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Influence of isoproterenol and calcium on cadmium- or lead-induced negative inotropy related to cardiac myofibrillar protein phosphorylations in perfused rat heart
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
55,
8- 17 (1980)
Influence of lsoproterenol and Calcium on Cadmium- or Lead-Induced Negative lnotropy Related to Cardiac Myofibrillar Protein Phosphorylations in Perfused Rat Heart STEPHEN *Nuclear
Magnetic
J. KoPP**’
AND MICHAEL
BARANYT
Resonance Laboratory, Chicago College of Osteopathic Medicine. and tDepartment of Biological Chemistry, University of Illinois at the Medical Center. Chicago. Illinois 60612
Received December
Chicago,
Illinois
60615,
12, 1979; accepted March 20, 1980
Influence of Isoproterenol and Calcium on Cadmium- or Lead-Induced Negative Inotropy Related to Cardiac Myofibrillar Protein Phosphorylations in Perfused Rat Heart. KOPP, S. J., AND BARANY, M. (1980). Toxicol. Appl. Pharmacol. 55, 8-17. The individual and combined physiological and myofibrillar protein phosphorylation effects of the negative inotropic agents, cadmium (3 x lo-” mM) and lead (3 x lo-” mM), and the positive inotropic agents isoproterenol (7 x lo-’ M) and calcium (6.5 mM) were examined in isolated modified perfused rat heart preparations to evaluate possible causal associations between cadmium- and lead-induced changes in myofibrillar protein phosphorylations and altered inotropic responsiveness. Cadmium and lead alone depressed cardiac contractility and only the phosphorylation of the myosin light chain-2 (LC-2). These effects were attenuated in the presence of isoproterenol and elevated extracellular calcium; however, cadmium and lead inhibited both the positive inotropic activation of the heart by calcium and isoproterenol and the concomitant increase in phosphorylations of the purported cardioregulatory phosphoproteins, LC-2 and the troponin inhibitory subunit (TN-I). Positive chronotropic responses to the p-adrenergic agonist were unaffected by cadmium and lead. These results suggest that the negative inotropic effects of cadmium and lead are related to depressed phosphorylation of LC-2 and TN-I. Furthermore, the present findings suggest that cadmium and lead may alter myofibrillar protein phosphorylation mechanisms by antagonizing sarcolemmal calcium translocation processes; however, these results do not preclude the possibility of direct intracellular antagonisms of calcium-dependent processes by these heavy metal ions.
Functional and metabolic processes of the mammalian heart are vulnerable to changes in extracellular and/or intracellular ionic composition (Opie, 1969). Thus, environmental stressors (e.g., heavy metals) which modify the chemical composition of this tissue are postulated to induce detectable physiologic and biochemical changes in the myocardium. Since isolated perfused mammalian heart techniques afford the opportunity to critically assess physiological responses of the 1 To whom correspondence
should be addressed.
0041-008X/80/100008-10$02.00/0 Copyright All rights
heart to diverse chemical insults under controlled conditions, this methodology has been utilized extensively in physiological and toxicological investigations (Hawley and Kopp, 1975; Hess and Gabel, 1979; Kopp and Hawley, 1976; Kopp et al., 1978a,b; Levine et al., 1976; Marsh, 1975). Previous studies have demonstrated that submicro- and micromolar (3 parts per billion (ppb), 0.03-3 parts per million (ppm)) cadmium and 0.3-3 ppm lead concentrations (i) selectively impair the excitation processes of the cardiac conduction system-cadmium depresses atrioventricular node con-
0 1980 by Academx Press. Inc. of reproduction in any form reserved.
8
HEART INOTROPY-MYOFIBRIL
ductivity, while lead depresses His-Purkinje cell excitability (Hawley and Kopp, 1975; Kopp and Hawley, 1976; Kopp et al., 1978a,b), and (ii) depress cardiac contractility (Kopp et al., 1978a,b). The in vitro findings have been corroborated in in vivo controlled diet studies involving cadmium or lead intake from drinking water (5 ppm) for 15-24 months (Kopp et al., 1978c, 1979a,b). Several molecular processes have been implicated as contributing factors to the regulation of chemical-mechanical energy transduction in heart muscle, including protein phosphorylation mechanisms involving a sarcoplasmic membrane constituent, phospholamban (Katz et al., 1975; Lindemann et af., 1978), sarcolemma membrane proteins (Drummond and Severson, 1979; Lindemann et al., 1978; Tada et al., 1979; Walsh et al., 1979; Wollenberger and Will, 1978), and the myofibrillar proteins: the inhibitory subunit of troponin (TN-I) (England, 1975, 1976; Solar0 et al., 1976) and the myosin light chain-2 (LC-2) (Frearson et al., 1976; Kopp and Barany, 1979: Lebowitz et al., 1976). Age- and concentration-dependent cadmium or lead accumulation in the heart has been shown to alter myocardial contractility in chronically fed experimental animals (Kopp et al., 1979b). The present investigations were undertaken to (i) evaluate the physiological responses of the heart and concomitant myofibrillar phosphorylation processes to lead or cadmium alone and in combination with inotropic (elevated extracellular calcium or /3adrenergic-isoproterenol) stimulation under controlled isolated perfused heart conditions; and (ii) correlate directly modified phosphorylation of specific myofibrillar regulatory proteins with altered inotropic responsiveness to determine possible causal associations. METHODS The heart perfusions were performed essentially as described previously (Kopp er a/. , 1978a). Heparin-
PHOSPHORYLATION
9
ized albino Sprague-Dawley rats (200-300 g) of random sex were sacrificed by cervical dislocation and the hearts (N = 40) were excised and chilled in precooled perfusion solution (lo”C), immediately stopping heart contractile activity. While the heart was in this medium, the aorta was cannulated and tied for retrograde perfusion, superfluous connective and adipose tissues were removed, and a single open silk (4-O) loop was sutured through the heart, 5 mm from the apex for recording systolic (peak active) tension. The cannulated heart was then perfused, as described previously (Kopp et al., 1978a), with a perfusate containing 152 mM NaCl, 5.4 mM KC], 1.05 mM MgC&, 1.9 mM CaCl,, 5.6 mM glucose, and 10 mM Tris-HCl buffer, pH 7.35, at 35°C for 30 min during which time electrocardiographic (ECG) and mechanical events stabilized, indicating optimal tissue reoxygenation. A constant diastolic load of 4.0 g was maintained by adjusting the position of the force displacement transducer. Cardiac electrical and mechanical events were recorded with a Grass Model 79C polygraph at paper speeds of 25 and 100 mm/set for accurate signal resolution. Only stable preparations characterized by a rhythmic spontaneous heart rate and stable mechanical activity were studied. Following the 30-min equilibration period, the hearts were equilibrated for 5 min with the experimental perfusate: Control (N = 9), 6 x lO-3 mM CdCl* (N = 3) 6 x lo-’ mM Pb3(CSH50,)2 (N = 3), 6.5 mM CaCl, (N = 4), 7 x 10m7M isoproterenol hydrochloride (N = 4). 6 x IO+ mM CdCl, + 6.5 mM CaCl* (N = 5) 6 x 10m4mM Pb,(C,H,O,), + 6.5 mM CaCl, (N = 5). 6 X 10m3mM CdCl, + 7 x 10m7M isoproterenol hydrochloride (N = 4), and 6 x lo-” mM Pb3(CfiHS07)2 + 7 x lo-’ M isoproterenol hydrochloride (N = 3) at the identical temperature, pressure, and pH (7.35). This exposure duration proved to be sufficient to induce maximal changes in the variables which were measured. At the conclusion of this perfusion period the radioactive experimental perfusate containing 1 mCi carrier-free 32P per liter at the identical temperature, pressure, and pH was turned on and the nonradioactive experimental perfusate turned off. This radioactive perfusion continued for 40 min and was followed by an ll-min washout perfusion with the nonradioactive experimental perfusate which removed 99.6% of the remaining noncovalently bound extracellular 32P (Kopp and Barany, 1979). Contractile and electrical parameters were recorded for analysis at IO-min intervals. Since the perfusion apparatus was a closed system, the flow rate of the heart during the radioactive perfusion was calculated as the difference between the initial perfusate volume and the remaining volume plus the dead space in the system. The percentage 32P uptake by the heart from the perfusate was determined as the ratio of total heart radioactivity to the total radioactivity exposed to the heart.
