and [H + 1, which is not what one would expect if the previously high pC0, had simply been a result of metabolic acidosis.
CO2 + HZ0 -HCO,-
+IH’I
1080
S. C. Dennis From glycogen turnover?
Some protons
might
also be generated
by an
et al. increased ischemia
turnover of (Equation 8):
(8)
glycogen
during
MgATP2
1
MgUTP*
I 1 glucosd- 1-P2I
Pi’- + glycogen 1‘
qH+
-
+ MgUDP-AUDP-glucoseZ*‘Lpi*
While elevated Pi, AMP and CAMP concentrations activate glycogen breakdown in ischemic tissue, build-up of “n-metabolized glucose- 1 -phosphate favors glycogen reformation [27, 311. Net proton production from a glycogen : glucose- 1 -phosphate cycle, however, is largely dependent on the mechanism of ATP re-synthesis. Where ATP is mainly regenerated by residual oxidative phosphoryl-
(9)
ation (Equation4), regional ischemia consumed.
as may be the case in [31], most of the protons are
From triglyceride-fatty acid recycling? Catecholamine-stimulated triglyceride lipolysis and fatty acid activation may also produce protons during ischemia (Equation 9):
3 MgATP2
- + 3 CoA* -
I
triglyceride
) 3 FFAI
+ glycerol +
3 acyl CoA* +--i 3Mg2’
(‘Assumes that inorganic pyrophosphatase and long-chain acyl CoA synthetase activities are similar.) According to the above equation, each triglyceride molecule converted to fatty acyl CoA could yield 6protons as previously argued [13]. Similarly, the release and activation of 2long-chain fatty acids from the degradation of membrane phosphoglyceride, late in ischemia, could form 5protons. How-
+ 3AMP2-
+ 6Pi*-’
+
l 3H’
3H+
ever, the following additional factors must also be considered. First, long-chain fatty acids may not be fully ionized in the cytosol, in which case, less protons would be generated than is supposed. Second, NADH reoxidation via glucose to glycerol-3-phosphate conversion promotes triglyceride : fatty acid cycling [30] and, in this cycle, the protons generated in the formation of fatty acyl CoA are taken up in the re-synthesis of ADP from AMP (Equation 10):
1081
Editorial (10)
3CoA’-
3 MgATP2
+ P;2-
Under these circumstances, produced by the provision
only one proton is of glycerol-3-phos-
- + 3 COA’ -
phate to the cycle from the glycolytic (Equation 11):
pathway
triglyceride turnover
0.5 glucose + MgATP2
- ->
Hence, far fewer protons are produced by this cycle than originally thought [13]. Only in severe ischemia is there a possibility of some proton production from triglyceride-fatty acid cycling. In moderate ischemia, where more than one out of six ADP’s is oxidatively rephosphorylated [31], the operation of a triglyceride:fatty acid cycle will consume, rather than produce protons.
From intracellular
calcium accumulation?
During early myocardial ischemia, the concentration of cytosolic calcium rises, according to a number of investigators who used different techniques [I, 24, 411. The mechanism of the intracellular Ca* + increase is unknown but it could be due to the reduction in the diastolic membrane potential in ischemic tissue. A less negative resting membrane potential would decrease the transsarco-
T
glycerol-P*-
+ MgADP-
+ ) H+ 1
-
lemmal efflux of Ca* + via the 3Na.+/Ca*+ exchange that is driven by the net inward movement of positive charge [I, 341. Early falls in cytosolic pH may therefore also arise from intracellular Ca*+ accumulation in ischemic tissue. Several groups have shown that Ca*+ and H+ ions compete for common intracellular binding sites [II, 26, 451 and, depending on the relative Kd values of those sites for Ca2 + and H + ion binding, between 0 and 2 protons could be displaced by each Ca2 + ion. With continued ischemia, however, further rises in cytosolic Ca2+ concentration are probably prevented by a decrease in the influx of Ca*+ . Protons accumulating in the interstitial space compete with Ca* + for access to the voltage-gated Ca2 + channels [22] and the 3Na+/Ca2+ exchange [33]. A low pH also depresses fast responses [17, 211 and promotes electrical uncoupling [ 7, 291.
1082
S. C. Dennis From net adenine nucleotide degradation?
In addition to slowing conduction and reducing the influx of Ca2+ for the “triggering” of contraction, proton accumulation also decreases cross-bridge cycling. Acidification of the cytoplasm reduces the activation of actin by Ca2+ [IZ] and slows the myosin ATPase [5]. In terms of cell survival, a rapid fall in cytosolic pH may therefore be of some benefit to ischemic tissue, by helping to decrease contractility and, thereby, retarding the breakdown of ATP [371. Decreased consumption of ATP by crossbridge cycling probably also slows the rate of further acidification in the ischemic myo-
et d.
cardium. With less ATP turnover, proton formation from glycolytic ATP re-synthesis will be decreased and CO1 accumulation from residual mitochondrial respiration will be reduced. Continuing acidification is also likely to be eventually limited by the inhibition of glycolysis at low pH [28] and the loss of mitochondrial function in late ischemia [18]. At this point, a further fall in pH could also be caused by the release of protons and phosphate from the net breakdown of ATP [2 1. Despite the conservative effects of contractile failure, there is a gradual accumulation and leakage of adenosine, inosine and hypoxanthine from adenine nucleotide degradation in ischemic cells (Equation 12):
(12) (Mg2 +) + MgATP2 1
-
inosine -+ NH*+ .L hypoxanthine +ribose
Although the formation of 3 H+ + 3 HP042from net ATP breakdown is slow in comparison to the production of protons from glycolytic ATP turnover and CO* accumulation, it is likely to contribute to the acidification of the myocardium in the later stages of ischemia. Once the intracellular pH is well below the 6.9pKa’ value for inorganic phosphate, even modest increases in the H2P04-/HP042ratio will raise the cytoplasmic proton concentration.
