Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol.

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP--kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase--MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.

Alterations in myocardial cytoskeletal and regulatory protein expression following a single Doxorubicin injection.

Immunochemical and ultrastructural studies of the rat heart after a single injection of doxorubicin (2.2 or 0.44 mg/kg) were performed. Ventricles were taken for the study 2 h and 3 weeks after injection. The light and electron microscopy and immunohistochemical determination of collagens of I, III, and IV types and fibronectin using specific antibodies were implied. Quantitive immunoblotting was used to analyze the expression levels of cytoskeletal and extracellular matrix proteins such as desmin, tubulin, vinculin, fibronectin, kinase-related protein (KRP or telokin), and smooth muscle/nonmuscle myosin light-chain kinase (MLCK). Doxorubicin (2.2 mg/kg) did not influence the relative volume and structure of collagen network but distinctly reduced the density of fibronectin distribution and decreased the content of tubulin, fibronectin, MLCK, and KRP. After 3 weeks, an increased density and extension of collagen network were observed, indicating the development of diffuse fibrosis whereas the content of tubulin and KRP increased above control level by 50 +/- 2.3% and 20 +/- 5.2%, correspondingly. Similar but less pronounced alterations were observed following the administration of 0.44 mg/kg doxorubicin. The content of MLCK after both doses consistently remained about 30% below its level in untreated animals. Isolated chick embryo cardiomyocytes subjected to doxorubicin responded by a 26% increase in KRP expression 4 days after whereas the level of tubulin expression remained unchanged. Thus, the damage of myocardium after a single injection of a therapeutic dose of doxorubicin was followed by an increased expression of selected cytoskeletal and extracellular matrix proteins, suggesting their involvement in cardiac reparation.

Smooth muscle myosin filament assembly under control of a kinase-related protein (KRP) and caldesmon.

Kinase-related protein (KRP) and caldesmon are abundant myosin-binding proteins of smooth muscle. KRP induces the assembly of unphosphorylated smooth muscle myosin filaments in the presence of ATP by promoting the unfolded state of myosin. Based upon electron microscopy data, it was suggested that caldesmon also possessed a KRP-like activity (Katayama et al., 1995, J Biol Chem 270: 3919-3925). However, the nature of its activity remains obscure since caldesmon does not affect the equilibrium between the folded and unfolded state of myosin. Therefore, to gain some insight into this problem we compared the effects of KRP and caldesmon, separately, and together on myosin filaments using turbidity measurements, protein sedimentation and electron microscopy. Turbidity assays demonstrated that KRP reduced myosin filament aggregation, while caldesmon had no effect. Additionally, neither caldesmon nor its N-terminal myosin binding domain (N152) induced myosin polymerization at subthreshold Mg2+ concentrations in the presence of ATP, whereas the filament promoting action of KRP was enhanced by Mg2+. Moreover, the amino-terminal myosin binding fragment of caldesmon, like the whole protein, antagonizes Mg(2+)-induced myosin filament formation. In electron microscopy experiments, caldesmon shortened myosin filaments in the presence of Mg2+ and KRP, but N152 failed to change their appearance from control. Therefore, the primary distinction between caldesmon and KRP appears to be that caldesmon interacts with myosin to limit filament extension, while KRP induces filament propagation into defined polymers. Transfection of tagged-KRP into fibroblasts and overlay of fibroblast cytoskeletons with Cy3KRP demonstrated that KRP colocalizes with myosin structures in vivo. We propose a new model that through their independent binding to myosin and differential effects on myosin dynamics, caldesmon and KRP can, in concert, control the length and polymerization state of myosin filaments.