The Raf-MEK-ERK Cascade Represents a Common Pathway for Alteration of Intracellular Calcium by Ras and Protein Kinase C in Cardiac Myocytes

Abstract

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating 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-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function. Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating 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-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function. Cardiac hypertrophy commonly develops in response to hemodynamic pressure overload (1Wankerl M. Schwartz K. J. Mol. Med. 1995; 73: 487-496Crossref PubMed Scopus (56) Google Scholar). The participation of Ras in the development of hypertrophy is suggested by the observations that hypertrophic stimuli activate Ras, dominant-negative Ras mutants inhibit the hypertrophic response, and overexpression of constitutively active Ha-RasV12 elicits hypertrophic growth (2Thorburn A. Thorburn J Chen S.-Y. Powers S. Shubeita H.E. Feramisco J.R. Chien K.R. J. Biol. Chem. 1993; 268: 2244-2249Abstract Full Text PDF PubMed Google Scholar, 3Hunter J.J. Tanaka N. Rockman H.A. Ross Jr., J. Chien K.R. J. Biol. Chem. 1995; 270: 23173-23178Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 4Bogoyevitch M.A. Sugden P.H. Int. J. Biochem. Cell Biol. 1996; 28: 1-12Crossref PubMed Scopus (71) Google Scholar). Downstream effectors of Ras, particularly kinases of the Raf-MEK-ERK and MEKK1-JNKK-JNK pathways, may also participate in these growth processes (4Bogoyevitch M.A. Sugden P.H. Int. J. Biochem. Cell Biol. 1996; 28: 1-12Crossref PubMed Scopus (71) Google Scholar). Hypertrophic and failing hearts feature reduced contractile calcium transients with prolonged decay, myofibrillar disarray, and reduced expression of SERCA2, an enzyme responsible for reuptake of calcium into the sarcoplasmic reticulum (1Wankerl M. Schwartz K. J. Mol. Med. 1995; 73: 487-496Crossref PubMed Scopus (56) Google Scholar, 5Wang J. Flemal K. Qiu Z. Ablin L Grossman W. Morgan J.P. Am. J. Physiol. 1994; 267: H918-H924PubMed Google Scholar, 6Bailey B.A. Dipla K. Li S. Houser S.R. J. Mol. Cell. Cardiol. 1997; 29: 1823-1835Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Exposure of cultured cardiac myocytes to PKC-activating phorbol esters also results in prolonged calcium transients and reduced SERCA2 (7Hartong R. Villarreal F.J. Giordano F. Hilal-Dandan R. McDonough P.M. Dillmann W.H. J. Mol. Cell. Cardiol. 1996; 28: 2467-2477Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Qi M. Bassani J.W. Bers D.M. Samarel A.M. Am. J. Physiol. 1996; 271: H1031-H1039PubMed Google Scholar), and PKC is elevated in hypertrophic hearts (9Gu X. Bishop S.P. Circ. Res. 1994; 75: 926-931Crossref PubMed Scopus (158) Google Scholar). The PKC effectors responsible for the altered regulation of [Ca2+]i have not been identified, but an intriguing possibility may be Raf, which can be activated by either PKC or Ras (4Bogoyevitch M.A. Sugden P.H. Int. J. Biochem. Cell Biol. 1996; 28: 1-12Crossref PubMed Scopus (71) Google Scholar, 10Zou Y. Komuro I. Yamazaki T. Aikawa R. Kudoh S. Shiojima I. Hiroi Y. Mizuno T. Yazaki Y. J. Biol. Chem. 1996; 271: 33592-33597Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). To address this possibility, the present study was undertaken to assess the effects of Ras, PKC, and related ERK activators on cardiac [Ca2+]i. Neonatal rat ventricular myocytes were dissociated and transfected via electroporation as described previously (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). To prepare myocytes for indo-1 measurements, 9 × 106 cells/transfection were electroporated and split into two 35-mm wells containing