No abstract

Abstract-Three major mammalian mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), p38, and c-Jun NH 2 -terminal protein kinase (JNK), have been identified in the cardiomyocyte, but their respective roles in the heart are not well understood. The present study explored their functions and cross talk in ischemia/reoxygenation (I/R)-induced cardiac apoptosis. Exposing rat neonatal cardiomyocytes to ischemia resulted in a rapid and transient activation of ERK, p38, and JNK. On reoxygenation, further activation of all 3 mitogen-activated protein kinases was noted; peak activities increased (fold) by 5.5, 5.2, and 6.2, respectively. Visual inspection of myocytes exposed to I/R identified 18.6% of the cells as showing morphological features of apoptosis, which was further confirmed by DNA ladder and terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL). Myocytes treated with PD98059, a MAPK/ERK kinase (MEK1/MEK2) inhibitor, displayed a suppression of I/R-induced ERK activation, whereas p38 and JNK activities were increased by 70.3% and 55.0%, respectively. In addition, the number of apoptotic cells was increased to 33.4%. With pretreatment of cells with SB242719, a selective p38 inhibitor, or SB203580, a p38 and JNK2 inhibitor, I/RϩPD98059-induced apoptotic cells were reduced by 42.8% and 63.3%, respectively. Hearts isolated from rats treated with PD98059 and subjected to global ischemia (30 minutes)/reoxygenation (1 hour) showed a diminished functional recovery compared with the vehicle group. Coadministration of SB203580 attenuated the detrimental effects of PD98059 and significantly improved cardiac functional recovery. The data taken together suggest that ERK plays a protective role, whereas p38 and JNK mediate apoptosis in cardiomyocytes subjected to I/R, and the dynamic balance of their activities is critical in determining cardiomyocyte fate subsequent to reperfusional injury. (Circ Res. 2000;86:692-699.)

The extracellular signal-regulated kinase (ERK) pathway is activated by hypertrophic stimuli in cardiomyocytes. However, whether ERK plays an essential role or is implicated in all major components of cardiac hypertrophy remains controversial. Using a selective MEK inhibitor, U0126, and a selective Raf inhibitor, SB-386023, to block the ERK signaling pathway at two different levels and adenovirus-mediated transfection of dominant-negative Raf, we studied the role of ERK signaling in response of cultured rat cardiomyocytes to hypertrophic agonists, endothelin-1 (ET-1), and phenylephrine (PE). U0126 and SB-386023 blocked ET-1 and PE-induced ERK but not p38 and JNK activation in cardiomyocytes. Both compounds inhibited ET-1 and PEinduced protein synthesis and increased cell size, sarcomeric reorganization, and expression of ␤-myosin heavy chain in myocytes with IC 50 values of 1-2 M. Furthermore, both inhibitors significantly reduced ET-1-and PE-induced expression of atrial natriuretic factor. In cardiomyocytes transfected with a dominant-negative Raf, ET-1-and PE-induced increase in cell size, sarcomeric reorganization, and atrial natriuretic factor production were remarkably attenuated compared with the cells infected with an adenovirus-expressing green fluorescence protein. Taken together, our data strongly support the notion that the ERK signal pathway plays an essential role in ET-1-and PE-induced cardiomyocyte hypertrophy.

1 Urotensin-II (U-II) and its receptor (UT) represent novel therapeutic targets for management of a variety of cardiovascular diseases. To test such hypothesis, it will be necessary to develop experimental animal models for the manipulation of U-II/UT receptor system. The goal of this study was to clone mouse and primate preproU-II and UT for pharmacological pro®ling. 2 Monkey and mouse preproU-II genes were identi®ed to encode 123 and 125 amino acids. Monkey and mouse UT receptors were 389, and 386 amino acids, respectively. Genomic organization of mouse genes showed that the preproU-II has four exons, while the UT receptor has one exon. 3 Although initially viewed by many exclusively as cardiovascular targets, the present study demonstrates expression of mouse and monkey U-II/UT receptor mRNA in extra-vascular tissue including lung, pancreas, skeletal muscle, kidney and liver. 4 Ligand binding studies showed that [ 125 I]h U-II bound to a single sites to the cloned receptors in a saturable/high a nity manner (K d 654+154 and 214+65 pM and B max of 1011+125 and 497+68 fmol mg 71 for mouse and monkey UT receptors, respectively). Competition binding analysis demonstrated equipotent, high a nity binding of numerous mammalian, amphibian and piscine U-II isopeptides to these receptors (K i =0.8 ± 3 nM). Fluorescein isothiocyanate (FITC) labelled U-II, bound speci®cally to HEK-293 cells expressing mouse or monkey UT receptor, con®rming cell surface expression of recombinant UT receptor. 5 Exposure of these cells to human U-II resulted in an increase in intracellular [Ca 2+ ] concentrations (EC 50 3.2+0.8 and 1.1+0.3 nM for mouse and monkey UT receptors, respectively) and inositol phosphate (Ip) formation (EC 50 7.2+1.8 and 0.9+0.2 nM for mouse and monkey UT receptors, respectively) consistent with the primary signalling pathway for UT receptor involving phospholipase C activation.