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In response to oxidative stress, the pathogenesis of a number of cardiovascular events and several genes are stimulated by extracellular signal-regulated kinases (ERK1/2). Biphasic (early, 10 min; and delayed, 120 min) ERK1/2 activation by H 2 O 2 , a reactive oxygen species, was observed in cultured neonatal rat cardiomyocytes. We investigated the hypothesis that the delayed activation of ERK1/2 depends on a factor secreted by oxidative stress (FSO). The delayed activation was inhibited by calphostin C, a protein kinase C inhibitor. Conditioned medium (CM) obtained from cells stimulated with H 2 O 2 induced rapid and monophasic ERK1/2 activation, which was not inhibited by calphostin C. In contrast, calphostin C-pretreated CM did not activate ERK1/2. Macrophage migration inhibitory factor (MIF) was one of the candidate FSOs activating ERK1/2. The existence of MIF in CM, the recombinant MIF-stimulated ERK1/2 rapid activation, and anti-MIF neutralizing antibodyinduced inhibition of the delayed activation implied that MIF could be the FSO. Pretreatment of cardiomyocytes with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor did not suppress the MIF secretion, although it prevented the ERK1/2 activation by H 2 O 2 . These results indicate that MIF is secreted from cardiomyocytes as a result of oxidative stress and activates ERK1/2 through a MEK1/2-dependent mechanism, although the secretion is not regulated by ERK1/2 but by protein kinase C.

Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiac hypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload without the influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, we examined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, and protein synthesis using heart perfusion model. The hearts isolated from Sprague-Dawley rats were perfused according to the Langendorff technique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated by its complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by its autophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fos expression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fos expression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failed to affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-induced acceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.

It is not certain whether activation of the Ras/mitogen-activated protein (MAP) kinase pathway is involved in cardiac hypertrophy.3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as lovastatin, prevent farnesylation of the Ras protein, which is critical for Ras's membrane localization and function. Therefore, the present study was undertaken to investigate the role of the Ras pathway, which is linked to mevalonate metabolism, in the mechanism of stretch-induced myocyte hypertrophy.Myocytes isolated from 1-to 2-day-old rats were cultured at 4.1 X 106 cells per well in a deformable silicon dish and incubated with serum-free medium for 7 days. The cultures were stretched by 15% on culture day 4. Stretch increased the RNA/DNA ratio by 20% to 26% on culture days 5 and 6 and the protein/DNA ratio by 18% to 20% on culture days 6 and 7. Stretch accelerated rates of protein synthesis by 24% on culture day 6. Stretch increased protein kinase C (PKC) activity, MAP kinase activity, and c-fos mRNA expression. A selective PKC inhibitor, calphostin C (1 X 10-6 M), prevented the stretch-induced increase in PKC activity, but lovastatin (7.5 X 10-6 M) did not. Lovastatin as well as calphostin C partially but significantly inhibited the stretch-induced increases in MAP kinase activity, c-fos mRNA expression, and protein synthesis. Pretreatment with both lovastatin and calphostin C completely inhibited the increases in these variables caused by stretch. Lovastatin as well as calphostin C prevents stretch-induced cardiac hypertrophy. These results suggest that mechanical stretch may activate the Ras pathway, which is linked to mevalonate metabolism, in cultured neonatal rat heart cells. (Hypertens Res 1998; 21: 109-119)

These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.

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