fibronectin-coated glass coverslips for plating. Cells to be analyzed for morphology were plated as described previously (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). After 18–20 h in serum-containing plating medium, the cells were rinsed and refed with serum-free maintenance medium (Dulbecco's modified Eagle's medium supplemented with 1 nm T3, 5 μg/ml transferrin, 1 μg/ml insulin, and 0.1 ng/ml selenium). Unless otherwise stated, cells were kept in maintenance medium for 48 h prior to analysis. pDCR Ha-RasV12 (activated Ha-Ras), pDCR Ha-RasV12S35, and pDCR Ha-RasV12C40 were obtained from D. Bar-Sagi (SUNY, Stony Brook, NY). RSV-Raf-1 BXB (codes for activated Raf-1 kinase) and RSV-C4B (codes for the amino terminus of Raf-1 kinase) were from U. Rapp (University of Wurzburg, Wurzburg, Germany). pCMV5 MEKKCOOH (codes for activated MEKK-1) was from G. Johnson (University of Colorado, Denver, CO). pEXV3-MAPKK1-Ala-217 (referred to as “dnMEK”) was obtained from S. Fuller (Imperial College, London, UK) and codes for a mutant MAPKK1 (synonymous with MEK1), which can act in a dominant-negative fashion because it is poorly phosphorylated by Raf (12Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar). PKAC was from M. Muramatsu (DNAX Research Institute, Palo Alto, CA). These constructs utilize the highly effective CMV, 1The abbreviations used are: CMVcytomegalovirusPKCprotein kinase CGFPgreen fluorescence proteinPMAphorbol 12-myristate 13-acetatePIphosphatidylinositolSRsarcoplasmic reticulumMAPKmitogen-activated protein kinaseJNKc-Jun NH2-terminal kinase. SV40, or Rous sarcoma virus promoters to drive expression of the regulatory protein. The amount of DNA used per transfection depended upon the constructs. The transfection markers pGreen Lantern-1 (Life Technologies, Inc.) and CMV-β-galactosidase were used at 4 μg or 9 μg/transfection, respectively. Within each experiment, the DNA amounts per transfection were equalized with empty vector DNA such as pCEP. Although transfection efficiency is relatively low with this cell type (averaging approximately 5%), the use of β-galactosidase and GFP allowed analysis of individual transfected myocytes for morphology, cell structures, and calcium transients. cytomegalovirus protein kinase C green fluorescence protein phorbol 12-myristate 13-acetate phosphatidylinositol sarcoplasmic reticulum mitogen-activated protein kinase c-Jun NH2-terminal kinase. Rat SERCA2 cDNA (nucleotides −267/3726, numbered relative to the start codon) in pBluescript was digested with ApaI and re-ligated, deleting 254 base pairs of the 5′ noncoding sequence SERCA2 (−13/3726). Next, the plasmid was linearized with DraIII at nucleotide 3076 of SERCA2 (located in the 3′ noncoding region of the SERCA2 cDNA), and the overhang was filled with T4 polymerase, generating a blunt end. The plasmid was subsequently digested with KpnI, and the SERCA2 encoding fragment was inserted into the adenoviral shuttle vector, pACCMV.pLpA, at the KpnI/blunted HindIII sites. The resulting vector (pACCMV.pLpA-SERCA2) contains SERCA2(−13/3076) inserted between the CMV promoter and the SV40 polyadenylation sequence. Cardiac myocytes were fixed and stained for β-galactosidase, actin, and SERCA2 as published (7Hartong R. Villarreal F.J. Giordano F. Hilal-Dandan R. McDonough P.M. Dillmann W.H. J. Mol. Cell. Cardiol. 