Protons:
Where
Do They
Go To?
So far, we have described the metabolic responses of the myocardium to ischemia and have argued that protons are mainly produced from glycolytic ATP turnover, CO, retention and, later, net ATP degradation (Table 1). In
the following sections, attention is focused on the accumulation of protons in the ischemic myocardium .
To the intracellular
bu&s
During the early restriction of coronary blood flow, protons are first buffered by intracellular protein histidine residues and by inorganic phosphate. Inorganic phosphate is the major intracellular buffer and is especially important in ischemic tissue. As creatine phosphate is broken down to help maintain ATP levels (Equation 5), inorganic phosphate accumulation [31] increases intracellular buffering capacity. Depending on the pH, rapid inorganic phosphate release may even produce a transient alkalosis (Table 1).
Elditorial TABLE
1.
The potential ischemia
contribution
of different
1083
metabolic
pathways
to proton
Protons starting
Process Early
ATP
HPO,*
breakdown
and re-synthesis
- accumulation
Residual
ATP
Carbon
dioxide
from
breakdown
ATP
Possible
glycolytic
Release
and activation
Possible
triglyceride:
Possible H,PO,-
net creatine
from
breakdown
phosphate
residual
of
- 1 to oa
phosphorylation
0 +1
phosphorylation
+2
via glycolysis
acids from
arising
triglyceride
from
gluamate
oxidation
+1 + 3 to 6b
lipolysis
acid cycling
-5tol’
glycogen:
glucose-
1 -P cycling
+ 1 too’
formation
from
acid
net ATP
+3toO”
degradation
indicate
that the production
ionization
in the cytosol
or consumption or ‘the
predominant
To the interstitial S/MC~ Some
0
breakdown
oxidative
+ H + accumulation of fatty
phosphate
via oxidative
and re-synthesis
NADH
creatine
the
protons
buffered
inorganic
(13)
mechanism
(Equation
Cytoplasm clH’
of protons
depends on either “the pH, of ATP re-synthesis.
phosphate may the reversible
by
+ HP04*-+
during
per mol of substrate
fatty
Superscripts fatty
from
and re-synthesis
retention
Increased
chain
formation
H2P04-
then
bthe degree of long
leave the ischemic Na+ - H2P04-
cells via symport
13):
Interstitium ) > > Na’
liEI lactate -
Phosphate ischemia
efflux is transiently [32] and by acidosis
t-w.
Unbuffered
initially
changer
leave
and
intracellular
and
accelerated by mild hypoxia
protons
via the sarcolemmal
H + -lactate -
can also /H + exco-transporter Na+
) 1
(Equation 13). Cytostolic proton accumulation allosterically activates the exchange of intracellular protons for extracellular Na+ [23, 361 and displaces the [H + ][lactate -1 in/out equilibrium towards proton + lactate release [8, 91. As ischemia progresses, however, further
1084
S. C. Dennis
proton efflux is likely to be opposed by interstitial proton accumulation. Because the buffering capacity of the extracellular space is less than that of the intracellular space, increasing acidosis severely decreases the normal trans-sarcolemmal proton gradient [6, 471. Loss of the cell membrane proton gradient slows H + and lactate- ion release by >60% [ 401 and reduces Na + /H + exchange by >80 % [351. To the blood Ultimate rates of proton release from ischemic tissue are therefore probably determined by the washout of protons from the interstitial space. In regional ischemia, for instance, the
(14)
et al. greatest proton accumulation is seen in tissue receiving the least collateral circulation [ 181. A limited clearance of protons from ischemic tissue is also suggested from studies in which coronary blood flow is suddenly restored. On reperfusion, the prompt washout of extracellular protons is thought to lead to a rapid efflux of accumulated intracellular protons with an eventual Ca*+ overload and evidence of tissue injury such as arrhythmias [IO]. An accelerated exchange of intracellular protons for extracellular Na + would result in a secondary influx of Ca2+ via the 3Na2/Ca2+ exchanger [19, 201. Calcium-proton interactions could then release more protons into the cytoplasm and promote further Ca2 + entry via the Na + -H + -Ca2 + exchangers (Equation 14):
Cytoplasm
Interstitium
Na+
interaction Ca*+
The operation of a Na + -H + -Ca2 + exchange might also explain why Ca2+ influx on reperfusion is (i)not fully blocked by Ca2+ channel antagonists and (ii)not associated with net Na + efflux [38]. A Na + -H + -Ca2 + exchange is also consistent with the observation that arrhythmias are decreased by more gradual reperfusion [48]. A slower washout of accumulated extracellular protons would limit the rate of Na + /H + exchange and allow more of the incoming Na’ to leave via the Na + /K + -ATPase rather than by the 3Na + /Ca2 + exchanger. In conclusion, ischemic acidosis is
predominantly due to a retention of protons from glycolytic ATP turnover, CO, accumulation and, eventually, net ATP breakdown. While tissue remains ischemic, a fall in tissue pH is probably of benefit in that it depresses cardiac contractility and, thereby, slows the breakdown of ATP. Whenever coronary blood flow is suddenly restored, however, protons rapidly leaving cells in exchange for Na+ may cause a secondary influx of Ca2+ via the 3Na +/Ca2 + exchange, with the risk of cytosolic Ca2 + overload.
