J. exp. Biol. (198a), 96, 4°5-4ia
With 3 figures
Printed m Great Britain
405
pH,, CONTRACTILITY AND Ca-BALANCE UNDER
HYPERCAPNIC ACIDOSIS IN THE MYOCARDIUM
OF DIFFERENT VERTEBRATE SPECIES
BY H. GESSER AND E. J0RGENSEN
Department of Zoophysiology, University of Aarhus, DK-8000
Aarkus C, Denmark
(Received 6 May 1981)
SUMMARY
The influence of hypercapnic acidosis upon the heart was examined in
four vertebrate species. The CO2 in the tissue bath was increased from
2-7 to 15 % at 12 °C for flounder (Platichthys flesus) and cod (Gadus morhua)
and from 3 to 13% at 22 °C for turtle (Pseudemys scripta) and rainbow
trout (Salmo gairdneri).
During hypercapnia, as previously described, there was a decline and
recovery of contractility in heart strips of flounder and turtle, and a sustained
decrease in cod and rainbow trout.
At high COg the increase in contractile force following increases yi the
extracellular Ca-concentration were smaller for the cod myocardium than
for the other myocardia.
The intracellular pH (pHJ, measured with the DMO method, in heart
strips of turtle and trout was significantly lowei at high than at low C0 2 .
This acidifying effect expressed as the increase in the intracellular concentration of hydrogen ions was larger in the turtle than in the trout myocardium.
Intracellular Ca-activity, measured by efflux of "Ca from preloaded
heart strips, was unaffected by high C0 2 in trout, but was raised in the other
three species.
Thus the ability to counteract the negative inotropic effect of hypercapnia
is apparently not due to cellular buffering or extrusion of hydrogen ions.
More probably it involves (a) a release of intracellular Ca; (b) a positive
inotropic effect of an increase in intracellular Ca-activity.
INTRODUCTION
In general, the contractility of the vertebrate heart is depressed by hypercapnic
acidosis. This appears to be due mainly to an antagonism between hydrogen ions and
the inotropic effect of Ca2+ (Fabiato & Fabiato, 1978; Williamson et al. 1976).
However, the myocardium of some vertebrates recovers contractility when the
hypercapnia has lasted for a few minutes (Foex & Fordham, 1972; Gesser & Poupa,
1978, 1979; Poupa, Gesser & Johansen, 1977). Evidence was obtained that this
recovery involves a release of intracellular Ca-stores making more Ca available to the
contractile system (Gesser & Poupa, 1978). In mammalian cardiac cells a large
406
H. GESSER AND E. J0RGENSEN
transient decrease in pH^ followed by a partial recovery has been observed during
hypercapnia (Ellis & Thomas, 1976). Thus, alternative explanations based on a
removal of hydrogen ions from the cytoplasm cannot be excluded.
In this paper, these tentative explanations are further examined in heart tissue of
flounder, turtle, cod and rainbow trout by measuring the influence of hypercapnia on
the pH^ on the intracellular Ca-activity and on the relationship between the extracellular Ca2+ concentration and the contractility. The myocardia of flounder and turtle,
in contrast to the other two, efficiently recover contractility during hypercapnia
(Poupa e£ a/. 1977; Gesser & Poupa, 1979).
MATERIALS AND METHODS
Heart strips were isolated from flounder (PleuronectesflesusL.), cod (Gadus
morhua L.), rainbow trout (Salmo gairdneri Richardson), and diving turtle (Pseudemys
scripta L.). Each animal was killed by decapitation. Strips with a diameter less than
1 mm were rapidly prepared from the cardiac ventricle and used for recording of
contractile force, pHj or *5Ca-efnux. For the cardiac muscle of flounder and cod the
physiological solution had a temperature of 12 °C and was composed of 150 mMNaCl,
5-25 mM-KCl, i-8o mM-MgSO4. It was bubbled with a mixture of 85% Oa and
either 2-7% CO2 and 12-3% Na or 15% CO2. For rainbow trout and turtle heart the
experimental temperature was 22 CC and the solution was composed of 125 mM-NaCl,
5-14 mM-KCl, 0-94 mM-MgSO4 and bubbled with 87% O2, and either 3 or 13 % CO2
(with 10% N a in the case of 3 % CO2). Both solutions contained in addition 30 mMNaHCO3, 1 mM-NaH2PO4, 1 mM-CaCl2 and 5 mM-glucose. The gas mixture was
delivered by two gas mixing pumps (Wtisthoff 1 M-301/a-F) arranged in serial. The
pH of both Ringers was 76 at low CO2 and 6-9 at high. The Ca2+ concentration of the
bath was increased by adding different volumes of 1 M-CaCla. Radiochemicals were
"Ca ( ~ 0-5 Ci/mmol) (Danish Atomic Energy Commission, Isotope Laboratory,
Rise), P*C]DMO (5-5-dimethyloxazolidine-2-4-dione-2-14C, 55 /iCi/mmol) and
PHJinulin (1-21 Ci/mmol) (Radiochemical Centre Amersham, England).
