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G McClellan

Publications and source records attributed to G McClellan.

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

Oxygen transport and intracellular bioenergetics on stimulated cat skeletal muscle.

A unique multiparameter recording of skeletal muscle bioenergetics, biochemistry and biomechanics has permitted determination of novel relationships among hemodynamics, cellular high-energy metabolites and mitochondrial bioenergetics in feline skeletal muscle. The study utilizes 31P NMR, NIR, and NADH fluorescence spectrophotometry, biochemical assays and muscle performance. Seven cats were anesthetized and mechanically ventilated. Calf muscles were stimulated through sciatic nerve electrical stimulation and tension was monitored by a strain gauge connected to the Achilles tendon. We stimulated the muscle to produce several workloads up to Vmax. We also changed FiO2 from normoxia to hypoxia for each %Vmax. From these results, the most sensitive indicators of cellular hypoxia leading to a reduction in muscle performance can be determined. Hemoglobin deoxygenation generally does not correlate with cellular hypoxia, although when the HbO2 drops below 30% saturation there is an increased incidence of cellular hypoxia. The [ADP], which is known to regulate mitochondrial function, has a close relation to the work, not to the hypoxia. On the other hand, the mitochondrial NADH does respond to cellular PO2. The degree of oxidation (NADH decrease) due to the ATP flux shifts with oxygen availability in mild to moderate hypoxia (at FiO2 down to 9%). As cellular hypoxia causes decreases in muscle performance (moderate to severe hypoxia), NADH is being reduced rather than oxidized with increasing workloads.

Analysis of Variance↗
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↗
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

Cardiac endothelial cells modulate contractility of rat heart in response to oxygen tension and coronary flow.

The aim of this study was to determine if endothelial cells in the heart release substances into the coronary perfusion medium that modify the contractility of myocardial cells. To assay the effects on the contractility of cardiac muscle of fluid that has passed through the coronary vasculature, a new method has been developed based on the cascade principle used to study vascular smooth muscle function. The coronary venous effluent from an isolated perfused working heart was collected periodically, and after reoxygenation it was used as the bathing medium for trabeculae isolated from the endocardial surface of another heart. The coronary venous effluent changed the contraction of the isolated trabeculae. The amplitude and the direction of the change depended on the degree of oxygen saturation of the coronary effluent before it was reoxygenated and the rate of coronary flow at the time the effluent was collected. The response of the trabecula to the coronary effluent was substantially altered by damaging the endocardial endothelium with a 1-second exposure to 0.5% Triton X-100 in Krebs' solution. It was completely eliminated by damaging endothelial cells in both the perfused heart producing the effluent and the trabecula on which the effluent was assayed. Therefore, endothelial cells are required for the presence of cardioactive substances in the coronary effluent. The production of a labile endothelium-derived upregulating (positively inotropic) factor and a more stable endothelium-derived downregulating (negatively inotropic) factor has been demonstrated and appears to account for all of the changes in myocardial contractility produced by the coronary effluent. Neither of the endothelium-derived substances demonstrated in the isolated perfused heart is nitric oxide or endothelin. The concentration of the endothelium-derived upregulating factor is sensitive to oxygen tension, whereas the concentration of the endothelium-derived downregulating factor is sensitive to the rate of coronary flow but not oxygen tension. The coronary effluent appears to contain substances that stimulate secretion by the endothelial cells (preendothelial factors) as well as substances that have been produced by the endothelial cells (endothelial factors). The results indicate that during the passage of perfusion medium through the coronary vasculature upregulating and downregulating factors are added to the perfusate in relative concentrations that depend at least in part on local tissue PO2 and the rate of coronary flow. In the intact heart, this mechanism could operate to maintain balance between energy supply and work performed.

Animals↗
PubMed1992

Endothelial cells regulate cardiac contractility.