References 1 ALLEN, D.G., ORCHARD, C.H. Intracellular Ca*’ concentration during hypoxia and metabolic inhibition mammalian ventricular muscle. J Physiol (Lond) 339, 107-122 (1983). 2 BERNE, R. M., RUBIO, R. Adenine nucleotide metabolism in the heart. Circ Res 34 (SupplS), 109-120 (1974).
in
Editorial
1085
3 BLATTER, L. A., MCGUIGAN, J. A. S. Free intracellular magnesium concentration in ferret ventricular muscle measured with ion-selective microelectrodes. Quart J Exp Physiol 71, 467-473 (1986). 4 CARAFOLI, E. The homeostasis of calcium in heart cells. J Mol Cell Cardiol 17, 203-212 (1985). 5 COOKE, R., FRANKS, K., LUCIANI, G. B., PATE, E. The inhibition ofrabbit skeletal muscle contraction by hydrogen ions and phosphate. J Physiol (Lond) 395, 77-97 (1988). 6 DE HAMFTINNE, A. Intracellular pH and surface pH in skeletal and cardiac muscle measured with a doublebarrelled pH microelectrode. Pfliigers Arch 386, 121-126 (1980). 7 DE MELLO, W. C. Intercellular communication in cardiac muscle. Circ Res 51, l-9 (1982). 8 DENNIS, S. C., KOHN, M. C., ANDERSON, G. J., GARFINKEL, D. Kinetic analysis of monocarboxylate uptake into perfused rat hearts. J Mol Cell Cardiol 17, 987-995 (1985). 9 DENNIS, S. C., MCCARTHY, J., KEDING, B., OPIE, L. H. Lactate efflux from the ischemic myocardium: the influence of pH. J Mol Cell Cardiol 18 (Suppl l), 346~ (1986) Abs. 10 DENNIS, S. C., COETZEE, W. A., CRAGOE JR., E. J,, OPIE, L. H. Effects of proton buffering and of amiloride derivatives on reperfusion arrhythmias in isolated rat hearts: possible evidence for an arrhythmogenic role of Na+ IH ’ exchange. Circ Res 66, 1156-1159 (1990). 11 ELLIS, D., MACLEOD, K. T. Sodium-dependent control of intracellular pH in Purkinje fibres of sheep heart. J Physiol (Lond) 359, 81-105 (1985). 12 FABIATO, A., FABIATO, F. Effects of pH on the myotilaments and sarcoplasmic reticulum of skinned cells of cardiac and skeletal muscles. J Physiol (Lond) 276, 233-255 (1978). 13 GEVERS, W. Generation of protons by metabolic processes in heart cells. J Mel Cell Cardiol9, 867-874 (1977). 14 GEVERS, W. Reply to letter by D. R. Wilkie. J Mol Cell Cardiol 11, 328-330 (1979). 15 HESS, P., METZGER, P., WEINGART, R. Free magnesium in sheep, ferret and frog striated muscle at rest measured with ion-selective micro-electrodes. J Physiol 333, 173-188 (1982). 16 JACOBUS, W. E., TAYLOR, G. J., HOLLIS, D. P., NUNNALLY, R. L. Phosphorus nuclear magnetic resonance ofperfused working rat hearts. Nature (Lond) 265, 756-758 (1977). 17 KAGIYAMA, Y., HILL, J. L., GETTES, L. S. Interaction of acidosis and increased extracellular potassium on action potential characteristics and conduction in guinea pig ventricular muscle. Circ Res 51, 614-623 (1982). 18 KHURI, S. F., KLONER, R. A., KARAFFA, S. A., et al. The significance of the late fall in myocardial pC0, and its relationship to myocardial pH after regional coronary occlusion in the dog. Circ Res 56, 537-547 (1985). 19 KIM, D., CRAGOE JR., E. J., SMITH, T. W. Relations among sodium pump inhibition, Na-Ca and Na-H exchange activities, and Ca-H interaction in cultured chick heart cells. Circ Res 60, 185-193 (1987). 20 KIM, D., SMITH, T. W. Cellular mechanisms underlying calcium-proton interactions in cultured chick ventricular cells. 1 Phvsio1398. 391-410 (1988). i., WILD;, A., JAN~E, M. J., DURRER, D., YAMADA, K. Combined effects of hypoxia, hyperkalaemia and 21 Ko&A, acidosis on membrane action potential and excitability of guinea pig ventricular muscle. J Mol Cell Cardiol 16, 246-259 (1984). protons on the electrical activity of single ventricular cells. Pfliigers Arch 22 KURACHI. Y. The effects of intracellular 394, 264-270 (1982). exchange system in cardiac cells: its biochemical and 23 LAZDUNSKI, M., FRELIN, C., VIGNE, P. The sodium-hydrogen pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. J Mol Cell Cardiol 17, 1029-1042 (1985). 