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

Abstract

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.

Regulation of human heart contractility by essential myosin light chain isoforms.

“…Recent X-ray crystallographic analysis demonstrated that both essential and regulatory MLC are associated with the neck region of the myosin-S1 fragment (7). In the human heart, five different MLC isoforms exist which are expressed in a tissue-specific manner: there are three regulatory (phosphorylatable) myosin light chains, namely two ventricular-specific (VLC-2 and VLC-2*) and an atrial-specific (ALC-2) (8,9). The ventricular regulatory light chains are monophosphorlyated each, while the ALC-2 may be mono-and diphosphorylated (9).…”

“…In the human heart, five different MLC isoforms exist which are expressed in a tissue-specific manner: there are three regulatory (phosphorylatable) myosin light chains, namely two ventricular-specific (VLC-2 and VLC-2*) and an atrial-specific (ALC-2) (8,9). The ventricular regulatory light chains are monophosphorlyated each, while the ALC-2 may be mono-and diphosphorylated (9). Two essential myosin light chain isoforms are produced by two different genes, namely a VLC-1 which is the same isoform as the MLC-1s present in adult slow skeletal muscle, and an atrial-specific (ALC-1) isoform (10).…”

In vitro motility assay of atrial and ventricular myosin from pig

“…The ventricular myosin has two forms of regulatory light chains (VLC-2 and VLC-2*), both of which can be phosphorylated. The corresponding atrial light chain (ALC-2) can be both mono-and double-phosphorylated [Morano et al, 1989]. The relative phosphorylation levels were 25-30% for each light chain.…”

“…Atrium and Ventricle* Atrium Light chain ALC-2U ALC-2P ALC-2PP % 7 5 Ϯ 8 20 Ϯ 6 5 Ϯ 2 Ventricle Light chain VLC-2U VLC-2P VLC-2*U VLC-2*P % 8 7 Ϯ 5 13 Ϯ 5 86 Ϯ 6 14 Ϯ 6 *Separations were as described by Morano et al [1989]. For the atrium, the unphosphorylated (U), the phosphorylated (P) and the double-phosphorylated (PP) variants of the light chain 2 (ALC-2) were separated.…”

“…The gels were stained with Coomassie blue R-250 and scanned to determine the relative contents of myosin and actin. Myosin light chain phosphorylation levels of atrial and ventricular myosin preparations were investigated by two-dimensional gel electrophoresis using a mini gel system (Bio-Rad, Richmond, CA), as described by Morano et al [1989]. The gels were stained and scanned, and the phosphorylation was calculated as the area under the intensity peaks.…”