The contractile force of cardiac strips developed upon electrical stimulation
(12 pulses/min) was recorded under isometric conditions as described previously
(Gesser & Poupa, 1978). The strips were stretched until no further increase in contractile force was observed, and they were allowed to stabilize for at least 15 min.
The pHi of unstretched, resting cardiac strips of turtle and trout was determined
from the distribution of DMO between the extracellular and intracellular water
(Wadell & Butler, 1959). The strips were kept for 30-60 min at low CO2 in an unlabelled Ringer. They were then transferred to a Ringer (0-5 ml/strip) containing
0-2 /iCi [MC]DMO and 1 fiCi pHJinulin together with 1 mM of the unlabelled forms
of DMO and inulin. The Ringer was aerated with either high or low CO2. To allow the
substances to attain steady-state distribution, incubation lasted 30-60 min for turtle
and 60-90 min for trout. Thereafter the strips were washed for less than a second in an
unlabelled Ringer, blotted with filter paper and cut into two pieces. After weighing,
one piece was dried for about 24 h at 110 °C to obtain its total water content. The other
was used for radioactivity determinations, after first boiling in 2 ml distilled water for
about 45 min. After the addition of 200 (A 50% trichloric acid (TCA) and centrifugaj
i contractility in different vertebrate species
407
22 °C
15%CO 2
Flounder (7)
13%CO2
Turtle (6)
Cod (6)
I Turtle (5)
~\ Flounder (7)
Trout(5)
13%CO2
Trout(5)
15%CO 2
Cod (6)
10
Time (min)
Fig. 1. Contractile force of electrically stimulated myocardial strips at low and high CO,.
Number of animals in brackets.
tion, 1 ml of the solution was transferred to a vial for scintillation counting and mixed
with 10 ml of a solution obtained by mixing 0-53 1 toluene, 0-33 1 Triton-x-100,
0-13 1 ethanol and 1-33 g Permablend III (Packard). 200 /A of the radioactive incubation solutions were added to 1800/d water and prepared for radioactivity determinations as above with boiling etc. Radioactivity was measured by scintillation spectrophotometry (Beckman LS 250). The pr^ was obtained knowing the total tissue water
and using inulin as an extracellular marker. The pKx of D M 0 was calculated using
the formula given by Malan, Wilson & Reeves (1976).
Intracellular Ca-activity was measured by efflux of 48Ca (Ashley, Caldwell & Lowe,
1972; Schatzmann, 1973) in resting, unstretched strips. Each strip was incubated for
2 h in 1 ml Ringer with 4 /*Ci "Ca/ml. After being washed three times for 20 min in
about 15 ml of an unlabelled Ringer, it was transferred through a series of baths of 2 m
Ringer (for a total of 10 min) at either high or low CO2. Finally the strip was digested
overnight in 30% H2O2 at n o °C. The residual was dissolved in 2 ml H2O. To this
solution and to each of the 2 ml portions of the incubation series 0-2 ml 50 % TCA
was added. One ml of each solution was transferred to a plastic vial containing 10 ml
of the Triton-1 oo/toluene scintillation solution. From measurements of the radioactivity the efflux rate was calculated as the fraction of the i5 Ca 2+ contained in the
tissue washed out per minute.
The results are presented as mean + SE, and Student's f-test was applied to evaluate
differences. The level of significance was 0-05.
408
H . GESSER AND E . J0RGENSEN
3001—
Flounder
22 °C
2-7% CO 2
Cod
Trout3%C02
Turtle 3% COj
Flounder
Turtle 13%COj
Trout 13%CO 2
15%C0j
Cod
20
5
5
mM-Ca3+
mM-Ca2+
Fig. a. Relation b e t w e e n contractile force and extracellular Ca-concentration at l o w and high
C O , . Forces were measured after 30 m i n at 1 ml Ca, and after 10 m i n for each successive
increase in Ca level. N u m b e r of animals as in Fig. 1.
RESULTS
Contractile force under hypercaprdc acidosis
The contractile force developed upon electrical stimulation of myocardial strips
decreased rapidly, to around 7 0 % in 5 min, after CO a was increased (Fig. 1). Myocardia of cod and trout then continued to lose force, while those from flounder and turtle
recovered force. The flounder myocardium achieved a level well above the original.
Contractile force and the extracellular Cai+
At both high and low CO 2 , greater force was developed as Ca 2+ concentrations were
raised, especially in the range 1-3 mM, which is probably the physiological range
(Prosser, 1973; Ruben & Bennett, 1981). In cod and trout high CO 2 depressed the
inotropic effect of Ca*+. At r mM-Ca 2+ in flounder and turtle high CO a enhanced the
force. At higher Ca 2 + concentrations, high CO 2 depressed the force for flounder but
had no significant effect for turtle (Fig. 2).
The pHj of myocardia from turtle and trout was lower at high than at low CO 2
(Table 1). The intracellular concentration of hydrogen ions rose b y i i ' 4 ( ± i * i ) x
io" 8 M for turtle and 6-4 ( ± 01) x io" 8 M for trout. Thus, the acidifying effect of the
increase in CO 2 was larger for the turtle than for the trout myocardium.
contractility in different vertebrate species
409
Table 1. pHit total and extracellular water content given as % of tissue wet weight, and
intracellular concentration of hydrogen ions ((H+)ic) calculated as antilog —pH, at low
and high COt for cardiac tissue of turtle and trout
The pHj of a tissue sample was calculated using the values of total and extra-cellular water from
the same sample. Values are mean ± SE ; n = number of animals.)
