614
Brief Communication
Modulation of Crossbridge Kinetics by Myosin Isoenzymes in Skinned Human Heart Fibers I.
Morano, C. Bletz, R. Wojciechowski, and J.C. Ruegg
Skinned fibers from the normal human heart with the f3-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2' sensitivity than skinned fibers with the a-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (gapp), myosin heavy chain isoenzymes
seem
to have different crossbridge turnover kinetics. Modulation of gapp by myosin
heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. gapp was independent of Ca2+. (Circulation Research 1991;68:614-618) In
the steady state, isometric force (F) of a muscle fiber can be described by the equation': F=F' ntot.fapp/(fapp +gapp)
(1)
where F' is the force generation per crossbridge, n,o, is the total amount of cycling crossbridges per half sarcomere, fapp is the rate constant for the transition from non-force-generating into force-generating crossbridge states, and gapp is the rate constant for the transition of force-generating into non-force-generating crossbridge states. The expression fapp/ (fapp+gapp) represents the fraction of cycling crossbridges in the force-generating states. For activation levels up to approximately 20% of maximal tension, Ca> seems to enhance n,0t (recruitment2). At higher activation levels, however, Ca> increases tension by changing crossbridge turnover kinetics,3 namely by increasing fapp.l Similarly, phosphorylation of myosin phosphorylatable light chains increases fapp and, therefore, isometric tension.4-6 The level of isometric ATPase activity during isometric steady-state tension can be described by the equation': ATPase = ntot.s fapp gapp (fapp + gapp)
(2)
From the Department of Physiology II, University of Heidelberg, FRG. Supported by the Deutsche Forschungsgemeinschaft (Mo 362/ 4-1). Address for correspondence: Ingo Morano, Department of Physiology II, University of Heidelberg, I.N.F. 326, D-6900, Heidelberg, FRG. Received April 12, 1990; accepted October 9, 1990.
where s is the number of half sarcomeres. Tension cost is obtained by dividing Equation 2 by Equation 1, which yields gapp.s/F'. Since under isometric steadystate conditions s is constant and F' of a- and ,B-myosin heavy chain (MHC) is considered to be equal (see "Discussion"), the relation of ATPase activity and tension is, therefore, proportional to gapp. From Equation 1, it can then easily be derived that isometric tension as well as Ca2 sensitivity of muscle fibers can be modulated by changing gapp: an increasing value of gapp would predict a decrease in force and Ca2 sensitivity, whereas a decreasing value would cause the opposite effect. This prediction is illustrated as a simple simulation in Figure 1: n (the fraction of crossbridges in the force-generating state [fapp/(fapp+gapp)]) is plotted against fapp (arbitrarily taking maximum fapp as 5.s-1) at two different values of gapp (namely, 1s -l and 2.s-1); this relation between force (proportional to n) and Ca'+ (which determines fapp)' at high gapp value is shifted to the left. Interestingly, Ca2+ sensitivity of skinned atrial fibers rich in ca-MHC and atrial-specific myosin light chains (AMLCs) is lower than that of ventricular fibers with the ,3-MHC and ventricular-specific MLC (V-MLC) of human and pig hearts.7 We tested the hypothesis that the different Ca2+ sensitivity of skinned atrial and ventricular fibers is indeed associated with a different value for gapp. Therefore, both isometric ATPase activity and isometric tension of skinned atrial and ventricular fibers of a normal human heart were determined to obtain the relation between ATPase and force at different Ca> concentrations. To avoid influences of different myosin P light chain phosphorylation levels, which affect Ca21 sensitivi-
Downloaded from http://circres.ahajournals.org/ at Monash University on March 7, 2015
Morano et al Crossbridge Kinetics in Human Heart % (n)
-9app =1*s1
__-gapp =2.s-S fQpp topps9opp
]
5 2 3 1 4 FIGURE 1. Simulation of tension-calcium curves at two different values of the rate constant for the transition of force-generating into non-force-generating crossbridge states (gapp): 1Ps' and 2 s' (where s is the number of half sarcomeres). Tension is proportional to n, which is equivalent to fappl(fapp +gapp) (where fapp is the rate constant for the transition fiom non-force-generating into force-generating crossbridge states and is determined by Ca2'). Tension is expressed as percentage of the maximal value of n given at an arbitrarily chosen maximal fapp value of 5.s'. n,, and F' =1, where n,o, is the total amount of cycling crossbridges per half sarcomere and F' is the force generation per crossbridge, as described by the equation for isometric force (F) of a muscle fiber: F = F'.ntOt fapp/ (fapp +gapp). 0
