G alpha 13 stimulates gene expression and increases cell size in cultured neonatal rat ventricular myocytes.

OBJECTIVES: Constitutively-active G alpha 13 causes permissive cell types to proliferate or undergo phenotypic transformation implying a role for G13 in the control of cell growth. Cardiac myocytes are terminally-differentiated cells which respond to growth stimuli by increasing in size rather than by cell division. The objective of this study was to determine whether constitutively-active G alpha 13 is able to induce a hypertrophic phenotype in cardiac myocytes. METHODS: Cultured neonatal rat ventricular myocytes were transiently transfected with an expression vector (pRC/RSV) encoding wild-type G alpha 13 or constitutively-active G alpha 13Q226L. Effects on transcription were monitored by co-transfected luciferase (LUX) reporter genes under the control of promoters responsive to hypertrophic stimuli. Cell size was determined by planimetry. RESULTS: Transfection of neonatal myocytes with G alpha 13Q226L, but not wild-type G alpha 13, stimulated ANF638LUX and ANF3003LUX expression to 3.0 +/- 0.3- and 4.3 +/- 0.6-fold of the control, respectively. Likewise, G alpha 13Q226L stimulated vMLC250LUX and vMLC2700LUX expression to 3.9 +/- 1.0- and to 7.7 +/- 1.7-fold of controls, respectively, but there was relatively little effect of G alpha 13Q226L on c-fos-SRE- and beta-MHC promoter activity. The effects of G alpha 13Q226L on ANF3003LUX were inhibited by expression of C3 exoenzyme. Wild-type G alpha 13 and G alpha 13Q226L increased myocyte area from 869 +/- 43 micron 2 in control tranfections to 1287 +/- 64 micron 2 and 1278 +/- 59 microns, respectively. CONCLUSION: We conclude that G alpha 13Q226L is able to induce gene expression and morphological changes associated with a hypertrophic response in cardiac myocytes and that the transcriptional effects may be mediated through a Rho-dependent mechanism.

c-Jun N-terminal kinase-interacting protein 1 inhibits gene expression in response to hypertrophic agonists in neonatal rat ventricular myocytes.

G(q)-coupled receptor agonists, such as endothelin-1 (ET-1) and phenylephrine (PE), initiate a hypertrophic response in cardiac myocytes that is characterized by increased expression of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), skeletal muscle alpha-actin (SkalphaA) and ventricular myosin light chain-2 (vMLC2). ET-1 and PE activate both the extracellular signal-regulated kinases and c-Jun N-terminal kinases (JNKs) in cardiac myocytes, but the extent to which each contributes to the hypertrophic response is uncertain. Here we have used the JNK-binding domain of JNK-interacting protein 1 (JIP-1), a cytosolic scaffold protein that binds to JNK and inhibits its signalling when overexpressed, to assess the contribution of JNK activation to the hypertrophic response. Expression of JIP-1 inhibited the increase in ANF, beta-MHC, SkalphaA and vMLC2 reporter gene expression in response to ET-1 (by 45-86%) and PE (by 56-60%). However, activation of these reporter genes by PMA, which does not activate JNK significantly in myocytes, was much less affected by overexpression of JIP-1. JIP-1 also failed to inhibit reporter gene activation in response to constitutively active Ras or Raf, but attenuated reporter gene activation induced by a constitutively active mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1), an upstream kinase that preferentially activates JNKs, by 50%. Overexpression of JIP-1 also significantly reduced the increase in cell area in response to PE from 63% to 56%, but had no effect on the increase in cell size in response to ET-1 (38%). These results suggest that activation of the JNK pathway contributes to the transcriptional and morphological responses to G(q) receptor-coupled hypertrophic agonists.

Stimulation of gene expression in neonatal rat ventricular myocytes by Ras is mediated by Ral guanine nucleotide dissociation stimulator (Ral.GDS) and phosphatidylinositol 3-kinase in addition to Raf.

Treatment of cultured neonatal ventricular myocytes with oncogenic Ras increases their size and stimulates the re-expression of genes which are normally restricted to the fetal stage of ventricular development, including atrial natriuretic factor (ANF) and skeletal muscle (SkM)-alpha-actin. To determine which signalling pathways mediate these responses, myocytes were transfected with oncogenic (V12) Ras mutants which interact selectively with different effectors and their effects on luciferase (LUX) reporter plasmids were examined. V12 human Ras (V12HRas), itself, activated ANF-LUX 9. 6-fold, whereas mutants of V12HRas, which selectively stimulate Ral guanine nucleotide dissociation stimulator (Ral.GDS) (E37G), c-Raf (D38E) and phosphatidylinositol 3-kinase (PI-3-K; Y40C) enhanced ANF-LUX expression 3.0-, 3.7- and 1.7-fold respectively. The full response of ANF-LUX to V12HRas was restored by using a combination of the individual effector domain mutants. Likewise, SkM-alpha-actin-LUX expression was activated 12.0-, 3.5-, 4.5- and 3. 0-fold by V12HRas, E37G, D38E and Y40C respectively, and a similar pattern of activation was also observed using a c-fos serum-response element-LUX reporter gene. Cell size was also increased by each of the mutants, but simultaneous expression of all three mutant constructs was needed to reconstitute the full effect of V12HRas on cell size (50% increase). Transfection with a constitutively active mutant of PI-3-K (p110K227E) stimulated ANF-LUX, SkM-alpha-actin-LUX, c-fos-serum-response element-LUX and Rous sarcoma virus-LUX by 3.1-, 3.2-, 2.1- and 2.9-fold respectively, but the co-transfected cytomegalovirus-beta-galactosidase reporter gene was activated to a similar extent (1.9-fold). These results suggest that Raf, Ral.GDS and PI-3-K can all transduce transcriptional responses to V12HRas, but that the specific induction of genes associated with the hypertrophic response is not mediated through PI-3-K.