24 LEE, H., SMITH, N., MOH~BIR, R., CLUSIN, W. T. Cytosolic calcium transients from the beating mammalian heart. Proc Nat1 Acad Sci USA 84, 7793-7797 (1987). 25 MEDINA, G., ILLINGWORTH, J, Some factors affecting phosphate transport in a perfused rat heart preparation. Biothem J 188, 297-311 (1980). 26 MEECH, R. W., THOMAS, R. C. Effect of measured calcium chloride injections on the membrane potential and internal pH of snail neurons. J Physiol (Lond) 298, 111-129 (1980). 27 NEELY, J. R., MORGAN, H. E. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Ann Rev Physio136, 413-459 (1974). 28 NEELY, J. R., WHITMER, J. T., RovE~-~o, M. J. Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. Circ Res 37, 733-741 (1975). ofjunctional conductance on proton calcium and magnesium ions in cardiac 29 NOMA, A., TSUBOI, N. Dependence paired cells of guinea pig. J Physiol382, 193-211 (1987). of free fatty acids, glucose and catecholamines in acute myocardial infarction. Am J Cardiol 30 OPIE, L. H. Metabolism 36, 938-953 (1975). 31 OPIE, L. H. Effects of regional ischemia on metabolism of glucose and fatty acids. Relative rates of aerobic and anaerobic energy production during myocardial infarction and comparison with effects of anoxia. Circ Res 38 (Suppl l), 52-74 (1976). 32 OPIE, L. H., THOMAS, M., OWEN, P., SHULMAN, G. Increased coronary venous inorganic phosphate concentrations during experimental myocardial ischemia. Am J Cardiol30, 503-513 (1972). 33 PHILIPSON, K. D., BERSOHN, M. M., NISHIMOTO, A. Y. Effects of pH on Na+ -Ca* + exchange in canine cardiac sarcolemmal vesicles. Circ Res 50, 287-293 (1982). exchange in plasma membrane vesicles. Ann Rev Physio147, 561-571 (1985). 34 PHILIPSON, K. D. Sodium-calcium 35 PIERCE, G. N., PHILIPSON, K. D. Na+-H + exchange in cardiac sarcolemmal vesicles. Biochim Biophys Acta 818, 109-l 16 (1985).
1086
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et PI.
36 PIWNICA-WORMS, D., JACOB, R., SHIGETO, N., HORRES, C. R., LIEBERMAN, M. Na/H exchange inculturedchick heart cells: secondary stimulation of electrogenic transport during recovery from intracellular acidosis. J Mol Cell Cardiol 18, 1109-1116 (1986). 37 POOLE-WILSON, P. A. Measurement of myocardial intracellular pH in pathological states. J Mol Cell Cardiol 10, Xl-526 (1978). 38 POOLE-WILSON, P. A., HARDING, D. P., BOURDILLON, P. D. V., TONES, M. A. Calcium out of control. J Mol Cell Cardiol 16, 175-187 (1984). 39 Roos, A., BORON, W. F. Intracellular pH. Physiol Rev 61, 296-434 (1981). 40 SPENCER, J. L., LEHNINGER, A. L. L-lcatate transport in Ehrlich ascites tumorcells. Biochem J 154,405-414 (1976). 41 STEENBERGEN, C., MURPHY, E., LEVY, L., LONDON, R. E. Elevation of cytosolic free Ca* + concentration early in myocardial ischemia in perfused rat heart. Circ Res 60, 700-707 (1987). 42 STEENBERGEN, C., MURPHY, E., LEVY, L. A., LONDON, R. E. Increase in cytosolic free magnesium during ischemia. Circulation 80, II-19 (1989) Abs. 43 TAEGTMEYER, H. Metabolic responses to cardiac hypoxia: increased production of succinate by rabbit papillary muscles. Circ Res 43, 808-815 (1978). 44 VAGHY, P. L. Role of mitochondrial oxidative phosphorylation in the maintenance of intracellular pH. J Mol Cell Cardiol 11,937-940 (1979). 45 VAUGHAN-JONES, R. D., LEDERER, W. J., EISNER, D.A. Ca*’ ions can affect intracellular pH in mammalian cardiac muscle. Nature 301, 522-524 (1983). 46 WILKIE, D. R. Generation of protons by metabolic processes other than glycokysis in muscle cells: A critical view. J Mol Cell Cardiol 11, 325-330 (1979). 47 WILLIAMSON, J. R., SCHAPFER, S. W., FORD, C., SAFER, B. Contribution oftissue acidosis to ischemic injury in the perfused rat heart. Circulation 53 (Suppl I), 3-14 (1976). 48 YAMAZAKI, S., FUJIBAYASHI, Y., RAJAGOPALAN, R. E., MEERBAUM, S., CORDAY, E. Effects of staged versus sudden reperfusion after acute coronary occlusion in the dog. J Am Ccl Cardiol 7, 564-572 (1986).
