George McClellan

Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle

In contrast to skeletal muscle isoforms of myosin binding protein C (MyBP-C), the cardiac isoform has 11 rather than 10 fibronectin or Ig modules (modules are identified as C0 to C10, NH2 to COOH terminus), 3 phosphorylation sites between modules C1 and C2, and 28 additional amino acids rich in proline in C5. Phosphorylation between C1 and C2 increases maximum Ca-activated force (Fmax), alters thick filament structure, and increases the probability of myosin heads on the thick filament binding to actin on the thin filament. Unphosphorylated C1C2 fragment binds to myosin, but phosphorylation inhibits the binding. MyBP-C also binds to actin. Using two types of immunoprecipitation and cosedimentation, we show that fragments of MyBP-C containing C0 bind to actin. In low concentrations C0-containing fragments bind to skinned fibers when the NH2 terminus of endogenous MyBP-C is bound to myosin, but not when MyBP-C is bound to actin. C1C2 fragments bind to skinned fibers when endogenous MyBP-C is bound to actin but not to myosin. Disruption of interactions of endogenous C0 with a high concentration of added C0C2 fragments produces the same effect on contractility as extraction of MyBP-C, namely decrease in Fmax and increase in Ca sensitivity. These results suggest that cardiac contractility can be regulated by shifting the binding of the NH2 terminus of MyBP-C between actin and myosin. This mechanism may have an effect on diastolic filling of the heart.

Changes in Cardiac Contractility Related to Calcium-Mediated Changes in Phosphorylation of Myosin-Binding Protein C

Ca ions can influence the contraction of cardiac muscle by activating kinases that specifically phosphorylate the myofibrillar proteins myosin-binding protein C (MyBP-C) and the regulatory light chain of myosin (RLC). To investigate the possible role of Ca-regulated phosphorylation of MyBP-C on contraction, isolated quiescent and rhythmically contracting cardiac trabeculae were exposed to different concentrations of extracellular Ca and then chemically skinned to clamp the contractile system. Maximum Ca-activated force (F(max)) was measured in quiescent cells soaking in 1) 2.5 mM Ca for 120 min, 2) 1.25 mM for 120 min, or 3) 1.25 mM for 120 min followed by 10 min in 7.5 mM, and 4) cells rhythmically contracting in 2.5 mM for 20 min. F(max) was, respectively, 21.5, 10.5, 24.7, and 32.6 mN/mm(2). Changes in F(max) were closely associated with changes in the degree of phosphorylation of MyBP-C and occurred at intracellular concentrations of Ca below levels associated with phosphorylation of RLC. Monophosphorylation of MyBP-C by a Ca-regulated kinase is necessary before beta-adrenergic stimulation can produce additional phosphorylation. These results suggest that Ca-dependent phosphorylation of MyBP-C modulates contractility by changing thick filament structure.

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.

Multiple Forms of Cardiac Myosin-binding Protein C Exist and Can Regulate Thick Filament Stability

Although absence or abnormality of cardiac myosin binding protein C (cMyBP-C) produces serious structural and functional abnormalities of the heart, function of the protein itself is not clearly understood, and the cause of the abnormalities, unidentified. Here we report that a major function of cMyBP-C may be regulating the stability of the myosin-containing contractile filaments through phosphorylation of cMyBP-C. Antibodies were raised against three different regions of cMyBP-C to detect changes in structure within the molecule, and loss of myosin heavy chain was used to monitor degradation of the thick filament. Results from Western blotting and polyacrylamide gel electrophoresis indicate that cMyBP-C can exist in two different forms that produce, respectively, stable and unstable thick filaments. The stable form has well-ordered myosin heads and requires phosphorylation of the cMyBP-C. The unstable form has disordered myosin heads. In tissue with intact cardiac cells, the unstable unphosphorylated cMyBP-C is more easily proteolyzed, causing thick filaments first to release cMyBP-C and/or its proteolytic peptides and then myosin. Filaments deficient in cMyBP-C are fragmented by shear force well tolerated by the stable form. We hypothesize that modulation of filament stability can be coupled at the molecular level with the strength of contraction by the sensitivity of each to the concentration of calcium ions.

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.

ELECTRON PROBE ANALYSIS OF THE SARCOPLASMIC RETICULUM AND VACUOLATED T‐TUBULE SYSTEM OF FATIGUED FROG MUSCLES*

In fatigued frog muscle fibers vacuolation has been observed with light microscopy.lJ In the course of investigating the ultrastructure and composition of these vacuoles with electron probe analysis. we were able to determine the calcium content of the in situ terminal cisternae in fatigued muscle. Bundles of 812 fibers of frog semitendinosus were stimulated by periodic tetani of 40-50 shockdsec during 0.3 sec every sec until tension declined to baseline. Light microscopy of single fatigued fibers showed vacuoles and an approximately 80% increase in fiber volume. When fatigue was established, the muscles were shot into supercooled Freon 22 and frozen sections of approximately 100 nm thickness were cut at -130°C on a modified LKB cryoultramicrotome without any cryoprotectants or fixation.3 The sections were dried at Torr below -80°C. Quantitative electron probe analysis of the cryo sections was performed as described p r e ~ i o u s l y . ~ Vacuoles, frequently located in longitudinal rows between the mitochondria and occasionally along the Z lines, were observed in electron micrographs of the cryo sections. These vacuoles contained high concentrations of NaC1. Pretreatment with strophanthidin c r ouabain ( IW4 M) did not prevent the appearance, extent, or high NaCl content of the vacuoles; therefore, it is unlikely that vacuolation was due to a ouabain-sensitive Na pump. Freeze-substitution studies of fatigued muscles showed the same distribution of vacuoles as in cryosections and permitted their identification as being membrane bound, continuous with the T-tubules, and involving predominately the longitudinal T-system. The cytoplasmic elemental concentrations measured with large diameter probes (0.5-10 wm) in fatigued muscles were (mmoleslkg dry wt, mean +SEM) Na 140 * 8, Mg 47 i 3, P 290 t 3, S 224 t 2. CI 133 t 2, K 332 i 3 , Ca 8 i I (FIGURE I ) . The relatively high fiber Ca content is in agreement with the reported increase in chemically measured Ca in fatigued muscles.6

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).

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.