1996; 28: 2467-2477Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). An fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody was utilized to visualize the anti-SERCA2 primary antibody. Actin was visualized using rhodamine phalloidin (Molecular Probes), according to the manufacturer's instructions. Cells were loaded with indo-1 as described previously (13McDonough P.M. Stella S.L. Glembotski C.C. J. Biol. Chem. 1994; 269: 9466-9472Abstract Full Text PDF PubMed Google Scholar). Indo-1 measurements were made in air-compatible Dulbecco's modified Eagle's medium at room temperature at 20 Hz. GFP-expressing myocytes, corresponding to myocytes that had taken up the GFP expression vector during the transfection procedure, were identified by monitoring fluorescence with an excitation wavelength of 475 nm. Excitation was then switched to 355 nm (optimal for indo-1), and indo-1 fluorescence was recorded from the cytoplasmic region of the myocyte. In the absence of indo-1, GFP-expressing myocytes were nonfluorescent at the excitation wavelength of 355 nm, indicating that GFP does not interfere with measurement of indo-1. Although indo-1 fluorescence is related to free calcium concentration via well established equilibrium relationships, exact calibration of the signal in is J.W. Bassani Bers D.M. J. 1995; Full Text PDF PubMed Scopus Google the results thus as the between the fluorescence at and or as the at and at the of the and respectively. were transfected with the constructs and a expression vector of were for the and for ERK in as described (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). myocytes with empty expression were or and in Ha-RasV12 transfection in an in cell and the cells a highly morphology Although myocytes stained for actin, the were not 1 The absence of in the myocytes does not represent a of the because myofibrils were identified in myocytes this in myocytes to of or transfection with the (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar, P.M. Glembotski C.C. J. Biol. Chem. 267: Full Text PDF PubMed Google Scholar). Ha-RasV12 had such a on morphology, it was of to expression to regulation of such as SERCA2, also be were cotransfected with Ha-RasV12 with the cells were then fixed and stained for β-galactosidase and SERCA2, which were visualized with or fluorescein isothiocyanate-conjugated secondary respectively. myocytes SERCA2 that was to myocytes and In contrast, myocytes transfected with Ha-RasV12 for SERCA2 and SERCA2 were and the of the were via and myocytes, and and the for Ha-RasV12 transfection were Although and SERCA2 expression may not be related because of such as these that Ha-RasV12 transfection SERCA2 The results suggested that the regulation of [Ca2+]i be modified by To this myocytes were cotransfected with the GFP expression with the Ha-RasV12 expression then loaded with indo-1, and for fluorescence during the of transfected with 1 μg of the Ha-RasV12 expression plasmid in indo-1 and a of decay and Transfection with μg of Ha-RasV12 prolonged decay and increased the for the calcium transients to transfection with μg of Ha-RasV12 also increased the indo-1 transfected with of the 20 myocytes transfected with μg of Ha-RasV12 in calcium a for GFP and These results with of SERCA2, because reduced calcium from the and the decay phase of the calcium of ERK effectors on in cultured ventricular response 20 1 μg of to μg of to to to to and μg of to to μg of to Raf and μg of to to to μg of to to to 1 μg of to to μg of dnMEK 5 dnMEK to to BXB Raf and μg of BXB Raf to to μg of dnMEK BXB Raf dnMEK and to to μg of dnMEK 5 dnMEK to to PKAC μg of PKAC to to PKAC μg of to and μg of to to to to μg SERCA2 9 to to to to to SERCA2 C and μg of BXB Raf to to μg of SERCA2 4 BXB Raf SERCA2 to BXB to BXB the indo-1 prior to the for myocytes at Hz. the indo-1 by each the between the start and the of the the of decay, which was by the decay phase of each to a function. and were to the corresponding for each and and cell the each the for the of cells in was using the to to to to BXB to in a the indo-1 prior to the for