Mol Cell Cardiol
23, 1077-1086 (1991)
EDITORIAL
Protons
in Ischemia:
Where
Do They
S. C. Dennis’,
Come
W. Gevers*
From;
Where
Do They
Go To?
and L. H. Opie’
1Bioenergetics of Exercise, 2Cell Biology ofAtherosclerosis and 31schemic Heart Disease Research Units of the Medical Research Counciland the University of Cape Town, Cape Town, South Africa (Received 22 June 1990, accepted in revisedform 20 March 1991)
It is commonly thought that the accumulation of lactic acid from anaerobic glycolysis is the cause of an undesirable acidosis during myocardial ischemia. In 1977, Gevers [13] suggested that the major source of protons during anaerobic metabolism was not so much the formation of lactic acid as the turnover of glycolytically produced ATP. The purpose of this Editorial is to re-evaluate this proposal and expand it, taking into account how ischemia and reperfusion might influence proton transport across cell membranes. To understand the formation of protons in the under-perfused myocardium requires emphasis that a total cessation of coronary blood flow is not a prerequisite for myocardial ischemia. A complete absence of blood flow rarely occurs clinically and, even under experimental conditions with multiple coronary artery ligations, there is still some collateral circulation to the ischemic zone [31]. Myocardial ischemia more commonly arises from a partial restriction of coronary blood flow and, this has two immediate consequences. One is that the supply of O2 for energy production is decreased. The other is that the removal of metabolites such as lactate, CO, and protons is impaired. Depending on the degree of 0, demand and on the severity of the reduction in coronary blood flow, intracellular pH declines from the normal values of around 7.2 [39] to values as Please address all correspondence Observatory 7925, South Africa. 0022-2828/91/091077
+ 06 $03.00/O
to: L. H.
Opie,
Heart
low as 6.3 [31] or even 5.7 [16]. From the reported physiochemical and metabolic buffering capacity of mammalian cardiac muscle [39], this fall of around 1 pH unit corresponds to an acid-load of 51-77 mmol H + per liter of tissue H,O.
Protons: Where Do They Come From? In considering the production of protons in the ischemic myocardium, it is important to appreciate that there is a relatively high (0.4-S. 5 mM) free cytoplasmic Mg* + concentration in cardiac tissue [3, 151. This, together with the fact that intracellular [Mg2 + ] rises when ATP levels fall 1421, strongly suggests that all of the ATP4- and ADP3 - is bound to Mg2 + and is therefore fully ionized at cytosolic pH [13, II]. Another point to note is that the pKa’ of inorganic phosphate is 6.9 and the normal intracellular pH is around 7.2 [39]. From these values, it can be calculated that, before acidosis, 72 % of the cytosolic inorganic phosphate is in the HP04’ - form [II, 46).
Not from lactic acid When intracellular [Mg2 + ] and pH are taken into account and the likely charges on ATP, ADP, inorganic phosphate and lactate are Research
Unit,
University
of Cape
Town,
0 1991 Academic
Medical
Press
School,
Limited
1078
S. C. Dennis
summed, it is found that the glycolytic pathway does not produce a large net gain of protons [13]. Anaerobic glycolysis has as its
(1)
end-product mainly lactic acid molecules
lactate anions rather than (Equation 1):
- + 2 Pi2- + 2 lactate - + 2 MgATP2
glucose + 2 MgADP
The metabolism of a hexose unit of glycogen to 2lactate molecules and 3 MgATP molecules also does not produce a net gain of protons. In
Nat from continued gbco&tic formation of NADH+H’ Contrary to assumptions often made, a continued conversion of glucose or glycogen to pyruvate- and NADH + H + is also unlikely to contribute to ischemic acidosis. Mitochondrial pyruvate metabolism and cytoplasmic NADH + H+ re-oxidation are linked. For the tricarboxylate cycle to proceed, the 2-0x0glutarate ( = ketoglutarate) leaving the mitochondria in exchange for malate has to be regenerated by the malate/aspartate shuttle.
-
fact, when 3 MgATPs are generated by the glycolytic pathway, a proton is consumed (Equation 2):
+ 3 P:- 3 2 lactate-
(2)
(3)
et al.
+ 3 MgATPs-
NADH and protons formed from glycolysis are therefore either taken into the mitochondria together with pyruvate or are used in the reduction of pyruvate to lactate. The only possible contribution to acidosis from continued glycolytic NADH + H + production is when glutamate is oxidized [43]. In the conversion of glutamate to succinate, pyruvate is diverted to alanine and, under circumstances, some glycolytic these NADH+H+ formation could persist (Equation 3):
Glycolysis
P;- + MgADPMgATP* F pyruvate
+ NAD’
- + NADH
+
q
glutamate2-oxoglutarate*-7
F alanine Cytoplasm Mitochondrion NADH+
+ MgGDPNADH
+ P:- -
+ MgGTP2
- +--’ ” succinate2 -
EditOrid Increases however, protons. NADH NADH cytosolic reaches
in NADH and H+ concentrations, provide only a transient source of Without a re-oxidation of + H+ to NAD+ , further glycolytic + H+ formation is prevented and the [NADH + H + ]/[NAD+ ] ratio simply a new steady-state.
