481
Journal of Physiology (1990), 422, pp. 481-497 With 8 figures Printed in Great Britain
DECREASED SENSITIVITY OF CONTRACTION TO CHANGES OF INTRACELLULAR pH IN PAPILLARY MUSCLE FROM DIABETIC RAT HEARTS
BY D. LAGADIC-GOSSMANN AND D. FEUVRAY From the Laboratoire de Physiologie Comparee et Laboratoire de Biomembranes et des Ensembles Neuronaux Associe au Centre National de la Recherche Scientifique, Universite Paris XI, 91405 Orsay, France
(Received 28 June 1989) SUMMARY
1. The relationship between intracellular pH (pHi) and contractile activity was investigated in papillary muscles isolated from right ventricle of normal and streptozotocin (STZ)-induced diabetic rats. pHi changes induced by 20 mm-NH4Cl were recorded with H+-sensitive microelectrodes. 2. An increase in pHi of 0-20 pH units on exposure to NH4C1 led to an increase of the maximum developed tension, which was 70788+57-5% (mean + S.E. of mean, n = 10) of control in normal muscles and 271 + 16-3 % (n = 10) in diabetic muscles. On the other hand, acidosis induced by NH4C1 withdrawal was associated with a fall in developed tension to 48'2 + 67 % of control in diabetic muscles, as compared to 79-2 + 8 % in normal muscles. 3. The decrease in tension associated with acidosis was rapidly followed (in 2 min) by a transient redevelopment of force, which peaked at 80-2 + 8-6 % of control in the diabetic nmuscles as compared to 1535 + 11-7 % in normal papillary muscles. The peak of this secondary positive inotropy coincided in both groups of muscles with the maximum decrease of pHi, i.e. - 0-40 + 0-02 and - 0-28 + 0-04 pH units in diabetic and normal muscles, respectively. 4. Caffeine (10 mm), which had a marked positive inotropic effect in both groups of muscles, abolished the transient recovery of tension occurring after NH4C1 withdrawal. Ryanodine (2 /tM) which had a marked negative inotropic effect on both normal and diabetic papillary muscles, also suppressed the transient recovery of tension. 5. The presence of amiloride (1 mm) during acidosis induced by NH4C1 withdrawal abolished the observed differences in developed tension, in particular the transient recovery of tension, between normal and diabetic muscles, as it abolished the differences in the amplitude of pHi decrease and in the time course of pHi recovery. 6. The presence of 2',4'-dichlorobenzamil amiloride (40 ,tM) significantly and similarly delayed and reduced the amplitude of transient recovery of tension in both normal and diabetic papillary muscles. 7. We conclude that STZ-induced diabetes induces a decrease in pHi sensitivity of contractile force. This may be the consequence of a change in sarcoplasmic reti-
MS 7794
16
PHY 422
482
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
culum (SR) composition and function, and may also indirectly result from changes in Na+-H+ exchange activity, particularly during intracellular acidosis. INTRODUCTION
Chronic diabetes mellitus is frequently associated with depressed cardiac function, even in the absence of atherosclerotic coronary disease (Fein & Sonnenblick, 1985). Most of the experimental studies concerning the alterations of mechanical function associated with diabetes have been performed using drug-induced diabetic rats. Fein, Kornstein, Strobeck, Capasso & Sonnenblick (1980) have shown that isolated papillary muscles from streptozotocin (STZ)-induced diabetic rats have a decreased shortening velocity and slowed rate of relaxation. Also, a decreased binding and uptake of Ca2+ by sarcoplasmic reticulum (SR) has been shown in diabetic rat hearts (Penpargkul, Fein, Sonnenblick & Scheuer, 1981; Ganguly, Pierce, Dhalla & Dhalla, 1983; Lopaschuk, Katz & McNeill, 1983). However, these abnormalities were probably not the only factors responsible for diabetes-induced myocardial depression (Tahiliani & McNeill, 1986). Other studies have pointed out a decrease in the sensitivity of diabetic hearts to external Ca2+(Bielefeld, Pace & Boshell, 1983; Sauviat & Feuvray, 1986). Another significant defect in diabetes is the increase in free fatty acid uptake and utilization, and the inhibition of glucose transport across the cell membrane due to lack of insulin (Randle, Garland, Hales, Newsholme, Denton & Pogson, 1966). The resulting decrease in myocardial glucose utilization (Feuvray, Idell-Wenger & Neely, 1979) might be associated with a change in proton production. However, we have recently shown that there was no difference between the steady-state intracellular pH (pHi) values recorded in papillary muscles from diabetic or normal rat hearts. However, differences between diabetic and normal muscles were found in the regulation of pHi where diabetes had induced a decrease in the activity of the amiloride-sensitive Na+-H+ exchange (Lagadic-Gossmann, Chesnais & Feuvray, 1988). By its influence on both pHiand Na+, the activity of the Na+-H+ exchange may well participate in the control of cardiac contractility (Vaughan-Jones, Eisner & Lederer, 1987). Indeed, close relationships have been demonstrated between the mechanisms controlling Ca 2+ and pHi (Deitmer & Ellis, 1980; Bers & Ellis, 1982; Vaughan-Jones, Lederer & Eisner, 1983). In particular, the membrane control of Ca1+ and pHi in heart relies upon a common ion (i.e. Na+) through the activities of both the Na+-H+ and the Na+-Ca2+ exchangers (Vaughan-Jones, 1988). Therefore, a decrease in the activity of the former may have indirect consequences on the contractile activity of diabetic hearts by altering the amount of Caf2 triggering the release of Ca2+ from the SR (Fabiato, 1985 a). The present study was designed to investigate the effect of diabetes on the relationship between pHi and contractile activity. We also examined the influence of transmembrane ionic exchangers and the possible role of the SR. The experiments were carried out on papillary muscles of normal and STZ-induced diabetic rat hearts. The results show that diabetes induces a decrease in the sensitivity of contractile force to a change in pHi. This may be the consequence of an altered SR function
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 483 associated with diabetes, or may indirectly result from decreased Na+-H+ exchange activity, particularly during intracellular acidosis. METHODS
The methods used here were similar to those described previously (Lagadic-Gossmann et al. 1988). Briefly, male Wistar rats weighing 250-300 g were fasted overnight and made diabetic by the injection of streptozotocin (STZ, Sigma, St Louis, MO, USA; 40 mg/kg) into the femoral vein. Streptozotocin-treated and age-matched control animals were maintained on the same diet until they were used 4-5 weeks later. The diabetic state was assessed by measurement of non-fasting glucose concentration in blood samples collected at the time of heart excision. Mean glucose levels were 9-5 + 0 7 and 35-7 + 2-1 mm for normal and diabetic rats, respectively. Rats were anaesthetized with pentothal (5 mg/100 g body weight, i.p.). The heart was removed rapidly and rinsed in Krebs bicarbonate buffer solution (see below) at 36 °C. A thin uniform papillary muscle (< 1 mm in diameter, 2-3 mm in length; same size in normal and diabetic groups) was dissected from the right ventricle, pinned to the bottom of the experimental chamber and superfused with Krebs bicarbonate buffer at 36+1 °C at a rate of 8-4 ml/min. The fluid volume around the papillary muscle was approximately 1-4 ml. The preparation was allowed to equilibrate for at least 30 min in the control buffer solution before recordings were obtained. Measurements of intracellular pH Measurements of pHi were made in superficial cells of quiescent preparations, using resin-filled H+-selective microelectrodes (Ammann, Lanter, Steiner, Schulthess, Shijo & Simon, 1981). The microelectrodes were connected to a high-impedance electrometer (FD 223, World Precision Instruments) and monitored via a pen recorder.
Tension measurements The papillary muscle was mounted horizontally between a fixed hook and a tension transducer, and was superfused continuously with Krebs bicarbonate buffer solution at 36 +1 °C. The isometric mechanical activity was displayed on a Gould paper recorder. After 30 min of superfusion, the muscle was stretched stepwise to maximum length (Lmax). Both field and focal stimulation were used. The muscle was stimulated at 1 Hz (8-10 ms duration pulses, twice threshold) either between two platinum wires positioned parallel to the muscle length or focally by punctate electrodes. Tension and pHi were not recorded simultaneously because of vigorous contractions of the rat papillary muscles which make stable impalements by microelectrodes very difficult. Therefore, pHi changes were recorded in experiments similar to those in which tension was measured, but without stimulation. In these experiments muscle length was not systematically fixed. However, each muscle had similar resting tension and we observed that steady-state pH, values were not affected by changing muscle length in control superfusion conditions.
Solutions The control Krebs bicarbonate buffer solution used in the experiments contained (mM): NaCl, 118; KCl, 4-8; CaCl2, 1-75; MgCl2.6H2O, 12; KH2PO4, 12; NaHCO3, 25; EGTA, 0 5; glucose, 10; sucrose, 15. This solution was equilibrated with 95 % 02-5 % CO2 to give a pH of 7-4. Intracellular alkalinizations and acidifications were induced by the NH4+ method (Boron & De Weer, 1976). In solutions which contained 20 mM-NH4Cl, the NaCl concentration was decreased to compensate for osmotic changes. The NH4Cl exposure lasted 15 min. Amiloride (Sigma, 1 mM) was used to block Na+-H+ exchange (Benos, 1982); 2',4'-dichlorobenzamil amiloride (DCB, given by Dr D. Escande, Rhone Poulenc France; 40 /uM) was used to inhibit Na+-Ca2+ exchange; caffeine (Sigma; 10 mM) and ryanodine (Calbiochem, France; 2 /tM) were used as inhibitors of the SR. Statistics Results are reported as the mean + S.E. of mean of n preparations. Comparisons between groups were made using Student's t test. Slopes of linear regressions were also compared using Student's 16-2
484
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
t test. A value for P of 0 05 was chosen as the threshold to reject the null hypothesis that there was no difference in means between groups. RESULTS
The effect of diabetes on the response of tension to ammonium chloride Addition of NH4Cl to the superfusing solution causes an intracellular alkalinization and its removal causes an intracellular acidification (Boron & De Weer, 1976) with A
20 mM-NH4CI
Tension 1 mNc --
bcd
a
e
6-7
pHi 71[ 7.37.5 L
'5 min'
B 20 mM-NH4CI Tension 1 mN[
bLcI I
a
pH
I
I
I
e
6.5 6.7 6.9
723 7.5
'5 min Fig. 1. The effect of exposure to, and withdrawal of, NH4Cl on tension and pHi of normal (A) and diabetic (B) papillary muscles. In each panel, traces show: developed tension (top) and pHi (bottom). The period during which NH4C1 was added to the superfusion solution is indicated above the record. Note the different scale for tension in each panel. Specific points are indicated by letters (a-e) on the top traces of each panel.
