In the course of MG-dependent ATP splitting by heart actomyosin, an "energy rich" actomyosin-ADP complex is formed, which promotes the incorporation of phosphate 32P into ATP in myofibrils. The rate of this ATP-phosphate exchange reaction depends on the extent of actin-myosin overlap which can be decreased by stretching glycerinated muscle fibres. In heart muscle, the calcium-ion dependence of this reaction is similar to that of the actomyosin ATPase, the tension, and "immediate fibre stiffness" (which is "hookean" and which is a measure for the number of myosin cross-bridges attached to and interacting with actin). These findings suggest that calcium increases the amount of "contractile" actomyosin-ADP complexes. The proportionality between tension and ATPase activity further suggests that the rate-limiting step of the cross-bridge cycle (which determines the molecular turnover number, the "Wechselzahl" of the ATPase) is only little affected by calcium ions. These ions act by recruiting more bridges rather than by accelerating their reactions. In addition, the depressing effect of inorganic phosphate on the contractile tension and its presumable role in energetic insuffciency will be discussed.

Actomyosin↗

PubMedDate not supplied

Glutathione alters calcium responsiveness of cardiac skinned fibers.

The glutathione status of cardiac muscle, that is the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) changes in certain forms of cardiomyopathy and during reperfusion of ischemic heart. Here we show that it also affects the sensitivity of contractile proteins to calcium. GSH (4 mM) increased the pCa50 for force development in skinned fibers by 0.2 pCa units and increased force by 44% +/- 5.4% at pCa 5.6 whereas GSSG (4 mM) decreased it by 54% +/- 17.8% at pCa 5.6. Half maximal activations and inhibitions were seen with 4 mM GSH or GSSG, respectively. In contrast to GSH, the reducing agent dithiothreitol at 5 mM had no activating effect. Our results suggest that the loss of contractility observed after a reperfusion of the ischemic heart may, at least in part, be due to a decreased responsiveness of the contractile proteins due to an altered glutathione status.

Animals↗

PubMedDate not supplied

Inorganic phosphate inhibits contractility and ATPase activity in skinned fibers from human myocardium.

During hypoxic heart failure, inorganic phosphate (Pi) accumulates. We report the effects of Pi on force development and on myofibrillar ATPase-activity of human skinned atrial fibers, both at normal and at reduced levels of Mg-ATP. Pi (10 mM) depressed force production at maximal calcium activation (pCa 4.3) by about 40%. At higher pCa values (pCa 5.6), force inhibition was even more pronounced, but at low concentrations of Mg-ATP (10 microM), Pi was less effective. In contrast to contractile force, myofibrillar ATPase was only inhibited by about 10% at pCa 4.3, whereas it could be inhibited by 40-50% at submaximal calcium activation (pCa 5.6). As Pi inhibited contractile force more than ATPase activity, the ratio of ATPase-activity to force (tension cost) was increased by inorganic phosphate. ATPase-activity and tension cost were significantly reduced by lowering Mg-ATP concentration to 10 microM, whereas contractile force was less affected. Pi did not affect ATPase under these conditions at 10 mM Mg-ATP. Pi also shifted the calcium-force relationship towards higher Ca++ concentrations, that is, it decreased calcium sensitivity. In contrast, the calcium sensitivity of myofibrillar ATPase was less affected. These findings suggest that inorganic phosphate may affect the myocardium by altering crossbridge kinetics rather than the calcium affinity of troponin-C. Because of its inhibitory effect on myofibrillar ATPase, inorganic phosphate may be partly cardioprotective in the hypoxic myocardium. However, this "energy sparing' effect is probably offset by the greater "tension cost' that decreases the "efficiency' of tension maintenance in the presence of inorganic phosphate.

Adenosine Triphosphatases↗

PubMedDate not supplied

Myosin P-light chain isoenzymes in the human heart: evidence for diphosphorylation of the atrial P-LC form.

We studied myosin light chains (LC) of human atrium and ventricle of normal and diseased individuals by a high-resolution 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique. Atrial LCs (ALC-1, ALC-2 (= P-LC)) revealed both higher molecular weights and lower isoelectric points (IEP) than their ventricular counterparts (VLC-1, VLC-2 (= P-LC)). Different P-LC forms with their distinct myosin isoenzymes have been designated as P-LC-polymorphism and myosin P-LC isoenzymes, respectively. In the dephosphorylated state two VLC-2 forms (VLC-2 and VLC-2*) with the same MW and different IEP, but only one ALC-2 form, were found. In the partially phosphorylated state ALC-2 appeared to be single- and double-phosphorylated (three spots in the 2D-PAGE), whereas the two VLC-2 forms appeared to be single-phosphorylated each (four spots in the 2D-PAGE). Phosphoryl-transfer from ATP to the P-LC forms was studied using skinned fibers incubated with MLCK (myosin light chain kinase) and (gamma-32P)ATP. Ventricular myosin P-LC isoenzyme pattern was usually the same in normal and diseased patients: the VLC-2 to VLC-2* ratio was approx. 70/30, but in one patient with valvular heart disease (VHD) the relation was 55/45 (shift to the VLC-2* form). In hypertrophied atria of VHD patients a shift of the myosin P-LC isoenzyme pattern to the VLC-2* form occurred, too.

Adult↗