Intracellular acidosis and contractility in the normal and ischemic heart as examined by 31P NMR

Journal

of Molecular and Cellular Cardiology (1982) 14, Sup~l. 3, 13-20.

Intracellular

Acidosis and Contractility in the Normal Heart as Examined by UP NMR*

and Ischemic

William E. Jacobus t : 11, Ira H. Pores fl, Scott K. Lucas 0, Myron L. Weisfeldt t and John T. Flaherty t From the t Peter Belfeer Laboratory for Myocardial Research, Department of Medicine and the $ Department of Physiological C~ist~, Th Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA WILLIAM E. JACOBUS, Iru H. PORES, &x-r K. LUCAS, MYRON L. WEISFELDT AND J.OHN T. FLAHERTY. Intracellular Acidosis and Contractility in the Normal and Ischemic Heart as examined by a’P NMR. Journal of Molecular and Cellular Cardiolou (1982) 14, &ppl. 3, 13-20. 31P was used to investigate correlations between intracellular pH (pHi) and myocardial contractility in the normal and ischemic isolated, perfused isovolumic rabbit heart. Intracellular pH was calculated from the chemical shift ofPi in hearts perfused with phosphate-free buffer. Normal intracellular pH was 7.22 -C 0.02 (n = 15). To calibrate the relationship between pHi and left ventricular developed pressure (LVDP), respiratory acidosis was induced by mixing 65% 0,: 30% N,: 5% CO, with 65% 0,: 35% CO, using Krebs buffer containing 24 mM HCO,. The results show that a 0.22 pH unit acidification correlates with a 50% reduction in LVDP. The correlation between pHi and LVDP was also studied in two models of ischemia: total global ischemia and steady state partial ischemia (50% reduction of LVDP). In both ischeniid conditions, a 50% lowering in LVDP correlated with only a 0.09 pH unit acidification. Thus, while intracellular acidosis may account for 40 to 50% of the depression of LVDP oberved during the early phases of ischemia, other factor must also play a role. Mass spectrometry was used to examine the potential regulatory role of tissue oxygen (PmOP). The model of steady-state partial ischemia was employed. Changes in LVDP and MVO, correlated quite closely with reductions in coronary flow. However, up to a 50% reduction in flow, pHi remained near normal, and tissue P,Op was normal or slightly elevated. These latter results suggest that an efficient autoregulatory mechanism controls both function and MVO, in close parallel to changes in flow. As a result, the metabolic supply/demand balance is maintained. However, beyond a 50% reduction in flow, this mechanism fails and metabolic indicies of ischemia are expressed. KEY WORDS: Intracellular pH; Acidosis; Myocardial hearts; 3’P NMR; Mass spectrometry; Wall tension.

contractility;

Ischemia;

Respiratory

acidosis;

Perfused

Introduction One of the most striking aspects of myocardial ischemia is the almost instantaneous compensatory reduction in ventricular performance observed with decreases in coronary arterial flow. In the intact animal, some degree of coronary arterial autoregulation protects against moderate changes in flow. However, in the isolated, non-blood perfused working heart even a very modest change in perfusion pressure or flow results in immediate changes in contractility. While this phenomenon has long

been recognized [ZZ], we still remain in considerable darkness concerning the mechanism(s) which so critically coordinate ventricular function to coronary flow. At the molecular or metabolic level, two hypotheses have received considerable attention [IS]. The first suggests that ischemic “power failure” is associated with the lack of supply of some requisite metabolite. Candidates in this category include oxygen, ATP and phosphocreatine. On the other

*‘This work was supported by United States Public Health ServiceGrants HL-22080, HL-I 7655 and HL-19414, and from the Susan II. Clayton Fund, the Johns Hopkins University School of Medicine. ]I Established Investigator of the American Heart Association, and author to whom request for reprints should be addressed at: 1385 Blalock Building, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Maryland 21205, USA. 5 Current Address: University of Oklahoma Health Science Center, Department of Surgery, P.O. Box 2690 I, Oklahoma City, Oklahoma 73190, USA. f[Current Address: Newark Beth Israel Medical Center, 201 Lyons Avenue Newark, New Jersey 07112, USA.

