Acta physiol. scand. 1975. 93.295-309 From the Department of Pharmacology, University of Lund, Sweden
Acid-Base Changes and Excitation-Contraction Coupling in Rabbit Myocardium. I. Effects on Isometric Tension Development at Different Contraction Frequencies BY
M. J~HANNSSON'and E. N~LSSON Received I August 1974
Abstract J~HANNSSON,M. and E. NILSSON. Acid-base changes and excitation-contraction coupling in rabbit myocardium. I. Effects on isometric tension development at different contraction frequencies. Acta physiol. scand. 1975. 93. 295-309. The effects of changes in acid-base parameters on the active force of isolated rabbit papillary muscles were studied at contraction frequencies of 12, 60 and 120/min. When extracellular p H was lowered from 7.4 to 7.0 and 6.7 in a bathing solution buffered with 10 mM histidine, the active force decreased at all contraction frequencies studied. After parallel increases of HCO; concentration (up to 47 mM) and Pco, at a constant extracellular pH of 7.4 the active force of the muscle increased at low and decreased at high contraction frequencies. None of these effects can be attributed to catecholamine release or to altered extracellular concentration of ionized calcium. The inotropic effects produced by bicarbonate were not reproducible by methyl sulfate (47 m M ) or propionate (47 mM). It is concluded that: 1. a lowering of the extracellular p H has a negative inotropic effect at all frequencies, 2. HCO; has a positive inotropic effect that is most pronounced at low contraction frequencies and 3. CO, has a negative inotropic effect exceeding that produced by the mere reduction in extracellular pH. The cellular mechanisms involved in the various inotropic effects are discussed. Key words: Rabbit heart, isometric force, contraction frequency, acidosis, HCO;,
Pcoz
From previous studies on isolated mammalian myocardial preparations it is not possible to form a clear opinion of how myocardial contractility is affected by different acid-base changes. It has been concluded that extracellular acidosis, within the patho-physiological range, does not affect contractility (Cingolani et al. 1970). Extracellular acidosis has also been found to cause a negative inotropic effect (Vaughan Williams and Whyte 1967). Evidence has been presented that a parallel rise in P,,, and HCO, concentration (at constant extracellular pH) does not alter the mechanical performance of isolated myocardial preparations (Vaughan Williams and Whyte 1967), that it induces a transient negative Present address: Department of Pharmacology, University of Iceland, Reykjavik, Iceland.
295
296
M. J ~ H A N N S S O NAND E. NILSSON
inotropic effect (Pannier and Leusen 1968) or causes a permanent depression of contractility (Cingolani et al. 1970). These discrepancies in results may be attributed to differences in both the myocardial preparation used (atrial or ventricular muscle and/or different species), and in the experimental conditions. An obvious difficulty in using a HCO;/CO, buffer system is the fact that extracellular pH, HCOi or P,,, cannot be altered individually, as at least one of the other two parameters will have to be changed simultaneously. Vaughan Williams and Whyte (1967) devised a statistical analysis to determine whether the inotropic effects observed after changes in P,,, or HCO; concentration are due to alterations of these parameters per se or to concomitant changes of extracellular pH. A more direct way of obtaining information about inotropic effects induced by extracellular pH-changes would be to use a non-bicarbonate buffer solution. In the present work the effects of extracellular acidosis on the mechanical performance of isolated papillary muscles were studied by varying the pH of a solution containing histidine as a buffer. With the histidine solution (of pH 7.4) as a control it was also possible to analyse the inotropic effects of parallel changes in HCO; concentration and Pco2 at a constant extracellular pH and to elucidate the effects caused by a separate change of HCO, concentration and P,,,.
