title: Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998.
abstract: We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions.
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: L E
authors.initials: LE
authors.lastname: Dobrunz
authors.forename: P H
authors.initials: PH
authors.lastname: Backx
authors.forename: D T
authors.initials: DT
authors.lastname: Yue
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D016257:Fura-2; D006352:Heart Ventricles; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008433:Mathematics; D008955:Models, Cardiovascular; D009200:Myocardial Contraction / Q000187:drug effects / Q000502:physiology*; D011804:Quinolines / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D012518:Sarcomeres / Q000187:drug effects / Q000502:physiology; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000502:physiology; D013829:Thiadiazines / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D002316:Cardiotonic Agents; D011804:Quinolines; D013829:Thiadiazines; C070609:EMD 53998; D007545:Isoproterenol; D002118:Calcium; D016257:Fura-2