no change of pHe. Figure 1 shows representative superimposed recordings of tension and pHi in a normal papillary muscle (Fig. 1A) and in a diabetic papillary muscle (Fig. 1B) before, during and after exposure to 20 mm-NH4Cl. In agreement with earlier studies (Lagadic-Gossmann et al. 1988), we found no difference in steady-state pHi values between the two groups of muscles (7 07 + 0-02 and 7 06 + 0-01, n = 10, for normal and diabetics, respectively). Likewise, no statistical difference appeared in developed tension between normal (I13 +0-1 mN, n = 10) and diabetic (105 + 012 mN, n = 10) papillary muscles superfused in control conditions. This
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 485 agrees with previous observations by others (Fein et al. 1980; Takeda, Nakamura, Hatanaka, Ohkubo & Nagano, 1988). The addition and subsequent withdrawal of NH4Cl led to transient changes of pHi which were accompanied by marked changes of developed tension in both types of 800 700 c-
o2
600500 400 300-
Co
a,
cc
200
100* 0AI Control
a
b
c
d
e
Fig. 2. Amplitude of developed tension, relative to its level in control superfusion solution, at specific points (a-e) indicated in Fig. 1, in normal (1) and diabetic (-) papillary muscles. Error bars show S.E.M. The number of separate experiments was ten. * Significantly different (P < 0001) responses between normal and diabetic muscles.
muscles although to a different degree (Fig. 1). The initial alkalosis on exposure to NH4C1 was associated with a rapid increase of developed tension. As pHi became less alkaline during the sustained exposure to NH4C1, there was also a decrease in tension. Conversely, the transient acidification on removal of NH4Cl was accompanied by an immediate decrease of tension. However, this decrease was interrupted by a rapid transient redevelopment of tension which reached a maximum after 2 min and then slowly recovered towards control values. Such a sequence of events has already been described in ferret papillary muscles under comparable experimental conditions (Orchard, 1987). In our study these events were qualitatively similar in papillary muscles of both normal and diabetic rat hearts, but their amplitude differed significantly. Thus, as shown in Fig. 2, the maximum developed tension (a) in response to the same increase in pHi (- + 0-20 pH units) during the NH4Cl pulse was only 271 + 16-3 % of control (n = 10) in diabetic muscles against 707-8 + 57.5% of control (n = 10) in normal muscles. Yet the maximum tension, although considerably less in the diabetics, was reached with the same half-time (ti) as in normal muscles (47-8 + 28 and 48-2 + 31 s, respectively). At the end of the exposure (b) a significant difference in developed tension still persisted between the two groups. Removal of NH4+ caused a rapid intracellular acidification, the amplitude of which was significantly greater in the diabetic muscles. Moreover, the subsequent recovery from acidification was slower in the diabetics, due to the reduced activity of the Na+-H+ exchange (Lagadic-Gossmann et al. 1988). Again, the decrease of developed tension (c in Figs 1 and 2) which appeared concomitant with acidosis induced by NH4+ withdrawal was different in its amplitude in the two types of muscles. Developed tension fell to 79-2 + 8% of control (n = 10) in normal muscles and to 48-2 + 6-7 % (n = 10) in the diabetics. However, when compared with tension values before NH4Cl withdrawal, the fall of tension was 53 %, associated with an acidification of
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
486 A
10 T a = 4.95: r = 0-99 c
0 .°4 c
0
> _
1
a = 3.16: r= 0.98
0
-j
0.1 10.3
0-1
0.2
-0.2
-0.1
0
-0.3
-0.4
ApHi B 10-
+ 10 mM-caffeine
a = 3.29: r = 0.96
C
0
._4
C
0
0
2
-W
1-
a = 3.52: r= 0.98
0
-J
0-1 0-2
0.3
-0.1
0
0-1
-02
-0.3
-0.4
ApHi C
10 r
+
C 0 co
c 0
a
1-
=
5.55:
r=
2 pM-ryanodine
0.98
0 0 L-
0.1
a
=
6-68:
r=
0.95
0
-J
0.01 0.3
0.2
0.1
0
-0.1
-0.2
ApHi Fig. 3. For legend see facing page.
-0.3
-0.4
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 487 0-2 pH units in normal muscles, and also 53 % for a decrease of 0 3 pH units in the diabetics. In addition, the decrease of tension in diabetic muscles was delayed as compared to that of normal papillary muscles, as shown by ti values (24-2 + 1-3 s vs. 18 7 + 0 7 s, P < 0 001). Furthermore, the secondary transient recovery of developed tension (d in Figs 1 and 2) was less abrupt in the diabetics. It appeared with a t1 of 755+5-8 s vs. 66+2-3 s (P < 0-001) in normal muscles, and peaked at only 80-2 + 8-6 % of control, as compared to 153-5 + 11-7 % in papillary muscles from normal hearts. It is worth noting that the peak of secondary transient tension recovery coincided, in both groups of muscles, with the maximum decrease of pHi, i.e. -040+0-02 pH units (n = 6) and -028+0X04 pH units (n = 6) in diabetic and normal muscles, respectively. The minimum of developed tension (e in Figs 1 and 2) which then followed the transient recovery was, once again, different in diabetic preparations as compared to normal (47 + 3.9 and 66-2 + 3-4 % of control, respectively) (P < 0-001). We then studied the relationship between developed tension and pHi for both normal and diabetic papillary muscles in control superfusion solution. Figure 3A shows that in both cases, there was, as described in cardiac Purkinje fibres (Vaughan-Jones et al. 1987), an approximately linear relationship between the plot of logarithmic tension and pHi. However, the slope was significantly smaller in the diabetic muscles (- 3) as compared to normal (- 5; P < 001). Therefore, the same change of pHi, in the range studied, had less effect on the developed tension of diabetic muscles than on that of normal muscles. These results support the hypothesis of a decrease in pHi sensitivity of contractile force induced by diabetes in rat papillary muscle. -
-
The effect of sarcoplasmic reticulum inhibitors on the response of tension to ammonium chloride in normal and diabetic papillary muscles It was shown by Fabiato (1985a) in skinned cardiac cells that an increase of pH enhanced Ca2+ accumulation into the SR, and consequently the amplitude of a subsequent Ca2+ release, while a decrease of pH had the opposite effects. These results suggested to us that the decrease in sensitivity of force in diabetic papillary muscles, may, at least partly, result from an altered response of the SR to pHi changes. We investigated this possibility by using the SR inhibitors caffeine (Weber & Herz, 1968) and ryanodine (Sutko & Willerson, 1980). For each muscle, normal (Fig. 4A) and diabetic (Fig. 4B), the top record shows the effect of exposure to and subsequent withdrawal of NH4C1 upon developed tension under control conditions. The bottom trace shows recordings from the same muscles Fig. 3. Relationship between developed tension and changes in pHi on semilogarithmic coordinates for normal (i) and diabetic (X) papillary muscles, in control superfusion solution (A), in the presence of caffeine (B), in the presence of ryanodine (C). The ordinate represents relative developed tension recorded in ten normal and ten diabetic muscles (A) and four normal and four diabetic muscles (B and C). The abscissa is the change of pHi from steady-state control level, either during the application of NH4C1 (positive values recorded each minute) or just after removal of NH4C1 (negative values, at point c of Fig. 1). Each point is the mean pH, value for six normal and diabetic muscles (A, B and C). The lines drawn through the points were fitted by regression analysis. a is the slope of the relationship and r the correlation coefficient.
488
D. LAGADIC-GOSSMANN AND D. FEUVRA Y A
20 mM-NH4CI
1 mN C
__ 20 mM-NH4CI 10 mM-caffeine
1
mN[
MW 56 min B 20 mM-NH4CI 1 mN C
20 mM-NH4CI 10 mM-caffeine
1 mNE_ 5 min
Fig. 4. The effect of caffeine on developed tension during exposure to and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 10 mM-caffeine (bottom record).
when NH4Cl was added and subsequently withdrawn from the superfusate in the presence of 10 mM-caffeine. Caffeine itself had marked positive inotropic effects in both groups, so that tension increased to an average of 350-3 + 48-6 % of control (n = 4) and 325 + 55.7 % of control (n = 4) in normal and diabetic muscles, respectively. With the addition of NH4C1, developed tension increased such that the maximum reached (for a corresponding similar increase of pHi in both groups) in this condition was not significantly different between normal (524 + 657 % of control, n = 4) and diabetic (457+1014 % of control, n = 4) muscles. Moreover, caffeine abolished the secondary transient recovery of tension that occurred after NH4C1 withdrawal. Developed tension was then decreased to a minimum of 70-3+1388% (n = 4) of control in normal muscles and of 42-9 + 6-9 % (n = 4) in diabetic muscles. Results in these experimental conditions (i.e. in presence of caffeine) are summarized by the fact that we found no significant difference in the slopes of the log(tension)-pH1 relationships between normal and diabetic papillary muscles (Fig. 3B).
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 489 A 20 mM-NH4CI
1 mNC
_
-
20 mM-NH4CI 2 /M-ryanodine 1 mN[
5 min B
20 mM-NH4CI 1 mNr
20 mM-NH4CI 2 juM-ryanodine 1 mN
[__ 5 min
Fig. 5. The effect of ryanodine on developed tension during exposure to and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 2 /LM-ryanodine (bottom record).
Ryanodine (2/tM) was used as a more specific SR inhibitor (Bers, Bridge & McLeod, 1987). This substance has no effect on Ca21 sensitivity of myofilaments (Fabiato, 1985b). Figure 5 shows that ryanodine had a marked negative inotropic effect on both normal and diabetic papillary muscles, leading to a fall of developed tension to 6-49 + 1P96 % of control (n = 4) in normal muscles, and to 7417 + 1P87 % of control (n = 4) in the diabetics. The changes of developed tension during exposure to and withdrawal of NH4CI, were similar in both groups of muscles in the presence of ryanodine. At the maximum NH4+-induced alkalinization, the increase in developed tension was not significantly different between both groups of muscles (101-2 + 14-6 % and 15-2 + 10-2 % in the normal and diabetics respectively, as compared to control tension, i.e. without ryanodine, n = 4). The acidosis induced by NH4+ withdrawal was accompanied by a decrease of tension to 5-2 + 1-6 % of control (n = 4) in normal and to 641 + 1-6% of control (n = 4) in diabetic preparations. Moreover, as observed with caffeine, there was no secondary transient recovery of tension. Therefore, as shown in Fig. 3 C, the presence of ryanodine, like that of caffeine,
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
490
rendered the tension-pHi relationship of diabetic papillary muscles identical to that of normal muscles. These results suggest that some diabetes-induced change in the SR might explain the differences that we observed in the pHi sensitivity of tension between diabetic and normal papillary muscles. 1.8
16 3a = -3.34: r =0.99 1.4
*C
1.2 02 0.6 0.4
a=-1 .07: r=0.99
0.2
0
-(.4
-0.3
-0.2
-0.1
0
ApH; Fig. 6. Relationship between tension recovery (after maximum transient positive inotropy, point d of Fig. 1) and pHi recovery (after maximum acidification) following NH4Cl withdrawal, in normal (@) and diabetic (X) muscles. The lines drawn through the points were fitted by regression analysis. (ApHi: n = 6 for both groups of muscles; tension: n = 10 for normal and diabetic muscles).