Methods Preparation and mounting
Isolated papillary muscles of rabbits were used. The rabbits (weight 0.8-1.5 kg) were heparinized before sacrifice, and the heart was immediately removed and opened in oxygenated perfusion solution. The papillary muscle and its tendon was dissected from the right ventricle together with a small portion of the ventricle wall near the base of the muscle. Platinum loops were firmly tied with silk thread to the tendon and to the piece of the ventricular wall, as closely as possible to the insertions of the muscle. The length of the preparations, as determined with a microscope (10 fi magnification) varied within the range 3 to 6 mm and the largest diameter of the muscles was 0.3-0.9 mm. The preparation was mounted horizontally in a jacketed, temperature controlled bath (volume 1.5 ml) with the ventricle end connected to a hook and the tendon end attached to a semiconductor strain gauge transducer (Kyowa). The force transducer had a compliance of 1.3 ,um/mN and a resonant frequency of approximately 500 Hz with the preparation mounted. A linear response was obtained for forces between 0.2 and 50 mN. The signal from the transducer was displayed on a Tektronix 502 A oscilloscope and an Elema Schhander Mingograph (frequency response 500 Hz). The muscle was stimulated by passing current through two pairs of platinum wire electrodes placed on one side of the preparation perpendicular to the long axis of the muscle (distance between two cathodes was 2 mm). The pulse duration was 2 ms and the stimulus intensity was 5 0 % above the threshold value. The resting length of the muscle was chosen approximately 5 % below the length found to be optimal for active force. Perfusion of muscle bath
Prior to its inflow into the muscle chamber, the perfusion solution was equilibrated in a thermostated glass jar with the gas or gas mixture indicated in Table 1. The pH of the solution was adjusted before the perfusion was started and controlled during the perfusion (see below). The rectangular muscle bath was perfused at a rate of 2-4 ml/min to give 90% exchange in about 80 s. Temperature control. Temperature within the muscle chamber was kept constant at 37"iOS"Cand was monitored by means of a thermistor in the muscle bath. In any given experiment, temperature variation was less than j O . 3 T . It was specially checked that this value was not exceeded during shift from one buffer solution to another.
297
ACID-BASE CHANGES IN MYOCARDIUM. I
TABLEI. Composition of solutions. mM
zeroHCOT
lowHCO;
mediumHC03
NaCl
145
142
128
98
98
17
47
47
NaHCO,
3.4
high-HCOT high HCOT- C,H,COOlow Ca++
98
CH,SO;
98
MgCla KCI
I .5
1 .5
1.5
1.5
1.5
I .5
5
5
5
5
5
5
5
CaCI, (added)
2.0
2.0
2.0
2.4
2.0
2.4
2.0
relative (Cat+)
1.oo
-
1.03
I .02
0.75
I-Histidine
10
10
10
10
10
d-Glucose
10
10
10
10
10
NaC,H,COO
0.97
10
10
10
47 47 100% 0
1
80% 0 1%
2
co,
19% N, PH
*
0.98
10
NaCH,SO, Gas
1.5
7.42 7.0 6.7
7.4
80% 0, 80% O2 80% 0, 5 % CO, 13.5 % co, 13.5% COs 6.5% Na 15% N, 6.5% N, 7.4
7.4
7.4
100% 0,
7.4
1000, 0,
7.4
Control solution.
Cheniicals and solutions. The composition of the different solutions used is given in Table 1. Chemicals were of analytical grade and the water was double distilled in borosilicate glass. Sodium methyl sulphate was purchased from Hopkin and Williams Ltd. The gas mixtures were analyzed by the manufacturer and the concentrations stated were found t o be correct to -10.3 %. The p H of all solutions was adjusted by adding HCI or NaOH. During each experiment, p H was controlled intermittently and was not allowed to vary by more than k0.02 units. Histidine was chosen as a buffer because it was found to reduce the C a t + concentration of the solutions to only a small degree. In 3 experiments omission of histidine (10 mM) from the ‘medium-HCO;’ solution increased the Ca++ concentration by 6.5 % (range 5.7-7.5 %). In 4 control experiments it was demonstrated that the addition of 10 mM histidine to the ‘medium-HCOY’ solution, did not alter the isometric force of papillary muscles at contraction frequencies 12, 60and 120 beats/min. Determination of Ca++ concentration
It is well known that many ions can bind calcium and therefore the calcium ion concentration of the solutions was measured with a calcium sensitive electrode (Orion model 99-20) (Moore 1970). In Table I, the Cat+ concentration of the various solutions is given in units of the Cat+ concentration present in the ‘zero - HCO;’ solution. The C a t + concentration of the ‘zero - HCO.1’ solution was found not to be influenced by pH-alterations within the range 6.7-7.4. The Cat+ concentration was nearly identical in the ‘zeroHCOF’ solution and in the ‘medium-HC0;’-solution. However, an increase in HCOF concentration from 17 to 47 mM reduced Ca++ concentration by approx. 25 %. In order to reach the same Ca++concentration in the ‘high-HC08’ as in the ‘zero-HCO;’ solution, extra CaCI, was added to the latter solution. Reserpinization of animals
Reserpine (Serpasila, CIBA) was given intraperitoneally, 4 mg/kg, 18-24 h before the sacrifice of the animal. It has been demonstrated that most of the noradrenaline of sympathetic nerve terminals of the heart disappear after such a pretreatment of the animals (Spilker and Cervoni 1969).