Endothelial cells lining the lumen of blood vessels contain the receptors for many substances that alter the contractile tone of smooth muscle in the walls of the blood vessels. In response to their interaction with the signal substances, the endothelial cells release vasoactive factors that modify the contractile state of the vascular smooth muscle. This study was conducted to determine if endothelial cells can also modulate the contraction of cardiac muscle cells and contribute to the physiological regulation of the heart. The venous effluent from the coronary circulation of an isolated perfused working heart was reoxygenated and used to superfuse a trabecula isolated from the right ventricle of another heart. The peak tension and the duration of the contraction of the trabecula were reversibly altered by the effluent fluid. The change in the contraction of the trabecula during its exposure to coronary effluent was inhibited by selectively damaging the endothelial cells in the trabecula before the application of the coronary effluent. The magnitude and direction of the effect of the coronary venous effluent were sensitive to the metabolic and mechanical conditions under which the isolated perfused heart was contracting at the time the effluent was collected. These observations indicate that cardiac tissue can release a substance or substances into the coronary circulation that induce the production of cardioactive factors by endothelial cells.

Animals↗
PubMed1992

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.

Adenosine Triphosphatases↗
PubMed1991

A physiological basis for variation in the contractile properties of isolated rat heart.

1. The maximum Ca(2+)-activated force, maximum velocity of unloaded shortening and both Ca(2+)- and actin-activated ATPase activities of myosin have been measured in detergent-skinned preparations of isolated bundles of rat right ventricle after exposure of the intact tissue to different conditions of superfusion, mechanical activity and temperature. 2. Maximum Ca(2+)-activated force per unit cross-sectional area decreases with increasing cross-sectional area, and, in the absence of electrical stimulation, with the duration of superfusion. Maximum velocity of unloaded shortening is not influenced by these differences. 3. Actin-activated ATPase activity of myosin decreases as cross-sectional area increases and duration of superfusion increases, but the extent of the decrease in enzymatic activity is less than that of developed force. Ca(2+)-activated ATPase activity is independent of these differences. 4. Actin-activated ATPase activity in cryostatic sections of quickly frozen tissue is not uniform across the transverse section. In thin bundles, it is highest in the centre and lowest at the edge of the section, which correspond, respectively, to the centre and the surface of the tissue bundle. Exposure of the tissue section to 1 microM-cyclic AMP increases the actin-activated ATPase activity of myosin with the largest increase in activity occurring at or near the surface of the bundle. 5. Ca(2+)-activated ATPase activity of myosin is uniform across the transverse section and is not changed by cyclic AMP. 6. Electrical stimulation, elevated Ca2+ concentration in the superfusion medium, or isoprenaline partially or completely reverse the decline in maximum Ca(2+)-activated force produced by prolonged superfusion of the bundle before its skinning. 7. These observations are similar in many ways to those made on frog skeletal muscles by Elzinga, Howarth, Rull, Wilson & Woledge (1989a). An explanation based on the existence of a physiological mechanism for regulating the properties of force generators is proposed. Regulation of the attachment of the cross-bridge to an actin filament may be the basis for the regulatory mechanism.

Actins↗
PubMed1991

Application of positron emission tomography to neurological oncology.

PET has a promising role in neuroradiology for accurate diagnosis and prognostication of malignant tumors as well as differential diagnosis of radiation necrosis and recurrent tumors. Particularly, PET has proven its ability to accurately differentiate radiation necrosis from recurrent brain tumor. Active tumors have accelerated glycolysis, and a remarkable accumulation of FDG radiotracer in high grade brain tumors is evident on PET images. Tumor metabolism also proportionally increases with increasing pathologic grades of brain tumor, and accelerated tumor metabolism indicates a poor prognosis for the tumor.

Adult↗
PubMed1989

A calcium independent on-off switch for cardiac force generators.

Developed force and ATPase activity of actomyosin in cardiac muscle are regulated not only by the concentration of cytosolic calcium, but also by the state of the contractile proteins. In this study, it has been shown that cardiac actomyosin ATPase, even in the presence of adequate Ca, can exist in an inactive state. Micromolar cyclic AMP activates the ATPase, inducing substantial enzymatic activity. Both active and inactive forms of myosin can co-exist in the same cells. Mammalian hearts appear to contain a physiological mechanism for altering the response of actomyosin to optimal concentrations of Ca.

Animals↗
PubMed1988

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 a transition between a Ca unresponsive and a Ca responsive force generator. A 21,000 dalton protein that reproduces the effect of beta adrenergic stimulation on myosin has been isolated.

Adenosine Triphosphatases↗