Andrea Weisberg

Relation Between Crossbridge Structure and Actomyosin ATPase Activity in Rat Heart

Cardiac myofilaments contain proteins that regulate the interaction between actin and myosin. In the thick filament, there are several proteins that may contribute to the regulation of the contraction. The myosin binding protein C, or C protein, has 4 sites that can be phosphorylated by a Ca2+-calmodulin-controlled kinase, protein kinase A or protein kinase C. Using electron microscopy and optical diffraction, we examined the structure of thick filaments isolated from rat ventricles with either the alpha or beta isoform of myosin heavy chain (MHC) and the effect of specific phosphorylation of C protein on the structure. In thick filaments with alpha-MHC, crossbridges were clearly visible. Phosphorylation of C protein by protein kinase A extended the crossbridges from the backbone of the filament, changed their orientation, increased the degree of order of the crossbridges, and decreased the flexibility of the crossbridges. Crossbridges in filaments with beta-MHC were less ordered and apparently more flexible. Phosphorylation of C protein in beta-MHC-containing filaments did not extend the crossbridges and did not alter degree of order or flexibility. The relative flexibility of the crossbridges inferred from the optical diffraction pattern correlated well with the rate of ATP hydrolysis by actomyosin. These results suggest that (1) crossbridge flexibility is an important parameter in setting the rate of crossbridge cycling, and (2) C protein-mediated control of the position and flexibility of crossbridges may regulate actomyosin ATPase activity by modifying the kinetics of crossbridge cycling.

Multiple structures of thick filaments in resting cardiac muscle and their influence on cross-bridge interactions.

Based on two criteria, the tightness of packing of myosin rods within the backbone of the filament and the degree of order of the myosin heads, thick filaments isolated from a control group of rat hearts had three different structures. Two of the structures of thick filaments had ordered myosin heads and were distinguishable from each other by the difference in tightness of packing of the myosin rods. Depending on the packing, their structure has been called loose or tight. The third structure had narrow shafts and disordered myosin heads extending at different angles from the backbone. This structure has been called disordered. After phosphorylation of myosin-binding protein C (MyBP-C) with protein kinase A (PKA), almost all thick filaments exhibited the loose structure. Transitions from one structure to another in quiescent muscles were produced by changing the concentration of extracellular Ca. The probability of interaction between isolated thick and thin filaments in control, PKA-treated preparations, and preparations exposed to different Ca concentrations was estimated by electron microscopy. Interactions were more frequent with phosphorylated thick filaments having the loose structure than with either the tight or disordered structure. In view of the presence of MgATP and the absence of Ca, the interaction between the myosin heads and the thin filaments was most likely the weak attachment that precedes the force-generating steps in the cross-bridge cycle. These results suggest that phosphorylation of MyBP-C in cardiac thick filaments increases the probability of cross-bridges forming weak attachments to thin filaments in the absence of activation. This mechanism may modulate the number of cross-bridges generating force during activation.

Effect of Endothelin-1 on Actomyosin ATPase Activity: Implications for the Efficiency of Contraction

Endothelin is a powerful inotropic peptide that increases isometric force in isolated papillary muscle and the extent of shortening in isolated single cardiac myocytes. Its mechanism of action has been variously attributed to increased Ca2+ activation, increased Ca2+ sensitivity of the contractile proteins, and increased intracellular pH, but the physiological function of the changes in cardiac performance remains obscure. In this study, the effects of endothelin-1 on both force development and the kinetics of contraction have been examined. Isometric force, actomyosin ATPase activity, and unloaded shortening velocity were measured. The effects were dose dependent. From 1 to 50 pmol/L endothelin-1 did not alter force development in isolated trabeculae with intact endothelial cells, but actomyosin ATPase activity was increased. Between 100 pmol/L and 10 nmol/L endothelin-1 raised isometric force, decreased actomyosin ATPase activity, and decreased unloaded shortening velocity. The reduction in ATPase activity was progressively enhanced as sarcomere length was increased from 1.9 t0 2.4 microns. These results indicate that the effects of endothelin-1 on the force of contraction and the rate of ATP hydrolysis are not tightly coupled and are changed in the opposite directions by endothelin-1 over most of its effective dose-range. This raises the possibility that endothelin-1 may increase the economy of contraction. A novel function of endothelin may be the modulation of the efficiency of contraction, particularly when increased preload raises the contractile work of the heart.

Contractile proteins in myocardial cells are regulated by factor(s) released by blood vessels.

The importance of perfusion of the coronary vasculature in the regulation of ATPase activity of myosin in rat myocardial cells has been studied. Quantitative histochemistry was used to determine the activity of the enzyme among cells in tissues that had been either perfused through the coronary system or superfused over the surface of the tissue. Enzymatic activity was measured in cryostatic sections from three different preparations: 1) hearts frozen immediately after removal from the animal; 2) isolated hearts frozen after they had been perfused through the coronary circulation; and 3) isolated papillary muscles or trabeculae that had been superfused after dissection and then frozen. ATPase activity was measured in the isolated tissues at different times after dissection. Both calcium- and actin-activated myosin ATPase activities were uniform among cells in both the ventricles of the hearts frozen immediately after dissection and those that had been perfused through the coronary system. In the superfused tissues, although calcium-activated myosin ATPase activity was uniform, actin-activated ATPase activity was not uniform for about 90 minutes after the dissection, the period required for stabilization of the contraction. The pattern of nonuniformity was complex. In all bundles the lowest enzymatic activity was found in the most superficial cells. In very thin bundles, the cells in the center had the highest activity. In the medium and thicker bundles, there were three concentric zones of actin-activated ATPase activity, the superficial zone with the lowest activity, an intermediate zone with high activity, and a central zone with lower activity. Within each zone, the activity was often greatest in myocardial cells immediately next to blood vessels even though the blood vessels had not been perfused. The transverse distribution of ATPase activity of myosin could be explained by a mechanism in which cells in blood vessels (presumably endothelium) release a substance that upregulates myosin ATPase activity, with the rate of release being related to the local oxygen tension. A downregulating substance may also be produced. The period of stabilization of the contraction coincides with the time during which the pattern of actomyosin ATPase activity is nonuniform. These data suggest that the contractile proteins are regulated by a substance produced by blood vessels in proportion to the local PO2, and possibly in relation to shear force on the vascular endothelium.

