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Biomedical subjects

S Winegrad

Publications and source records attributed to S Winegrad.

At least 55 records · Page 3Linked to original sources
PubMed1983

Regulation of calcium sensitivity in perforated mammalian cardiac cells.

Sarcolemmal perforations can be produced in bundles of rat right ventricular cells by either perfusion of the heart or soaking of the bundles with a solution containing 10 mM EGTA. All cells are affected and lose approximately 40% of the surface membrane. In these cells it is possible to show cAMP regulation of contractility (maximum Ca-activated force) without cAMP regulation of Ca sensitivity (pCa for 50% of maximum Ca-activated force). Therefore, the target molecule for cAMP is different for the two regulatory systems. Both regulatory systems can be slowly washed out of the cell by 10 mM EGTA solution but not by relaxing or contraction solutions. A model for regulation of Ca sensitivity is proposed.

Animals↗
PubMed1983

Cyclic AMP regulation of myosin isozymes in mammalian cardiac muscle.

Hyperpermeable cells from rat heart contain a cAMP-dependent system that can increase the maximum Ca-activated force (contractility) of the contractile proteins. In two different conditions where the relative concentration of the myosin isozymes changes, i.e., hypothyroidism and aging, the size of the increase in contractility from activation of the cAMP-regulated system varies closely with the relative concentration of V1, the isozyme of myosin with the greatest Ca- and actin-activated ATPase activity. The existence of another system for the regulation of the slow isozyme V3 has been demonstrated, and it may be inhibited by beta-adrenergic activity. The possibility of cAMP-dependent myosin regulation of contraction in addition to Ca regulation of troponin is considered. Phosphorylation of the contractile proteins themselves is not required for the increased contractility.

Animals↗
PubMed1983

Energy transport from mitochondria to myofibril by a creatine phosphate shuttle in cardiac cells.

In hyperpermeable cardiac cells, in which the surface membrane has been made highly permeable to small molecules and ions, resting tension increases when the concentration of ATP falls below 200 microM. Peak resting tension occurs in 10 microM ATP and equals 60% of maximum Ca-activated force in 5 mM ATP. The mitochondria in hyperpermeable cells can maintain an ATP concentration above 200 microM if supplied with O2, substrate, ADP, and inorganic phosphate (Pi). Removal of ATP from the bathing solution does not increase resting tension as long as creatine phosphate is present. However O2, substrate, and Pi cannot lower resting tension in the absence of ATP and creatine phosphate. These results are interpreted as evidence for adenine nucleotide tightly bound to the myofibrils and a creatine phosphate shunt of energy from the mitochondria to the myofibrils.

Adenosine Diphosphate↗
PubMed1983

Regulation of cardiac contractile proteins by phosphorylation.

Several of the contractile proteins of the heart can be phosphorylated, but in studies with isolated proteins only phosphorylation of the inhibitory subunit of troponin (TnI) produces a major change in the properties of the contractile system. As TnI is phosphorylated, the concentration of calcium required for activation of contraction is increased. Phosphorylation of the tropomyosin-binding subunit of troponin (TnT) or of the light chain of myosin fails to change ATPase activity of the isolated protein system. Phosphorylation of TnI is stimulated by the beta-adrenergic system and inhibited by the cholinergic system. Maximum calcium-activated force produced by the contractile system can be increased in hyperpermeable cardiac cells by cyclic AmP (cAMP) or agents that stimulate cAMP synthesis. This change in the contractile system, which appears to be part of the physiological response to beta-adrenergic stimulation, is mediated by phosphorylation of an intermediate that then modifies the contractile system. Phosphorylation of the contractile proteins is not involved.

Adenosine Triphosphate↗
PubMed1982

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.

Adenosine Triphosphatases↗
PubMed1980

Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells.

Bundles of cells from rat right ventricular myocardium were made "hyperpermeable" by an overnight soak in 10 mM EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764). In this preparation the cytoplasmic concentration of Ca++ and ATP could be controlled while sarcolemmal receptors and enzymes were retained. The Ca sensitivity of the tissues (as indicated by the pCa for 50% maximum activation) was altered to different extents in the presence of [32Pgamma]ATP by treatment with cyclic nucleotides, catecholamines, or a low concentration of nonionic detergent. The proteins of the tissue were then isolated by SDS-polyacrylamide gel electrophoresis, and the identity of 32P-labeled proteins was determined. The Ca sensitivity is inversely related to the relative amount of 32P incorporated into the inhibitory subunit of troponin (TNI). Extrapolation of the relation to the lowest Ca sensitivity observed gives a stoichiometry of about 0.8 mol PO4 per mol TNI. These results support the hypothesis that Ca sensitivity of cardiac myofibrils is regulated by a phosphrylation of TNI that is stimulated by cyclic AMP (cAMP) and inhibited by cGMP.

Adenosine Triphosphate↗
PubMed1980

Cyclic nucleotide regulation of the contractile proteins in mammalian cardiac muscle.