10
KOPP AND BARANY
Upon completion of the wash perfusion period, the hearts were removed from the cannula and immediately immersed in iced extraction buffer for myotibril extraction. The hearts were observed to stop in diastole after a maximum of a single contraction. This methodology (Kopp and Barany, 1979) yielded consistent reproducible results concerning the phosphorylation of myofibril proteins during cardiac diastole. Tritondeoxycholate-purified cardiac myofibrils free of mitochondrial, sarcolemmal, and sarcoplasmic reticulum membrane contaminants were prepared from these hearts using the procedures of Solar0 ef al. (1971) and Martin ef al. (1977) modified as described elsewhere (Kopp and B&any, 1979). The specific [32P]phosphocreatine radioactivity was determined (Schliselfeld, 1974), the purified myofibrils were fractionated by electrophoresis, the protein bands identified and the [3ZP]phosphate incorporation into the individual myofibrillar proteins quantitated, as detailed previously (Kopp and Barany, 1979). Calculations
and Statistical
Methods
The heart rates were calculated from the R-R interval of the electrocardiogram. The flow rate during the 40-min 32P perfusion was measured as milliliter per minute perfused through the heart. The flow rates were normalized by expressing the values as microliter per heart cycle. All physiological parameters (mechanical and electrical events, 32P uptakes and flow rates) represent the average events during the 40-min 3zP Least Squares Linear Eqh y= 0.0041
Regression x t II 50
.
15
. l
.
‘k
.:.**
. .
:.. .
. . . .
-
.
.
:;*i
l. .
l
.**
IO
.
. l
..-l . *. .
Active Tension (4
.
:
5
ll,ll.Sl,# 100
150
Spontaneous
),,, 200
Heart
S,, 250
Ratebts,4nii
FIG. 1. Graph depicting independent force (active tension&frequency (heart rate) relationship derived from a population of spontaneously beating rat hearts (range 133-237 beats/mitt). Least squares linear regression slope is shown.
20
I
d
1'5
3’0 PerfusIon
FIG. 2. Graph depicting the of the electrocardiogram) and tive tension) stability of control equilibration perfusion period. regression slopes are shown.
45
60
lime(mln)
electrical (PR interval mechanical (peak achearts during the postLeast squares linear
perfusion period. The reported values for the physiological and radioactivity measurements represent the mean + SE for each experimental group. To compensate for the slight differences in gel recoveries, myofibril concentrations applied to the gels and specific radioactivities of the [32P]phosphate in phosphocreatine encountered among the various individual heart experiments, all measured gel recoveries, total gel protein concentrations and specific radioactivities for each heart experiment were adjusted to 75%, 600 pg, and 3 x IO6 cpm/Fmol, respectively. The resultant small normalization factor for each experiment was used to adjust individual myofibril protein gel radioactivity values to their equivalent normalized values. This normalization procedure was necessary to evaluate statistically the quantitative differences in myofibril protein zone radioactivity caused by the specific experimental perfusates studied. The incorporation of [32P]phosphate into the myofibrillar proteins was calculated from the following data: Heart myofibrils contain 50% myosin and 20% actin (Katagiri and Morkin, 1974). Cardiac myosin consists of two heavy chains with apparent molecular weights of 200,000 daltons each (Siemankowski and Dreizen, 1978), two light chains of 27,000 daltons, and two light chains of 19,000 daltons (Frearson and Perry, 1975). The molecular weight of cardiac actin is 42,000 daltons (Leger et al., 1975). The molar ratio of actin:tropomyosin is 4.4: 1, and the molar ratio of actin:TN-T:TN-I is 4.3:1: 1. These ratios were calculated by us from staining intensities of the protein
HEART INOTROPY-MYOFIBRIL TABLE PHYSIOLOGICAL COMBINED
1
PARAMETERS DURING 32P PERFUSION OF NEGATIVE (CADMIUM OR LEAD) AND POSITIVE
Heart perfusion
N
Control 6 x lo-” mM CdCI, 6 x lo-” mM Pb,(C,H,O,), 6.5 mM CaC& 6 x 10m3InM CdCle + 6.5 mM CaCI,, 6 x IO-” mM Pb,(C,H,O,), + 6.5 mM CaCI, 7 X 10-j M isoproterenol 6 x 1O-3 mM CdClr + 7 x IO-’ M isoproterenol 6 Y lo-’ mM Pb,(C,H,O,), + 7 x IO-’ M isoproterenol
9 3 3 4 5 5 4
Note. Values ” Significantly ’ Significantly ’ Significantly ” Significantly I’ Significantly I Significantly v Significantly
represent different different different different different different different
11
PHOSPHORYLATION
RAT HEARTS UNDER THE INFLUENCE OF SINGLE OR (ISOPROTERENOL
Active tension (8) 12.4 4.4 0.6 21.4 11.7 11.2 18.8
2 t t t r + -t
OR CALCIUM)
Spontaneous heart rate (beatsimin)
0.8 0.5” 0.2” 0.2” 1.6”,” 0.4’ 0.7”
1672 120 k 147* 188 k 132 + 202 k 315 k
4
8.8 t 0.6”J
_152 k
3
10.2 + 1.6”,O
7 lob 5 15 10 19 26”
INOTROPIC
Perfusion flow rate (PI/heart cycle) 50 48 81 48 59 34 48
f t 2 k k + t
5 7 2” 7 3 2’ 4
AGENTS
Percentage OsPuptake 7.2 8.0 6.5 6.0 7.0 8.5 5.1
k k t t t t +
0.4 1.2 0.2 0.6 0.7 0.6 0.4’
fy.lt
38 k 1
4.9 t 0.31
264 it 14”,”
39 + 3’
7.2 k 0.4
mean t SE. from control. p < 0.001. from control. p < 0.01. from 6.5 mM CaC&, p < 0.01. from 6 X 10m9mM CdCI,, p < 0.01. from 6 x lo-” mM Pb,(C,H,O,),. p < 0.001. from control, p < 0.02. from 7 x 10m7M isoproterenol. p < 0.01.
bands on gels measured with a Zeineh soft laser scanning densitometer, and they are in good agreement with those which we calculated from the data of Katagiri and Morkin (1974). We used an apparent molecular weight of 68,000 daltons for cardiac tropomyosin (Cummins and Perry. 1974). 38,000 daltons for cardiac TN-T (Brekke and Greaser, 1976), and 29,500 daltons for cardiac TN-I. This latter value is somewhat higher than the 28.000 daltons described by other investigators (Cole and Perry, 1975: Berson et al., 1978). Student’s t test of the means was used to evaluate statistically the significance of the results. A value of p < 0.02 was accepted as significant. Least squares linear regression analyses were performed to ascertain significant correlations between changes in phosphorylation and the myocardial contractile state.
(Fig. 1). The functional stability of the control heart perfusions with respect to contractility and excitability is demonstrated in Fig. 2. These results indicated that the 40-min 32P perfusion time used in these experiments did not alter or contribute to instability in these heart preparations which would produce erroneous phosphorylation results. Thus, in the experimental perfusions altered cardiac contractility was directly attributable to the influence of the experimental perfusates alone.
Calcium
Inotropic
Activation
RESULTS Force (active tension) and frequency (spontaneous heart rate) are effectively independent of one another within the range of heart rate changes detected in these studies
The physiological effects and changes in myofibril protein phosphorylation induced by cadmium and lead perfusions alone relative to control are shown in Tables 1 and 2 and Fig. 3.
