J. C. Rüegg

Skinned fibers of human atrium and ventricle: myosin isoenzymes and contractility.

Different myosin isoenzymes of pig and human atrium and ventricle and rat ventricle were characterized by two approaches: pyrophosphate polyacrylamide gel electrophoresis (PP-PAGE) and analysis of the myosin P light chains by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We further investigated the relation between atrial and ventricular myosin isoenzymes of human, pig, and rat, and the maximum (unloaded) shortening velocity (Vmax and the Ca2+ sensitivity of chemically skinned fibers of the same species. The myosin isoenzymes of both human and pig atrium comigrated in the PP-PAGE with rat V2 isomyosin, whereas the ventricle of human and pig comigrated with rat V3 In both human and pig ventricle, a myosin P light chain polymorphism exists (two phosphorylatable P light chains with the same molecular weight but different isoelectric points). In contrast, we found no P light chain polymorphism in the atrium of human and pig and in the ventricle of rat (one phosphorylatable P light chain only). A correlation exists between Vmax, Ca2+ sensitivity, and atrium- and ventricle-specific myosin isoenzymes of human and pig. Vmax was determined by the slack-test method. Plots of Al versus At of atrial and ventricular skinned fibers were well fitted by a single straight line up to Δl = 15% and Δl = 13%, respectively. Vmax of skinned ventricular fibers was lower than Vmax of skinned atrial fibers in both human and pig. Ca2+ sensitivity of skinned fibers of ventricle, however, was higher than Ca2+ sensitivity of atrial skinned fibers in both human and pig.

Regulation of myosin heavy chain expression in the hearts of hypertensive rats by testosterone.

Stroke-prone spontaneously hypertensive rats were used for our investigation of the influence of prepubertal gonadectomy and testosterone substitution on blood pressure, cardiac hypertrophy, and the expression of ventricular myosin heavy chain (MHC) isoenzymes at different developmental stages. Blood pressure and the degree of cardiac hypertrophy were decreased by castration and increased by testosterone substitution. We found the same relative distributions of MHC isoforms on the protein level (investigated by pyrophosphate electrophoresis) and on the messenger RNA level (investigated by the polymerase chain reaction). Castration favored the expression of the beta-MHC form, and testosterone substitution enhanced the expression of the alpha-MHC form. These effects were more pronounced in 8-week-old than in 14-16-week-old animals. We conclude that testosterone regulates cardiac MHC expression on a pretranslational level. This regulation is independent of hemodynamic load or cardiac hypertrophy.

Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease

In the present study, the Ca2(+)-sensitivity and myosin light chain patterns of skinned fibers of right atrium and left papillary muscles of 27 patients suffering from mitral valve disease (MVD, moderate heart failure), ischemic cardiomyopathy (ICM, severe heart failure), dilated cardiomyopathy (DCM, severe heart failure), and coronary heart disease (CHD, no heart failure, no atrial hypertrophy) were investigated. Myosin light chains of both chemically skinned and intact samples were studied by two-dimensional gel electrophoresis (2D-PAGE). Ca2(+)-sensitivity of ventricular fibers was about 0.14 pCa-units higher than that of atrial fibers in all groups except dilated cardiomyopathy where this difference was markedly diminished (only 0.06 pCa-units). Generally, Ca2(+)-sensitivity of skinned ventricular fibers was the same among the different heart diseases. Skinned atrial fibers from patients with dilated cardiomyopathy, however, were significantly (about 0.08 pCa-units) more sensitive for Ca2+ than those of the other groups (coronary heart disease, mitral valve disease or ischemic cardiomyopathy) which showed similar Ca2(+)-tension relationships. Ventricle-specific P-light chain forms could be observed in atrial samples from patients of all groups, whereas no atrium-specific light chain forms were detectable in any ventricular sample. It is concluded that there is no difference in Ca2(+)-sensitivity of the ventricular contractile elements of the human heart in different heart diseases. In atrial myocardium, there is an increased Ca2(+)-sensitivity of skinned fibers from hearts with dilated cardiomyopathy which is probably related to an elevation of right atrial pressure.

Increased calcium sensitivity of chemically skinned human atria by myosin light chain kinase.