ty,8,9 atrial and ventricular tissues were incubated in cardioplegic solution for 3 hours at +4°C before skinning, a procedure that has been shown to dephosphorylate the myosin P light chain completely.9
Materials and Methods Skinned atrial (auricle) and ventricular (papillary muscle) fibers were prepared from a normal human heart (the patient died by motorcycle accident). The fibers were kindly provided by Prof. E. Erdmann, Munich. They were prepared as previously described7 using Triton X-100 as a detergent. Mechanical and optical setups were as described.10 Fibers were mounted between the force transducer and vibrator by microsyringes. Initially, fibers were stretched slightly so that resting tension was just threshold. This corresponds to a sarcomere length of approximately 1.95 gm, as judged by laser diffraction in control experiments. ATPase of the fibers (approximately 4 mm in length with a 150-,um cross section) was determined during isometric steady-state tension using an NADH-coupled optical assay method'0"1': ATP->ADP (reaction a), phospho(enol)pyruvate+ ADP->pyruvate+ATP (reaction b), and pyruvate+ NADH+H-lactate+NAD+ (reaction c). Reaction b is catalyzed by pyruvate kinase; reaction c, by lactate dehydrogenase. In this assay, the decrease of NADH fluorescence (excitation, 340 nm; emission, 470 nm) corresponds to the ATP consumption of the fiber and was used as a measure of isometric ATPase activity. This activity was corrected for basal ATPase
615
activity obtained under relaxation conditions. Isometric tension of fibers was determined by an optoelectronic force transducer system. Relaxation solution contained (mM) imidazole 20, Na2ATP 10, NaN3 5, EGTA 5, MgCl2 12.5, phospho(enol)pyruvate 5, NADH 0.6, and P',P5-di(adenosine-5')pentaphosphate (myokinase inhibitor) 0.2, together with 100 units/ml pyruvate kinase and 125 units/ml lactate dehydrogenase, pH 7.0 at 25°C. The contraction solution contained CaEGTA (5 mM) instead of EGTA. Free Ca21 concentration was determined by calculator programs designed for experiments in skinned muscle cells.12 Statistics Values are expressed as mean±SEM. Statistical comparisons were performed by Student's t test. pCa-tension relations were fitted by the Hill equa-
tion'3: Y=[Ca2+ ]H/(pCa50) +[Ca2+]H (3) where Y is the fractional force, pCA50 is the Ca2` concentration giving half-maximal activation, and H is an index of cooperativity. Results Results of simultaneous measurements of isometric ATPase and tension are shown in Figure 2, left panel. In these original experiments, force generated at maximal Ca2' activation (pCa 4.3) by atrial and ventricular fibers was 1,193 mg/mm2 and 2,774 mg/ mm2, respectively. ATPase activity of the same fibers was 76.4 ,uM NADH/(s.mm3) and 50.62 ,uM NADH/ (s_mm3), respectively. On average, force generated by atrial and ventricular skinned fibers was 1,430 mg/ mm2 and 2,150 mg/mm2, respectively (means of six fibers each; SEM
Brief Communication
Modulation of Crossbridge Kinetics by Myosin Isoenzymes in Skinned Human Heart Fibers I.
Morano, C. Bletz, R. Wojciechowski, and J.C. Ruegg
Skinned fibers from the normal human heart with the f3-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2' sensitivity than skinned fibers with the a-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (gapp), myosin heavy chain isoenzymes
seem
to have different crossbridge turnover kinetics. Modulation of gapp by myosin
heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. gapp was independent of Ca2+. (Circulation Research 1991;68:614-618) In
the steady state, isometric force (F) of a muscle fiber can be described by the equation': F=F' ntot.fapp/(fapp +gapp)
(1)
where F' is the force generation per crossbridge, n,o, is the total amount of cycling crossbridges per half sarcomere, fapp is the rate constant for the transition from non-force-generating into force-generating crossbridge states, and gapp is the rate constant for the transition of force-generating into non-force-generating crossbridge states. The expression fapp/ (fapp+gapp) represents the fraction of cycling crossbridges in the force-generating states. For activation levels up to approximately 20% of maximal tension, Ca> seems to enhance n,0t (recruitment2). At higher activation levels, however, Ca> increases tension by changing crossbridge turnover kinetics,3 namely by increasing fapp.l Similarly, phosphorylation of myosin phosphorylatable light chains increases fapp and, therefore, isometric tension.4-6 The level of isometric ATPase activity during isometric steady-state tension can be described by the equation': ATPase = ntot.s fapp gapp (fapp + gapp)
(2)
From the Department of Physiology II, University of Heidelberg, FRG. Supported by the Deutsche Forschungsgemeinschaft (Mo 362/ 4-1). Address for correspondence: Ingo Morano, Department of Physiology II, University of Heidelberg, I.N.F. 326, D-6900, Heidelberg, FRG. Received April 12, 1990; accepted October 9, 1990.