298
M. J ~ H A N N S S O NAND E. NILSSON
Experimental procedures and evaluation of data An equilibration time of 60 min was allowed before the experiment was started. During the equilibration period the preparation was paced to contract at a frequency of 60/min. This stimulation frequency was also used during the whole experiment except for brief periods of stimulation at frequencies of I2 o r l20/ rnin. After the equilibration period the isometric twitch response was recorded at 12, 60 and 120 contractions/ min in the control solution (pH 7.4). in the test solution, and in the control solution again. The muscle bath was perfused with a given solution for at least I5 min before changing the stimulation frequency to 12 o r 120/min. The active force a t a frequency of 12/min was measured after attainment of steady state responses which occurred 3-5 rnin after switching to this frequency. At 120 stimulations/min the optimum isometric tension was measured, as generally no steady state was reached at this frequency. The optimum occurred within 1-3 min after altering the stimulation rate from 60 to 120/min. The steady state tension a t 60 stimulations/min was recorded before and after determining the isometric twitch amplitude at a frequency of 12 o r 120/min. Thus, in each solution the preparation was paced in the following order: 60, 12, 60, 120 and 60/min. The isometric twitch tension obtained at 12 and 120 contractions/min in a given solution is given as a fraction of the mean value of the twitch amplitude at 60 stimulations/min determined before and after the test period. In order to compare the peak isometric twitch tension in the control solution and in any of the test solutions the following procedure was used. The mean value of the twitch tension obtained in the control solution at 60 stimulations/min before and after perfusion with the test solution was taken as reference. Mean values of peak twitch tension at 12 and 120 stimulations/min in the control solution, as well as twitch amplitudes at all frequencies in the test solution, are given as fractions of the reference tension. In this way slow changes of contractility of the muscles with time could be allowed for. Only in experiments with the 'high-HCOT' solution was a slight, but consistent, irreversible deterioration of the preparation observed. In the figures, isometric tension is shown as meanfS.E. The statistical evaluation of inotropic effects of a test solution at a given contraction frequency was based upon the difference between the isometric force in the test solution and that in the control solution. A t-test based on paired observations was used.
Results A. Influence of extracellular p H on the isometric twitch
In the analysis of the effects of extracellular pH-changes on isometric twitch tension, solutions buffered with histidine alone at pH 7.4, 7.0 and 6.7 were used. The calcium concentration of the histidine solutions did not vary with pH within the range studied (see Methods). Measurements at pH 7.0 and 6.7 were started 15-30 rnin after a change from pH 7.4.When extracellular pH was lowered from 7.4 to 7.0 or 6.7,the active force of the papillary muscles (measured at 60 stimulations/min) diminished slowly and reached a steady state 1&15 rnin after alteration of pH. Recordings obtained up to 90 rnin after a lowering of pH revealed no further change in peak isometric tension. Fig. 1 shows the peak isometric force of the muscles at stimulation frequencies 12, 60 and 120/min. The mean value of the isometric tension at a stimulation frequency of 60/min in the solution of pH 7.4 is taken as unity (see Methods). As demonstrated in Fig. 1, a lowering of pH within the range 7.4-6.7caused a reduction of the isometric tension at all contraction frequencies studied. However, extracellular pH-changes did not alter the relative time course (kinetics) of the isometric twitch. Fig. 2 shows traces of typical isometric twitches at pH 7.4,and 6.7.Note that time to peak tension and the duration of the relaxation phase is not significantly affected by the pHchange.