Histochemical detection of specific isozymes of myosin in rat ventricular cells.

A histochemical method for distinguishing isozymes of myosin in rat ventricles has been developed. The procedure involves preincubation in pH 10.5, which inhibits Ca-activated ATPase of the V3 isozyme but not the V1 isozyme of myosin. The specificity of the technique has been demonstrated by comparison of results in hearts from young euthyroid and hypothyroid rats, in which the predominant isozymes are, respectively, V1 and V3. The technique is capable of detecting as small a change in the relative amount of V1 as 15% of the total myosin. Isoenzymes appear to be uniformly distributed within each ventricular cell. There is only a small difference in the content of V1 among the cells in a ventricular chamber of hearts from young euthyroid and hypothyroid rats, but in the period of rapid transition of isozyme content after thyroidectomy, there is considerable heterogeneity of V1 concentration among the cells. The functional implications of the mixture of isozymes is discussed.

Variable Calcium Sensitivity of the Mammalian Cardiac Contractile System

Activation of the contraction in cardiac muscle occurs as a result of a rise in the concentration of calcium in the immediate vicinity of the myofibrils. The sources of the calcium for this rise are the extracellular space and the sarcoplasmic reticulum. Within a specific range of concentration, in the vicinity of 1µ molar, the amount of force that is generated is dependent on the amplitude of the calcium concentration. The amplitude of the contraction, however, is also dependent on the affinity of the regulatory protein, troponin, for calcium. Developed force rises from zero to maximum with a change in concentration of calcium of approximately tenfold, but the specific concentration range at which this occurs is dependent upon the properties of troponin, in particular, the affinity of the calcium binding site on one of the three subunits of the regulatory protein. A change in the range of calcium concentration that initiates contraction as a result of modification of the calcium-binding characteristics of troponin can be considered an alteration in calcium sensitivity. This can occur without any change in maximum calcium activity (Figure 4-1).

Effect of left atrial filling pressure on the activity of specific myosin isozymes in rat heart.

One of the fundamental properties of cardiac muscle is the increase in force generated and work performed with a rise in the resting length of the tissue. There are data to indicate that length-dependent responses of electromechanical coupling and calcium binding by troponin are part of the basis for the pressure-volume relation in the heart. In this study, the contribution of changes in the functional properties of the contractile proteins independent of modification in electromechanical coupling has been examined. Isolated working hearts containing either a mixture of myosin heavy chain (MHC) isozymes (alpha[fast] and beta [slow]) or exclusively the fast MHC have been subjected to left atrial filling pressures (LAPs) between 5 and 20 cm H2O. After 40 minutes at a given LAP, the heart was quickly frozen. The relative activities of calcium- and actin-activated ATPase of V1 and V3 myosin, containing alpha- and beta-MHC, were measured in cryostatic sections of the heart by quantitative histochemistry under conditions for which the concentration of calcium would not be limiting. In hearts containing both isozymes of myosin, the relative enzymatic activity of each isozyme of myosin varied with LAP. At low LAP, V1 was primarily responsible for the enzymatic activity, but as LAP increased the relative contribution of V1 decreased and that of V3 increased. The change in the calcium- and actin-activated activities of the enzyme with change in LAP occurred within 5 minutes and was reversible. In spite of the apparent substitution of enzymatic activity of V3 for V1, total myosin ATPase activity did not decline, but instead remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Ca-independent regulation of cardiac myosin *

Calcium-independent regulation of the contractile proteins of cardiac muscle has been studied using hyperpermeable cells from rat ventricles and sections of quickly-frozen rat hearts. These preparations have been used to study maximum Ca-activated force, myosin ATPase activity and the maximum velocity of unloaded shortening. Beta adrenergic activity increases the amount of force and the ATPase activity in accordance with the concentration of the V1 isozyme of myosin. V3 activity is decreased at the same time. In tissues containing only V1, there is no change in maximum velocity in response to beta adrenergic stimulation. These results indicate that beta adrenergic stimulation recruits V1 force generators and probably regulates the transition between a Ca unresponsive and a Ca responsive force generator. This type of regulation provides the cell with the ability to operate along many different force-velocity relations.

The Properties of Cardiac Contractile Proteins Are Modulated by Autonomic Innervation

The force generators in myocardial cells of rats and rabbits can exist in three different states: (a) relaxed and calcium unresponsive; (b) relaxed and calcium responsive: and (c) contracted. The transition between the two calcium responsive states is produced by the abrupt rise in the concentration of calcium ions during activation by depolarization of the surface membrane. The transition between the two relaxed states is controlled by the β-adrenergic system. Stimulation converts calcium unresponsive to calcium responsive force generators by causing the release of a 21,000 dalton regulatory factor from intracellular sites on a membrane. The factor then interacts with myosin. The regulatory system can distinguish between V1, and V2, myosins, producing a calcium responsive state in the former and a calcium unresponsive state in the latter. The result of β-adrenergic activity is, therefore, an increase in the faster and a decrease in the slower force generators. As a result individual cardiac cells can have many different force-velocity relations.