12
KOPP
AND
The physiological responses of hearts perfused with elevated calcium alone, or elevated calcium in combination with cadmium or lead are presented in Table 1. Cadmium or lead ions significantly depressed cardiac contractility and calcium-activated positive inotropy. Other physiological indices were similar to those findings detected for elevated calcium alone. This elevated extracellular calcium concentration did, howthe negative inotropic ever, attenuate effects of cadmium or lead alone. In conjunction with these findings, the significantly increased phosphorylation of the various myofibril proteins induced by elevated perfusate calcium levels were significantly depressed by cadmium or lead (Table 2). Although the myosin light chain-2 phosphorylation in response to cadmium + calcium treatment remained depressed relative TABLE
BARANY
to control as well, the phosphorylation of the other myofibril proteins from the lead + calcium and cadmium + calcium hearts were comparable to control values. Zsoproterenol
Activation
The cardiological indices of hearts perfused with isoproterenol alone or in combination with lead or cadmium are shown in Table 1. Cadmium and lead ions inhibited the isoproterenol stimulated positive inotropy; however, the Padrenergic receptor-mediated augmentation in heart rate was not altered significantly by the presence of either heavy metal in the perfusate. Other physiological responses were comparable to isoproterenol treatment alone. These findings suggest the existence of functional myocardial P-receptors during heart per2
SPECIFIC [32P]P~~~~~~~~ INCORPORATION INTO VARIOUS CARDIAC MYOFIBRILLARPROTEINS OF PERFUSED RAT HEART MOLE[~*P]PHOSPHATE/MOLE SPECIFIC MYOFIBRIL PROTEIN Heart
perfusions
CO”tKd 6 x IO-’ ITIM CdCi, 6 x IO-’ nm Pb,(CdW’,h 6.5 tnhd CaCI, 6 x 1Om3 rn~ CdCP + 6.5 nm C&I, 6 x lo-’ rn~ Pb,(C&O,), + 6.5 ,m.t CaCl, 7 x 10-7 M isoproterenol 6 x IO-s nm CdCI, +7x IO’M isoproterenol 6 x 10.’ IIIM Pb,(C.Hs0~)r + 7 x 10-7 M isoproterenol
N
Tropomyosin”
Actin
9 3
0.089 f 0.008 0.074 f 0.008
0.022 2 0.005 0.021 * 0.002
0.064 0.069
+ 0.014 +- 0.011
0.053 2 0.006 0.048 + o.cm
0.029 0.030
3 4
0.066 2 0.009 0.166 * 0.017’
0.023 2 0.002 0.048 -c 0.008’
0.050 t 0.008 0.138 k 0.020’
0.062 -t 0.006 0.171 + 0.011”
0.021 + 0.003 0.042 f 0.008
0.042 2 0.002’ 0.177 f 0.021”
5
0.072 T 0.004’
0.024
-c 0.002
0.059 z 0.003’
0.064 f 0.004’
0.029 f 0.002
0.085 -t 0.008”~
5
0.069
0.016 -c 0.002
0.035 z 0.003’
0.030
0.026
z 0.002
0.092
e O.OWJ
4
0.107 + 0.025
0.031
-’ 0.007
0.075
0.212 z 0.02S”
0.031 t 0.002
0.167
? 0.014”
4
0.059 2 0.002
0.019
f 0.002
0.038 2 0.003
0.0842 o.ow*~’
0.023
0.095 2 o.cw
3
0.046 2 0.004”
0.017 _’ 0.003
0.049 k 0.004’
0.065 f 0.014’
0.019 f 0.003
+ 0.005’
TN-T”
Note. Values represent mean + SE. ” TM, fropomyosin (MW 68,ooO daltons). D TN-T, Tropomyosin binding subunit of troponin (MW 38,000 daltons) r TN-I. Troponin inhibitory subunit (MW 29,500 daltons). d LC-I. Myosin light chain-l (MW 27,ooO daltons). s LC-2, Myosin light chain-2 (MW 19,000 d&tons). ’ Significantly different from control, p < 0.02. ” Significantly different from control, p < 0.001. h Significantly different from control, p < 0.01. I Significantly different from 6.5 rn~ C&I,, p < 0.01. ’ Significantly different from 6 x 10e4 IIIM Pb,(C,H,O,),, p < 0.02. * Significantly different from 6 X 1Om3mM CdCI,. p < 0.01. ’ Significantly different from 7 x IO-’ M isoprotercnol. p < 0.001.
r 0.011
TN-I’
f O.cKw’
LC-I” z 0.007 + 0.004
t 0.001
LC-2’ 0.116 0.087
0.087
k 0.006 + 0.007
f 0.007”‘J
HEART INOTROPY-MYOFIBRIL
l
CONTROL
PHOSPHORYLATION
13
(N=9)
o 6 x 1O-3 mM CdC12 (N=3) -
. 6 x~O-~,M . s*n”ka”t. I. sl.@“iunt.
Pb$Cdrate)p P< Il.02 P
tqooo 1 .c E 8 5 E 2 0
c
5,000
-
1,000
-
Iy *cm TN-T
Molecular
TN-1 LC-I
LC-2
Weigll
FIG. 3. The radioactivity distribution profiles of Triton-deoxycholate-purified rat heart myofibrillar proteins electrophoresed on SDS-urea gels. The average protein band counts reflect changes induced by cadmium or lead relative to control. The ordinate (counts120 min) represents normalized values for equivalence in gel protein concentrations, specific activities of [32P]phosphocreatine and gel recoveries as detailed in the text. Beef heart tropomyosin (TM), troponin (TN-T, tropomyosin-binding subunit; TN-I, inhibitory subunit), and myosin (LC-1, light chain-l; LC-2, light chain-21 were used to identify the proteins in the various zones. Actin was identified by its molecular weight.
fusions with cadmium or lead ions. In addition the significantly elevated phosphorylation of LC-2 induced by isoproterenol was inhibited completely and the phosphorylation of TN-I was attenuated by cadmium or lead + isoproterenol perfusions (Table 2 and Fig. 4). The phosphorylations of other myofibrillar proteins were not altered by isoproterenol treatment alone or combined perfusions with cadmium or lead. A complete correlation of contractile state measured as active tension only with myosin light chain-2 [32P]phosphate incorporation was detected in response to the various experimental perfusates, as shown in Fig. 5. The least squares linear regression equation
depicting this relationship was determined to be y = 0.0062~ + 0.0367 with a correlation coefficient = 0.88. These results suggest that the depressed myosin light chain-2 phosphorylation detected in these studies was a direct factor contributing to the cadmium- or lead-induced negative inotropy and inhibition of calcium or isoproterenol activated positive inotropy in the heart. DISCUSSION Myocardial responses to extrinsic (e.g., hormonal) and intrinsic (e.g., myogenic) physiological effecters may be mediated or modulated through a common pathway involving the differential phosphorylation of
14
KOPP AND BARANY
A 7 X U7 M ISOPROTERENOL (N=4) Y 6 X 1O-3 mM CdCI, + 7 X 1O-7 M ISOPRO l 6 X 10m4rnM Pb3(Citrale)2 + 7X 10m7 M ” SlrnLfirml. P
FIG. 4. The radioactivity distribution profiles of Triton-deoxycholate purified rat heart myofibrillar proteins electrophoresed on SDS-urea gels. The average protein band counts reflect the effects of isoproterenol alone and isoproterenol in combination with either cadmium or lead. See text and Fig. 2 legend for details.