299
ACID-BASE CHANGES IN MYOCARDIUM. I
Acid-Base Changes and Excitation-Contraction Coupling in Rabbit Myocardium. I. Effects on Isometric Tension Development at Different Contraction Frequencies BY
M. J~HANNSSON'and E. N~LSSON Received I August 1974
Abstract J~HANNSSON,M. and E. NILSSON. Acid-base changes and excitation-contraction coupling in rabbit myocardium. I. Effects on isometric tension development at different contraction frequencies. Acta physiol. scand. 1975. 93. 295-309. The effects of changes in acid-base parameters on the active force of isolated rabbit papillary muscles were studied at contraction frequencies of 12, 60 and 120/min. When extracellular p H was lowered from 7.4 to 7.0 and 6.7 in a bathing solution buffered with 10 mM histidine, the active force decreased at all contraction frequencies studied. After parallel increases of HCO; concentration (up to 47 mM) and Pco, at a constant extracellular pH of 7.4 the active force of the muscle increased at low and decreased at high contraction frequencies. None of these effects can be attributed to catecholamine release or to altered extracellular concentration of ionized calcium. The inotropic effects produced by bicarbonate were not reproducible by methyl sulfate (47 m M ) or propionate (47 mM). It is concluded that: 1. a lowering of the extracellular p H has a negative inotropic effect at all frequencies, 2. HCO; has a positive inotropic effect that is most pronounced at low contraction frequencies and 3. CO, has a negative inotropic effect exceeding that produced by the mere reduction in extracellular pH. The cellular mechanisms involved in the various inotropic effects are discussed. Key words: Rabbit heart, isometric force, contraction frequency, acidosis, HCO;,
Pcoz
From previous studies on isolated mammalian myocardial preparations it is not possible to form a clear opinion of how myocardial contractility is affected by different acid-base changes. It has been concluded that extracellular acidosis, within the patho-physiological range, does not affect contractility (Cingolani et al. 1970). Extracellular acidosis has also been found to cause a negative inotropic effect (Vaughan Williams and Whyte 1967). Evidence has been presented that a parallel rise in P,,, and HCO, concentration (at constant extracellular pH) does not alter the mechanical performance of isolated myocardial preparations (Vaughan Williams and Whyte 1967), that it induces a transient negative Present address: Department of Pharmacology, University of Iceland, Reykjavik, Iceland.
295
296
M. J ~ H A N N S S O NAND E. NILSSON
inotropic effect (Pannier and Leusen 1968) or causes a permanent depression of contractility (Cingolani et al. 1970). These discrepancies in results may be attributed to differences in both the myocardial preparation used (atrial or ventricular muscle and/or different species), and in the experimental conditions. An obvious difficulty in using a HCO;/CO, buffer system is the fact that extracellular pH, HCOi or P,,, cannot be altered individually, as at least one of the other two parameters will have to be changed simultaneously. Vaughan Williams and Whyte (1967) devised a statistical analysis to determine whether the inotropic effects observed after changes in P,,, or HCO; concentration are due to alterations of these parameters per se or to concomitant changes of extracellular pH. A more direct way of obtaining information about inotropic effects induced by extracellular pH-changes would be to use a non-bicarbonate buffer solution. In the present work the effects of extracellular acidosis on the mechanical performance of isolated papillary muscles were studied by varying the pH of a solution containing histidine as a buffer. With the histidine solution (of pH 7.4) as a control it was also possible to analyse the inotropic effects of parallel changes in HCO; concentration and Pco2 at a constant extracellular pH and to elucidate the effects caused by a separate change of HCO, concentration and P,,,.
