THE RAF-MEK-ERK CASCADE REPRESENTS A COMMON PATHWAY FOR ALTERATION OFINTRACELLULAR CALCIUM BY RAS AND PROTEIN-KINASE-C IN CARDIAC MYOCYTES

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade , may participate in the development of cardiac hypertrophy, a conditi on characterized by diminished and prolonged contractile calcium trans ients. To directly examine the influence of this pathway on intracellu lar calcium ([Ca2+](i)), cardiac myocytes were cotransfected with effe ctors of this pathway and with green fluorescent protein, which allowe d the living transfected myocytes to be identified and examined for [C a2+](i) via indo-1. Transfection with constitutively active Pas (Ha-Ra s(V12)) increased cell size, decreased expression of the myofibrils an d the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+](i) transients, Sim ilar effects on [Ca2+](i) were obtained with Ha-Ras(V12S35), a Ras mut ant that selectively couples to Raf, and with constitutively active Ra f. in contrast, Ha-Ras(V12C40), a Ras mutant that activates the phosph atidylinositol 3-kinase pathway, had a lesser effect. The PKC-activati ng phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+](i) transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+](i). The effects of Ha-Ras(V12) and Raf on [Ca2+]i were also cou nteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+](i) via the Raf-MEK-ERK cascade, and this pathway may r epresent a critical determinant of cardiac physiological function.