critical regulatory proteins present in the mammalian myocardium (Greengard, 1978: Nathanson, 1977). The phosphoproteins with purported regulatory actions in the control of cardiac contractility include the myofibrillar proteins, TN-I (troponin inhibitory subunit) (England, 1975, 1976; Solar0 et al., 1976; Ezrailson et al., 1977) and LC-2 (myosin light chain-2) (Frearson et al., 1976; Lebowitz et al., 1976); a sarcoplasmic protein, phospholamban (Katz et al., 1975; Lindemann et al., 1978; Tada et al., 1979); and several sarcolemmal membrane proteins (Lindemann et al., 1978; Wollenberger and Will, 1978; Walsh et al., 1979). Investigations concerned with the physiological implications of altered LC-2 phosphorylation suggest that LC-2 phosphorylation may enhance myosin-actin interactions, and thereby facilitate cross-bridge
formation (Bat-any and Barany, 1980). Thus, the magnitude of LC-2 phosphorylation in the heart during diastole has been postulated to predetermine directly the force of active tension generated by the heart in the ensuing contraction (Kopp and Barany, 1979). Previously, a direct linear relationship was described between active tension formation and LC-2 phosphorylation which was represented by the least squares linear regression equation: mole[32P]phosphate/ mole LC-2 = O.O066[active tension (g)] + 0.0437 (Kopp and Barany, 1979). This relationship is consistent with the findings of the present study which were expressed in the least squares linear regression equation: mole[32P]phosphate/mole LC-2 = 0.0062 x [active tension (g)] + 0.0367. The slope and intercept discrepancies between these equa-
HEART INOTROPY-MYOFIBRIL
PHOSPHORYLATION
15
1
IO
50
IO0 Actw
Tsnsh
150
ZOO
(g)
FIG. 5. Correlation between peak active tension and LC-2 phosphorylation. Symbols represent results from the following experimental conditions: W, control; 0, Pb; 0, Cd; A, isoproterenol; A, isoproterenol + Cd; A, isoproterenol + Pb; 0, Ca; q , Ca + Pb; and b, Ca + Cd. Least squares linear regression equation describing this correlation is mol(3zP)phosphate/mo1 light chain-2 = 0.0062 (active tension) + 0.0367 with a correlation coefficient of 0.88. The graphed values represent the mean -C SE.
tions are minor and within the experimental error of the applied techniques. The present investigations have demonstrated that cadmiumand lead-induced negative inotropy is attenuated in the presence of isoproterenol or elevated extracellular calcium; however, these heavy metal ions inhibit the positive inotropy associated with calcium or isoproterenol activation. These physiological responses parallel directly the changes in LC-2 phosphorylation. Elevated extracellular calcium concentrations presumably augment myocardial contractility through a direct stimulation of calcium-dependent processes induced by elevated ionized sarcoplasmic calcium levels during the excitation-contraction coupling process (Keely and Corbin, 1977; Wollenberger and Will, 1978). This increased free calcium in the sarcoplasm may activate directly the calcium-dependent protein kinases causing an increased phosphorylation of the cardioregulatory protein effecters (e.g., LC-2, TN-I, phospholamban) (Reddy et al., 1977; Wollenberger.and Will, 1978)) thereby promoting increased cardiac contractility. The subcellular mechanisms
attributed to p-adrenergic agonist activity purportedly involve the P-receptor-hormone complex formation which activates the adenylate cyclase-cyclic AMP-protein kinase system and induces concomitant intracellular (i) elevation in sarcoplasmic ionized calcium concentrations; (ii) enhanced calcium transport; (iii) increased calcium binding; and/or (iv) augmented calcium sensitivity (Wollenberger and Will, 1978). If cadmium and lead act to impair calcium utilization, intracellularly, a plausible interpretation consistent with present findings would suggest that cadmium and lead ions may antagonize transmembrane calcium transport processes either directly by displacing calcium ions from membrane binding sites (Langer er al., 1974), or indirectly by inhibiting the phosphorylation of sarcolemma proteins required for calcium ion influx. The partial reversal of the negative inotropic effects of cadmium and lead by elevated extracellular calcium is postulated to represent a competitive mass action effect, while the partial reversal by isoproterenol may reflect an enhanced intracellu-
16
KOPP AND BARtiNY
lar utilization or sensitivity to existing calcium, independent of increased calcium permeation. This isoproterenol-augmented sensitivity to calcium may explain the increased TN-I phosphorylation above control observed in cadmium + isoproterenol-treated hearts. The positive chronotropic responses in the presence of cadmium and lead with isoproterenol are not consistent with the premise that cadmium and lead necessarily alter adenylate cyclase activity at the concentrations studied. Cadmium and lead depression of cardiac contractility is partially reversible by isoproterenol and elevated extracellular calcium. Altered phosphorylation of the myofibrillar protein, myosin LC-2 which is purportedly a cardioregulatory phosphoprotein, paralleled changes detected in active tension. The present findings suggest the possibility of extracellular or intracellular antagonisms of calcium-dependent processes by these heavy metal ions. ACKNOWLEDGMENTS This research was supported by grants from the United States National Institutes of Health (NS-12172). the Muscular Dystrophy Association, and the National Institutes of Health National Research Service Award l-F32 ES 05112 TOX from the National Institute of Environmental Health Sciences (S.J.K.). We wish to thank Mr. Robert Kelly and Ms. Ruth Zelkha for the fine graphic art illustrations presented in the figures. We wish to extend a special thanks to Ms. Rose Sage, Mrs. Majorie Pole, and Ms. Laura Gloger for their painstaking devotion to the preparation of this manuscript.
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BREKKE, C. .I., AND GREASER, M. L. (1976). Separation and characterization of the troponin components
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150,669-671.
HESS, M. E., AND GABEL, B. E. (1979). Dissociation between cardiac cyclic AMP and myocardial contractility induced by verapamil, calcium and magnesium ions. Cardiology 64, 75-86. KATAGIRI, T., AND MORKIN, E. (1974). Studies on the substructure of myosin in cardiac hypertrophy. Characterization of light chains. Biochim. Biophys.
Acta
342, 262-274.
KATZ, A. M., TADA, M., AND KIRCHBERGER, M. A. (1975). Control of calcium transport in the myocardium by the cyclic AMP-protein kinase system. in Advances in Cyclic Nuclear Research (P. Greengard and G. A. Robison, eds.), Vol. 5, pp. 453-472. Academic Press, New York. KEELEY, S. L., AND CORBIN, J. D. (1977). Involvement of CAMP-dependent protein kinase in the regulation of heart contractile force. Amer. J. Physiol. 233, H269- H275.
HEART
INOTROPY-MYOFIBRIL
KOPP, S. J., AND HAWLEY, P. L. (1976). Factors influencing cadmium toxicity in A-V conduction system of isolated perfused rat heart. Toxicol. Appl. Pharmacol. 37, 53 l-544. KOPP, S. J., BAKER, J. C., D’AGROSA, L. S., AND HAWLEY, P. L. (1978a). Simultaneous recording of His bundle electrogram, electrocardiogram, and systolic tension from intact modified Langendorff rat heart preparations. I. Effects of perfusion time, cadmium and lead. Toxicol. Appl. Pharmacol. 46, 475-487.
KOPP, S. J., BAKER, J. C., AND D’AGROSA, L. S. (1978b). Simultaneous recording of His bundle electrogram. electrocardiogram, and systolic tension from intact modified Langendorff rat heart preparations. II. Dose-response relationship of cadmium. Toxicol. Appl.
Pharmacol.
46, 489-497.
KOPP, S. J., AND BARANY, M. (1979). Phosphorylation of the 19,000-dalton light chain of myosin in perfused rat heart under the influence of negative and positive inotropic agents. J. Viol. Chem. 254, 12007-12012. KOPP. S. J., FISCHER, V. W., ERLANGER. M., PERRY, E. F., AND PERRY, H. M., JR. (1978~). Electrocardiographical, biochemical and morphological effects of chronic low level cadmium feeding on the rat heart. Proc. Sot. Exp. Biol. Med. 159,339-345. KOPP,
S.
J.,
GLONEK,
T..
ERLANGER,
M.,
PERRY.
F.. BARANY, M., AND PERRY, H. M., JR. (1979a). Altered myocardial function and metabolism induced by chronic, low level cadmium feeding in the rat. In Trace Substances in Environmental Health (D. D. Hemphill, ed.), Vol. 13. pp. 446-452. Univ. of Missouri Press, Columbia. KOPP, S. J., GLONEK, T., ERLANGER, M., PERRY, E. F., BARANY, M., AND PERRY, H. M., JR. (1979b). Hypertension and cardiac changes in rats fed cadmium and/or lead. Clin. Res. 27, 468A. LANCER, G. A., SERENA, S. D., AND NUDD, L. M. (1974). Cation exchange in heart cell culture: correlation with effects on contractile force. J. Mol. Cell. Cardiol. 6, 149- 161. LEBOWITZ, E. A., THIBAULT, L. E., AND ADELSTEIN, R. S. (1976). Phosphorylation of cat myocardial myosin and M-line-protein and enhancement by norepinephrine. Circulation 54(11), 113. E.