The contractile system of rat cardiac muscle that has been made hyperpermeable by soaking the tissue in EGTA (McClellan and Winegrad. 1978. J. Gen. Physiol. 72:737-764) can be probed directly with Ca buffer from the bathing solution without significant interference from either sarcoplasmic reticulum or mitochondria on the Ca concentration. Changes in Ca-activated force are due therefore to changes in the properties of the contractile system itself and not to regulation of Ca concentration. The addition of cAMP, cGMP, and GTP, guanylyl imidodiphosphate (GMP-PNP), or epinephrine to the bath does not alter maximum Ca-activated force, but when these drugs are added with 1% nonionic detergent to the bath, contractility increases by as much as 180%. An inhibitor of phosphodiesterase must be present for the inotropic effect of cAMP but not cGMP, GTP, GMP-PNP, or epinephrine. The inotropic response to cAMP is independent of the Ca sensitivity of the contractile system, but guanine nucleotides enhance contractility only when Ca sensitivity is not high. The inotropic effect of epinephrine is inhibited to a large extent by cGMP but not by GMP-PNP. These data can be explained by a model in which contractility is enhanced by a cAMP-regulated phosphorylation that can be controlled through the beta-receptor adenylate cyclase complex in the sarcolemma. The regulation involves two reactions, one a phosphorylation and a second that occurs in the presence of detergent. Phosphorylation of neither the myosin light chain nor the inhibitory subunit of troponin appears to be involved in this mechanism for regulating contractility.

Animals↗
PubMed1979

The measurement and dynamic implications of thin filament lengths in heart muscle.

1. The lengths of the thin filaments in amphibian and mammalian cardiac muscle have been determined from electron micrographs of serial transverse sections. Thin filament lengths in frog atrial trabeculae range from 0.8 to greater than 1.3 micrometers, with a maximum possible error of 0.14--0.15 micrometer. In rat atrial tissue the span is from 0.6 to more than 1.1 micrometer, whereas in rat papillary muscle the breadth of the distribution is much narrower, from 0.9 to greater than 1.1 micrometer. Double overlap of thin filaments should, therefore, exist over a wide range of sarcomere lenghts. Thin filaments from opposite halves of a sarcomere accommodate each other by flexing up to an angle of about 2 degrees and moving from the trigonal position among the thick filaments to the centre of the region between two thick filaments. Such rearrangement probably contributes to the internal resistance to shortening in the muscle. 2. Except for the variation in thin filament lengths, the over-all morphology of the cardiac sarcomere is generally similar to that found in skeletal muscle. Thick filaments in heart muscle are uniform in length, and their profiles change along their lengths. They are generally round in the M band, triangular adjacent to the M band, round again in the overlap region, and either round or triangular near the tapered tips. The M bridges in rat cardiac tissue link neighbouring thick filaments to form a symmetric hexagonal array, whereas in the frog atrium, the M bridge connexions are incomplete and often form isolated triangular clusters. 3. Computed sarcomere length-developed tension curves were calculated using the thin filament length distributions and the assumptions basic to the sliding filament theory of muscle contraction. The curves for atrial tissue have plateau regions approximately as wide as the one-half micron variation in thin filament length. 4. Work done against the internal loads during systole may be stored as potential energy and released during diastole to produce sarcomeric re-extension.

Animals↗
PubMed1978

The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle.

Treatment of rat ventricular cells with 10 mM EGTA makes the sarcolemma highly permeable to small ions and molecules without removing its restriction of the diffusion of larger molecules or inactivating all of its enzymatic functions. These hyperpermeable cardiac cells have been used to study the regulation of the range of concentration of Ca over which activation of the contractile proteins occurs (Ca sensitivity). The Ca sensitivity can varied from three- to sixfold without any significant alteration in the general shape of the relation between force and Ca concentrations. Although cyclic nucleotides in concentrations of 10(-9) to 10(-5) M do not influence Ca sensitivity, in the presence of a phosphodiesterase inhibitor, cGMP increases and cAMP decreases Ca sensitivity. Treatment of the hyperpermeable cells with a nonionic detergent raises Ca sensitivity as does removal of the phosphate donor by complete substitution of CTP for ATP. These data indicate that Ca sensitivity is probably modulated by a cAMP-dependent phosphorylation that decreases Ca sensitivity. The sarcolemma is required for this reaction to take place. The effect of this reaction is antagonized by a cGMP-dependent reaction occurring inside the cell. Studies involving the perfusion of the heart with and without epinephrine before the exposure to EGTA indicate that epinephrine can regulate this system of control of Ca sensitivity. The functional considerations of this regulatory system are discussed.

Aging↗
PubMed1978

Force generation among cells in the relaxing heart.

When an isolated bundle of mammalian heart cells has been soaked for several hours in a solution containing 3 mM EGTA, a calcium chelator, 2 important changes in the tissue occur: (1) the surface membrane becomes very permeable to small ions and molecules so that the contractile proteins can be directly activated by Ca ions added to the bathing solution; and (2) all intercalated discs open. In this preparation direct Ca activation can produce the transmission of even greater forces among the cells than can be achieved by electrical stimulation of the intact bundle in the presence of catecholamines, high Ca and low Na. Some structure other than the intercalated discs must be transmitting the force. A careful examination of the ultrastructure of the mammalian myocardium has revealed a highly organized network of microfilaments which run between the basement membranes of adjacent cells and from the basement membranes to the collagen fibers. This hitherto undescribed network has all the structural appearances of a major force-bearing structure, and it is probably the primary pathway for force transmission during systole as well as the restoration to the diastolic configuration during relaxation.

Animals↗