W. E. Jacobus

14

hand, diminished flow reduces washout. Therefore, toxic metabolites may accumulate which feedback to inhibit contraction. Likely candidates for this second hypotheses are lactate and H+ (acidosis). The importance of intracellular pH in the regulation of cell function has recently been reviewed by Roos and Boron [18], who state that the first observation that acidosis depresses contractility can be traced to the writings of Sir Isaac Newton. Recently, Steenbergen et al. [,?I] have concluded that the ischemic decline in pressure development was the result of the combined effects of intracellular and extracellular acidosis, with little or no contribution from a change in cellular energy status. To investigate the relative importance of these two hypotheses, we have used the biophysical technique of 31P nuclear magnetic resonance [7]. From the phosphorus NMR spectrum of heart, we are able to obtain data concerning the tissue quan-

Materials

et al.

tities of ATP, phosphocreatine, ADP, and Pi. In 1973, Moon and Richards [16] demonstrated that the chemical shift of Pi could be used to estimate tissue pH. Since then the method has been successfully employed in studies of skeletal muscle (3, 4,5, 12) and the perfused heart (8,9,10,11,13,19) as well as other organs [7]. Because the method is noninvasive, non-destructive and flow independent, it offers considerable advantages to biopsy methods for metabolite analysis, and acid or base distribution methods for pHi determination [18]. Our results show a rather tight coupling between intracellular pH and contractility. Nevertheless, during ischemia acidosis is insufficient to fully account for the contractile depression. Other factors must participate in the negative inotropic effect of ischemia. Among these factors could be changes in Ca2+ influx, gap junction alterations, and/or the “garden hose” effect on an “internal Starling” mechanism.

and Methods

Heart perfusion Hearts from New Zealand white female rabbits (1.2-2.0 kg) were perfused in a modified Langendroff model previously described in detail [s]. The phosphate-free Krebs Ringers bicarbonate buffer solution contained 117 mM NaCl, 6 rnbr KCl, 3.0 mM CaCl,, 1 .O rnM MgSO,, 0.6 mM EDTA, 16.7 rnhl glucose, 24 mM bicarbonate at a pH of 7.46 when gassed with 95% 0,/5% CO,. Since the perfusate is phosphate free, all 31P NMR signals must

arise from tissue metabolites. Functional stability was equivalent to hearts perfused with phosphate containing perfusate. The hearts were paced at a rate of 150 to 170 beats/min. Isovolumic ventricular pressure was measured with a latex balloon positioned in the left ventricle and connected to a Statham P 23 Db transducer. Control end diastolic pressure was set at 10 mmHg.

Nuclear magnetic resonance 3’P NMR spectra were obtained at 72.89 MHz using a Bruker WH-180 spectrometer. The probe diameter was 25 mm; the stability of the magnetic field was such that field/frequency lock was not required. The instrument was operated in the pulsed Fourier transform mode and was interfaced to a Bruker 1080 computer. Proton decoupled

spectra were obtained from transients following 45” pulses delivered at 2 set intervals. The data were collected with a 4k data table at a 3000 Hz spectral width. Typical spectra required 200-400 transients. More details concerning data accumulation and experimental conditions are provided in the Figure legends.

Estimation of tissuepH Measurement of intracellular from the chemical shift (&) following equation: pH;

pH was determined of the Pi peak by the

= pK - log-.

s,

-

.a

IjB 0

To minimize effects of tissue inhomogenity, chemical shift values were determined relative to the phosphocreatine resonance. Under these conditions, the constants in the equation are: pK = 6.90; 8,& = 3.290 and 8B = 5.805. The validation of these numbers has been reported [s].

Intracellular

pH and contractility

with 31P NMR

15

Tissue mass spectrometly Measurement of tissue gasses were done by mass spectrometry methods standard in this laboratory [N]. Briefly, a 22-gauge Teflon coated needle is inserted tangentiaily into the free-wall of the left ventricle. The tissue gas mixture is withdrawn across the teflon membrane, through stainless steel

tubing, and into the mass spectrometer. Because metal cannot be inserted into the NMR magnetic field, the mass spectrometry data were accumulated from hearts treated in exact parallel to those studied in the NMR. Further details of the mass spectrometry method have been reported [14].

Results and Discussion Calibration

of intracellularpH

In preparation for our studies on ischemia, it was first essential to determine the relationship between intracellular acidosis and the depression of myocardial contractility. As a calibration study we have used the model of respiratory acidosis. Five rabbit hearts were used in this protocol. The hearts were initially perfused with normal phosphate-free Krebs Ringer’s bicarbonate buffer, gassed with 65% 02: 30% NP: 5% CO,, at a buffer pH of 7.48. A 400-p&e NMR spectrum was obtained and the Pi chemical shift suggested that tissue pHi was 7.22 +0.02, Figure 1 (a). A combination glass pHelectrode was then placed in the buffer reservoir and a second gas used to lower buffer pH. This gas contained 65% O?: 35% CO,. The flow rate of the second tank was adjusted in a step-wise sequence to reduce buffer pH to values of 7.3, 7.2, 7.05, 6.9,6.8