Methods Preparation and mounting
Isolated papillary muscles of rabbits were used. The rabbits (weight 0.8-1.5 kg) were heparinized before sacrifice, and the heart was immediately removed and opened in oxygenated perfusion solution. The papillary muscle and its tendon was dissected from the right ventricle together with a small portion of the ventricle wall near the base of the muscle. Platinum loops were firmly tied with silk thread to the tendon and to the piece of the ventricular wall, as closely as possible to the insertions of the muscle. The length of the preparations, as determined with a microscope (10 fi magnification) varied within the range 3 to 6 mm and the largest diameter of the muscles was 0.3-0.9 mm. The preparation was mounted horizontally in a jacketed, temperature controlled bath (volume 1.5 ml) with the ventricle end connected to a hook and the tendon end attached to a semiconductor strain gauge transducer (Kyowa). The force transducer had a compliance of 1.3 ,um/mN and a resonant frequency of approximately 500 Hz with the preparation mounted. A linear response was obtained for forces between 0.2 and 50 mN. The signal from the transducer was displayed on a Tektronix 502 A oscilloscope and an Elema Schhander Mingograph (frequency response 500 Hz). The muscle was stimulated by passing current through two pairs of platinum wire electrodes placed on one side of the preparation perpendicular to the long axis of the muscle (distance between two cathodes was 2 mm). The pulse duration was 2 ms and the stimulus intensity was 5 0 % above the threshold value. The resting length of the muscle was chosen approximately 5 % below the length found to be optimal for active force. Perfusion of muscle bath
Prior to its inflow into the muscle chamber, the perfusion solution was equilibrated in a thermostated glass jar with the gas or gas mixture indicated in Table 1. The pH of the solution was adjusted before the perfusion was started and controlled during the perfusion (see below). The rectangular muscle bath was perfused at a rate of 2-4 ml/min to give 90% exchange in about 80 s. Temperature control. Temperature within the muscle chamber was kept constant at 37"iOS"Cand was monitored by means of a thermistor in the muscle bath. In any given experiment, temperature variation was less than j O . 3 T . It was specially checked that this value was not exceeded during shift from one buffer solution to another.
297
ACID-BASE CHANGES IN MYOCARDIUM. I
TABLEI. Composition of solutions. mM
zeroHCOT
lowHCO;
mediumHC03
NaCl
145
142
128
98
98
17
47
47
NaHCO,
3.4
high-HCOT high HCOT- C,H,COOlow Ca++
98
CH,SO;
98
MgCla KCI
I .5
1 .5
1.5
1.5
1.5
I .5
5
5
5
5
5
5
5
CaCI, (added)
2.0
2.0
2.0
2.4
2.0
2.4
2.0
relative (Cat+)
1.oo
-
1.03
I .02
0.75
I-Histidine
10
10
10
10
10
d-Glucose
10
10
10
10
10
NaC,H,COO
0.97
10
10
10
47 47 100% 0
1
80% 0 1%
2
co,
19% N, PH
*
0.98
10
NaCH,SO, Gas
1.5
7.42 7.0 6.7
7.4
80% 0, 80% O2 80% 0, 5 % CO, 13.5 % co, 13.5% COs 6.5% Na 15% N, 6.5% N, 7.4
7.4
7.4
100% 0,
7.4
1000, 0,
7.4
Control solution.
Cheniicals and solutions. The composition of the different solutions used is given in Table 1. Chemicals were of analytical grade and the water was double distilled in borosilicate glass. Sodium methyl sulphate was purchased from Hopkin and Williams Ltd. The gas mixtures were analyzed by the manufacturer and the concentrations stated were found t o be correct to -10.3 %. The p H of all solutions was adjusted by adding HCI or NaOH. During each experiment, p H was controlled intermittently and was not allowed to vary by more than k0.02 units. Histidine was chosen as a buffer because it was found to reduce the C a t + concentration of the solutions to only a small degree. In 3 experiments omission of histidine (10 mM) from the ‘medium-HCO;’ solution increased the Ca++ concentration by 6.5 % (range 5.7-7.5 %). In 4 control experiments it was demonstrated that the addition of 10 mM histidine to the ‘medium-HCOY’ solution, did not alter the isometric force of papillary muscles at contraction frequencies 12, 60and 120 beats/min. Determination of Ca++ concentration
It is well known that many ions can bind calcium and therefore the calcium ion concentration of the solutions was measured with a calcium sensitive electrode (Orion model 99-20) (Moore 1970). In Table I, the Cat+ concentration of the various solutions is given in units of the Cat+ concentration present in the ‘zero - HCO;’ solution. The C a t + concentration of the ‘zero - HCO.1’ solution was found not to be influenced by pH-alterations within the range 6.7-7.4. The Cat+ concentration was nearly identical in the ‘zeroHCOF’ solution and in the ‘medium-HC0;’-solution. However, an increase in HCOF concentration from 17 to 47 mM reduced Ca++ concentration by approx. 25 %. In order to reach the same Ca++concentration in the ‘high-HC08’ as in the ‘zero-HCO;’ solution, extra CaCI, was added to the latter solution. Reserpinization of animals
Reserpine (Serpasila, CIBA) was given intraperitoneally, 4 mg/kg, 18-24 h before the sacrifice of the animal. It has been demonstrated that most of the noradrenaline of sympathetic nerve terminals of the heart disappear after such a pretreatment of the animals (Spilker and Cervoni 1969).