(H+)<<, x io 8
pH4
13%
Tissue
water
3%
13%
Extracellular
water
3 % CO,
13% CO,
3%
3%
13%
7-i 3 ±o-O3
n =• 12
6-74±oo6
n= 8
7-710-6
n = 12
Turtle
19-613-2
n= 8
77±i
8o±2
n •= 12 n = 8
37±2
n = 12 n = 8
7'34±o-2i
n = 14
6-99±o-c>9
n=i7
4 - 8±o-4
n = i4
Trout
II-2±I-6
n = i7
75±3
72±i
n = 22 n = 15
*4±i
2o±i
n = i ; n ° 20
The total and the extracellular water were not affected by the difference in CO2
except that the extracellular water of the trout myocardium was somewhat lower at
high CO2.
u
Ca-efflux
Increase in CO2 resulted in an increased "Ca-efflux rate in all myocardia except for
that of trout (Fig. 3).
DISCUSSION
As observed earlier the myocardium generally loses force when made hypercapnic.
In some myocardia, however, like those of flounder and turtle, contractile force is
recovered in spite of a continuing acidosis (Fofix & Fordham, 1972; Gesser & Poupa,
1978,1979; Poupa et al. 1977).
Mammal myocardial cells have been found to respond to hypercapnia by a sharp
decrease in pH4 followed after a few minutes by a partial recovery (Ellis & Thomas,
1976). Fry & Poole-Wilson (1981) used this observation in explaining their finding of a
partial recovery of force in the hypercapnic myocardium of guinea pig and rabbit.
More generally they suggested that the myocardial contractile force under respiratory
or metabolic acidosis mainly varies in parallel with the pH i . The results of the present
study, however, suggest that the force recovery under hypercapnia is not solely due
to a recovery of pH4.
Hypercapnia induced a lasting acidification of the myocardium, which was more
pronounced in the turtle than in the trout. This is compatible with the finding of a
lower non-bicarbonate buffer value in the turtle than in the trout myocardium
(Damm Hansen & Gesser, 1980). In spite of these differences the turtle myocardium,
in contrast to that of trout, recovered force efficiently under hypercapnia.
It has earlier been hypothesized that hypercapnia causes a release of intracellular
Ca-stores. This would result in an enlarged pool of Caa+ available for contraction,
counteracting the negative inotropic effects of hydrogen ions (Gesser & Poupa, 1978).
is hypothesis is supported by the estimated increase in intracellular Ca-activity in
H . GESSER AND E . J0RGENSEN
410
0010
Flounder
^
1o
Cod
0-005
n °7
tJ
2-7-15%COi
e
u.
2-7-15%COi
0010
I
S
0005 -
60
Time (min)
Fig. 3. uCa-efflux of myocardial strip at low CO, (O) and high CO, ( • ) . n = number of
animals.
myocardia of flounder and turtle, both of which showed efficient force recovery, and
by the lack of change in the trout myocardium, which did not recover.
Observations for the cod heart, however, do not support the above hypothesis.
During hypercapnia no recovery of force could be observed in spite of a sharp rise in
the estimated concentration of intracellular Ca. Perhaps factors other than the
cytoplasmic Ca-activity limit the contractile force developed by the hypercapnic cod
myocardium. This is supported by the finding that the positive inotropic effect of an
increased extracellular Ca-concentration was much less expressed in the cod myocardium than in the other myocardia under hypercapnia.
i contractility in different vertebrate species
411
E
At concentrations of extracellular Ca + above 3 mM the force of the flounder
myocardium attained a maximal value well below those recorded at low C0 a . Thus,
above a certain level of force development in this tissue, hypercapnia results in an
inhibition of the contractility which cannot be removed by Ca2+. This is in accordance
with the previous finding of a more expressed recovery at a low than at a high extracellular Ca-concentration (Gesser & Poupa, 1979). At variance with this situation the
force of the turtle myocardium levelled off at similar values at low and high CO2. In
the trout myocardium no levelling off of force could be observed within the Ca-range
tested.
The hypothesis that the recovery of force under hypercapnia is due to an increased
intracellular Ca-activity must, however, be viewed cautiously. Hess & Weingardt
(1980) found a simultaneous decrease in both Ca-activity and pFL, in sheep Purkinje
cells during 5 min of hypercapnia. It should, however, be recalled that the recovery
of force can only be observed for hypercapnic periods lasting more than about 5 min.
A release of let us say mitochondrial Ca (Gesser & Poupa, 1978) may be a relatively
slow process.
To conclude, the variation in ability to maintain myocardial force under hypercapnic acidosis does not appear to be due to a varying ability to regulate intracellular
pH. Rather it appears to depend on a varying release of intracellular Ca2+-stores
combined with a varying inotropic response to increases in the cytoplasmic Caactivity.
This study was supported by the Danish Natural Science Research Council.
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