and uentricularperformance and 6.7. At each value, buffer pH was stabilized for a period of at least 15 to 20 min. As buffer pH w.as reduced we noted a depression in contractility. During these periods of depressed stable performance, a series of 400 pulse NMR spectra were obtained. The signed-to-noise ratio of these spectra was 100 to 1 for phosphocreatine, 50 to 1 for ATP and 25 to 1 for Pi. The chemical shift of Pi was used to estimate tissue intracellular pH. The changes in intracellular pH (pHi) as a function of buffer pH are shown in Figure 1 (a), in which we see that over the buffer pH range of 7.48 to 6.70, intracellular pH falls from 7.22 to a value of6.93. ,4t each buffer pH value, we also ndted ventricular performance. Therefore, it was possible to correla.te pHi with left ventricular contractility. The correlation between NMR determined pH,

(b)

6.9’

I 6.8

I 7.0

I 7.2 Buffer

pH

I 7.4

L___

I 0.3 ApH

change

FIGURE 1. Effects of respiratory acidosis on intracellular pH and ventricular performance. (a) Buffer pH is step-wise reduced by mixing 65% 02: 35% CO, with the normal gas, 65% 02: 30% N,: 5% COP. NMR spectra (400 pulse) were obtained at each buffer pH value, and intracellular pH determined from the chemical shift of Pi. (b) Correlation between intracellular acidosis and left ventricular developed pressure (LVDP). LVDP was calculated as a percent of control function from measurements obtained with a balloon inserted in the left ventricle, see Methods. Control LVDP was always greater than 100 mmHg.

16

W. E. Jacobus

and ventricular performance is shown in Figure 1 (b). Left ventricular developed pressure (LVDP) was calculated as a percent of control performance. The data ofFigure 1 (b) show a quite linear relationship between the depression of LVDP and intracellular acidosis. From Figure l(b) it can be esti-

Contractility

mated that a 50% depression of LVDP correlated with a 0.22 $3 unit acidification. In other weds, a 50% fall in function is associated with a pH, shift from 7.22 to 7.00. These data clearly show a very tight coupling between pHi shifts and the contractile state of the ventricle.

and pHi during

Two models were used to examine the relationship between pHi and contractility in the ischemic myocardium. The first was total global ischemia, achieved by cross-clamping the aortic perfusion line and inducing a no-flow state. The protocol for these experiments is illustrated in Figure 2(a). The NMR spectrometer was programmed to obtain three consecutive spectra, here designated as Files 01,02 and 03. These are only 10 pulse spectra, requiring only 20 set per file. Between the first and second spectrum, the aortic line was clamped, and after 40 set of ischemia reflow was established, indicated as Release. This provided us with data time-averaged during the control period, and at 10 and 30 set of ischemia. The performance change observed during a typical determination are shown in the lower panel. One observes that shortly after reperfusion was initiated, performance returned to its control state. Therefore, in order to obtain sufficient

PROTOCOL

et al.

ischemia

signal, this protocol was repeated 20 times on the same heart. LVDP data showed no significant changes in control performance during this series of experiments, and the response of the heart to ischemia was also uniform. Therefore, data from these 20 consecutive experiments were summated by the computer to obtain 3 individual 200 pulse spectra with excellent signal-to-noise. This, then, was a condition of transient ischemia. The second ischemic protocol was that of partial, steady-state ischemia (Figure 2(b)). In these experiments, perfusion pressure was reduced sufficient to depress contractility by 50%. This was achieved simply by lowering the level of the perfusate reservoir. If intracellular acidosis fully accounts for the negative inotropic effect of ischemia [21], we anticipated from the data of Figure 1 (b) that we would observe a 0.22 pH unit acidification in this protocol. This, however, was

FOR TOTAL

GLOBAL

ISCHEMA Release

Occlusion

(a)

Files Time

Data points

>

I”“secl

[El

c::::!::!:::I::‘::: -20

(set)

:!:::+!:l+:l:::‘l -10

(Rapid

trace)

0 Time kec)

IO

20

30

40

R

Intracellular

pH and contractility

with 31P NMR

(b) HZ/Cm 200 100

50A. CONT.