SummaryWe investigated the influence of myosin P-LC phosphorylation catalysed by calcium/calmodulin-dependent myosin light chain kinase (MLCK) on the tension-pCa relation of chemically skinned human atrial fibres. MLCK-induced increased myosin P-LC phosphorylation sensitized human atrial skinned fibres for calcium by 0.11 pCa-units in patients with valvular heart disease, and by 0.05 to 0.07 pCa-units in patients with coronary heart disease. The MLCK effect could be antagonized by a light chain phosphatase. The protein phosphatase ocadaic acid (OA) had no influence on the tension-pCa relation of skinned human atrial fibres and had no potentiating effect together with MLCK. The MLCK preparation used in this study was from bovine ventricle and revealed a KM of 1.8×10−5 M and a Vmax of 822 nmol Pi/min/mg using purified bovine ventricular myosin-LCs as substrate.

Myosin isoenzymes of vascular smooth and cardiac muscle in the spontaneously hypertensive and normotensive male and female rat: a comparative study.

Cardiac hypertrophy in hypertensive subjects, its biochemical markers, and functional consequences are of great clinical importance but still unclear. We observed a shift of the ventricular isomyosin of adult spontaneously hypertensive (H) rats of both sexes to the V3 form and a decreased myofibrillar ATPase activity in the H animals when compared to normotensive (N) controls. Compared to the male H rats, age-matched female H animals revealed a lower blood pressure, the same or even an elevated magnitude of cardiac hypertrophy, a different ventricular isomyosin pattern, and a higher myofibrillar ATPase activity. In female H rats the V1 and V3 isomyosins were equally distributed (35% V1 and 35% V3), but in male H animals the V3 was predominant (24% V1 and 45% V3). The Ca2+: -regulated Mg2+:-dependent myofibrillar ATPase of the rat ventricle correlated positively with the amount of V1 when measured at pCa 5 (maximum activation). At submaximum Ca2+-concentrations (pCa 6.9–:5.9) the myofibrillar ATPase activities were not changed with the proportion of V1. The cooperativity of the Ca2+:-activation of the myofibrillar ATPase increased with increasing amount of V1 (Hill-coefficient 3.7 with 100% V1) and decreased with increased proportion of V3 (Hill-coefficient 1.3 at 45% V3). Two myosin isoenzymes were detected in the aorta of rats, a slow migrating (S2) and a fast migrating (S1) form having both a higher mobility than the ventricular isomyosins. Only one band was observed in the portal vein, which revealed the same mobility as S2. Hypertension did not change the appearance of these vascular smooth muscle isomyosins neither in male nor in female animals.

Modulation of crossbridge kinetics by myosin isoenzymes in skinned human heart fibers.

Skinned fibers from the normal human heart with the beta-myosin heavy chain (ventricular fibers) revealed both a higher force generation per cross section and a higher Ca2+ sensitivity than skinned fibers with the alpha-myosin heavy chain (atrial fibers). The relation between isometric ATPase activity and isometric tension of atrial fibers was higher than that of ventricular fibers. Since the ATPase-tension relation equals the rate constant for the transition from force-generating into non-force-generating crossbridge states (g(app)), myosin heavy chain isoenzymes seem to have different crossbridge turnover kinetics. Modulation of g(app) by myosin heavy chain isoenzymes could explain the different contractile behavior of atrial and ventricular fibers. g(app) was independent of Ca2+.

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

SummaryWe investigated the influence of myosin P-light chain phosphorylation by Ca2+-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (vmax) of chemically skinned heart fibres of the pig.Submaximum Ca2+ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca2+ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (vmax) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca2+ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of32P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.

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

SummaryWe 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 (γ-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.

Investigations on glycerinated cardiac muscle fibres in relation to the problem of regulation of cardiac contractility — effects of Ca++ and c-AMP)

Alterations in myocardial contractile force and maximum unloaded shortening velocity (Vmax) occurring in the course of isometric twitch contraction and with changes in inotropism are assumed to be mediated by changes in intracellular Ca++ and/or c-AMP concentration. In the present study, the influences of Ca++ and cyclic AMP upon the contractility of briefly glycerinated myocardial preparations are described. It is shown that Ca++ ions affect tension and Vmax, as measured by rectangular releases in length, in different concentration ranges. This suggests that, besides the number of attached crossbridges regulated by Ca++ binding to troponin C, a Ca++-dependent phosphorylation of the P-light chain of myocardial myosin may be involved in the regulation of Vmax. Cyclic AMP, on the other hand, induces phosphorylation of troponin I, thereby reducing the sensitivity of tension to Ca++.

Glutathione alters calcium responsiveness of cardiac skinned fibers

SummaryThe 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 ba 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 dithiotreitol at 5 mM had no activating effect. Our results suggest that the loss of contractility observed after a reperfusion of the ischemic heart my, at least in part, be due to a decreased responsiveness of the contractile proteins due to an altered glutathione status.