LUGER. J. J., BERSON, SCHWARTZ. K.,
G..
DELCAYRE, C., LUGER, J., STEPHENS,
KLOTZ,
M.,
C.. AND
B. (1975). Heart contractile proteins. Biochimie 51, 1249- 1273. LEVINE, R. M., HAUGAARD, N., AND HESS, M. E. (1976). Opposing actions of calcium and magnesium ions on the metabolic effects of epinephrine in rat heart. Biochem. Pharmacol. 25, 1963-1969. SWYNGHEDAUW,
17
PHOSPHORYLATION
LINDEMANN, J. P., JONES, L. R., BESCH, H. R., JR., AND WATANABE, A. M. (1978). Isoproterenolinduced phosphorylation of membrane proteins in intact myocardium. Circdation 58(H), 21. MARSH, N. A. (1975). The effects of divalent cations on the isolated chicken heart. J. Physiol. 256, 17-18P.
MARTIN, A. F., RABINOWITZ, M., BLOUGH, R., PRIOR, G., AND ZAK, R. (1977). Measurements of half-life of rat cardiac myosin heavy chain with leucyl-t RNA used as precursor pool. J. Biol. Chem. 252, 3422-3429.
NATHANSON, J. A. (1977). Cyclic nucleotides and nervous system function. Physiol. Rev. 57, 157-256. OPIE, L. H. (1969). Metabolism of the heart in health and disease. Amer. Heart .I. 77, 100-122. REDDY, Y. S., PITTS, B. J. R., AND SCHWARTZ, A. (1977). Cyclic AMP-dependent and independent protein kinase phosphorylation of canine cardiac myosin light chains. J. MO/. Cell. Cardiol. 9, 501-513. SCHLISELFELD, L. H. (1974). Binding of adenyl imidodiphosphate, an analog of adenosine triphosphate, to myosin and heavy meromyosin. J. Bid. Chem.
249, 4985-4989.
SIEMANKOWSKI,
R.
F.,
AND
DREIZEN,
P.
(1978).
Canine cardiac myosin with special reference topressure overload cardiac hypertrophy. I. Subunit composition.
J. Biol.
Chem.
253, 8648-8658.
R. J., MOIR, A. J. G., AND PERRY, S. V. (1976). Phosphorylation of troponin I and the ino-
SOLARO,
tropic heart.
effect Nature
of adrenaline in the (London) 262,615-617.
perfused
rabbit
SOLARO, R. J., PANG, D. C., AND BRIGGS, F. N. (1971).
The
Triton
X-100. Biochim.
purification
of cardiac Biophys.
myotibrils Acta 245,
with 259-
262.
TADA, M., OHMORI, F., YAMADA, M., AND ABE, H. (1979). Mechanism of the stimulation of Ca*+dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3’:5’-monophosphate-dependent protein kinase. Role of the 22,000-dalton protein. J. Biol.
Chem.
254, 319-326.
WALSH, D. A., CLIPPINCER, M. S., SIVARAMAKRISHNAN, S., AND MCCULLOUGH, T. E. (1979). Cyclic adenosine
monophosphate
dependent
and
inde-
pendent phosphorylation of sarcolemma membrane proteins in perfused rat heart. Biochemistr?, 18, 871-877. WOLLENBERGER, A., AND WILL, H. (1978). Protein kinase-catalyzed
membrane
phosphorylation
and its
possible relationship to the role of calcium in the adrenergic regulation of cardiac contraction. Life Sri.
22, 1159-
1178.
AND
APPLIED
PHARMACOLOGY
55,
8- 17 (1980)
Influence of lsoproterenol and Calcium on Cadmium- or Lead-Induced Negative lnotropy Related to Cardiac Myofibrillar Protein Phosphorylations in Perfused Rat Heart STEPHEN *Nuclear
Magnetic
J. KoPP**’
AND MICHAEL
BARANYT
Resonance Laboratory, Chicago College of Osteopathic Medicine. and tDepartment of Biological Chemistry, University of Illinois at the Medical Center. Chicago. Illinois 60612
Received December
Chicago,
Illinois
60615,
12, 1979; accepted March 20, 1980
Influence of Isoproterenol and Calcium on Cadmium- or Lead-Induced Negative Inotropy Related to Cardiac Myofibrillar Protein Phosphorylations in Perfused Rat Heart. KOPP, S. J., AND BARANY, M. (1980). Toxicol. Appl. Pharmacol. 55, 8-17. The individual and combined physiological and myofibrillar protein phosphorylation effects of the negative inotropic agents, cadmium (3 x lo-” mM) and lead (3 x lo-” mM), and the positive inotropic agents isoproterenol (7 x lo-’ M) and calcium (6.5 mM) were examined in isolated modified perfused rat heart preparations to evaluate possible causal associations between cadmium- and lead-induced changes in myofibrillar protein phosphorylations and altered inotropic responsiveness. Cadmium and lead alone depressed cardiac contractility and only the phosphorylation of the myosin light chain-2 (LC-2). These effects were attenuated in the presence of isoproterenol and elevated extracellular calcium; however, cadmium and lead inhibited both the positive inotropic activation of the heart by calcium and isoproterenol and the concomitant increase in phosphorylations of the purported cardioregulatory phosphoproteins, LC-2 and the troponin inhibitory subunit (TN-I). Positive chronotropic responses to the p-adrenergic agonist were unaffected by cadmium and lead. These results suggest that the negative inotropic effects of cadmium and lead are related to depressed phosphorylation of LC-2 and TN-I. Furthermore, the present findings suggest that cadmium and lead may alter myofibrillar protein phosphorylation mechanisms by antagonizing sarcolemmal calcium translocation processes; however, these results do not preclude the possibility of direct intracellular antagonisms of calcium-dependent processes by these heavy metal ions.
Functional and metabolic processes of the mammalian heart are vulnerable to changes in extracellular and/or intracellular ionic composition (Opie, 1969). Thus, environmental stressors (e.g., heavy metals) which modify the chemical composition of this tissue are postulated to induce detectable physiologic and biochemical changes in the myocardium. Since isolated perfused mammalian heart techniques afford the opportunity to critically assess physiological responses of the 1 To whom correspondence
should be addressed.
0041-008X/80/100008-10$02.00/0 Copyright All rights
heart to diverse chemical insults under controlled conditions, this methodology has been utilized extensively in physiological and toxicological investigations (Hawley and Kopp, 1975; Hess and Gabel, 1979; Kopp and Hawley, 1976; Kopp et al., 1978a,b; Levine et al., 1976; Marsh, 1975). Previous studies have demonstrated that submicro- and micromolar (3 parts per billion (ppb), 0.03-3 parts per million (ppm)) cadmium and 0.3-3 ppm lead concentrations (i) selectively impair the excitation processes of the cardiac conduction system-cadmium depresses atrioventricular node con-
0 1980 by Academx Press. Inc. of reproduction in any form reserved.