298
M. J ~ H A N N S S O NAND E. NILSSON
Experimental procedures and evaluation of data An equilibration time of 60 min was allowed before the experiment was started. During the equilibration period the preparation was paced to contract at a frequency of 60/min. This stimulation frequency was also used during the whole experiment except for brief periods of stimulation at frequencies of I2 o r l20/ rnin. After the equilibration period the isometric twitch response was recorded at 12, 60 and 120 contractions/ min in the control solution (pH 7.4). in the test solution, and in the control solution again. The muscle bath was perfused with a given solution for at least I5 min before changing the stimulation frequency to 12 o r 120/min. The active force a t a frequency of 12/min was measured after attainment of steady state responses which occurred 3-5 rnin after switching to this frequency. At 120 stimulations/min the optimum isometric tension was measured, as generally no steady state was reached at this frequency. The optimum occurred within 1-3 min after altering the stimulation rate from 60 to 120/min. The steady state tension a t 60 stimulations/min was recorded before and after determining the isometric twitch amplitude at a frequency of 12 o r 120/min. Thus, in each solution the preparation was paced in the following order: 60, 12, 60, 120 and 60/min. The isometric twitch tension obtained at 12 and 120 contractions/min in a given solution is given as a fraction of the mean value of the twitch amplitude at 60 stimulations/min determined before and after the test period. In order to compare the peak isometric twitch tension in the control solution and in any of the test solutions the following procedure was used. The mean value of the twitch tension obtained in the control solution at 60 stimulations/min before and after perfusion with the test solution was taken as reference. Mean values of peak twitch tension at 12 and 120 stimulations/min in the control solution, as well as twitch amplitudes at all frequencies in the test solution, are given as fractions of the reference tension. In this way slow changes of contractility of the muscles with time could be allowed for. Only in experiments with the 'high-HCOT' solution was a slight, but consistent, irreversible deterioration of the preparation observed. In the figures, isometric tension is shown as meanfS.E. The statistical evaluation of inotropic effects of a test solution at a given contraction frequency was based upon the difference between the isometric force in the test solution and that in the control solution. A t-test based on paired observations was used.
Results A. Influence of extracellular p H on the isometric twitch
In the analysis of the effects of extracellular pH-changes on isometric twitch tension, solutions buffered with histidine alone at pH 7.4, 7.0 and 6.7 were used. The calcium concentration of the histidine solutions did not vary with pH within the range studied (see Methods). Measurements at pH 7.0 and 6.7 were started 15-30 rnin after a change from pH 7.4.When extracellular pH was lowered from 7.4 to 7.0 or 6.7,the active force of the papillary muscles (measured at 60 stimulations/min) diminished slowly and reached a steady state 1&15 rnin after alteration of pH. Recordings obtained up to 90 rnin after a lowering of pH revealed no further change in peak isometric tension. Fig. 1 shows the peak isometric force of the muscles at stimulation frequencies 12, 60 and 120/min. The mean value of the isometric tension at a stimulation frequency of 60/min in the solution of pH 7.4 is taken as unity (see Methods). As demonstrated in Fig. 1, a lowering of pH within the range 7.4-6.7caused a reduction of the isometric tension at all contraction frequencies studied. However, extracellular pH-changes did not alter the relative time course (kinetics) of the isometric twitch. Fig. 2 shows traces of typical isometric twitches at pH 7.4,and 6.7.Note that time to peak tension and the duration of the relaxation phase is not significantly affected by the pHchange.
299
ACID-BASE CHANGES IN MYOCARDIUM. I