The effect of amiloride on transient recovery of tension following acidosis induced by withdrawal of ammonium The maximum transient redevelopment of tension corresponded with the maximum acidification in both groups of muscles, as described above (Fig. 1). However, the peak of transient tension recovery in diabetic papillary muscles was slightly delayed and markedly attenuated as compared to normal. Figure 6 shows that there was a good correlation between the time courses of tension recovery and of pHi recovery from maximum acidification, in both normal and diabetic muscles. It has been shown previously that recovery of pHi from an intracellular acid load is largely due to the amiloride-sensitive Na+-H+exchange (Deitmer & Ellis, 1980; Piwnica-Worms, Jacob, Horres & Lieberman, 1985). In this study, the presence of 1 mM-amiloride (Fig. 7) suppressed the transient redevelopment of force, such that tension rapidly reached an average minimum of 24-2 + 3-3 % (n = 4) and 20-5 + 7 % of control (n = 3) in normal and diabetic muscles, respectively. These values were not significantly different but were significantly lower than the minimum values reached under these conditions in muscles in the absence of amiloride. In addition, minimum tension was reached, in the presence of amiloride, with similar ti values for the two groups of muscles (85 + 7-9 s in normal and 106-6 + 24 s in diabetic muscles). Therefore, the presence of amiloride during an intracellular acid load abolished the
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 491 20 mM-NH4CI
A
Tension_
1 mM-amiloride
_
1 mN c 6.5-
6.71 pHi
6.9
-
7-1 73 7
51
5 minT 20 mM-NH4CI
B
1 mM-amiloride
Tension 1 mN C:
6.5 6.7
pHi 6.97.1
5 min
Fig. 7. The effect of 1 mM-amiloride on developed tension and pH, following NH4Cl withdrawal, in a normal (A) and in a diabetic muscle (B).
differences observed in developed tension between both types of muscles. In particular, amiloride abolished the differences observed in transient recovery of tension, in the amplitude of pHi decrease and in the time course of pH, recovery from acidosis (Fig. 7). These results demonstrate that transient recovery of tension must be related to the activity of the Na+-H+ exchange, this exchange being normally particularly stimulated by an intracellular acidification (Grinstein & Rothstein, 1986). In this respect, the decrease in the activity of the exchange induced by diabetes (LagadicGossmann et al. 1988) would account for the smaller redevelopment of force observed in diabetic papillary muscles upon acidification induced by NH4+.
The effect of 2',4'-dichlorobenzamil amiloride on transient recovery of tension following acidosis induced by withdrawal of ammonium It has been suggested that the positive inotropy which appears when Na+-H+ exchange is operational, would result from a secondary stimulation of the Na+-Ca2+ exchange, which elevates Ca1+ and, consequently, cardiac contractility
492
D. LAGADIC-GOSSMANN AND D. FEUVRA Y A
20 mM-NH4CI
1 mN
20 mM-NH4CI 40 iM-DCB
1 mNc
_
-~~~~IA
5 min 20 mM-NH4CI
B
1 mN[
_
_
_
20 mM-NH4CI 40 uM-DCB
1 mNC 5 min
Fig. 8. The effect of DCB on developed tension during exposure and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 40 ,sM-DCB (bottom record). DCB had no effect on control tension.
(Vaughan-Jones, 1988). In an attempt to evaluate the role of Na+-Ca2+ in transient redevelopment of tension during an internal acid load, we used DCB, an amiloride derivative, which is known to be a potent inhibitor of this ionic exchange (Frelin, Barbry, Vigne, Chassande, Cragoe & Lazdunski, 1988). Figure 8 shows that the presence of 40 ,tM-DCB in the superfusing solution accentuated the immediate decrease of tension that appeared on removal of NH4C1. Developed tension was thus decreased by the same order of magnitude in both types of preparations (61-5 + 7-8 % and 53-3 + 1-2 %, n = 3, in normal and diabetic muscles, respectively). This decrease occurred, for both types of muscles, with identical ti values (- 40 s). Furthermore, the presence of DCB significantly and similarly reduced and delayed the amplitude of the secondary redevelopment of force in both normal and diabetic papillary muscles. The amplitude reached was then only 747 + 2 % (n = 3) of that obtained in the absence of DCB in normal muscles, and 766 + 8-2 % (n = 3) in the diabetics, whereas the mean ti for reaching the maximum
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 493 increased up to 137 + 12 s and to 175 + 32-8 s (not significant) for normal and diabetic muscles, respectively. These results appear to be in favour of the hypothesis of a participation of Na+-Ca2+ exchange in transient redevelopment of tension during an intracellular acidification. DISCUSSION
The results of this study show a decreased response of contractile force to changes of pHi in papillary muscles from STZ-induced diabetic rat hearts. This effect appears to be related in part to some defect in SR function, since the tension-pHi relationship in diabetic muscles became identical to that of normal muscles in the presence of SR inhibitors. Furthermore, our results obtained in both normal and diabetic preparations agree with the original observation, in ferret papillary muscles (Orchard, 1987), of a transient redevelopment of force following the initial decrease in tension with acidosis. However, this secondary transient positive inotropy was markedly attenuated in the diabetics, and it could be suppressed in the two groups of muscles by SR inhibitors and also by blockage of the sarcolemmal Na+-H+ exchange. The decrease in pHi sensitivity of developed tension in diabetic papillary muscles It has been shown in skinned cardiac cells of rat ventricle (Fabiato & Fabiato, 1978), as well as in intact sheep cardiac Purkinje fibres (Vaughan-Jones et al. 1987), that a fall in pHi reduces tension production while an increase in pHi has the opposite effect. In the present study, changes of + 0-3 units around steady-state pHi produced graded changes of tension in both normal and diabetic papillary muscles. However, a quantitative analysis of these changes clearly shows a lower sensitivity of tension to variations in pHi in diabetic muscles (Fig. 3A). For instance, an alkalinization of 0-2 units increases tension by 590% in normal muscles and only by 230 % in the diabetics. Conversely, an acidification of 0-2 units decreases tension by 47 % of control in diabetic muscles compared to 63 % in normal muscles. Since pHi and tension were not recorded simultaneously in our study, one may argue that steady-state pHi or NH4+-induced changes in pHi may be different in stimulated muscles as compared to quiescent muscles. Indeed, several studies have shown that stimulation produces an intracellular acidification and an increase of intracellular Na+and Ca2+(Bountra, Kaila & Vaughan-Jones, 1988a, b). Moreover, differences may occur in the metabolic response to stimulation, between normal and diabetic muscles, which may affect intracellular pH and the Na+-H+ exchange mechanism. We assume that these differences have negligible effects on our results since Bountra & Vaughan-Jones (1986) have shown, with simultaneous measurements of pHi and force during stimulation in guinea-pig papillary muscles, that the transient positive inotropic effect and the acidification were linked. There may be several reasons for the difference between the pHi sensitivity of normal and diabetic muscles. It may result from diabetes-induced changes in the Ca2+ sensitivity of the myofilaments or alteration of some membrane processes regulating Ca1 . Although a decrease of Ca2+ binding to troponin C under acidosis is plausible (Solaro, Lee, Kentish & Allen, 1988), it has been considered as an unlikely
494
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
explanation for the effects of the pH change on the tension-Ca2+ relationship (Fabiato, 1985a). We investigated the second hypothesis by studying the influence of a major component of CaF+ regulating processes i.e. the SR. Although both caffeine and ryanodine are known to interfere with SR function through completely different mechanisms (Weber & Herz, 1968; Sutko & Willerson, 1980), they both abolished the difference in contractile response to pHi changes between normal and diabetic muscles (Fig. 3B and C). In this respect, it should be noted that we did not observe any effect of caffeine or ryanodine on pHi (data not shown), in agreement with previous studies (Cannell, Vaughan-Jones & Lederer, 1985; Orchard, 1985). Several studies (Penpargkul et al. 1981; Ganguly et al. 1983; Lopaschuk et al. 1983) have shown a decreased ability of diabetic rat cardiac SR to transport Ca2+. This defect appeared to be a consequence of insulin deficiency and associated metabolic changes (Feuvray et al. 1979; Lopaschuk et al. 1983). It has been suggested that a change in the environment surrounding the Ca2+-stimulated ATPase protein may influence its activity (Ganguly et al. 1983). Indeed, the Ca2+ affinity of this pump is strongly dependent on H+ activity (Grassi de Gende, 1988), and there is a competition between Ca2+ and H+ for the high-affinity binding sites that are directly involved in active Ca2+ transport (Levitsky & Benevolensky, 1986). The influence of H+ is such that under high pH, Ca2+ accumulation into the SR and its subsequent release are increased, whereas a low pH has the converse effect (Fabiato, 1985a). In this context, we can reasonably assume that in diabetic rat hearts, which rely essentially on long-chain fatty acids as a metabolic fuel (Randle et al. 1966) and accumulate long-chain fatty acyl derivatives (Feuvray et al. 1979), the SR membrane environment and composition will be affected (Ganguly et al. 1983; Lopaschuk, Eibschutz, Katz & McNeill, 1984). In particular, this could result in a smaller number of, or less accessible, high-affinity Ca2+-binding sites being available for competition with H+. The transient redevelopment offorce following the initial decrease in tension with acidosis in diabetic papillary muscles The initial decrease in tension upon acidosis induced by NH4+ withdrawal was rapidly followed (within 2 min) by a transient redevelopment of force. This was observed in both normal and diabetic papillary muscles. Yet the time course and the amplitude of this transient recovery of tension in diabetic muscles differed markedly from that in normal muscles. In fact, it developed more slowly in the diabetics, as shown by a 15 % greater value of ti, and the maximum amplitude was about half that in normal muscles. A comparable transient positive inotropy has been found in normal Purkinje fibres (Vaughan-Jones et al. 1987) and heart muscle (Orchard, 1987). The latter study showed that this early recovery of tension was due to increased Ca2+ release from the SR, occurring secondarily to an increase in resting Ca?+. Our results show that not only do caffeine and ryanodine suppress the transient positive inotropy in normal muscles (Orchard, 1987), but have the same effect in diabetic muscles. Moreover, our results demonstrate: (i) that the maximum transient redevelopment of force corresponded with the maximum acidification in both groups of muscles and (ii) that the time courses of tension recovery and of pHi recovery were linearly related in both preparations (Fig. 6). Since pHi recovery from an acid load
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 495 depends essentially on the activity of Na+-H+ exchange (Deitmer & Ellis, 1980; Piwnica-Worms et al. 1985), the smaller amplitude of the transient positive inotropy, as well as the slowing down of pHi recovery and of tension recovery in diabetic muscles, may well indicate a primary role of this exchange in the transient redevelopment of force. This appeared to be confirmed by the fact that inhibition of the exchange by amiloride during the acid load suppressed the transient redevelopment of force in both normal and diabetic preparations. In the present study, we did not measure intracellular Na+ activity. However, work performed on other tissues (Deitmer & Ellis, 1980; Piwnica-Worms et al. 1985) has shown that the peak of acidosis induced by NH4+ withdrawal coincided with a rise in aNa, probably mediated by Na+-H+ exchange. This increase in aNa could then be sufficient to activate the Na+-Ca2+ exchanger and lead to an increase in CaW+ (Bers & Ellis, 1982). We approached the possible role of a secondary activation of the Na+-Ca2+exchange by using DCB. Although the results are not as clear-cut as those obtained in the presence of SR inhibitors or the Na+-H+ blocker, DCB induced a significant delay and reduction in the amplitude of the transient tension redevelopment. Therefore our results are in line with the suggestion (Orchard, 1987) that an increase in W+, through stimulation of SR Ca2+loading, may induce a larger release of Ca2+, thus overcoming the direct inhibitory effect of acidosis. In diabetic papillary muscles, the decrease in Na+-H+ exchange activity compared to normal muscles upon acidosis (Lagadic-Gossmann et al. 1988) would then lead to less of an increase of Ca1+ and consequently to a smaller release of Ca2+ from the SR. Alternatively, the greater acidification upon NH4+ withdrawal due to decreased activity of the Na+-H+ exchanger may reduce the SR Ca2+ trigger, either indirectly through an effect on Na+-Ca2+exchange (Philipson, Bersohn & Nishimoto, 1982) or more directly through the SR Ca2+-ATPase (Levitsky & Benevolensky, 1986). In addition, a smaller Ca2+ release from the SR in diabetic muscles may result from an alteration in SR membrane composition and function. In conclusion, STZ-induced diabetes induces: (i) a decrease in the sensitivity of contractile force to a change in pH1; this may be the consequence of an altered SR membrane composition and function, although other possibilities such as a change in the Ca2+sensitivity of myofilaments cannot be discounted; (ii) a marked attenuation of the transient positive inotropy which follows the initial decrease in tension associated with acidosis; this may indirectly result from the decrease in the activity of the sarcolemmal Na+-H+exchange and its consequences on membrane systems regulating CaV. We wish to thank Ian Findlay for his helpful comments on an earlier version of the manuscript, Jean-Michel Chesnais and Philippe Jourdon for valuable discussion and Francoise James for her technical assistance. This work was supported in part by a grant from the Association Frangaise contre les Myopathies. REFERENCES
AMMANN, D., LANTER, F., STEINER, R. A., SCHULTHESS, P., SHIJO, Y. & SIMON, W. (1981). Neutral carrier based hydrogen ion selective microelectrode for extra- and intracellular studies. Analytical Chemistry 53, 2267-2269.
