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S Winegrad

Publications and source records attributed to S Winegrad.

At least 19 recordsLinked to original sources
PubMed2004

Effect of cardiac myosin-binding protein C on stability of the thick filament.

In contrast to skeletal muscle isoforms of myosin-binding protein C (MyBP-C), the cardiac isoform has 11 rather than 10 modules (labeled C0-C10, N-C terminus), three phosphorylation sites between C1 and C2, and 28 additional amino acids in C5. Within the C5-C10 region of cardiac MyBP-C (cMyBP-C) there are interactions between C5 and C8 as well as C7 and C10. Isolated skinned cardiac trabeculae were incubated with one of three recombinant fragments of cMyBP-C to interfere with interactions of endogenous C5. 2-10 microM C5 or C5-containing peptide fragments of cMyBP-C reversibly reduced Ca sensitivity without extracting myofibrillar protein. C2-C4 fragments had no effect. This result indicated that the region of cMyBP-C that contains C5 maintains a specific structural arrangement of myosin that helps set its contractile properties. Greater than 10 microM C5 caused skinned trabeculae to lose a substantial amount of cMyBP-C and some myosin heavy chain, resulting in irreversible decline in maximum Ca-activated force. MyBP-C appears to stabilize the structure of the thick filament and modulate the way in which myosin heads extend to the thin filament.

Actin Cytoskeleton↗
PubMed2003

Effect of extraction of myosin binding protein C on contractility of rat heart.

Human hearts with reduced or mutant myosin binding protein C (MyBP-C) undergo hypertrophy and dilation, suggesting that reduction or alteration of MyBP-C interferes with normal contraction. Extraction of 60-70% of MyBP-C over 1 h from a mechanically disrupted cardiac myocyte has been shown to increase Ca sensitivity but does not appear to impair development of maximum Ca-activated force (Fmax). To determine whether loss of MyBP-C over a longer period of time will decrease force development in a reversible manner, MyBP-C has been extracted from chemically skinned rat cardiac trabeculae for 1-4 h, and force production, Ca sensitivity, and thick filament structure were measured. Although extraction of MyBP-C for 1 h did not alter Fmax, after 4 h, myosin heads became disordered and Fmax decreased. At this point, incubation of the trabeculae with rat cardiac MyBP-C in a relaxing solution reversed the decline in Fmax and most of the change in order of myosin heads. Extraction of MyBP-C appears to produce a change in the orientation of myosin heads that is associated with a decreased ability of the contractile system to develop force.

Animals↗
PubMed2001

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.

Actins↗
PubMed2001

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.

Adrenergic beta-Agonists↗
PubMed1999

Self-protection by cardiac myocytes against hypoxia and hyperoxia.

Cardiac muscle must maintain a continuous balance between its energy supply and work performed. An important mechanism involved in achievement of this balance is cross talk via chemical signals between cardiac myocytes and the cardiac muscle vascular system. This has been demonstrated by incubating isolated cardiac myocytes in different concentrations of oxygen and then assaying the conditioned media for vasoactive substances on isolated aortic rings and small-resistance arteries. With increasing oxygen concentrations above 6%, cardiac myocytes produce increasing amounts of angiotensin I, which is converted to angiotensin II by the blood vessel. The angiotensin II stimulates vascular endothelial cells to secrete endothelin and increase vascular tone. Below 6% oxygen, cardiac myocytes secrete adenosine, which acts directly on vascular smooth muscle to block the effect of alpha-adrenergic agonists and reduce vascular tone. In an intact heart, the net effect of these 2 regulatory systems would be the maintenance of oxygen concentration within a narrow range at the cardiac myocytes. By acting as oxygen sensors, cardiac myocytes modulate vascular tone according to the needs of the myocytes and reduce potential problems of hypoxia and extensive formation of reactive oxygen species.

Adenosine↗
PubMed1999

Cardiac myosin binding protein C.