myocytes at Hz. the indo-1 by each the between the start and the of the the of decay, which was by the decay phase of each to a function. and were to the corresponding for each and and cell the each the for the of cells in was using the Raf and phosphatidylinositol kinase the Ras effectors in To between these pathways, Ha-RasV12 mutants have been identified that selectively to Raf or 3-kinase T. M.A. Bar-Sagi D. 1996; 271: PubMed Scopus Google Scholar, P. P.H. A. D. P. A. J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). a the of these in the growth and [Ca2+]i to Ha-RasV12, the of and Ha-RasV12C40 to the Raf-MEK-ERK pathway was to myocytes were cotransfected with with Ha-RasV12, Ha-RasV12S35, or Ha-RasV12C40, and in kinase were on Cotransfection with Ha-RasV12 in a of ERK transfection with a In contrast, transfection with Ha-RasV12C40 not activate at activates the Raf-MEK-ERK pathway, it is a effective of ERK this is with P. P.H. A. D. P. A. J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and be the effects of these Ras mutants on cell growth and [Ca2+]i. were transfected with amounts of or Ha-RasV12C40 and analyzed for cell and [Ca2+]i. myocytes were but were myocytes transfected with Ha-RasV12 myocytes were in to Transfection with modified [Ca2+]i in a to the effects of Ha-RasV12, with and and and Transfection with Ha-RasV12C40 also modified but the of Ha-RasV12C40 was to the of decay, and the of decay was that by the relative effects of the Ras mutants on with the of these mutants on ERK that the Raf-MEK-ERK pathway may as a via which [Ca2+]i may be altered by Ras The with that the 3-kinase pathway also participate in the of [Ca2+]i but to a lesser the Raf-MEK-ERK Raf Ras to ERK it was of to the effects of constitutively active Raf on [Ca2+]i. transfection results in of ERK and cell but does not expression or (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar). myocytes but increased and prolonged decay for from cell and The of by with the results obtained with Ha-RasV12 and and to the contractile by pressure overload hypertrophy J. Flemal K. Qiu Z. Ablin L Grossman W. Morgan J.P. Am. J. Physiol. 1994; 267: H918-H924PubMed Google Scholar, 6Bailey B.A. Dipla K. Li S. Houser S.R. J. Mol. Cell. Cardiol. 1997; 29: 1823-1835Abstract Full Text PDF PubMed Scopus (55) Google Scholar). transfection with a of that activates ERK in this cell type (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar), also modified the calcium transients in a to Ha-RasV12 and a and and not but for a of the of the pathway in the regulation of cardiac [Ca2+]i by Ras and Raf, the effects of with dnMEK on [Ca2+]i were Cotransfection of dnMEK with Ha-RasV12 the of Ha-RasV12 on to and inhibited the of Ha-RasV12 on decay and The of dnMEK to the effects of Ha-RasV12 is with the that Ha-RasV12 [Ca2+]i via the Raf-MEK-ERK with the relative of Raf and in the ERK cascade, of dnMEK also inhibited the effects of on [Ca2+]i C and the transients to of cultured cardiac myocytes with protein kinase phorbol esters activates ERK M.A. Sugden P.H. 1993; PubMed Scopus Google Scholar, P.M. Hanford D.S. Glembotski C.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), SERCA2, and decay (7Hartong R. Villarreal F.J. Giordano F. Hilal-Dandan R. McDonough P.M. Dillmann W.H. J. Mol. Cell. Cardiol. 1996; 28: 2467-2477Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 8Qi M. Bassani J.W. Bers D.M. Samarel A.M. Am. J. Physiol. 