496
D. LAGADIC-GOSSMANN AND D. FEUVRAY
BENOS, D. J. (1982). Amiloride: a molecular probe of sodium transport in tissues and cells. American Journal of Physiology 242, C131-145. BERS, D. M., BRIDGE, J. H. B. & MAcLEOD, K. T. (1987). The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures. Canadian Journal of Physiology and Pharmacology 65, 610-618. BERS, D. M. & ELLIS, D. (1982). Intracellular calcium and sodium activity in sheep heart Purkinje fibres. Effect of changes of external sodium and intracellular pH. Pfiugers Archiv 393, 171-178. BIELEFELD, D. R., PACE, C. S. & BOSHELL, B. R. (1983). Altered sensitivity of chronic diabetic rat heart to calcium. American Journal ofPhysiology 245, E560-567. BORON, W. F. & DE WEER, P. (1976). Intracellular pH transients in squid giant axons caused by C02, NH3 and metabolic inhibitors. Journal of General Physiology 67, 91-112. BOUNTRA, C., KAILA, K. & VAUGHAN-JONES, R. D. (1988a). Effect of repetitive activity upon intracellular pH, sodium and contraction in sheep cardiac Purkinje fibres. Journal of Physiology 398, 341-360. BOUNTRA, C., KAILA, K. & VAUGHAN-JONES, R. D. (1988b). Mechanism of rate-dependent pH changes in the sheep cardiac Purkinje fibre. Journal ofPhysiology 406, 483-501. BOUNTRA, C. & VAUGHAN-JONES, R. D. (1986). The influence of pH, on twitch tension in isolated guinea-pig papillary muscle. Journal of Physiology 382, 109P. CANNELL, M. B., VAUGHAN-JoNEs, R. D. & LEDERER, W. J. (1985). Ryanodine block of calcium oscillations in heart muscle and the sodium-tension relationship. Federation Proceedings 44, 2964-2969. DEITMER, J. W. & ELLIS, D. (1980). Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibres. Journal of Physiology 304, 471-488. FABIATO, A. (1985a). Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells. Cell Calcium 6, 95-108. FABIATO, A. (1985b). Effects of ryanodine in skinned cardiac cells. Federation Proceedings 44, 2970-2976. FABIATO, A. & FABIATO, F. (1978). Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal of Physiology 276, 233-255. FEIN, F. S., KORNSTEIN, L. B., STROBECK, J. E., CAPASSO, J. M. & SONNENBLICK, E. H. (1980). Altered myocardial mechanics in diabetic rats. Circulation Research 47, 922-933. FEIN, F. S. & SONNENBLICK, H. (1985). Diabetic cardiomyopathy. Progress in Cardiovascular Diseases 27, 255-270. FEUVRAY, D., IDELL-WENGER, J. A. & NEELY, J. R. (1979). Effects of ischemia on rat myocardial function and metabolism in diabetes. Circulation Research 44, 322-329. FRELIN, C., BARBRY, P., VIGNE, P., CHASSANDE, O., CRAGOE, E. J. JR & LAZDUNSKI, M. (1988). Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+-H+ antiport and the Na+-Ca2+ exchanger. Biochimie 70, 1285-1290. GANGULY, P. K., PIERCE, G. N., DHALLA, K. S. & DHALLA, N. S. (1983). Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. American Journal of Physiology 244, E528-535. GRASSI DE GENDE, A. 0. (1988). The effect of pH on the calcium dependence of calcium accumulation in dog cardiac muscle sarcoplasmic reticulum. Journal of Molecular and Cellular Cardiology 20, 1087-1093. GRINSTEIN, S. & ROTHSTEIN, A. (1986). Mechanisms of regulation of the Na+/H+ exchanger. Journal of Membrane Biology 90, 1-12. LAGADIC-GOSSMANN, D., CHESNAIS, J. M. & FEUVRAY, D. (1988). Intracellular pH regulation in papillary muscle cells from streptozotocin diabetic rats: an ion-sensitive microelectrode study. Pflilgers Archiv 412, 613-617. LEVITSKY, D. 0. & BENEVOLENSKY, D. S. (1986). Effects of changing Ca2+-to-H+ ratio on Ca2+ uptake by cardiac sarcoplasmic reticulum. American Journal of Physiology 250, H360-365. LOPASCHUK, G. D., EIBSCHUTZ, B., KATZ, S. & MCNEILL, J. H. (1984). Depression of calcium transport in sarcoplasmic reticulum from diabetic rats: lack of involvement by specific regulatory mediators. General Pharmacology 15, 1-5. LOPASCHUK, G. D., KATZ, S. & MONEILL, J. H. (1983). The effect of alloxan- and streptozotocin-
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 497 induced diabetes on calcium transport in rat cardiac sarcoplasmic reticulum. The possible involvement of long chain acylcarnitines. Canadian Journal of Physiology and Pharmacology 61, 439-448. ORCHARD, C. H. (1985). The effect of sarcoplasmic inhibitors on the response of ventricular muscle to acidosis. Biophysical Journal 47, 379a. ORCHARD, C. H. (1987). The role of the sarcoplasmic reticulum in the response of ferret and rat heart muscle to acidosis. Journal of Physiology 384, 431-449. PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H. & SCHEUER, J. (1981). Depressed cardiac sarcoplasmic reticular function from diabetic rats. Journal of Molecular and Cellular Cardiology 13, 303-309. PHILIPSON, K. D., BERSOHN, M. M. & NISHIMOTO, A. Y. (1982). Effects ofpH on Na'-Ca 2+exchange in canine cardiac sarcolemmal vesicles. Circulation Research 50, 287-293. PIWNICA-WORMS, D., JACoB, R., HORRES, C. R. & LIEBERMAN, M. (1985). Na/H exchange in cultured chick heart cells. pHi regulation. Journal of General Physiology 85, 43-64. RANDLE, P. J., GARLAND, P. B., HALES, C. N., NEWSHOLME, E. A., DENTON, R. M. & POGSON, C. I. (1966). Protein hormones interactions of metabolism and the physiological role of insulin. Recent Progress in Hormone Research 22, 1-48. SAUVIAT, M. P. & FEUVRAY, D. (1986). Electrophysiological analysis of the sensitivity to calcium in ventricular muscle from alloxan diabetic rats. Basic Research in Cardiology 81, 489-496. SOLARO, R. J., LEE, J. A., KENTISH, J. C. & ALLEN, D. G. (1988). Effects of acidosis on ventricular muscle from adult and neonatal rats. Circulation Research 63, 779-787. SUTKO, J. L. & WILLERSON, J. T. (1980). Ryanodine alteration of the contractile state of rat ventricular myocardium. Comparison with dog, cat, and rabbit ventricular tissues. Circulation Research 46, 332-343. TAHILIANI, A. G. & MCNEILL, J. H. (1986). Diabetes-induced abnormalities in the myocardium. Life Sciences 38, 959-974. TAKEDA, N., NAKAMURA, I., HATANAKA, T., OHKUBO, T. & NAGANO, M. (1988). Myocardial mechanical and myosin isoenzyme alterations in streptozotocin-diabetic rats. Japanese Heart Journal 29,455-463. VAUGHAN-JONES, R. D. (1988). Regulation of intracellular pH and contractility in cardiac muscle. Journal ofMolecular and Cellular Cardiology 20, S.33. VAUGHAN-JONES, R. D., EISNER, D. A. & LEDERER, W. J. (1987). Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibres. Journal of General Physiology 89, 1015-1032. VAUGHAN-JONES, R. D., LEDERER, W. J. & EISNER, D. A. (1983). Ca2+ ions can affect intracellular pH in mammalian cardiac muscle. Nature 301, 522-524. WEBER, A. M. & HERZ, R. (1968). The relationship between caffeine contracture of intact muscle and the effect of caffeine on isolated reticulum. Journal of General Physiology 52, 750-759.