From decreased mitochondn’al ATP Instead,
(4)
the metabolic T MgATP2
n-synthesis?
generation
‘M”Tg’-‘i”i+
-
phosphorylation
In very early ischemia, the rapid transfer of phosphate from creatine phosphate to ADP MgATP2 T
)Mgr-
Mg~glycolysis
arising from (Equation6):
its hydrolysis
+Mg-r-
Thus, the metabolic generation of protons during ischemia is primarily a result of the increased reliance on glycolysis for ATP resynthesis. From carbon dioxide retention? In the early stages of regional myocardial ischemia, another likely cause of acidification is the accumulation of CO2 from at least some mitochondrial respiration residual (Equation 7): Mitochondrial
respiration
-
generated
by ATP
‘i++P:-
hydrolysis
However, when ATP is formed by glycolysis rather than by mitochondrial metabolism or creatine phosphate breakdown, the protons
(7)
also takes up the protons breakdown (Equation 5):
-
creatine-P2-
(6)
during ischemia is more a consequence of the decrease in mitochondrial ATP production in relation to glycolytic ATP formation. During oxidative phosphorylation, the influx of protons into the mitochondria exceeds the outward “pumping” of protons by the electron transport chain [44] and most of the protons produced by ATP hydrolysis are probably utilized in its re-synthesis (Equation 4):
of protons
oxidative
(5)
1079
are not consumed
+r-
+m
Khuri et al. [18] showed that the increase in tissue pC02 from around 50 to 190mmHg during the first 30min of coronary artery occlusion is not entirely due to the 46 to 200 nmol/l rise in [H + ] displacing the HCOs-/CO2 equilibrium towards CO2 formation. As ischemia continued, there was a gradual decline in tissue pCOz and [H + 1, which is not what one would expect if the previously high pC0, had simply been a result of metabolic acidosis.
CO2 + HZ0 -HCO,-
+IH’I
1080
S. C. Dennis From glycogen turnover?
Some protons
might
also be generated
by an
et al. increased ischemia
turnover of (Equation 8):
(8)
glycogen
during
MgATP2
1
MgUTP*
I 1 glucosd- 1-P2I
Pi’- + glycogen 1‘
qH+
-
+ MgUDP-AUDP-glucoseZ*‘Lpi*
While elevated Pi, AMP and CAMP concentrations activate glycogen breakdown in ischemic tissue, build-up of “n-metabolized glucose- 1 -phosphate favors glycogen reformation [27, 311. Net proton production from a glycogen : glucose- 1 -phosphate cycle, however, is largely dependent on the mechanism of ATP re-synthesis. Where ATP is mainly regenerated by residual oxidative phosphoryl-
(9)
ation (Equation4), regional ischemia consumed.
as may be the case in [31], most of the protons are
From triglyceride-fatty acid recycling? Catecholamine-stimulated triglyceride lipolysis and fatty acid activation may also produce protons during ischemia (Equation 9):
3 MgATP2
- + 3 CoA* -
I
triglyceride
) 3 FFAI
+ glycerol +
3 acyl CoA* +--i 3Mg2’
(‘Assumes that inorganic pyrophosphatase and long-chain acyl CoA synthetase activities are similar.) According to the above equation, each triglyceride molecule converted to fatty acyl CoA could yield 6protons as previously argued [13]. Similarly, the release and activation of 2long-chain fatty acids from the degradation of membrane phosphoglyceride, late in ischemia, could form 5protons. How-
+ 3AMP2-
+ 6Pi*-’
+
l 3H’
3H+
ever, the following additional factors must also be considered. First, long-chain fatty acids may not be fully ionized in the cytosol, in which case, less protons would be generated than is supposed. Second, NADH reoxidation via glucose to glycerol-3-phosphate conversion promotes triglyceride : fatty acid cycling [30] and, in this cycle, the protons generated in the formation of fatty acyl CoA are taken up in the re-synthesis of ADP from AMP (Equation 10):
1081
Editorial (10)
3CoA’-
3 MgATP2
+ P;2-
Under these circumstances, produced by the provision
only one proton is of glycerol-3-phos-
- + 3 COA’ -
phate to the cycle from the glycolytic (Equation 11):
pathway
triglyceride turnover
0.5 glucose + MgATP2
- ->
Hence, far fewer protons are produced by this cycle than originally thought [13]. Only in severe ischemia is there a possibility of some proton production from triglyceride-fatty acid cycling. In moderate ischemia, where more than one out of six ADP’s is oxidatively rephosphorylated [31], the operation of a triglyceride:fatty acid cycle will consume, rather than produce protons.
From intracellular
calcium accumulation?