Hz 10000 Hz 5000 HZ 2500

8000 4000 2000

d

6000 3000

4000 2000

1500

1000

8. EC.

2000 1000 500

D. DIF. (B-A)

FIGURE 2. Effects of ischemia on intracellular pH and ventricular performance. (a) Protocol for transient, total global ischemia. Upper section indicates NMR program; lower panel presents performance data for a single 40 set ischemic time. Time 0 is time of occlusion. (b) Protocol for steady-state 50% reduction in LVDP. CONT = control spectrum; IS.3 = ischemic spectrum; REFL = reflow spectrum; DIF = difference spectrum (EA) subtracted by the computer. The peaks and abscissa are labelled in CONT spectrum: a = sugar phosphate; b = P,; c = glycerolphosphorylcholine; d = creatine phosphate; e = y.ATP and P.ADP; f = a.ATP and a.ADP; g= P.ATP. The peak downfield, labelled r, originates from methylenediphosphonic acid in the intraventricular balloon. Spectral width = 5000 Hz, with a 4k data table. Panel at lower right is performance data obtained during the early reflow period. (c). Correlation between pH; and LVDP during ischemia. Solid line is Ihe calibration line from Figure 1 (b).

not the case. During 50% steady-state ischemia, intracellular pH fell from 7.15 (Spectrum A), to 7.06 (Spectrum B), or by only 0.09 pH units. During reperfusion (Spectrum C), intracellular pH and function returned to control, pHi = 7.18. The fact that these hearts were subjected to ischemia of sufficient magnitude to evoke other contractile abnormalities is shown by the data at the lower right of Figure 2(a). In the control state, paced hearts normally relaxed to an end diastolic pressure of 4 mmHg. However, during early post ischemic reflow, end diastolic pressures as high as 20 mmHg were observed. When the pacer was turned off, end

ApH

change

diastolic pressure fell to 4 mmHg, indicating that incomplete relaxation was observed. Incomplete relaxation during early reflow is one of the known effects of ischemia on myocardiai performance. Thus, the presence of ischemia in these hearts is evidenced by contractile depression as well as

W. E. Jacobus

18

reflow abnormalities is diastolic properties. However, intracellular acidosis was clearly less than expected. To evaluate the role of high energy phosphate depletion during ischemia, a difference spectrum was obtained (Spectrum D). The computer subtracted the control Spectrum A from the ischemic Spectrum B. In control experiments a 10% change in metabolite content could be detected. However, in Spectrum D we see no changes, indicating that reductions in the high energy phosphate metabolites were minimal, or less than 10%. The summated pH data from the experiments presented in Figures 2(a) and 2(b) are shown in

et al.

Figure 2(c). The solid line is the calibration line determined by respiratory acidosis (Figure lb). These data show that for either condition of ischemia, a 50% fall in contractility is associated with only a 0.09 pH unit acidification. In conclusion, these data show that while changes in intracellular pH may account for between 40 to 50% of the early depression of contractility, other metabolic and/or physiological factors must also play an important regulatory role. The minimal changes in the high energy phosphates (Figure 2b, Spectrum D) suggests that these compounds may not be important regulatory metabolites [Zl] under these conditions.

Mass sbech romety From the perspective of heart energy metabolism, oxygen is the most necessary and also the most limited substrate, having no cytoplasmic reserve. It has been estimated that less than 5 set after coronary artery ligation, the tissue concentration of oxygen falls below the Km for cytochrome oxidase, and thus the electron transport chain becomes reduced. Oxidative phosphorylation, the source of 90% of contractile ATP, stops under these conditions. It was therefore quite logical that the changes in contractility observed at the onset of ischemia might parallel a decline in tissue oxygen tension, P,O,. To investigate this hypothesis we used the combined methods of NMR and mass spectrometry (Figure 3).

studies

The protocol for these experiments was similar to that used in Figure 2. Hearts were intitially perfused at a pressure of 80 mmHg. In a step-wise manner perfusion pressure was reduced from 80 to 20 mmHg, or, expressed a percentage reduction, to 25% of control pressure. In both panels of Figure 3, the line ofidentity is indicated by the dashed line. In Figure 3(a) we see that three parameters parallel the line of identity. These are: rates of coronary blood flow (CBF), left ventricular developed pressure (DP), and the rate of myocardial oxygen utilization, (MVO,). In other words, as perfusion pressure is diminished there is an almost linear decline in coronary blood flow. In conjunction, there is a similar decline in developed pressure,

(b)

‘v 4

pm02 pnlco2 7.16 7.14 7.12 7.10 7.06

Ia

7.06

01 80 100

FIGURE ischemia. in perfusion

I 70 88

I 60 75

I 50 63

3. Mass (a) Correlation pressure.

I 40 50

I 30 38

I 20 Perfusion 25 %Control

I pressure (mmHg) (perfusion pressure)