Journal of Physiology (1990), 422, pp. 481-497 With 8 figures Printed in Great Britain
DECREASED SENSITIVITY OF CONTRACTION TO CHANGES OF INTRACELLULAR pH IN PAPILLARY MUSCLE FROM DIABETIC RAT HEARTS
BY D. LAGADIC-GOSSMANN AND D. FEUVRAY From the Laboratoire de Physiologie Comparee et Laboratoire de Biomembranes et des Ensembles Neuronaux Associe au Centre National de la Recherche Scientifique, Universite Paris XI, 91405 Orsay, France
(Received 28 June 1989) SUMMARY
1. The relationship between intracellular pH (pHi) and contractile activity was investigated in papillary muscles isolated from right ventricle of normal and streptozotocin (STZ)-induced diabetic rats. pHi changes induced by 20 mm-NH4Cl were recorded with H+-sensitive microelectrodes. 2. An increase in pHi of 0-20 pH units on exposure to NH4C1 led to an increase of the maximum developed tension, which was 70788+57-5% (mean + S.E. of mean, n = 10) of control in normal muscles and 271 + 16-3 % (n = 10) in diabetic muscles. On the other hand, acidosis induced by NH4C1 withdrawal was associated with a fall in developed tension to 48'2 + 67 % of control in diabetic muscles, as compared to 79-2 + 8 % in normal muscles. 3. The decrease in tension associated with acidosis was rapidly followed (in 2 min) by a transient redevelopment of force, which peaked at 80-2 + 8-6 % of control in the diabetic nmuscles as compared to 1535 + 11-7 % in normal papillary muscles. The peak of this secondary positive inotropy coincided in both groups of muscles with the maximum decrease of pHi, i.e. - 0-40 + 0-02 and - 0-28 + 0-04 pH units in diabetic and normal muscles, respectively. 4. Caffeine (10 mm), which had a marked positive inotropic effect in both groups of muscles, abolished the transient recovery of tension occurring after NH4C1 withdrawal. Ryanodine (2 /tM) which had a marked negative inotropic effect on both normal and diabetic papillary muscles, also suppressed the transient recovery of tension. 5. The presence of amiloride (1 mm) during acidosis induced by NH4C1 withdrawal abolished the observed differences in developed tension, in particular the transient recovery of tension, between normal and diabetic muscles, as it abolished the differences in the amplitude of pHi decrease and in the time course of pHi recovery. 6. The presence of 2',4'-dichlorobenzamil amiloride (40 ,tM) significantly and similarly delayed and reduced the amplitude of transient recovery of tension in both normal and diabetic papillary muscles. 7. We conclude that STZ-induced diabetes induces a decrease in pHi sensitivity of contractile force. This may be the consequence of a change in sarcoplasmic reti-
MS 7794
16
PHY 422
482
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
culum (SR) composition and function, and may also indirectly result from changes in Na+-H+ exchange activity, particularly during intracellular acidosis. INTRODUCTION
Chronic diabetes mellitus is frequently associated with depressed cardiac function, even in the absence of atherosclerotic coronary disease (Fein & Sonnenblick, 1985). Most of the experimental studies concerning the alterations of mechanical function associated with diabetes have been performed using drug-induced diabetic rats. Fein, Kornstein, Strobeck, Capasso & Sonnenblick (1980) have shown that isolated papillary muscles from streptozotocin (STZ)-induced diabetic rats have a decreased shortening velocity and slowed rate of relaxation. Also, a decreased binding and uptake of Ca2+ by sarcoplasmic reticulum (SR) has been shown in diabetic rat hearts (Penpargkul, Fein, Sonnenblick & Scheuer, 1981; Ganguly, Pierce, Dhalla & Dhalla, 1983; Lopaschuk, Katz & McNeill, 1983). However, these abnormalities were probably not the only factors responsible for diabetes-induced myocardial depression (Tahiliani & McNeill, 1986). Other studies have pointed out a decrease in the sensitivity of diabetic hearts to external Ca2+(Bielefeld, Pace & Boshell, 1983; Sauviat & Feuvray, 1986). Another significant defect in diabetes is the increase in free fatty acid uptake and utilization, and the inhibition of glucose transport across the cell membrane due to lack of insulin (Randle, Garland, Hales, Newsholme, Denton & Pogson, 1966). The resulting decrease in myocardial glucose utilization (Feuvray, Idell-Wenger & Neely, 1979) might be associated with a change in proton production. However, we have recently shown that there was no difference between the steady-state intracellular pH (pHi) values recorded in papillary muscles from diabetic or normal rat hearts. However, differences between diabetic and normal muscles were found in the regulation of pHi where diabetes had induced a decrease in the activity of the amiloride-sensitive Na+-H+ exchange (Lagadic-Gossmann, Chesnais & Feuvray, 1988). By its influence on both pHiand Na+, the activity of the Na+-H+ exchange may well participate in the control of cardiac contractility (Vaughan-Jones, Eisner & Lederer, 1987). Indeed, close relationships have been demonstrated between the mechanisms controlling Ca 2+ and pHi (Deitmer & Ellis, 1980; Bers & Ellis, 1982; Vaughan-Jones, Lederer & Eisner, 1983). In particular, the membrane control of Ca1+ and pHi in heart relies upon a common ion (i.e. Na+) through the activities of both the Na+-H+ and the Na+-Ca2+ exchangers (Vaughan-Jones, 1988). Therefore, a decrease in the activity of the former may have indirect consequences on the contractile activity of diabetic hearts by altering the amount of Caf2 triggering the release of Ca2+ from the SR (Fabiato, 1985 a). The present study was designed to investigate the effect of diabetes on the relationship between pHi and contractile activity. We also examined the influence of transmembrane ionic exchangers and the possible role of the SR. The experiments were carried out on papillary muscles of normal and STZ-induced diabetic rat hearts. The results show that diabetes induces a decrease in the sensitivity of contractile force to a change in pHi. This may be the consequence of an altered SR function
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 483 associated with diabetes, or may indirectly result from decreased Na+-H+ exchange activity, particularly during intracellular acidosis. METHODS
The methods used here were similar to those described previously (Lagadic-Gossmann et al. 1988). Briefly, male Wistar rats weighing 250-300 g were fasted overnight and made diabetic by the injection of streptozotocin (STZ, Sigma, St Louis, MO, USA; 40 mg/kg) into the femoral vein. Streptozotocin-treated and age-matched control animals were maintained on the same diet until they were used 4-5 weeks later. The diabetic state was assessed by measurement of non-fasting glucose concentration in blood samples collected at the time of heart excision. Mean glucose levels were 9-5 + 0 7 and 35-7 + 2-1 mm for normal and diabetic rats, respectively. Rats were anaesthetized with pentothal (5 mg/100 g body weight, i.p.). The heart was removed rapidly and rinsed in Krebs bicarbonate buffer solution (see below) at 36 °C. A thin uniform papillary muscle (< 1 mm in diameter, 2-3 mm in length; same size in normal and diabetic groups) was dissected from the right ventricle, pinned to the bottom of the experimental chamber and superfused with Krebs bicarbonate buffer at 36+1 °C at a rate of 8-4 ml/min. The fluid volume around the papillary muscle was approximately 1-4 ml. The preparation was allowed to equilibrate for at least 30 min in the control buffer solution before recordings were obtained. Measurements of intracellular pH Measurements of pHi were made in superficial cells of quiescent preparations, using resin-filled H+-selective microelectrodes (Ammann, Lanter, Steiner, Schulthess, Shijo & Simon, 1981). The microelectrodes were connected to a high-impedance electrometer (FD 223, World Precision Instruments) and monitored via a pen recorder.
Tension measurements The papillary muscle was mounted horizontally between a fixed hook and a tension transducer, and was superfused continuously with Krebs bicarbonate buffer solution at 36 +1 °C. The isometric mechanical activity was displayed on a Gould paper recorder. After 30 min of superfusion, the muscle was stretched stepwise to maximum length (Lmax). Both field and focal stimulation were used. The muscle was stimulated at 1 Hz (8-10 ms duration pulses, twice threshold) either between two platinum wires positioned parallel to the muscle length or focally by punctate electrodes. Tension and pHi were not recorded simultaneously because of vigorous contractions of the rat papillary muscles which make stable impalements by microelectrodes very difficult. Therefore, pHi changes were recorded in experiments similar to those in which tension was measured, but without stimulation. In these experiments muscle length was not systematically fixed. However, each muscle had similar resting tension and we observed that steady-state pH, values were not affected by changing muscle length in control superfusion conditions.
Solutions The control Krebs bicarbonate buffer solution used in the experiments contained (mM): NaCl, 118; KCl, 4-8; CaCl2, 1-75; MgCl2.6H2O, 12; KH2PO4, 12; NaHCO3, 25; EGTA, 0 5; glucose, 10; sucrose, 15. This solution was equilibrated with 95 % 02-5 % CO2 to give a pH of 7-4. Intracellular alkalinizations and acidifications were induced by the NH4+ method (Boron & De Weer, 1976). In solutions which contained 20 mM-NH4Cl, the NaCl concentration was decreased to compensate for osmotic changes. The NH4Cl exposure lasted 15 min. Amiloride (Sigma, 1 mM) was used to block Na+-H+ exchange (Benos, 1982); 2',4'-dichlorobenzamil amiloride (DCB, given by Dr D. Escande, Rhone Poulenc France; 40 /uM) was used to inhibit Na+-Ca2+ exchange; caffeine (Sigma; 10 mM) and ryanodine (Calbiochem, France; 2 /tM) were used as inhibitors of the SR. Statistics Results are reported as the mean + S.E. of mean of n preparations. Comparisons between groups were made using Student's t test. Slopes of linear regressions were also compared using Student's 16-2
484
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
t test. A value for P of 0 05 was chosen as the threshold to reject the null hypothesis that there was no difference in means between groups. RESULTS
The effect of diabetes on the response of tension to ammonium chloride Addition of NH4Cl to the superfusing solution causes an intracellular alkalinization and its removal causes an intracellular acidification (Boron & De Weer, 1976) with A
20 mM-NH4CI
Tension 1 mNc --
bcd
a
e
6-7
pHi 71[ 7.37.5 L
'5 min'
B 20 mM-NH4CI Tension 1 mN[
bLcI I
a
pH
I
I
I
e
6.5 6.7 6.9
723 7.5
'5 min Fig. 1. The effect of exposure to, and withdrawal of, NH4Cl on tension and pHi of normal (A) and diabetic (B) papillary muscles. In each panel, traces show: developed tension (top) and pHi (bottom). The period during which NH4C1 was added to the superfusion solution is indicated above the record. Note the different scale for tension in each panel. Specific points are indicated by letters (a-e) on the top traces of each panel.