During early myocardial ischemia, the concentration of cytosolic calcium rises, according to a number of investigators who used different techniques [I, 24, 411. The mechanism of the intracellular Ca* + increase is unknown but it could be due to the reduction in the diastolic membrane potential in ischemic tissue. A less negative resting membrane potential would decrease the transsarco-
T
glycerol-P*-
+ MgADP-
+ ) H+ 1
-
lemmal efflux of Ca* + via the 3Na.+/Ca*+ exchange that is driven by the net inward movement of positive charge [I, 341. Early falls in cytosolic pH may therefore also arise from intracellular Ca*+ accumulation in ischemic tissue. Several groups have shown that Ca*+ and H+ ions compete for common intracellular binding sites [II, 26, 451 and, depending on the relative Kd values of those sites for Ca2 + and H + ion binding, between 0 and 2 protons could be displaced by each Ca2 + ion. With continued ischemia, however, further rises in cytosolic Ca2+ concentration are probably prevented by a decrease in the influx of Ca*+ . Protons accumulating in the interstitial space compete with Ca* + for access to the voltage-gated Ca2 + channels [22] and the 3Na+/Ca2+ exchange [33]. A low pH also depresses fast responses [17, 211 and promotes electrical uncoupling [ 7, 291.
1082
S. C. Dennis From net adenine nucleotide degradation?
In addition to slowing conduction and reducing the influx of Ca2+ for the “triggering” of contraction, proton accumulation also decreases cross-bridge cycling. Acidification of the cytoplasm reduces the activation of actin by Ca2+ [IZ] and slows the myosin ATPase [5]. In terms of cell survival, a rapid fall in cytosolic pH may therefore be of some benefit to ischemic tissue, by helping to decrease contractility and, thereby, retarding the breakdown of ATP [371. Decreased consumption of ATP by crossbridge cycling probably also slows the rate of further acidification in the ischemic myo-
et d.
cardium. With less ATP turnover, proton formation from glycolytic ATP re-synthesis will be decreased and CO1 accumulation from residual mitochondrial respiration will be reduced. Continuing acidification is also likely to be eventually limited by the inhibition of glycolysis at low pH [28] and the loss of mitochondrial function in late ischemia [18]. At this point, a further fall in pH could also be caused by the release of protons and phosphate from the net breakdown of ATP [2 1. Despite the conservative effects of contractile failure, there is a gradual accumulation and leakage of adenosine, inosine and hypoxanthine from adenine nucleotide degradation in ischemic cells (Equation 12):
(12) (Mg2 +) + MgATP2 1
-
inosine -+ NH*+ .L hypoxanthine +ribose
Although the formation of 3 H+ + 3 HP042from net ATP breakdown is slow in comparison to the production of protons from glycolytic ATP turnover and CO* accumulation, it is likely to contribute to the acidification of the myocardium in the later stages of ischemia. Once the intracellular pH is well below the 6.9pKa’ value for inorganic phosphate, even modest increases in the H2P04-/HP042ratio will raise the cytoplasmic proton concentration.
Protons:
Where
Do They
Go To?
So far, we have described the metabolic responses of the myocardium to ischemia and have argued that protons are mainly produced from glycolytic ATP turnover, CO, retention and, later, net ATP degradation (Table 1). In
the following sections, attention is focused on the accumulation of protons in the ischemic myocardium .
To the intracellular
bu&s
During the early restriction of coronary blood flow, protons are first buffered by intracellular protein histidine residues and by inorganic phosphate. Inorganic phosphate is the major intracellular buffer and is especially important in ischemic tissue. As creatine phosphate is broken down to help maintain ATP levels (Equation 5), inorganic phosphate accumulation [31] increases intracellular buffering capacity. Depending on the pH, rapid inorganic phosphate release may even produce a transient alkalosis (Table 1).
Elditorial TABLE
1.
The potential ischemia
contribution
of different
1083
metabolic
pathways
to proton
Protons starting
Process Early
ATP
HPO,*
breakdown
and re-synthesis
- accumulation
Residual
ATP
Carbon
dioxide
from
breakdown
ATP
Possible
glycolytic
Release
and activation
Possible
triglyceride:
Possible H,PO,-
net creatine
from
breakdown
phosphate
residual
of
- 1 to oa
phosphorylation
0 +1
phosphorylation
+2
via glycolysis
acids from
arising
triglyceride
from
gluamate
oxidation
+1 + 3 to 6b
lipolysis
acid cycling
-5tol’
glycogen:
glucose-
1 -P cycling
+ 1 too’
formation
from
acid
net ATP
+3toO”
degradation
indicate
that the production
ionization
in the cytosol
or consumption or ‘the
predominant
To the interstitial S/MC~ Some
0
breakdown
oxidative
+ H + accumulation of fatty
phosphate
via oxidative
and re-synthesis
NADH
creatine
the
protons
buffered
inorganic
(13)
mechanism
(Equation
Cytoplasm clH’
of protons
depends on either “the pH, of ATP re-synthesis.
phosphate may the reversible
by
+ HP04*-+
during
per mol of substrate
fatty
Superscripts fatty
from
and re-synthesis
retention
Increased
chain
formation
H2P04-
then
bthe degree of long
leave the ischemic Na+ - H2P04-
cells via symport
13):
Interstitium ) > > Na’
liEI lactate -
Phosphate ischemia
efflux is transiently [32] and by acidosis
t-w.