8
HEART INOTROPY-MYOFIBRIL
ductivity, while lead depresses His-Purkinje cell excitability (Hawley and Kopp, 1975; Kopp and Hawley, 1976; Kopp et al., 1978a,b), and (ii) depress cardiac contractility (Kopp et al., 1978a,b). The in vitro findings have been corroborated in in vivo controlled diet studies involving cadmium or lead intake from drinking water (5 ppm) for 15-24 months (Kopp et al., 1978c, 1979a,b). Several molecular processes have been implicated as contributing factors to the regulation of chemical-mechanical energy transduction in heart muscle, including protein phosphorylation mechanisms involving a sarcoplasmic membrane constituent, phospholamban (Katz et al., 1975; Lindemann et af., 1978), sarcolemma membrane proteins (Drummond and Severson, 1979; Lindemann et al., 1978; Tada et al., 1979; Walsh et al., 1979; Wollenberger and Will, 1978), and the myofibrillar proteins: the inhibitory subunit of troponin (TN-I) (England, 1975, 1976; Solar0 et al., 1976) and the myosin light chain-2 (LC-2) (Frearson et al., 1976; Kopp and Barany, 1979: Lebowitz et al., 1976). Age- and concentration-dependent cadmium or lead accumulation in the heart has been shown to alter myocardial contractility in chronically fed experimental animals (Kopp et al., 1979b). The present investigations were undertaken to (i) evaluate the physiological responses of the heart and concomitant myofibrillar phosphorylation processes to lead or cadmium alone and in combination with inotropic (elevated extracellular calcium or /3adrenergic-isoproterenol) stimulation under controlled isolated perfused heart conditions; and (ii) correlate directly modified phosphorylation of specific myofibrillar regulatory proteins with altered inotropic responsiveness to determine possible causal associations. METHODS The heart perfusions were performed essentially as described previously (Kopp er a/. , 1978a). Heparin-
PHOSPHORYLATION
9
ized albino Sprague-Dawley rats (200-300 g) of random sex were sacrificed by cervical dislocation and the hearts (N = 40) were excised and chilled in precooled perfusion solution (lo”C), immediately stopping heart contractile activity. While the heart was in this medium, the aorta was cannulated and tied for retrograde perfusion, superfluous connective and adipose tissues were removed, and a single open silk (4-O) loop was sutured through the heart, 5 mm from the apex for recording systolic (peak active) tension. The cannulated heart was then perfused, as described previously (Kopp et al., 1978a), with a perfusate containing 152 mM NaCl, 5.4 mM KC], 1.05 mM MgC&, 1.9 mM CaCl,, 5.6 mM glucose, and 10 mM Tris-HCl buffer, pH 7.35, at 35°C for 30 min during which time electrocardiographic (ECG) and mechanical events stabilized, indicating optimal tissue reoxygenation. A constant diastolic load of 4.0 g was maintained by adjusting the position of the force displacement transducer. Cardiac electrical and mechanical events were recorded with a Grass Model 79C polygraph at paper speeds of 25 and 100 mm/set for accurate signal resolution. Only stable preparations characterized by a rhythmic spontaneous heart rate and stable mechanical activity were studied. Following the 30-min equilibration period, the hearts were equilibrated for 5 min with the experimental perfusate: Control (N = 9), 6 x lO-3 mM CdCl* (N = 3) 6 x lo-’ mM Pb3(CSH50,)2 (N = 3), 6.5 mM CaCl, (N = 4), 7 x 10m7M isoproterenol hydrochloride (N = 4). 6 x IO+ mM CdCl, + 6.5 mM CaCl* (N = 5) 6 x 10m4mM Pb,(C,H,O,), + 6.5 mM CaCl, (N = 5). 6 X 10m3mM CdCl, + 7 x 10m7M isoproterenol hydrochloride (N = 4), and 6 x lo-” mM Pb3(CfiHS07)2 + 7 x lo-’ M isoproterenol hydrochloride (N = 3) at the identical temperature, pressure, and pH (7.35). This exposure duration proved to be sufficient to induce maximal changes in the variables which were measured. At the conclusion of this perfusion period the radioactive experimental perfusate containing 1 mCi carrier-free 32P per liter at the identical temperature, pressure, and pH was turned on and the nonradioactive experimental perfusate turned off. This radioactive perfusion continued for 40 min and was followed by an ll-min washout perfusion with the nonradioactive experimental perfusate which removed 99.6% of the remaining noncovalently bound extracellular 32P (Kopp and Barany, 1979). Contractile and electrical parameters were recorded for analysis at IO-min intervals. Since the perfusion apparatus was a closed system, the flow rate of the heart during the radioactive perfusion was calculated as the difference between the initial perfusate volume and the remaining volume plus the dead space in the system. The percentage 32P uptake by the heart from the perfusate was determined as the ratio of total heart radioactivity to the total radioactivity exposed to the heart.
10
KOPP AND BARANY
Upon completion of the wash perfusion period, the hearts were removed from the cannula and immediately immersed in iced extraction buffer for myotibril extraction. The hearts were observed to stop in diastole after a maximum of a single contraction. This methodology (Kopp and Barany, 1979) yielded consistent reproducible results concerning the phosphorylation of myofibril proteins during cardiac diastole. Tritondeoxycholate-purified cardiac myofibrils free of mitochondrial, sarcolemmal, and sarcoplasmic reticulum membrane contaminants were prepared from these hearts using the procedures of Solar0 ef al. (1971) and Martin ef al. (1977) modified as described elsewhere (Kopp and B&any, 1979). The specific [32P]phosphocreatine radioactivity was determined (Schliselfeld, 1974), the purified myofibrils were fractionated by electrophoresis, the protein bands identified and the [3ZP]phosphate incorporation into the individual myofibrillar proteins quantitated, as detailed previously (Kopp and Barany, 1979). Calculations
and Statistical
Methods
The heart rates were calculated from the R-R interval of the electrocardiogram. The flow rate during the 40-min 32P perfusion was measured as milliliter per minute perfused through the heart. The flow rates were normalized by expressing the values as microliter per heart cycle. All physiological parameters (mechanical and electrical events, 32P uptakes and flow rates) represent the average events during the 40-min 3zP Least Squares Linear Eqh y= 0.0041
Regression x t II 50
.
15
. l
.
‘k
.:.**
. .
:.. .
. . . .
-
.
.
:;*i
l. .
l
.**
IO
.
. l
..-l . *. .
Active Tension (4
.
:
5
ll,ll.Sl,# 100
150
Spontaneous
),,, 200
Heart
S,, 250
Ratebts,4nii
FIG. 1. Graph depicting independent force (active tension&frequency (heart rate) relationship derived from a population of spontaneously beating rat hearts (range 133-237 beats/mitt). Least squares linear regression slope is shown.
20
I
d
1'5
3’0 PerfusIon
FIG. 2. Graph depicting the of the electrocardiogram) and tive tension) stability of control equilibration perfusion period. regression slopes are shown.
45
60
lime(mln)
electrical (PR interval mechanical (peak achearts during the postLeast squares linear
perfusion period. The reported values for the physiological and radioactivity measurements represent the mean + SE for each experimental group. To compensate for the slight differences in gel recoveries, myofibril concentrations applied to the gels and specific radioactivities of the [32P]phosphate in phosphocreatine encountered among the various individual heart experiments, all measured gel recoveries, total gel protein concentrations and specific radioactivities for each heart experiment were adjusted to 75%, 600 pg, and 3 x IO6 cpm/Fmol, respectively. The resultant small normalization factor for each experiment was used to adjust individual myofibril protein gel radioactivity values to their equivalent normalized values. This normalization procedure was necessary to evaluate statistically the quantitative differences in myofibril protein zone radioactivity caused by the specific experimental perfusates studied. The incorporation of [32P]phosphate into the myofibrillar proteins was calculated from the following data: Heart myofibrils contain 50% myosin and 20% actin (Katagiri and Morkin, 1974). Cardiac myosin consists of two heavy chains with apparent molecular weights of 200,000 daltons each (Siemankowski and Dreizen, 1978), two light chains of 27,000 daltons, and two light chains of 19,000 daltons (Frearson and Perry, 1975). The molecular weight of cardiac actin is 42,000 daltons (Leger et al., 1975). The molar ratio of actin:tropomyosin is 4.4: 1, and the molar ratio of actin:TN-T:TN-I is 4.3:1: 1. These ratios were calculated by us from staining intensities of the protein
HEART INOTROPY-MYOFIBRIL TABLE PHYSIOLOGICAL COMBINED
1
PARAMETERS DURING 32P PERFUSION OF NEGATIVE (CADMIUM OR LEAD) AND POSITIVE
Heart perfusion
N
Control 6 x lo-” mM CdCI, 6 x lo-” mM Pb,(C,H,O,), 6.5 mM CaC& 6 x 10m3InM CdCle + 6.5 mM CaCI,, 6 x IO-” mM Pb,(C,H,O,), + 6.5 mM CaCI, 7 X 10-j M isoproterenol 6 x 1O-3 mM CdClr + 7 x IO-’ M isoproterenol 6 Y lo-’ mM Pb,(C,H,O,), + 7 x IO-’ M isoproterenol
9 3 3 4 5 5 4
Note. Values ” Significantly ’ Significantly ’ Significantly ” Significantly I’ Significantly I Significantly v Significantly
represent different different different different different different different
11
PHOSPHORYLATION
RAT HEARTS UNDER THE INFLUENCE OF SINGLE OR (ISOPROTERENOL
Active tension (8) 12.4 4.4 0.6 21.4 11.7 11.2 18.8
2 t t t r + -t
OR CALCIUM)
Spontaneous heart rate (beatsimin)
0.8 0.5” 0.2” 0.2” 1.6”,” 0.4’ 0.7”
1672 120 k 147* 188 k 132 + 202 k 315 k
4
8.8 t 0.6”J
_152 k
3
10.2 + 1.6”,O
7 lob 5 15 10 19 26”
INOTROPIC
Perfusion flow rate (PI/heart cycle) 50 48 81 48 59 34 48
f t 2 k k + t
5 7 2” 7 3 2’ 4
AGENTS
Percentage OsPuptake 7.2 8.0 6.5 6.0 7.0 8.5 5.1
k k t t t t +
0.4 1.2 0.2 0.6 0.7 0.6 0.4’
fy.lt
38 k 1
4.9 t 0.31
264 it 14”,”
39 + 3’
7.2 k 0.4
mean t SE. from control. p < 0.001. from control. p < 0.01. from 6.5 mM CaC&, p < 0.01. from 6 X 10m9mM CdCI,, p < 0.01. from 6 x lo-” mM Pb,(C,H,O,),. p < 0.001. from control, p < 0.02. from 7 x 10m7M isoproterenol. p < 0.01.