01 60 100

I 70 66

I 60 75

I 50 63

1 40 50

1 30 36

1 20 Perfusion 25 %Control

pressure (mmHg1 (perfusion pressure)

and carbon dioxide (P,CO,) as a function of studies of tissue oxygen (P,O,) offlow (CBF), developed pressure (DP) and rates ofoxygen consumption (MVO?) to changes (b) Correlation of tissue gas and NMR determined pHi as a function of perfusion pressure.

spectrometry

Intracellular

pH and contractility

which because of decreased ventricular work leads to a parallel decline in oxygen utilization. These results were not surprising. In contrast, a rather striking and unanticipated set of results was seen in the NMR and mass spectrometry data, Figure 3(b). If our working hypothesis had been correct, we would have predicted an immediate fall in tissue P,O,, a sharp rise in tissue P,CO,, with moderate changes in pHi. This was not observed. Initially P,,,O, increased while tissue P,CO, fell and pHi remained constant. At about a 25% reduction in perfusion pressure, PmO, started to decline and P&O, began to rise, still at constant pHi. The gasses continue to change until the crossover point, which occurred at about a 44% reduction in perfusion pressure or a 52% reduction in flow. These data suggest that up to a 50% reduction in flow, an efficient autoregulatory mechanism so specifically down-regulates contraction and oxygen utilization in response to decreasing flow, that oxygen supply exceeds oxygen demand. As a result, tissue P,O, actually rises presumably allowing aerobic metabolism to continue and intracellular pH to remain in balance. Beyond a 50% flow reduction, this mechanism fails. Contractile demands than exceed supply and metabolic indices of ischemia are expressed as high

with 31P NMR

19

P&O,, low P,Os and intracellular acidosis. Therefore, moderate reductions in coronary flow do not result in a supply/demand inbalance, even though left ventricular function is diminished. In summary, the data presented in this communication provide several new insights into the possible role of intracellular pH in the regulation 01 myocardial contractility. We have shown that small changes in intracellular pH may profoundly affect. mechanical performance (Figure 1). With respect to global ischemia, however changes in intracellular pH or metabolic parameters such as P,O,, P&O,, ATP or phosphocreatine cannot fully account foxthe stepwise decline of function. Clearly, other physiological regulatory mechanisms must come into play during severe or even moderate reductions in flow. These mechanisms may include a reduction of cell junction conductance [ZO], extracellular acidosis [JY], alterations in Ca2+ fluxes across the sarcolemmal membrane [17], or mechanical effects related to changes in wall thickness and thus in dialostic wall tension [I], the so called “garden hose” phenomenon [2]. The contribution of any or all of these mechanisms to the negative inotropic effect of ischemia awaits further experimental elucidation.

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KOBLER, W. & KATZ, A. M. Mechanism of early “pump” failure of the ischemic heart. Possible role ofadenosine triphosphate depletion and inorganic phosphate accumulation. American Journalof Cardiology, 40,467-47 1 (1977). MOON, R. B. & RICHARDS, J. H. Determination OfintracellularpH by 31P magnetic resonance. Journal ofBiological Chemistry, P46,7276-7278 (1973). POOLE-WILSON, P. A. & LANGER, G. A. Effects ofacidosis on mechanical function and Ca*+ exchange in rabbit myocardium. American.JournalofPhysiolo,~y, 236, H525-H533 (1979). Rbos, A. & BORON, w. F. In~ac&ula~pH. Physiological Reviews, 61, 296-434 (1981). SALHANY. I. M.. PIEPER. G. M.. Wu. S.. TODD. G. L.. CLAYTON. F. C. & ELIOT, R. S. 3LP nuclear maEnetic resonane ieasurements dfcardik pHin perfusedguinea-pig hearts: Journal of Molecub and Cellular Cardiology, 11, 601-610 (1979). SPRAY, D. C., HARRIS, A. L. & BENNETT, M. V. L. Gap junctional conductance is a simple and sensitive function ofintracellular pH. Science, 211,712-715 (1981). STEENBERGEN, C., DELLEUW, G., RICH, T. & WILLIAMSON, J. R. Effects ofacidosis and ischemia on contractility and intracellular pH of rat heart. Circulation Research, 41,84%358 (1977). TENNANT, R. & WIGGERS, C. J. The effect of coronary occulsion on myocardial contraction. American Journal of Physiology, 114,351~361 (1935).