Unbuffered
initially
changer
leave
and
intracellular
and
accelerated by mild hypoxia
protons
via the sarcolemmal
H + -lactate -
can also /H + exco-transporter Na+
) 1
(Equation 13). Cytostolic proton accumulation allosterically activates the exchange of intracellular protons for extracellular Na+ [23, 361 and displaces the [H + ][lactate -1 in/out equilibrium towards proton + lactate release [8, 91. As ischemia progresses, however, further
1084
S. C. Dennis
proton efflux is likely to be opposed by interstitial proton accumulation. Because the buffering capacity of the extracellular space is less than that of the intracellular space, increasing acidosis severely decreases the normal trans-sarcolemmal proton gradient [6, 471. Loss of the cell membrane proton gradient slows H + and lactate- ion release by >60% [ 401 and reduces Na + /H + exchange by >80 % [351. To the blood Ultimate rates of proton release from ischemic tissue are therefore probably determined by the washout of protons from the interstitial space. In regional ischemia, for instance, the
(14)
et al. greatest proton accumulation is seen in tissue receiving the least collateral circulation [ 181. A limited clearance of protons from ischemic tissue is also suggested from studies in which coronary blood flow is suddenly restored. On reperfusion, the prompt washout of extracellular protons is thought to lead to a rapid efflux of accumulated intracellular protons with an eventual Ca*+ overload and evidence of tissue injury such as arrhythmias [IO]. An accelerated exchange of intracellular protons for extracellular Na + would result in a secondary influx of Ca2+ via the 3Na2/Ca2+ exchanger [19, 201. Calcium-proton interactions could then release more protons into the cytoplasm and promote further Ca2 + entry via the Na + -H + -Ca2 + exchangers (Equation 14):
Cytoplasm
Interstitium
Na+
interaction Ca*+
The operation of a Na + -H + -Ca2 + exchange might also explain why Ca2+ influx on reperfusion is (i)not fully blocked by Ca2+ channel antagonists and (ii)not associated with net Na + efflux [38]. A Na + -H + -Ca2 + exchange is also consistent with the observation that arrhythmias are decreased by more gradual reperfusion [48]. A slower washout of accumulated extracellular protons would limit the rate of Na + /H + exchange and allow more of the incoming Na’ to leave via the Na + /K + -ATPase rather than by the 3Na + /Ca2 + exchanger. In conclusion, ischemic acidosis is
predominantly due to a retention of protons from glycolytic ATP turnover, CO, accumulation and, eventually, net ATP breakdown. While tissue remains ischemic, a fall in tissue pH is probably of benefit in that it depresses cardiac contractility and, thereby, slows the breakdown of ATP. Whenever coronary blood flow is suddenly restored, however, protons rapidly leaving cells in exchange for Na+ may cause a secondary influx of Ca2+ via the 3Na +/Ca2 + exchange, with the risk of cytosolic Ca2 + overload.
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in
Editorial
1085
3 BLATTER, L. A., MCGUIGAN, J. A. S. Free intracellular magnesium concentration in ferret ventricular muscle measured with ion-selective microelectrodes. Quart J Exp Physiol 71, 467-473 (1986). 4 CARAFOLI, E. The homeostasis of calcium in heart cells. J Mol Cell Cardiol 17, 203-212 (1985). 5 COOKE, R., FRANKS, K., LUCIANI, G. B., PATE, E. The inhibition ofrabbit skeletal muscle contraction by hydrogen ions and phosphate. J Physiol (Lond) 395, 77-97 (1988). 6 DE HAMFTINNE, A. Intracellular pH and surface pH in skeletal and cardiac muscle measured with a doublebarrelled pH microelectrode. Pfliigers Arch 386, 121-126 (1980). 7 DE MELLO, W. C. Intercellular communication in cardiac muscle. Circ Res 51, l-9 (1982). 8 DENNIS, S. C., KOHN, M. C., ANDERSON, G. J., GARFINKEL, D. Kinetic analysis of monocarboxylate uptake into perfused rat hearts. J Mol Cell Cardiol 17, 987-995 (1985). 9 DENNIS, S. C., MCCARTHY, J., KEDING, B., OPIE, L. H. Lactate efflux from the ischemic myocardium: the influence of pH. J Mol Cell Cardiol 18 (Suppl l), 346~ (1986) Abs. 10 DENNIS, S. C., COETZEE, W. A., CRAGOE JR., E. J,, OPIE, L. H. Effects of proton buffering and of amiloride derivatives on reperfusion arrhythmias in isolated rat hearts: possible evidence for an arrhythmogenic role of Na+ IH ’ exchange. Circ Res 66, 1156-1159 (1990). 11 ELLIS, D., MACLEOD, K. T. Sodium-dependent control of intracellular pH in Purkinje fibres of sheep heart. J Physiol (Lond) 359, 81-105 (1985). 12 FABIATO, A., FABIATO, F. Effects of pH on the myotilaments and sarcoplasmic reticulum of skinned cells of cardiac and skeletal muscles. J Physiol (Lond) 276, 233-255 (1978). 13 GEVERS, W. Generation of protons by metabolic processes in heart cells. J Mel Cell Cardiol9, 867-874 (1977). 14 GEVERS, W. Reply to letter by D. R. Wilkie. J Mol Cell Cardiol 11, 328-330 (1979). 15 HESS, P., METZGER, P., WEINGART, R. 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