bands on gels measured with a Zeineh soft laser scanning densitometer, and they are in good agreement with those which we calculated from the data of Katagiri and Morkin (1974). We used an apparent molecular weight of 68,000 daltons for cardiac tropomyosin (Cummins and Perry. 1974). 38,000 daltons for cardiac TN-T (Brekke and Greaser, 1976), and 29,500 daltons for cardiac TN-I. This latter value is somewhat higher than the 28.000 daltons described by other investigators (Cole and Perry, 1975: Berson et al., 1978). Student’s t test of the means was used to evaluate statistically the significance of the results. A value of p < 0.02 was accepted as significant. Least squares linear regression analyses were performed to ascertain significant correlations between changes in phosphorylation and the myocardial contractile state.
(Fig. 1). The functional stability of the control heart perfusions with respect to contractility and excitability is demonstrated in Fig. 2. These results indicated that the 40-min 32P perfusion time used in these experiments did not alter or contribute to instability in these heart preparations which would produce erroneous phosphorylation results. Thus, in the experimental perfusions altered cardiac contractility was directly attributable to the influence of the experimental perfusates alone.
Calcium
Inotropic
Activation
RESULTS Force (active tension) and frequency (spontaneous heart rate) are effectively independent of one another within the range of heart rate changes detected in these studies
The physiological effects and changes in myofibril protein phosphorylation induced by cadmium and lead perfusions alone relative to control are shown in Tables 1 and 2 and Fig. 3.
12
KOPP
AND
The physiological responses of hearts perfused with elevated calcium alone, or elevated calcium in combination with cadmium or lead are presented in Table 1. Cadmium or lead ions significantly depressed cardiac contractility and calcium-activated positive inotropy. Other physiological indices were similar to those findings detected for elevated calcium alone. This elevated extracellular calcium concentration did, howthe negative inotropic ever, attenuate effects of cadmium or lead alone. In conjunction with these findings, the significantly increased phosphorylation of the various myofibril proteins induced by elevated perfusate calcium levels were significantly depressed by cadmium or lead (Table 2). Although the myosin light chain-2 phosphorylation in response to cadmium + calcium treatment remained depressed relative TABLE
BARANY
to control as well, the phosphorylation of the other myofibril proteins from the lead + calcium and cadmium + calcium hearts were comparable to control values. Zsoproterenol
Activation
The cardiological indices of hearts perfused with isoproterenol alone or in combination with lead or cadmium are shown in Table 1. Cadmium and lead ions inhibited the isoproterenol stimulated positive inotropy; however, the Padrenergic receptor-mediated augmentation in heart rate was not altered significantly by the presence of either heavy metal in the perfusate. Other physiological responses were comparable to isoproterenol treatment alone. These findings suggest the existence of functional myocardial P-receptors during heart per2
SPECIFIC [32P]P~~~~~~~~ INCORPORATION INTO VARIOUS CARDIAC MYOFIBRILLARPROTEINS OF PERFUSED RAT HEART MOLE[~*P]PHOSPHATE/MOLE SPECIFIC MYOFIBRIL PROTEIN Heart
perfusions
CO”tKd 6 x IO-’ ITIM CdCi, 6 x IO-’ nm Pb,(CdW’,h 6.5 tnhd CaCI, 6 x 1Om3 rn~ CdCP + 6.5 nm C&I, 6 x lo-’ rn~ Pb,(C&O,), + 6.5 ,m.t CaCl, 7 x 10-7 M isoproterenol 6 x IO-s nm CdCI, +7x IO’M isoproterenol 6 x 10.’ IIIM Pb,(C.Hs0~)r + 7 x 10-7 M isoproterenol
N
Tropomyosin”
Actin
9 3
0.089 f 0.008 0.074 f 0.008
0.022 2 0.005 0.021 * 0.002
0.064 0.069
+ 0.014 +- 0.011
0.053 2 0.006 0.048 + o.cm
0.029 0.030
3 4
0.066 2 0.009 0.166 * 0.017’
0.023 2 0.002 0.048 -c 0.008’
0.050 t 0.008 0.138 k 0.020’
0.062 -t 0.006 0.171 + 0.011”
0.021 + 0.003 0.042 f 0.008
0.042 2 0.002’ 0.177 f 0.021”
5
0.072 T 0.004’
0.024
-c 0.002
0.059 z 0.003’
0.064 f 0.004’
0.029 f 0.002
0.085 -t 0.008”~
5
0.069
0.016 -c 0.002
0.035 z 0.003’
0.030
0.026
z 0.002
0.092
e O.OWJ
4
0.107 + 0.025
0.031
-’ 0.007
0.075
0.212 z 0.02S”
0.031 t 0.002
0.167
? 0.014”
4
0.059 2 0.002
0.019
f 0.002
0.038 2 0.003
0.0842 o.ow*~’
0.023
0.095 2 o.cw
3
0.046 2 0.004”
0.017 _’ 0.003
0.049 k 0.004’
0.065 f 0.014’
0.019 f 0.003
+ 0.005’
TN-T”
Note. Values represent mean + SE. ” TM, fropomyosin (MW 68,ooO daltons). D TN-T, Tropomyosin binding subunit of troponin (MW 38,000 daltons) r TN-I. Troponin inhibitory subunit (MW 29,500 daltons). d LC-I. Myosin light chain-l (MW 27,ooO daltons). s LC-2, Myosin light chain-2 (MW 19,000 d&tons). ’ Significantly different from control, p < 0.02. ” Significantly different from control, p < 0.001. h Significantly different from control, p < 0.01. I Significantly different from 6.5 rn~ C&I,, p < 0.01. ’ Significantly different from 6 x 10e4 IIIM Pb,(C,H,O,),, p < 0.02. * Significantly different from 6 X 1Om3mM CdCI,. p < 0.01. ’ Significantly different from 7 x IO-’ M isoprotercnol. p < 0.001.
r 0.011
TN-I’
f O.cKw’
LC-I” z 0.007 + 0.004
t 0.001
LC-2’ 0.116 0.087
0.087
k 0.006 + 0.007
f 0.007”‘J
HEART INOTROPY-MYOFIBRIL
l
CONTROL
PHOSPHORYLATION
13
(N=9)
o 6 x 1O-3 mM CdC12 (N=3) -
. 6 x~O-~,M . s*n”ka”t. I. sl.@“iunt.