1996; 271: H1031-H1039PubMed Google Scholar), it was of to the of dnMEK to inhibit the effects of on [Ca2+]i. with prolonged and and Transfection with dnMEK inhibited the of on and and which activates the and in this cell type M. Hilal-Dandan R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), may [Ca2+]i via of the Raf-MEK-ERK Transfection with a constitutively active M. K. K. Mol. Cell. Biol. PubMed Scopus Google Scholar), also prolonged decay of the transients not but for and this was inhibited by the dominant-negative J. H. J. P. J. Rapp J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). PKAC also in a These results that PKC may cardiac [Ca2+]i via the Raf transfection with Ha-RasV12 to of SERCA2 expression and of it was of to [Ca2+]i in myocytes cotransfected with expression for for of SERCA2 with in but not of the calcium transients and of SERCA2 not the of Ha-RasV12 but counteracted the effects of Ha-RasV12 on SERCA2 also a the of Ha-RasV12 SERCA2 by had on cell morphology and not the of Ha-RasV12 on cell Cotransfection with SERCA2 also the of on [Ca2+]i C and and These results with the that the pathway may in by SERCA2 the of SERCA2 to the in [Ca2+]i by Ha-RasV12 that Ha-RasV12 also [Ca2+]i by that of In the present study cultured rat ventricular myocytes were transfected with effectors of the and related pathways, with constructs the transfected myocytes to be identified and analyzed for cell and a was in which GFP was used as a transfection for living this the to the effects of the effectors on [Ca2+]i a physiological of to cardiac in and The obtained with that of Ha-RasV12 into cardiac myocytes elicits a growth response and hypertrophic expression (2Thorburn A. Thorburn J Chen S.-Y. Powers S. Shubeita H.E. Feramisco J.R. Chien K.R. J. Biol. Chem. 1993; 268: 2244-2249Abstract Full Text PDF PubMed Google Scholar), and the expression of Ha-RasV12 in with a in myofibrillar J.J. Tanaka N. Rockman H.A. Ross Jr., J. Chien K.R. J. Biol. Chem. 1995; 270: 23173-23178Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, K.R. J.J. Tanaka N. N. Ross Jr., J. Chien K.R. U. S. A. 1997; PubMed Scopus (56) Google Scholar). in the present transfection with an in cell with of the contractile a hypertrophic Am. J. PubMed Scopus Google Scholar, G. R. J. W. S. G. J. Am. Cardiol. PubMed Scopus Google Scholar). Transfection with Ha-RasV12 also altered expression of SERCA2, a calcium-regulatory enzyme in calcium reuptake during the decay phase of the contractile calcium which is also commonly in of pressure overload hypertrophy and (1Wankerl M. Schwartz K. J. Mol. Med. 1995; 73: 487-496Crossref PubMed Scopus (56) Google Scholar, 5Wang J. Flemal K. Qiu Z. Ablin L Grossman W. Morgan J.P. Am. J. Physiol. 1994; 267: H918-H924PubMed Google Scholar, 6Bailey B.A. Dipla K. Li S. Houser S.R. J. Mol. Cell. Cardiol. 1997; 29: 1823-1835Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Indo-1 measurements of [Ca2+]i that Ha-RasV12 transfection the magnitude of the contractile calcium transients and prolonged the decay phase of the a calcium that is with the of Ha-RasV12 to To the via which Ha-RasV12 expression encoding Ras mutants that selectively to Raf-MEK-ERK and 3-kinase were altered [Ca2+]i and cell in a with Ha-RasV12 Ha-RasV12C40, that the Raf-MEK-ERK pathway is in the growth and calcium to Ha-RasV12 The of on [Ca2+]i and cell was that of Ha-RasV12, indicating that Ras effectors Raf may also a in these to be is the to which Ha-RasV12 and activate Although is for the ERK pathway, it is not as effective a of ERK as the lesser of on [Ca2+]i with Ha-RasV12 to the lesser of on ERK Transfection with a constitutively active Raf also modified the magnitude of the the to the and decay, which in [Ca2+]i commonly in myocytes from hypertrophic and failing hearts J. Flemal K. Qiu Z. Ablin L Grossman W. Morgan J.P. Am. J. Physiol. 1994; 267: H918-H924PubMed Google Scholar, 6Bailey B.A. Dipla K. Li S. Houser S.R. J. Mol. Cell. Cardiol. 