Myosin binding protein C (MyBP-C) is one of a group of myosin binding proteins that are present in the myofibrils of all striated muscle. The protein is found at 43-nm repeats along 7 to 9 transverse lines in a portion of the A band where crossbridges are found (C zone). MyBP-C contains myosin and titin binding sites at the C terminus of the molecule in all 3 of the isoforms (slow skeletal, fast skeletal, and cardiac). The cardiac isoform also includes a series of residues that contain 3 phosphorylatable sites and an additional immunoglobulin module at the N terminus that are not present in skeletal isoforms. The following 2 major functions of MyBP-C have been suggested: (1) a role in the formation of the sarcomeric myofibril as a result of binding to myosin and titin and (2) in the case of the cardiac isoform, regulation of contraction through phosphorylation. The first is supported by the demonstrated effect of MyBP-C on the packing of myosin in the thick filament, the coincidence of appearance of sarcomeres and MyBP-C during myofibrillogenesis, and the defective formation of sarcomeres when the titin and/or myosin binding sites of MyBP-C are missing. The second is supported by the specific phosphorylation sites in cardiac MyBP-C, the presence in the thick filament of an enzyme specific for MyBP-C phosphorylation, the alteration of thick filament structure by MyBP-C phosphorylation, and the accompaniment of MyBP-C phosphorylation with all major physiological mechanisms of modulation of inotropy in the heart.

Animals↗
PubMed1998

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.

Actin Cytoskeleton↗
PubMed1998

Vascular endothelial cell-cardiac myocyte crosstalk in achieving a balance between energy supply and energy use.

In isolated perfused hearts, endothelial cells in the coronary arterial vascular system release substances that can alter the contractility of the cardiac myocytes. There are at least two different substances, one that increases and another that decreases the contractility of cardiac myocytes. The rate of release of these endothelial-derived cardioactive substances depends on the oxygen tension in the immediate vicinity of the cardiac myocytes. As the local oxygen tension increases the contractility changes in the same direction. The oxygen sensor in this regulatory system is the cardiac myocyte, which then releases substances that regulate the secretion of endothelin and a relaxant by endothelial cells. The result is a loop involving cross talk between coronary endothelial cells and cardiac myocytes to modulate cardiac contractility in accordance with the oxygen supply to the cardiac myocytes. Preliminary data suggest that the change in contractility is related to a change in structure and position of the cross bridge due to phosphorylation of a protein in the thick filament.

Animals↗
PubMed1997

Endothelial cell regulation of contractility of the heart.

Endocardial and coronary vascular endothelial cells release substances that modify the contraction of cardiac myocytes. The major and possibly the sole up-regulating substance is endothelin. Several down-regulating substances are secreted, but none has yet been specifically identified. The relative amounts of up- and down-regulating substances are related to tissue oxygen tension. As pO2 rises, the concentration of up- and down-regulating substances, respectively, increases and decreases. Endothelin increases isometric force and decreases actomyosin ATPase activity thus increasing the economy of conversion of chemical to hydrodynamic energy. Beta-adrenergic agonists increase ATPase activity through an endothelial cell-dependent mechanism, leading to decreased economy. Therefore, two endothelial cell-dependent systems exist for regulating contractile efficiency: One involving endothelin appears to optimize the contraction for efficiency; the other, the beta-adrenergic-mediated system, optimizes for power.

Animals↗
PubMed1996

Alteration of myosin cross bridges by phosphorylation of myosin-binding protein C in cardiac muscle.

In addition to the contractile proteins actin and myosin, contractile filaments of striated muscle contain other proteins that are important for regulating the structure and the interaction of the two force-generating proteins. In the thin filaments, troponin and tropomyosin form a Ca-sensitive trigger that activates normal contraction when intracellular Ca is elevated. In the thick filament, there are several myosin-binding proteins whose functions are unclear. Among these is the myosin-binding protein C (MBP-C). The cardiac isoform contains four phosphorylation sites under the control of cAMP and calmodulin-regulated kinases, whereas the skeletal isoform contains only one such site, suggesting that phosphorylation in cardiac muscle has a specific regulatory function. We isolated natural thick filaments from cardiac muscle and, using electron microscopy and optical diffraction, determined the effect of phosphorylation of MBP-C on cross bridges. The thickness of the filaments that had been treated with protein kinase A was increased where cross bridges were present. No change occurred in the central bare zone that is devoid of cross bridges. The intensity of the reflections along the 43-nm layer line, which is primarily due to the helical array of cross bridges, was increased, and the distance of the first peak reflection from the meridian along the 43-nm layer line was decreased. The results indicate that phosphorylation of MBP-C (i) extends the cross bridges from the backbone of the filament and (ii) increases their degree of order and/or alters their orientation. These changes could alter rate constants for attachment to and detachment from the thin filament and thereby modify force production in activated cardiac muscle.

Animals↗
PubMed1996

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.