Pb$Cdrate)p P< Il.02 P
tqooo 1 .c E 8 5 E 2 0
c
5,000
-
1,000
-
Iy *cm TN-T
Molecular
TN-1 LC-I
LC-2
Weigll
FIG. 3. The radioactivity distribution profiles of Triton-deoxycholate-purified rat heart myofibrillar proteins electrophoresed on SDS-urea gels. The average protein band counts reflect changes induced by cadmium or lead relative to control. The ordinate (counts120 min) represents normalized values for equivalence in gel protein concentrations, specific activities of [32P]phosphocreatine and gel recoveries as detailed in the text. Beef heart tropomyosin (TM), troponin (TN-T, tropomyosin-binding subunit; TN-I, inhibitory subunit), and myosin (LC-1, light chain-l; LC-2, light chain-21 were used to identify the proteins in the various zones. Actin was identified by its molecular weight.
fusions with cadmium or lead ions. In addition the significantly elevated phosphorylation of LC-2 induced by isoproterenol was inhibited completely and the phosphorylation of TN-I was attenuated by cadmium or lead + isoproterenol perfusions (Table 2 and Fig. 4). The phosphorylations of other myofibrillar proteins were not altered by isoproterenol treatment alone or combined perfusions with cadmium or lead. A complete correlation of contractile state measured as active tension only with myosin light chain-2 [32P]phosphate incorporation was detected in response to the various experimental perfusates, as shown in Fig. 5. The least squares linear regression equation
depicting this relationship was determined to be y = 0.0062~ + 0.0367 with a correlation coefficient = 0.88. These results suggest that the depressed myosin light chain-2 phosphorylation detected in these studies was a direct factor contributing to the cadmium- or lead-induced negative inotropy and inhibition of calcium or isoproterenol activated positive inotropy in the heart. DISCUSSION Myocardial responses to extrinsic (e.g., hormonal) and intrinsic (e.g., myogenic) physiological effecters may be mediated or modulated through a common pathway involving the differential phosphorylation of
14
KOPP AND BARANY
A 7 X U7 M ISOPROTERENOL (N=4) Y 6 X 1O-3 mM CdCI, + 7 X 1O-7 M ISOPRO l 6 X 10m4rnM Pb3(Citrale)2 + 7X 10m7 M ” SlrnLfirml. P
FIG. 4. The radioactivity distribution profiles of Triton-deoxycholate purified rat heart myofibrillar proteins electrophoresed on SDS-urea gels. The average protein band counts reflect the effects of isoproterenol alone and isoproterenol in combination with either cadmium or lead. See text and Fig. 2 legend for details.
critical regulatory proteins present in the mammalian myocardium (Greengard, 1978: Nathanson, 1977). The phosphoproteins with purported regulatory actions in the control of cardiac contractility include the myofibrillar proteins, TN-I (troponin inhibitory subunit) (England, 1975, 1976; Solar0 et al., 1976; Ezrailson et al., 1977) and LC-2 (myosin light chain-2) (Frearson et al., 1976; Lebowitz et al., 1976); a sarcoplasmic protein, phospholamban (Katz et al., 1975; Lindemann et al., 1978; Tada et al., 1979); and several sarcolemmal membrane proteins (Lindemann et al., 1978; Wollenberger and Will, 1978; Walsh et al., 1979). Investigations concerned with the physiological implications of altered LC-2 phosphorylation suggest that LC-2 phosphorylation may enhance myosin-actin interactions, and thereby facilitate cross-bridge
formation (Bat-any and Barany, 1980). Thus, the magnitude of LC-2 phosphorylation in the heart during diastole has been postulated to predetermine directly the force of active tension generated by the heart in the ensuing contraction (Kopp and Barany, 1979). Previously, a direct linear relationship was described between active tension formation and LC-2 phosphorylation which was represented by the least squares linear regression equation: mole[32P]phosphate/ mole LC-2 = O.O066[active tension (g)] + 0.0437 (Kopp and Barany, 1979). This relationship is consistent with the findings of the present study which were expressed in the least squares linear regression equation: mole[32P]phosphate/mole LC-2 = 0.0062 x [active tension (g)] + 0.0367. The slope and intercept discrepancies between these equa-
HEART INOTROPY-MYOFIBRIL
PHOSPHORYLATION
15
1
IO
50
IO0 Actw
Tsnsh
150
ZOO
(g)
FIG. 5. Correlation between peak active tension and LC-2 phosphorylation. Symbols represent results from the following experimental conditions: W, control; 0, Pb; 0, Cd; A, isoproterenol; A, isoproterenol + Cd; A, isoproterenol + Pb; 0, Ca; q , Ca + Pb; and b, Ca + Cd. Least squares linear regression equation describing this correlation is mol(3zP)phosphate/mo1 light chain-2 = 0.0062 (active tension) + 0.0367 with a correlation coefficient of 0.88. The graphed values represent the mean -C SE.
tions are minor and within the experimental error of the applied techniques. The present investigations have demonstrated that cadmiumand lead-induced negative inotropy is attenuated in the presence of isoproterenol or elevated extracellular calcium; however, these heavy metal ions inhibit the positive inotropy associated with calcium or isoproterenol activation. These physiological responses parallel directly the changes in LC-2 phosphorylation. Elevated extracellular calcium concentrations presumably augment myocardial contractility through a direct stimulation of calcium-dependent processes induced by elevated ionized sarcoplasmic calcium levels during the excitation-contraction coupling process (Keely and Corbin, 1977; Wollenberger and Will, 1978). This increased free calcium in the sarcoplasm may activate directly the calcium-dependent protein kinases causing an increased phosphorylation of the cardioregulatory protein effecters (e.g., LC-2, TN-I, phospholamban) (Reddy et al., 1977; Wollenberger.and Will, 1978)) thereby promoting increased cardiac contractility. The subcellular mechanisms
attributed to p-adrenergic agonist activity purportedly involve the P-receptor-hormone complex formation which activates the adenylate cyclase-cyclic AMP-protein kinase system and induces concomitant intracellular (i) elevation in sarcoplasmic ionized calcium concentrations; (ii) enhanced calcium transport; (iii) increased calcium binding; and/or (iv) augmented calcium sensitivity (Wollenberger and Will, 1978). If cadmium and lead act to impair calcium utilization, intracellularly, a plausible interpretation consistent with present findings would suggest that cadmium and lead ions may antagonize transmembrane calcium transport processes either directly by displacing calcium ions from membrane binding sites (Langer er al., 1974), or indirectly by inhibiting the phosphorylation of sarcolemma proteins required for calcium ion influx. The partial reversal of the negative inotropic effects of cadmium and lead by elevated extracellular calcium is postulated to represent a competitive mass action effect, while the partial reversal by isoproterenol may reflect an enhanced intracellu-
16
KOPP AND BARtiNY
lar utilization or sensitivity to existing calcium, independent of increased calcium permeation. This isoproterenol-augmented sensitivity to calcium may explain the increased TN-I phosphorylation above control observed in cadmium + isoproterenol-treated hearts. The positive chronotropic responses in the presence of cadmium and lead with isoproterenol are not consistent with the premise that cadmium and lead necessarily alter adenylate cyclase activity at the concentrations studied. Cadmium and lead depression of cardiac contractility is partially reversible by isoproterenol and elevated extracellular calcium. Altered phosphorylation of the myofibrillar protein, myosin LC-2 which is purportedly a cardioregulatory phosphoprotein, paralleled changes detected in active tension. The present findings suggest the possibility of extracellular or intracellular antagonisms of calcium-dependent processes by these heavy metal ions. ACKNOWLEDGMENTS This research was supported by grants from the United States National Institutes of Health (NS-12172). the Muscular Dystrophy Association, and the National Institutes of Health National Research Service Award l-F32 ES 05112 TOX from the National Institute of Environmental Health Sciences (S.J.K.). We wish to thank Mr. Robert Kelly and Ms. Ruth Zelkha for the fine graphic art illustrations presented in the figures. We wish to extend a special thanks to Ms. Rose Sage, Mrs. Majorie Pole, and Ms. Laura Gloger for their painstaking devotion to the preparation of this manuscript.
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