1997; 29: 1823-1835Abstract Full Text PDF PubMed Scopus (55) Google Scholar). the effects of Ha-RasV12 and were by with the Raf-MEK-ERK pathway as a for the of cardiac [Ca2+]i. the with dnMEK not the effects of Ha-RasV12 on this that Ras effectors Raf may also a that be the of transfection with dnMEK also inhibited the of the PKC-activating phorbol ester, on [Ca2+]i. with a dominant-negative Raf inhibited the of a constitutively active protein kinase C to [Ca2+]i. These that the Raf-MEK-ERK a of via which hypertrophic stimuli that activate Ras PKC to and of the cardiac contractile calcium transients. PKC activate Raf by directly it Y. Komuro I. Yamazaki T. Aikawa R. Kudoh S. Shiojima I. Hiroi Y. Mizuno T. Yazaki Y. J. Biol. Chem. 1996; 271: 33592-33597Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, W. G. R. H. H. D. Rapp 1993; PubMed Scopus Google Scholar), it been that PKC can activate Ras M.A. S. J. J. 1993; Google Scholar), which also subsequently to Raf and ERK is also to have hypertrophic effects on cardiac myocytes P.M. K. A. Chien K.R. J. Mol. Cell. Cardiol. Full Text PDF PubMed Scopus Google and to the and of the contractile in cultured rat myocytes J. Cell Google resulting in a to the effects of Ha-RasV12 have two via which ERK regulate SERCA2 to of the rat SERCA2 promoter Hilal-Dandan R. H. Dillmann 1997; PubMed Scopus Google Scholar), a of SERCA2 expression at the SERCA2 is reduced by phorbol esters M. Bassani J.W. Bers D.M. Samarel A.M. Am. J. Physiol. 1996; 271: H1031-H1039PubMed Google Scholar). in with also in S. R. R. T. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), that the regulation of by PKC be a for [Ca2+]i regulation in cardiac and cell The in the present study that with SERCA2 to for the effects of Ha-RasV12 and on [Ca2+]i is with that of cultured ventricular myocytes with a to the effects of on [Ca2+]i F.J. H. McDonough P.M. M. Dillmann W.H. 1997; PubMed Scopus Google Scholar). SERCA2 may not be the by which the [Ca2+]i is altered in hypertrophic of in pressure overload prior to the in SERCA2 M. Bers D.M. Samarel A.M. Am. J. Physiol. 1997; Google Scholar), and calcium transients may be reduced in cardiac myocytes obtained from hypertrophic hearts in which the is well loaded with calcium A.M. H. 1997; PubMed Scopus Google Scholar). been suggested that calcium in hypertrophic myocytes because of increased between calcium and at the A.M. H. 1997; PubMed Scopus Google Scholar). myofibrillar expression and the is with myofibrils J.R. J. Mol. Cell Cardiol. 1995; Full Text PDF PubMed Scopus Google Scholar), is a possibility Ha-RasV12 transfection and function. analysis of be to this ERK may to the maintenance of the cardiac it is to the of the related and in cardiac results that in cultured ventricular myocytes may be by contractile P.M. Hanford D.S. Glembotski C.C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google and that contractile and represent of cell growth and the contractile P.M. Glembotski C.C. J. Biol. Chem. 267: Full Text PDF PubMed Google Scholar, Y. J. Chien K.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Transfection with constitutively active of also cell and (11Zechner D. Thuerauf D.J. Hanford D.S. McDonough P.M. Glembotski C.C. J. Cell Biol. 1997; 139: 115-127Crossref PubMed Scopus (280) Google Scholar, Y. S. Ross Jr., J. J. Chien K.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in a with the phase of cardiac may participate in the development and maintenance of the cardiac and altered of these may to the development of or in morphology and The of dnMEK to inhibit the effects of Ha-RasV12 and on cardiac [Ca2+]i that of the Raf-MEK-ERK via or be an effective in the contractile with cardiac hypertrophy and R. and J. Thuerauf for