Adenosine Triphosphate↗
PubMed1995

Endothelin regulation of cardiac contractility in absence of added endothelin.

Endothelin has a positive inotropic effect on cardiac muscle, but its role in the regulation of contraction in cardiac tissue is not clear, inasmuch as there has been no demonstration of endothelin regulation of contractility in the absence of added endothelin. To address this question, the changes in contractility of isolated rat ventricular trabeculae produced by endothelin and by BQ-123, an endothelin receptor A antagonist, were measured in tissues with different levels of contractility resulting from bathing the tissues for different lengths of time. The effect of endothelin depended on the extent to which tension had declined from its peak level: the greater the decline, the larger the increase in developed force produced by endothelin. The effect of BQ-123 also depended on the extent to which force had declined. The effects of the addition of endothelin or BQ-123 indicate the presence of substantial regulation of contractility due to basal secretion of endothelin: the degree of endothelin activity is greater in cardiac tissue generating more tension. Damage to the endocardial endothelium from a brief exposure to Triton X-100 reduced the response to BQ-123. The response to BQ-123 depends on the number of functioning endothelial cells. From the extent of the effect of endothelin or BQ-123, it appears that as much as 50% of total force-generating capacity of the tissue is sensitive to endothelin produced by the endothelial cells in the isolated heart. This stimulation of contractility is gradually lost in the isolated cardiac tissue, contributing to the progressive decline in developed force with time.

Animals↗
PubMed1994

cAMP can raise or lower cardiac actomyosin ATPase activity depending on alpha-adrenergic activity.

Adenosine 3',5'-cyclic monophosphate (cAMP) or beta-adrenergic stimulation has been shown to increase actomyosin adenosinetriphosphatase (ATPase) activity in cardiac muscle. Because the major catecholamine transmitters have both alpha- and beta-adrenergic activity, the possibility of a role for alpha-adrenergic stimulation in the regulation of ATPase activity has been investigated. Histochemical measurement of actomyosin ATPase activity in quickly frozen rat hearts has been used as the assay of enzymatic function of the contractile proteins. The dose-response curve of ATPase activity to cAMP shows an increase in ATPase activity at a threshold concentration of 0.01 microM, a peak effect at 0.5-1.0 microM, and a decline beyond 1.5 microM to a level below control at 10 microM cAMP. Kinetic studies varying ATP concentration from 0.5 to 10 mM indicated the existence of multiple forms of actomyosin ATPase activity in the absence of cAMP and only one form with a higher maximum velocity in the presence of 1 microM cAMP. Apparently cAMP raises the enzymatic activity of the individual actomyosin molecule rather than increasing the number of active molecules. The addition of an alpha-adrenergic blocker had no significant effect in the absence of added cAMP, but in the presence of the cyclic nucleotide, 1 microM prazosin always produced a negative effect on ATPase activity. Over the entire range of 0.01-10 microM, cAMP lowered ATPase activity when the alpha-adrenergic antagonist was present. The integrity of the cAMP regulatory system was sensitive to the tissue oxygen tension at the time the heart was quickly frozen. At certain oxygen tension, the stimulatory component of the cAMP regulation was observed without any inhibitory component, suggesting that there are two relatively independent parts of the regulatory mechanism, an inhibitory and a stimulatory. In the presence of gamma-labeled [32P]ATP, 32P was incorporated into several proteins, including the inhibitory subunit of troponin (TNI), C protein, and the regulatory light chain of myosin. cAMP (1 microM) caused an increase in 32P labeling of TNI and C protein. The addition of prazosin with cAMP caused a decrease in the overall level of phosphorylation with specific dephosphorylation of C protein and TNI, the former to a degree similar to the decrease in actomyosin ATPase activity, the latter to a greater degree. These results indicate that alpha-adrenergic activity modulates the balance between kinase and phosphatase activity in the presence of cAMP, probably by inhibiting phosphatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗
PubMed1994

Endothelial cell storage and release of endothelin as a cardioregulatory mechanism.