no change of pHe. Figure 1 shows representative superimposed recordings of tension and pHi in a normal papillary muscle (Fig. 1A) and in a diabetic papillary muscle (Fig. 1B) before, during and after exposure to 20 mm-NH4Cl. In agreement with earlier studies (Lagadic-Gossmann et al. 1988), we found no difference in steady-state pHi values between the two groups of muscles (7 07 + 0-02 and 7 06 + 0-01, n = 10, for normal and diabetics, respectively). Likewise, no statistical difference appeared in developed tension between normal (I13 +0-1 mN, n = 10) and diabetic (105 + 012 mN, n = 10) papillary muscles superfused in control conditions. This
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 485 agrees with previous observations by others (Fein et al. 1980; Takeda, Nakamura, Hatanaka, Ohkubo & Nagano, 1988). The addition and subsequent withdrawal of NH4Cl led to transient changes of pHi which were accompanied by marked changes of developed tension in both types of 800 700 c-
o2
600500 400 300-
Co
a,
cc
200
100* 0AI Control
a
b
c
d
e
Fig. 2. Amplitude of developed tension, relative to its level in control superfusion solution, at specific points (a-e) indicated in Fig. 1, in normal (1) and diabetic (-) papillary muscles. Error bars show S.E.M. The number of separate experiments was ten. * Significantly different (P < 0001) responses between normal and diabetic muscles.
muscles although to a different degree (Fig. 1). The initial alkalosis on exposure to NH4C1 was associated with a rapid increase of developed tension. As pHi became less alkaline during the sustained exposure to NH4C1, there was also a decrease in tension. Conversely, the transient acidification on removal of NH4Cl was accompanied by an immediate decrease of tension. However, this decrease was interrupted by a rapid transient redevelopment of tension which reached a maximum after 2 min and then slowly recovered towards control values. Such a sequence of events has already been described in ferret papillary muscles under comparable experimental conditions (Orchard, 1987). In our study these events were qualitatively similar in papillary muscles of both normal and diabetic rat hearts, but their amplitude differed significantly. Thus, as shown in Fig. 2, the maximum developed tension (a) in response to the same increase in pHi (- + 0-20 pH units) during the NH4Cl pulse was only 271 + 16-3 % of control (n = 10) in diabetic muscles against 707-8 + 57.5% of control (n = 10) in normal muscles. Yet the maximum tension, although considerably less in the diabetics, was reached with the same half-time (ti) as in normal muscles (47-8 + 28 and 48-2 + 31 s, respectively). At the end of the exposure (b) a significant difference in developed tension still persisted between the two groups. Removal of NH4+ caused a rapid intracellular acidification, the amplitude of which was significantly greater in the diabetic muscles. Moreover, the subsequent recovery from acidification was slower in the diabetics, due to the reduced activity of the Na+-H+ exchange (Lagadic-Gossmann et al. 1988). Again, the decrease of developed tension (c in Figs 1 and 2) which appeared concomitant with acidosis induced by NH4+ withdrawal was different in its amplitude in the two types of muscles. Developed tension fell to 79-2 + 8% of control (n = 10) in normal muscles and to 48-2 + 6-7 % (n = 10) in the diabetics. However, when compared with tension values before NH4Cl withdrawal, the fall of tension was 53 %, associated with an acidification of
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
486 A
10 T a = 4.95: r = 0-99 c
0 .°4 c
0
> _
1
a = 3.16: r= 0.98
0
-j
0.1 10.3
0-1
0.2
-0.2
-0.1
0
-0.3
-0.4
ApHi B 10-
+ 10 mM-caffeine
a = 3.29: r = 0.96
C
0
._4
C
0
0
2
-W
1-
a = 3.52: r= 0.98
0
-J
0-1 0-2
0.3
-0.1
0
0-1
-02
-0.3
-0.4
ApHi C
10 r
+
C 0 co
c 0
a
1-
=
5.55:
r=
2 pM-ryanodine
0.98
0 0 L-
0.1
a
=
6-68:
r=
0.95
0
-J
0.01 0.3
0.2
0.1
0
-0.1
-0.2
ApHi Fig. 3. For legend see facing page.
-0.3
-0.4
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 487 0-2 pH units in normal muscles, and also 53 % for a decrease of 0 3 pH units in the diabetics. In addition, the decrease of tension in diabetic muscles was delayed as compared to that of normal papillary muscles, as shown by ti values (24-2 + 1-3 s vs. 18 7 + 0 7 s, P < 0 001). Furthermore, the secondary transient recovery of developed tension (d in Figs 1 and 2) was less abrupt in the diabetics. It appeared with a t1 of 755+5-8 s vs. 66+2-3 s (P < 0-001) in normal muscles, and peaked at only 80-2 + 8-6 % of control, as compared to 153-5 + 11-7 % in papillary muscles from normal hearts. It is worth noting that the peak of secondary transient tension recovery coincided, in both groups of muscles, with the maximum decrease of pHi, i.e. -040+0-02 pH units (n = 6) and -028+0X04 pH units (n = 6) in diabetic and normal muscles, respectively. The minimum of developed tension (e in Figs 1 and 2) which then followed the transient recovery was, once again, different in diabetic preparations as compared to normal (47 + 3.9 and 66-2 + 3-4 % of control, respectively) (P < 0-001). We then studied the relationship between developed tension and pHi for both normal and diabetic papillary muscles in control superfusion solution. Figure 3A shows that in both cases, there was, as described in cardiac Purkinje fibres (Vaughan-Jones et al. 1987), an approximately linear relationship between the plot of logarithmic tension and pHi. However, the slope was significantly smaller in the diabetic muscles (- 3) as compared to normal (- 5; P < 001). Therefore, the same change of pHi, in the range studied, had less effect on the developed tension of diabetic muscles than on that of normal muscles. These results support the hypothesis of a decrease in pHi sensitivity of contractile force induced by diabetes in rat papillary muscle. -
-
The effect of sarcoplasmic reticulum inhibitors on the response of tension to ammonium chloride in normal and diabetic papillary muscles It was shown by Fabiato (1985a) in skinned cardiac cells that an increase of pH enhanced Ca2+ accumulation into the SR, and consequently the amplitude of a subsequent Ca2+ release, while a decrease of pH had the opposite effects. These results suggested to us that the decrease in sensitivity of force in diabetic papillary muscles, may, at least partly, result from an altered response of the SR to pHi changes. We investigated this possibility by using the SR inhibitors caffeine (Weber & Herz, 1968) and ryanodine (Sutko & Willerson, 1980). For each muscle, normal (Fig. 4A) and diabetic (Fig. 4B), the top record shows the effect of exposure to and subsequent withdrawal of NH4C1 upon developed tension under control conditions. The bottom trace shows recordings from the same muscles Fig. 3. Relationship between developed tension and changes in pHi on semilogarithmic coordinates for normal (i) and diabetic (X) papillary muscles, in control superfusion solution (A), in the presence of caffeine (B), in the presence of ryanodine (C). The ordinate represents relative developed tension recorded in ten normal and ten diabetic muscles (A) and four normal and four diabetic muscles (B and C). The abscissa is the change of pHi from steady-state control level, either during the application of NH4C1 (positive values recorded each minute) or just after removal of NH4C1 (negative values, at point c of Fig. 1). Each point is the mean pH, value for six normal and diabetic muscles (A, B and C). The lines drawn through the points were fitted by regression analysis. a is the slope of the relationship and r the correlation coefficient.
488
D. LAGADIC-GOSSMANN AND D. FEUVRA Y A
20 mM-NH4CI
1 mN C
__ 20 mM-NH4CI 10 mM-caffeine
1
mN[
MW 56 min B 20 mM-NH4CI 1 mN C
20 mM-NH4CI 10 mM-caffeine
1 mNE_ 5 min
Fig. 4. The effect of caffeine on developed tension during exposure to and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 10 mM-caffeine (bottom record).
when NH4Cl was added and subsequently withdrawn from the superfusate in the presence of 10 mM-caffeine. Caffeine itself had marked positive inotropic effects in both groups, so that tension increased to an average of 350-3 + 48-6 % of control (n = 4) and 325 + 55.7 % of control (n = 4) in normal and diabetic muscles, respectively. With the addition of NH4C1, developed tension increased such that the maximum reached (for a corresponding similar increase of pHi in both groups) in this condition was not significantly different between normal (524 + 657 % of control, n = 4) and diabetic (457+1014 % of control, n = 4) muscles. Moreover, caffeine abolished the secondary transient recovery of tension that occurred after NH4C1 withdrawal. Developed tension was then decreased to a minimum of 70-3+1388% (n = 4) of control in normal muscles and of 42-9 + 6-9 % (n = 4) in diabetic muscles. Results in these experimental conditions (i.e. in presence of caffeine) are summarized by the fact that we found no significant difference in the slopes of the log(tension)-pH1 relationships between normal and diabetic papillary muscles (Fig. 3B).
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 489 A 20 mM-NH4CI
1 mNC
_
-
20 mM-NH4CI 2 /M-ryanodine 1 mN[
5 min B
20 mM-NH4CI 1 mNr
20 mM-NH4CI 2 juM-ryanodine 1 mN
[__ 5 min
Fig. 5. The effect of ryanodine on developed tension during exposure to and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 2 /LM-ryanodine (bottom record).
Ryanodine (2/tM) was used as a more specific SR inhibitor (Bers, Bridge & McLeod, 1987). This substance has no effect on Ca21 sensitivity of myofilaments (Fabiato, 1985b). Figure 5 shows that ryanodine had a marked negative inotropic effect on both normal and diabetic papillary muscles, leading to a fall of developed tension to 6-49 + 1P96 % of control (n = 4) in normal muscles, and to 7417 + 1P87 % of control (n = 4) in the diabetics. The changes of developed tension during exposure to and withdrawal of NH4CI, were similar in both groups of muscles in the presence of ryanodine. At the maximum NH4+-induced alkalinization, the increase in developed tension was not significantly different between both groups of muscles (101-2 + 14-6 % and 15-2 + 10-2 % in the normal and diabetics respectively, as compared to control tension, i.e. without ryanodine, n = 4). The acidosis induced by NH4+ withdrawal was accompanied by a decrease of tension to 5-2 + 1-6 % of control (n = 4) in normal and to 641 + 1-6% of control (n = 4) in diabetic preparations. Moreover, as observed with caffeine, there was no secondary transient recovery of tension. Therefore, as shown in Fig. 3 C, the presence of ryanodine, like that of caffeine,
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
490
rendered the tension-pHi relationship of diabetic papillary muscles identical to that of normal muscles. These results suggest that some diabetes-induced change in the SR might explain the differences that we observed in the pHi sensitivity of tension between diabetic and normal papillary muscles. 1.8
16 3a = -3.34: r =0.99 1.4
*C
1.2 02 0.6 0.4
a=-1 .07: r=0.99
0.2
0
-(.4
-0.3
-0.2
-0.1
0
ApH; Fig. 6. Relationship between tension recovery (after maximum transient positive inotropy, point d of Fig. 1) and pHi recovery (after maximum acidification) following NH4Cl withdrawal, in normal (@) and diabetic (X) muscles. The lines drawn through the points were fitted by regression analysis. (ApHi: n = 6 for both groups of muscles; tension: n = 10 for normal and diabetic muscles).
