Mitogen-activated protein kinases in the acute diabetic myocardium.

Diabetes mellitus (DM) causes myocardial remodeling on the subcellular level and alterations in the function of the cell membranes ion transport systems resulting in contractile dysfunction. The present study was aimed to investigate the expression and activation of mitogen-activated protein kinases (MAPKs) and their possible role in the acute diabetic rat hearts. Rats were injected with single dose of streptozotocin (45 mg/kg, i.v.), and after 1 week the disease was manifested by hyperglycemia and cardiac dysfunction. The Langendorff-perfused hearts were subjected to ischemia (5 or 30 min occlusion of LAD coronary artery). The protein pattern in cytosolic fraction of the heart tissue was determined after electrophoretic separation. The levels and activation of MAPKs were determined by Western blot analysis using specific antibodies. No differences between the diabetics and controls in the level of ERKs were found at baseline. However, in DM samples ERKs phosphorylation was markedly increased, and further changes occurred during ischemia. Also content of phoshorylated c-Raf kinase (an upstream activator of ERKs) was slightly increased at baseline conditions in the diabetic samples. In contrast, no significant changes in the contents and phosphorylation of p38-MAPK were observed at baseline. But some differences in the p38-MAPK phosphorylation were found during ischemia. The results show that differential pattern of protein kinase cascades activation in the diabetic hearts might be account for the modulation of their response to ischemia.

Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology.

Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene targeting or expression of constitutively active and dominant-negative forms of enzymes (adenovirus-mediated gene transfer). This review is focused on the regulatory role of MAPKs in the myocardium, with particular regard to their involvement in pathophysiological processes, such as myocardial hypertrophy and heart failure, ischemia/reperfusion injury, as well as in the mechanisms of cardioprotection. In addition, it summarizes current information on pharmacological modulations of MAPKs activity and their impact on the cardiac response to pathophysiological processes.

Involvement of the fibroblast growth factor system in adaptive and chemokine-induced arteriogenesis.

Fibroblast growth factors (FGFs) have been applied in a variety of therapeutic and experimental studies to improve collateral blood flow. However, the pathophysiological role and the temporospatial expression of the FGFs and their receptors during arteriogenesis have never been elucidated in vivo. Here, we report that collateral artery growth in its early phase is associated with an increased expression of FGF receptor-1 (FGFR-1) and syndecan-4 on mRNA and protein levels as well as with an increased kinase activity of FGFR-1 in a rabbit model of arteriogenesis. However, the mRNA levels of FGF-1 and -2 remained constant. Our data suggest that these growth factors are supplied by endothelial attracted monocytes that, in turn, produce and deliver the FGFs to growing collateral arteries. Monocyte chemoattractant protein-1-stimulated arteriogenesis was strongly reduced in rabbits by application of the FGF inhibitor polyanetholesulfonic acid, indicating that the monocyte-related arteriogenesis (as well as the unstimulated adaptation proper) is promoted by FGFs. In summary, this study shows that arteriogenesis is associated with an increased expression of the FGFRs at the site of the vessel, whereas the growth-promoting ligands are supplied by monocytes in a paracrine way.

Transcription inhibitor actinomycin-D abolishes the cardioprotective effect of ischemic reconditioning.

OBJECTIVE: Our previous studies have suggested a role of mitogen-activated protein kinases (MAPKs) in cardioprotection in the porcine heart. To investigate, whether this could be due to modification of transcriptional events we studied the influence of actinomycin-D (act-D), a known RNA-synthesis inhibitor on (i) ischemic preconditioning, (ii) (IP)-mediated cardioprotection, (iii) transcription factors levels and MAPKs activation. METHODS: The IP-design in our model included two cycles of 10' LAD occlusion (CO) and 10' reperfusion (RP), followed by 40' CO (index ischemia) and 60' RP. Act-D was infused intramyocardially (i.my.) or systemically (syst.) (0.05 or 0.12 mg/kg) during 15' before IP and during both RP cycles of the IP-protocol. The i.my. infusions occurred via four pairs of needles into the risk area (RA). RESULTS: Systemic infusion of act-D (0.05 mg/kg) before index ischemia significantly increased the IS from 54.0+/-2.5 to 78.5+/-3.8%. IP significantly reduced the IS to 2.5+/-0.8%. Syst. of act-D completely abolished the IP-induced cardioprotection. At a dose of 0.12 mg/kg the IS was 88.6+/-1.7% of the risk area; at 0.05 mg/kg IS was 65.6+/-1.5%. Local infusion of act-D reduced the IP-induced cardioprotection in a concentration dependent manner. Syst. or i.my. infusion of DMSO in KHB did not influence the IP-induced cardioprotection. Western blot analysis with phospho-specific antibodies showed a significant increase in phosphorylation of cytosolic ERK1/2 and SAPK/JNKs at the end of IP procedure and act-D treatment inhibited IP-induced activation of these MAPKs. By Western blot analysis using phospho-specific antibodies against c-Jun, ATF-2, Elk-1 and c-Myc we found increased phosphorylation of all these transcription factors in the myocardial risk area at the end of IP protocol and both local and systemic infusion of act-D significantly (P<0.05) inhibited this increased phosphorylation. Unlike UO, act-D had no influence on the Akt-pathway but inhibited the increased expression of S100 protein induced by IP. CONCLUSIONS: We demonstrate in vivo that act-D, completely cancelled the IP-induced cardioprotection. The influence of act-D on cardioprotection, transcription factors, and activities of ERKs and JNKs indicates a possible transcriptional role of these MAPKs signal transduction pathways during ischemia and in IP.