where s is the number of half sarcomeres. Tension cost is obtained by dividing Equation 2 by Equation 1, which yields gapp.s/F'. Since under isometric steadystate conditions s is constant and F' of a- and ,B-myosin heavy chain (MHC) is considered to be equal (see "Discussion"), the relation of ATPase activity and tension is, therefore, proportional to gapp. From Equation 1, it can then easily be derived that isometric tension as well as Ca2 sensitivity of muscle fibers can be modulated by changing gapp: an increasing value of gapp would predict a decrease in force and Ca2 sensitivity, whereas a decreasing value would cause the opposite effect. This prediction is illustrated as a simple simulation in Figure 1: n (the fraction of crossbridges in the force-generating state [fapp/(fapp+gapp)]) is plotted against fapp (arbitrarily taking maximum fapp as 5.s-1) at two different values of gapp (namely, 1s -l and 2.s-1); this relation between force (proportional to n) and Ca'+ (which determines fapp)' at high gapp value is shifted to the left. Interestingly, Ca2+ sensitivity of skinned atrial fibers rich in ca-MHC and atrial-specific myosin light chains (AMLCs) is lower than that of ventricular fibers with the ,3-MHC and ventricular-specific MLC (V-MLC) of human and pig hearts.7 We tested the hypothesis that the different Ca2+ sensitivity of skinned atrial and ventricular fibers is indeed associated with a different value for gapp. Therefore, both isometric ATPase activity and isometric tension of skinned atrial and ventricular fibers of a normal human heart were determined to obtain the relation between ATPase and force at different Ca> concentrations. To avoid influences of different myosin P light chain phosphorylation levels, which affect Ca21 sensitivi-
Downloaded from http://circres.ahajournals.org/ at Monash University on March 7, 2015
Morano et al Crossbridge Kinetics in Human Heart % (n)
-9app =1*s1
__-gapp =2.s-S fQpp topps9opp
]
5 2 3 1 4 FIGURE 1. Simulation of tension-calcium curves at two different values of the rate constant for the transition of force-generating into non-force-generating crossbridge states (gapp): 1Ps' and 2 s' (where s is the number of half sarcomeres). Tension is proportional to n, which is equivalent to fappl(fapp +gapp) (where fapp is the rate constant for the transition fiom non-force-generating into force-generating crossbridge states and is determined by Ca2'). Tension is expressed as percentage of the maximal value of n given at an arbitrarily chosen maximal fapp value of 5.s'. n,, and F' =1, where n,o, is the total amount of cycling crossbridges per half sarcomere and F' is the force generation per crossbridge, as described by the equation for isometric force (F) of a muscle fiber: F = F'.ntOt fapp/ (fapp +gapp). 0
ty,8,9 atrial and ventricular tissues were incubated in cardioplegic solution for 3 hours at +4°C before skinning, a procedure that has been shown to dephosphorylate the myosin P light chain completely.9
Materials and Methods Skinned atrial (auricle) and ventricular (papillary muscle) fibers were prepared from a normal human heart (the patient died by motorcycle accident). The fibers were kindly provided by Prof. E. Erdmann, Munich. They were prepared as previously described7 using Triton X-100 as a detergent. Mechanical and optical setups were as described.10 Fibers were mounted between the force transducer and vibrator by microsyringes. Initially, fibers were stretched slightly so that resting tension was just threshold. This corresponds to a sarcomere length of approximately 1.95 gm, as judged by laser diffraction in control experiments. ATPase of the fibers (approximately 4 mm in length with a 150-,um cross section) was determined during isometric steady-state tension using an NADH-coupled optical assay method'0"1': ATP->ADP (reaction a), phospho(enol)pyruvate+ ADP->pyruvate+ATP (reaction b), and pyruvate+ NADH+H-lactate+NAD+ (reaction c). Reaction b is catalyzed by pyruvate kinase; reaction c, by lactate dehydrogenase. In this assay, the decrease of NADH fluorescence (excitation, 340 nm; emission, 470 nm) corresponds to the ATP consumption of the fiber and was used as a measure of isometric ATPase activity. This activity was corrected for basal ATPase
615
activity obtained under relaxation conditions. Isometric tension of fibers was determined by an optoelectronic force transducer system. Relaxation solution contained (mM) imidazole 20, Na2ATP 10, NaN3 5, EGTA 5, MgCl2 12.5, phospho(enol)pyruvate 5, NADH 0.6, and P',P5-di(adenosine-5')pentaphosphate (myokinase inhibitor) 0.2, together with 100 units/ml pyruvate kinase and 125 units/ml lactate dehydrogenase, pH 7.0 at 25°C. The contraction solution contained CaEGTA (5 mM) instead of EGTA. Free Ca21 concentration was determined by calculator programs designed for experiments in skinned muscle cells.12 Statistics Values are expressed as mean±SEM. Statistical comparisons were performed by Student's t test. pCa-tension relations were fitted by the Hill equa-
tion'3: Y=[Ca2+ ]H/(pCa50) +[Ca2+]H (3) where Y is the fractional force, pCA50 is the Ca2` concentration giving half-maximal activation, and H is an index of cooperativity. Results Results of simultaneous measurements of isometric ATPase and tension are shown in Figure 2, left panel. In these original experiments, force generated at maximal Ca2' activation (pCa 4.3) by atrial and ventricular fibers was 1,193 mg/mm2 and 2,774 mg/ mm2, respectively. ATPase activity of the same fibers was 76.4 ,uM NADH/(s.mm3) and 50.62 ,uM NADH/ (s_mm3), respectively. On average, force generated by atrial and ventricular skinned fibers was 1,430 mg/ mm2 and 2,150 mg/mm2, respectively (means of six fibers each; SEM