The effect of amiloride on transient recovery of tension following acidosis induced by withdrawal of ammonium The maximum transient redevelopment of tension corresponded with the maximum acidification in both groups of muscles, as described above (Fig. 1). However, the peak of transient tension recovery in diabetic papillary muscles was slightly delayed and markedly attenuated as compared to normal. Figure 6 shows that there was a good correlation between the time courses of tension recovery and of pHi recovery from maximum acidification, in both normal and diabetic muscles. It has been shown previously that recovery of pHi from an intracellular acid load is largely due to the amiloride-sensitive Na+-H+exchange (Deitmer & Ellis, 1980; Piwnica-Worms, Jacob, Horres & Lieberman, 1985). In this study, the presence of 1 mM-amiloride (Fig. 7) suppressed the transient redevelopment of force, such that tension rapidly reached an average minimum of 24-2 + 3-3 % (n = 4) and 20-5 + 7 % of control (n = 3) in normal and diabetic muscles, respectively. These values were not significantly different but were significantly lower than the minimum values reached under these conditions in muscles in the absence of amiloride. In addition, minimum tension was reached, in the presence of amiloride, with similar ti values for the two groups of muscles (85 + 7-9 s in normal and 106-6 + 24 s in diabetic muscles). Therefore, the presence of amiloride during an intracellular acid load abolished the
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 491 20 mM-NH4CI
A
Tension_
1 mM-amiloride
_
1 mN c 6.5-
6.71 pHi
6.9
-
7-1 73 7
51
5 minT 20 mM-NH4CI
B
1 mM-amiloride
Tension 1 mN C:
6.5 6.7
pHi 6.97.1
5 min
Fig. 7. The effect of 1 mM-amiloride on developed tension and pH, following NH4Cl withdrawal, in a normal (A) and in a diabetic muscle (B).
differences observed in developed tension between both types of muscles. In particular, amiloride abolished the differences observed in transient recovery of tension, in the amplitude of pHi decrease and in the time course of pH, recovery from acidosis (Fig. 7). These results demonstrate that transient recovery of tension must be related to the activity of the Na+-H+ exchange, this exchange being normally particularly stimulated by an intracellular acidification (Grinstein & Rothstein, 1986). In this respect, the decrease in the activity of the exchange induced by diabetes (LagadicGossmann et al. 1988) would account for the smaller redevelopment of force observed in diabetic papillary muscles upon acidification induced by NH4+.
The effect of 2',4'-dichlorobenzamil amiloride on transient recovery of tension following acidosis induced by withdrawal of ammonium It has been suggested that the positive inotropy which appears when Na+-H+ exchange is operational, would result from a secondary stimulation of the Na+-Ca2+ exchange, which elevates Ca1+ and, consequently, cardiac contractility
492
D. LAGADIC-GOSSMANN AND D. FEUVRA Y A
20 mM-NH4CI
1 mN
20 mM-NH4CI 40 iM-DCB
1 mNc
_
-~~~~IA
5 min 20 mM-NH4CI
B
1 mN[
_
_
_
20 mM-NH4CI 40 uM-DCB
1 mNC 5 min
Fig. 8. The effect of DCB on developed tension during exposure and subsequent withdrawal of NH4Cl in a normal (A) and in a diabetic muscle (B). For each muscle, records were obtained in control superfusion solution (top record) and after 15 min exposure to 40 ,sM-DCB (bottom record). DCB had no effect on control tension.
(Vaughan-Jones, 1988). In an attempt to evaluate the role of Na+-Ca2+ in transient redevelopment of tension during an internal acid load, we used DCB, an amiloride derivative, which is known to be a potent inhibitor of this ionic exchange (Frelin, Barbry, Vigne, Chassande, Cragoe & Lazdunski, 1988). Figure 8 shows that the presence of 40 ,tM-DCB in the superfusing solution accentuated the immediate decrease of tension that appeared on removal of NH4C1. Developed tension was thus decreased by the same order of magnitude in both types of preparations (61-5 + 7-8 % and 53-3 + 1-2 %, n = 3, in normal and diabetic muscles, respectively). This decrease occurred, for both types of muscles, with identical ti values (- 40 s). Furthermore, the presence of DCB significantly and similarly reduced and delayed the amplitude of the secondary redevelopment of force in both normal and diabetic papillary muscles. The amplitude reached was then only 747 + 2 % (n = 3) of that obtained in the absence of DCB in normal muscles, and 766 + 8-2 % (n = 3) in the diabetics, whereas the mean ti for reaching the maximum
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 493 increased up to 137 + 12 s and to 175 + 32-8 s (not significant) for normal and diabetic muscles, respectively. These results appear to be in favour of the hypothesis of a participation of Na+-Ca2+ exchange in transient redevelopment of tension during an intracellular acidification. DISCUSSION
The results of this study show a decreased response of contractile force to changes of pHi in papillary muscles from STZ-induced diabetic rat hearts. This effect appears to be related in part to some defect in SR function, since the tension-pHi relationship in diabetic muscles became identical to that of normal muscles in the presence of SR inhibitors. Furthermore, our results obtained in both normal and diabetic preparations agree with the original observation, in ferret papillary muscles (Orchard, 1987), of a transient redevelopment of force following the initial decrease in tension with acidosis. However, this secondary transient positive inotropy was markedly attenuated in the diabetics, and it could be suppressed in the two groups of muscles by SR inhibitors and also by blockage of the sarcolemmal Na+-H+ exchange. The decrease in pHi sensitivity of developed tension in diabetic papillary muscles It has been shown in skinned cardiac cells of rat ventricle (Fabiato & Fabiato, 1978), as well as in intact sheep cardiac Purkinje fibres (Vaughan-Jones et al. 1987), that a fall in pHi reduces tension production while an increase in pHi has the opposite effect. In the present study, changes of + 0-3 units around steady-state pHi produced graded changes of tension in both normal and diabetic papillary muscles. However, a quantitative analysis of these changes clearly shows a lower sensitivity of tension to variations in pHi in diabetic muscles (Fig. 3A). For instance, an alkalinization of 0-2 units increases tension by 590% in normal muscles and only by 230 % in the diabetics. Conversely, an acidification of 0-2 units decreases tension by 47 % of control in diabetic muscles compared to 63 % in normal muscles. Since pHi and tension were not recorded simultaneously in our study, one may argue that steady-state pHi or NH4+-induced changes in pHi may be different in stimulated muscles as compared to quiescent muscles. Indeed, several studies have shown that stimulation produces an intracellular acidification and an increase of intracellular Na+and Ca2+(Bountra, Kaila & Vaughan-Jones, 1988a, b). Moreover, differences may occur in the metabolic response to stimulation, between normal and diabetic muscles, which may affect intracellular pH and the Na+-H+ exchange mechanism. We assume that these differences have negligible effects on our results since Bountra & Vaughan-Jones (1986) have shown, with simultaneous measurements of pHi and force during stimulation in guinea-pig papillary muscles, that the transient positive inotropic effect and the acidification were linked. There may be several reasons for the difference between the pHi sensitivity of normal and diabetic muscles. It may result from diabetes-induced changes in the Ca2+ sensitivity of the myofilaments or alteration of some membrane processes regulating Ca1 . Although a decrease of Ca2+ binding to troponin C under acidosis is plausible (Solaro, Lee, Kentish & Allen, 1988), it has been considered as an unlikely
494
D. LAGADIC-GOSSMANN AND D. FEUVRA Y
explanation for the effects of the pH change on the tension-Ca2+ relationship (Fabiato, 1985a). We investigated the second hypothesis by studying the influence of a major component of CaF+ regulating processes i.e. the SR. Although both caffeine and ryanodine are known to interfere with SR function through completely different mechanisms (Weber & Herz, 1968; Sutko & Willerson, 1980), they both abolished the difference in contractile response to pHi changes between normal and diabetic muscles (Fig. 3B and C). In this respect, it should be noted that we did not observe any effect of caffeine or ryanodine on pHi (data not shown), in agreement with previous studies (Cannell, Vaughan-Jones & Lederer, 1985; Orchard, 1985). Several studies (Penpargkul et al. 1981; Ganguly et al. 1983; Lopaschuk et al. 1983) have shown a decreased ability of diabetic rat cardiac SR to transport Ca2+. This defect appeared to be a consequence of insulin deficiency and associated metabolic changes (Feuvray et al. 1979; Lopaschuk et al. 1983). It has been suggested that a change in the environment surrounding the Ca2+-stimulated ATPase protein may influence its activity (Ganguly et al. 1983). Indeed, the Ca2+ affinity of this pump is strongly dependent on H+ activity (Grassi de Gende, 1988), and there is a competition between Ca2+ and H+ for the high-affinity binding sites that are directly involved in active Ca2+ transport (Levitsky & Benevolensky, 1986). The influence of H+ is such that under high pH, Ca2+ accumulation into the SR and its subsequent release are increased, whereas a low pH has the converse effect (Fabiato, 1985a). In this context, we can reasonably assume that in diabetic rat hearts, which rely essentially on long-chain fatty acids as a metabolic fuel (Randle et al. 1966) and accumulate long-chain fatty acyl derivatives (Feuvray et al. 1979), the SR membrane environment and composition will be affected (Ganguly et al. 1983; Lopaschuk, Eibschutz, Katz & McNeill, 1984). In particular, this could result in a smaller number of, or less accessible, high-affinity Ca2+-binding sites being available for competition with H+. The transient redevelopment offorce following the initial decrease in tension with acidosis in diabetic papillary muscles The initial decrease in tension upon acidosis induced by NH4+ withdrawal was rapidly followed (within 2 min) by a transient redevelopment of force. This was observed in both normal and diabetic papillary muscles. Yet the time course and the amplitude of this transient recovery of tension in diabetic muscles differed markedly from that in normal muscles. In fact, it developed more slowly in the diabetics, as shown by a 15 % greater value of ti, and the maximum amplitude was about half that in normal muscles. A comparable transient positive inotropy has been found in normal Purkinje fibres (Vaughan-Jones et al. 1987) and heart muscle (Orchard, 1987). The latter study showed that this early recovery of tension was due to increased Ca2+ release from the SR, occurring secondarily to an increase in resting Ca?+. Our results show that not only do caffeine and ryanodine suppress the transient positive inotropy in normal muscles (Orchard, 1987), but have the same effect in diabetic muscles. Moreover, our results demonstrate: (i) that the maximum transient redevelopment of force corresponded with the maximum acidification in both groups of muscles and (ii) that the time courses of tension recovery and of pHi recovery were linearly related in both preparations (Fig. 6). Since pHi recovery from an acid load
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pHi 495 depends essentially on the activity of Na+-H+ exchange (Deitmer & Ellis, 1980; Piwnica-Worms et al. 1985), the smaller amplitude of the transient positive inotropy, as well as the slowing down of pHi recovery and of tension recovery in diabetic muscles, may well indicate a primary role of this exchange in the transient redevelopment of force. This appeared to be confirmed by the fact that inhibition of the exchange by amiloride during the acid load suppressed the transient redevelopment of force in both normal and diabetic preparations. In the present study, we did not measure intracellular Na+ activity. However, work performed on other tissues (Deitmer & Ellis, 1980; Piwnica-Worms et al. 1985) has shown that the peak of acidosis induced by NH4+ withdrawal coincided with a rise in aNa, probably mediated by Na+-H+ exchange. This increase in aNa could then be sufficient to activate the Na+-Ca2+ exchanger and lead to an increase in CaW+ (Bers & Ellis, 1982). We approached the possible role of a secondary activation of the Na+-Ca2+exchange by using DCB. Although the results are not as clear-cut as those obtained in the presence of SR inhibitors or the Na+-H+ blocker, DCB induced a significant delay and reduction in the amplitude of the transient tension redevelopment. Therefore our results are in line with the suggestion (Orchard, 1987) that an increase in W+, through stimulation of SR Ca2+loading, may induce a larger release of Ca2+, thus overcoming the direct inhibitory effect of acidosis. In diabetic papillary muscles, the decrease in Na+-H+ exchange activity compared to normal muscles upon acidosis (Lagadic-Gossmann et al. 1988) would then lead to less of an increase of Ca1+ and consequently to a smaller release of Ca2+ from the SR. Alternatively, the greater acidification upon NH4+ withdrawal due to decreased activity of the Na+-H+ exchanger may reduce the SR Ca2+ trigger, either indirectly through an effect on Na+-Ca2+exchange (Philipson, Bersohn & Nishimoto, 1982) or more directly through the SR Ca2+-ATPase (Levitsky & Benevolensky, 1986). In addition, a smaller Ca2+ release from the SR in diabetic muscles may result from an alteration in SR membrane composition and function. In conclusion, STZ-induced diabetes induces: (i) a decrease in the sensitivity of contractile force to a change in pH1; this may be the consequence of an altered SR membrane composition and function, although other possibilities such as a change in the Ca2+sensitivity of myofilaments cannot be discounted; (ii) a marked attenuation of the transient positive inotropy which follows the initial decrease in tension associated with acidosis; this may indirectly result from the decrease in the activity of the sarcolemmal Na+-H+exchange and its consequences on membrane systems regulating CaV. We wish to thank Ian Findlay for his helpful comments on an earlier version of the manuscript, Jean-Michel Chesnais and Philippe Jourdon for valuable discussion and Francoise James for her technical assistance. This work was supported in part by a grant from the Association Frangaise contre les Myopathies. REFERENCES
AMMANN, D., LANTER, F., STEINER, R. A., SCHULTHESS, P., SHIJO, Y. & SIMON, W. (1981). Neutral carrier based hydrogen ion selective microelectrode for extra- and intracellular studies. Analytical Chemistry 53, 2267-2269.
