We tested the hypothesis that accumulation of H+ or inorganic phosphate (Pi) is responsible for the early contractile failure of hypoxia by measuring maximal Ca2+-activated pressure and 31P nuclear magnetic resonance spectra in Langendorff-perfused ferret hearts at 30 degrees C. Maximal Ca2+-activated pressure was identified by the saturation of pressure with respect to [Ca2+]o observed during tetani as [Ca2+]o was increased to 15 mM in HEPES-buffered, 100% O2-bubbled perfusate and during hypoxia induced by bubbling with room air or with 100% N2. Tetani were produced by pacing at 8-12 Hz following exposure to ryanodine (1-5 microM), an inhibitor of Ca2+ release from the sarcoplasmic reticulum, and were elicited once a minute to measure maximal Ca2+-activated pressure during acquisition of nuclear magnetic resonance spectra. An inverse correlation was observed between [Pi] and maximal Ca2+-activated pressure (r = -0.87 mean, n = 12), with an average decline of 8.6% in pressure per 1 mumol/g wet wt. increase in [Pi]. Intracellular pH (pHi) showed no significant correlation with maximal Ca2+-activated pressure (r = 0.49 mean, n = 12). Two other protocols, pacing at variable rates and gated measurements at two different times during the tetanus, were also used to correlate [Pi], pHi, and maximal Ca2+-activated pressure. These protocols confirmed the highly significant correlation between [Pi] and maximal Ca2+-activated pressure, as well as the lack of correlation with pHi. Acidosis induced by NH4Cl (20 mM) or by bubbling with 95% O2/5% CO2 was associated with less than 20% depression of maximal Ca2+-activated pressure in the pHi range down to 6.8, but much greater depression at lower pHi. The data are consistent with depression of maximal Ca2+-activated force during the early phase of hypoxia by Pi but not by H+.

Mechanism of early contractile failure during hypoxia in intact ferret heart: evidence for modulation of maximal Ca2+-activated force by inorganic phosphate.

The in vitro cardiac effects of DPI 201-106, a novel piperazinyl-indole, were investigated. DPI 201-106 produced concentration-dependent positive inotropic effects in guinea-pig and rat left atria, kitten, rabbit and guinea-pig papillary muscles and Langendorff perfused hearts of rabbits between 10−7 and 3×10−6 mol/l. During isometric twitches, contraction and relaxation phases were prolonged in guinea-pig left atria and right ventricular papillary muscles from kitten and guinea-pigs. Spontaneous sinus rate was decreased in right atria of guinea-pigs and rats. Coronary flow increased in rabbit isolated hearts. Functional refractory period was increased in left atria from guinea-pigs and rats with EC50 values of 1.7 and 0.24 μmol/l respectively. In electrophysiological measurements, DPI 201-106 prolonged the action potential duration (APD70) in guinea-pig papillary muscles up to 70% and in rabbit atria up to 120% at 3 μmol/l. Other action potential characteristics were not changed in guinea-pig papillary muscles but V
max was decreased in rabbit left atria. The electrophysiological as well as the positive inotropic effects were stereoselective with the activity residing in the S-enantiomer. DPI 201-106 increased the Ca2+-sensitivity of skinned fibres from porcine trabecular septomarginalis with an EC50 of 0.2 nmol/l. DPI 201-106 did not change cAMP levels in guinea-pig atria and rabbit papillary muscles. Slow action potentials were not induced by DPI 201-106 in partially depolarized guinea-pig papillary muscles. Phosphodiesterase activity of rat hearts was not inhibited by DPI 201-106 at pharmacologically relevant concentrations. The presence of propranolol did not influence the inotropic potency of DPI 201-106 in guinea-pig atria. In conclusion, DPI 201-106 represents a novel type of positive inotropic agents with a synergistic sarcolemmal and intracellular mechanism of action.

DPI 201-106, a novel cardioactive agent. Combination of cAMP-independent positive inotropic, negative chronotropic, action potential prolonging and coronary dilatory properties.

Vanadate and its significance in biochemistry and pharmacology

Vanadare and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle

The suppression of tension development by orthovanadate (Vi) was studied in mechanical experiments and by measuring the binding of radioactive Vi and nucleotides to glycerol-extracted rabbit muscle fibers. During active contractions, Vi bound to the cross-bridges and suppressed tension with an apparent second-order rate constant of 1.34 X 10(3) M-1s-1. The half-saturation concentration for tension suppression was 94 microM Vi. The incubation of fibers in Vi relaxing or rigor solutions prior to initiation of active contractions had little effect on the initial rise of active tension. The addition of adenosine diphosphate (ADP) and Vi to fibers in rigor did not cause relaxation. Suppression of tension only developed during cross-bridge cycling. After slow relaxation from rigor in 1 mM Vi and low (50 microM) MgATP concentration (0 Ca2+), radioactive Vi and ADP were trapped within the fiber. This finding indicated the formation of a stable myosin X ADP X Vi complex, as has been reported in biochemical experiments with isolated myosin. Vi and ADP trapped within the fibers were released only by subsequent cross-bridge attachment. Vi and ADP were preferentially trapped under conditions of cross-bridge cycling in the presence of ATP rather than in relaxed fibers or in rigor with ADP. These results indicate that in the normal cross-bridge cycle, inorganic phosphate (Pi) is released from actomyosin before ADP. The resulting actomyosin X ADP intermediate can bind Vi and Pi. This intermediate probably supports force. Vi behaves as a close analogue of Pi in muscle fibers, as it does with isolated actomyosin.

/pdf/suppression-of-muscle-contraction-by-vanadate-mechanical-and-40umu0uv5i.pdf

Suppression of muscle contraction by vanadate. Mechanical and ligand binding studies on glycerol-extracted rabbit fibers.

Covalent alterations of cardiac myofibrils by protein phosphorylation may alter the free Ca**-force relation in a way that would affect the relaxation properties of the heart independent of the sarcoplasmicfree Ca concentration. The clearest picture for such an alteration is emerging from studies of phosphorylation of troponin I. Troponin I is phosphorylated in situ as a specific response to p-adrenergic stimulation and in vitro phosphorylation of cardiac myofibrils results in a rightward shift of the free CA**-myofibrillar activation relation. The mechanism for this effect appears to be an alteration of calcium binding properties of myofibrillar TnC induced by phosphorylation of Tnl. This effect may be responsible, in part, for the relaxant effect of catecholamines. P-light chains of myosin are partially phosphorylated in beating hearts but there is as yet no clear indication that the covalent phosphate content of myosin changes with the physiological state of the heart. Vanadate, a transition state analogue of phosphate, present in most tissues including heart, may modulate relaxation of the heart by a non-covalent interaction with myosin. We found that vanadate ion inhibits isometric tension and immediate stiffness of chemically skinned heart muscle fibers with a Ki of 50 HM. Vanadate did not affect myofibrillar calcium binding or the Ca dependence of normalized isometric force development by the heart fibers. Vanadate also depressed shortening velocity of the chemically skinned fibers contracting with zero external load. Our data therefore indicate that vanadate may not only alter the affinity of cardiac myosin for actin but also the rate ofcross-bridge turnover.

M. J. Holroyde

Papers

Cardiac relaxation and myofibrillar interactions with phosphate and vanadate