Coronary vascular endothelial cells release substances into the coronary circulation that modify the contractile system of cardiac myocytes, and cardiac myocytes may release factors that modulate the secretion of cardioregulatory substances by endothelial cells. This regulatory loop is sensitive to the rate of coronary flow and tissue oxygen tension. In the present study, coronary venous effluent from isolated perfused hearts and the contents of the coronary vascular endothelial cells have been collected, the latter by disrupting the cells with coronary perfusion at high pressure. The relative amounts of upregulating and downregulating factors in both collections have been estimated by assaying their effects on the contractility of isolated cardiac trabeculas. The amount of upregulating factor stored in the endothelial cells is sensitive to the rate of coronary flow just before disruption of the cells. The quantity of endothelin in the coronary venous effluent and in the vascular endothelial cell contents was measured by radioimmunoassay and compared with the degree of upregulation of contractility produced by the two types of solutions. Upregulation was never produced in the absence of endothelin. The extent of the increase in contractility that was observed with endothelial cell contents correlated with the concentration of endothelin and was approximately the same as the increase in contractility from similar concentrations of endothelin added to standard Krebs' solution. The amount of the increase in contractility from coronary effluent could be accounted for by the concentration of endothelin in the effluent with the additional presence of some downregulating factor as well. The endothelin antagonist BQ123 inhibited the upregulation from coronary perfusate. It appears that endothelin alone can account for all of the upregulation of contractility produced by the vascular endothelial cells. Coronary flow, probably through shear forces, seems to regulate the production of endothelin possibly from an inactive precursor. Tissue oxygen tension appears to modulate the rate of release of the endothelin from endothelial cells though substances released by cardiac myocytes or other cells in the tissue. The downregulating factor is stored to a much smaller extent.

Animals↗
PubMed1993

Endothelial cells are required for the cAMP regulation of cardiac contractile proteins.

The contractile proteins in mammalian cardiac muscle are regulated by a cAMP-dependent reaction that alters the activity of the actomyosin ATPase. The ATPase activity of cardiac actomyosin has also been shown to depend on factors released by small arteries in the myocardial tissue. Endothelial cells have been implicated in the regulation of the contractile force developed by isolated cardiac tissue. To determine whether endothelial cells are required for the cAMP-dependent regulation of the contractile proteins, the effect of cAMP on the actomyosin ATPase activity was measured in cryostatic sections of isolated, quickly frozen rat ventricular trabeculae. In half of the trabeculae, the endocardial endothelial cells had been damaged by a 1-sec exposure to 0.5% Triton X-100. In trabeculae with intact endothelial cells, cAMP increased actomyosin ATPase activity toward an apparently maximum value. In trabeculae with damaged endothelial cells, cAMP did not change actomyosin ATPase activity. The coronary venous effluent from an isolated heart has already been shown to modify the maximum isometric force developed by an isolated trabecula. The extent to which the force of the isolated trabecula is changed by the coronary venous effluent is closely related to the degree to which cAMP has up-regulated the actomyosin ATPase activity in the isolated heart donating the coronary effluent: the greater the degree of up-regulation of ATPase activity, the greater the increase in force produced by the effluent. These results indicate that endothelial cells are required for the cAMP-dependent regulation of cardiac contractile proteins to function, and these results further suggest that the myocardium autoregulates by modulating the cAMP regulation of contractile proteins with endothelial-derived factors.

Animals↗
PubMed1993

Evidence for the existence of endothelial factors regulating contractility in rat heart.

Force developed by isolated papillary muscle decreases as the cross-sectional area increases. The basis for this decline in force is not clear in as much as theoretical considerations and experimental data have indicated that the rate of diffusion of oxygen into thin bundles should not be limiting. Decline of maximum Ca-activated force with increasing cross-sectional area of detergent skinned papillary muscle can be attributed to the accumulation of inorganic phosphate in the center of the bundle. In both cases, the bundle of intact cells with a possible limitation of diffusion of oxygen into the bundle and of skinned cells with a limitation of diffusion of P(i) outward, the lowest level of activity should be in the center of the bundle. We have used quantitative histochemistry for measuring Ca- and actin-activated myosin ATPase activity in cryostatic sections of rapidly frozen isolated trabeculae. The technique is very sensitive and has sufficient spatial resolution to resolve individual myofibrils. At different times after dissection, ventricular trabeculae were quickly frozen, transversely sectioned and Ca- and actin-activated myosin ATPase, measured in serial sections both without and with 1 microM cAMP in the assay solution. In none of over 40 trabeculae studied was there an inward gradient of actin-activated ATPase activity of myosin. The most superficial cells had very low enzymatic activity. Cyclic AMP decreased the gradient by raising the enzymatic activity of the less active cells more that the more active cells. Ca-activated myosin ATPase was always uniform across the transverse section.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