496
D. LAGADIC-GOSSMANN AND D. FEUVRAY
BENOS, D. J. (1982). Amiloride: a molecular probe of sodium transport in tissues and cells. American Journal of Physiology 242, C131-145. BERS, D. M., BRIDGE, J. H. B. & MAcLEOD, K. T. (1987). The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures. Canadian Journal of Physiology and Pharmacology 65, 610-618. BERS, D. M. & ELLIS, D. (1982). Intracellular calcium and sodium activity in sheep heart Purkinje fibres. Effect of changes of external sodium and intracellular pH. Pfiugers Archiv 393, 171-178. BIELEFELD, D. R., PACE, C. S. & BOSHELL, B. R. (1983). Altered sensitivity of chronic diabetic rat heart to calcium. American Journal ofPhysiology 245, E560-567. BORON, W. F. & DE WEER, P. (1976). Intracellular pH transients in squid giant axons caused by C02, NH3 and metabolic inhibitors. Journal of General Physiology 67, 91-112. BOUNTRA, C., KAILA, K. & VAUGHAN-JONES, R. D. (1988a). Effect of repetitive activity upon intracellular pH, sodium and contraction in sheep cardiac Purkinje fibres. Journal of Physiology 398, 341-360. BOUNTRA, C., KAILA, K. & VAUGHAN-JONES, R. D. (1988b). Mechanism of rate-dependent pH changes in the sheep cardiac Purkinje fibre. Journal ofPhysiology 406, 483-501. BOUNTRA, C. & VAUGHAN-JONES, R. D. (1986). The influence of pH, on twitch tension in isolated guinea-pig papillary muscle. Journal of Physiology 382, 109P. CANNELL, M. B., VAUGHAN-JoNEs, R. D. & LEDERER, W. J. (1985). Ryanodine block of calcium oscillations in heart muscle and the sodium-tension relationship. Federation Proceedings 44, 2964-2969. DEITMER, J. W. & ELLIS, D. (1980). Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibres. Journal of Physiology 304, 471-488. FABIATO, A. (1985a). Use of aequorin for the appraisal of the hypothesis of the release of calcium from the sarcoplasmic reticulum induced by a change of pH in skinned cardiac cells. Cell Calcium 6, 95-108. FABIATO, A. (1985b). Effects of ryanodine in skinned cardiac cells. Federation Proceedings 44, 2970-2976. FABIATO, A. & FABIATO, F. (1978). Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal of Physiology 276, 233-255. FEIN, F. S., KORNSTEIN, L. B., STROBECK, J. E., CAPASSO, J. M. & SONNENBLICK, E. H. (1980). Altered myocardial mechanics in diabetic rats. Circulation Research 47, 922-933. FEIN, F. S. & SONNENBLICK, H. (1985). Diabetic cardiomyopathy. Progress in Cardiovascular Diseases 27, 255-270. FEUVRAY, D., IDELL-WENGER, J. A. & NEELY, J. R. (1979). Effects of ischemia on rat myocardial function and metabolism in diabetes. Circulation Research 44, 322-329. FRELIN, C., BARBRY, P., VIGNE, P., CHASSANDE, O., CRAGOE, E. J. JR & LAZDUNSKI, M. (1988). Amiloride and its analogs as tools to inhibit Na+ transport via the Na+ channel, the Na+-H+ antiport and the Na+-Ca2+ exchanger. Biochimie 70, 1285-1290. GANGULY, P. K., PIERCE, G. N., DHALLA, K. S. & DHALLA, N. S. (1983). Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. American Journal of Physiology 244, E528-535. GRASSI DE GENDE, A. 0. (1988). The effect of pH on the calcium dependence of calcium accumulation in dog cardiac muscle sarcoplasmic reticulum. Journal of Molecular and Cellular Cardiology 20, 1087-1093. GRINSTEIN, S. & ROTHSTEIN, A. (1986). Mechanisms of regulation of the Na+/H+ exchanger. Journal of Membrane Biology 90, 1-12. LAGADIC-GOSSMANN, D., CHESNAIS, J. M. & FEUVRAY, D. (1988). Intracellular pH regulation in papillary muscle cells from streptozotocin diabetic rats: an ion-sensitive microelectrode study. Pflilgers Archiv 412, 613-617. LEVITSKY, D. 0. & BENEVOLENSKY, D. S. (1986). Effects of changing Ca2+-to-H+ ratio on Ca2+ uptake by cardiac sarcoplasmic reticulum. American Journal of Physiology 250, H360-365. LOPASCHUK, G. D., EIBSCHUTZ, B., KATZ, S. & MCNEILL, J. H. (1984). Depression of calcium transport in sarcoplasmic reticulum from diabetic rats: lack of involvement by specific regulatory mediators. General Pharmacology 15, 1-5. LOPASCHUK, G. D., KATZ, S. & MONEILL, J. H. (1983). The effect of alloxan- and streptozotocin-
DIABETIC RAT PAPILLARY MUSCLE CONTRACTION AND pH1 497 induced diabetes on calcium transport in rat cardiac sarcoplasmic reticulum. The possible involvement of long chain acylcarnitines. Canadian Journal of Physiology and Pharmacology 61, 439-448. ORCHARD, C. H. (1985). The effect of sarcoplasmic inhibitors on the response of ventricular muscle to acidosis. Biophysical Journal 47, 379a. ORCHARD, C. H. (1987). The role of the sarcoplasmic reticulum in the response of ferret and rat heart muscle to acidosis. Journal of Physiology 384, 431-449. PENPARGKUL, S., FEIN, F., SONNENBLICK, E. H. & SCHEUER, J. (1981). Depressed cardiac sarcoplasmic reticular function from diabetic rats. Journal of Molecular and Cellular Cardiology 13, 303-309. PHILIPSON, K. D., BERSOHN, M. M. & NISHIMOTO, A. Y. (1982). Effects ofpH on Na'-Ca 2+exchange in canine cardiac sarcolemmal vesicles. Circulation Research 50, 287-293. PIWNICA-WORMS, D., JACoB, R., HORRES, C. R. & LIEBERMAN, M. (1985). Na/H exchange in cultured chick heart cells. pHi regulation. Journal of General Physiology 85, 43-64. RANDLE, P. J., GARLAND, P. B., HALES, C. N., NEWSHOLME, E. A., DENTON, R. M. & POGSON, C. I. (1966). Protein hormones interactions of metabolism and the physiological role of insulin. Recent Progress in Hormone Research 22, 1-48. SAUVIAT, M. P. & FEUVRAY, D. (1986). Electrophysiological analysis of the sensitivity to calcium in ventricular muscle from alloxan diabetic rats. Basic Research in Cardiology 81, 489-496. SOLARO, R. J., LEE, J. A., KENTISH, J. C. & ALLEN, D. G. (1988). Effects of acidosis on ventricular muscle from adult and neonatal rats. Circulation Research 63, 779-787. SUTKO, J. L. & WILLERSON, J. T. (1980). Ryanodine alteration of the contractile state of rat ventricular myocardium. Comparison with dog, cat, and rabbit ventricular tissues. Circulation Research 46, 332-343. TAHILIANI, A. G. & MCNEILL, J. H. (1986). Diabetes-induced abnormalities in the myocardium. Life Sciences 38, 959-974. TAKEDA, N., NAKAMURA, I., HATANAKA, T., OHKUBO, T. & NAGANO, M. (1988). Myocardial mechanical and myosin isoenzyme alterations in streptozotocin-diabetic rats. Japanese Heart Journal 29,455-463. VAUGHAN-JONES, R. D. (1988). Regulation of intracellular pH and contractility in cardiac muscle. Journal ofMolecular and Cellular Cardiology 20, S.33. VAUGHAN-JONES, R. D., EISNER, D. A. & LEDERER, W. J. (1987). Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibres. Journal of General Physiology 89, 1015-1032. VAUGHAN-JONES, R. D., LEDERER, W. J. & EISNER, D. A. (1983). Ca2+ ions can affect intracellular pH in mammalian cardiac muscle. Nature 301, 522-524. WEBER, A. M. & HERZ, R. (1968). The relationship between caffeine contracture of intact muscle and the effect of caffeine on isolated reticulum. Journal of General Physiology 52, 750-759.
