Abstract
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 PE-induced protein synthesis and increased cell size, sarcomeric reorganization, and expression of beta-myosin heavy chain in myocytes with IC(50) values of 1-2 microm. 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.
Highlights
The extracellular signal-regulated kinase (ERK) pathway is activated by hypertrophic stimuli in cardiomyocytes
ET-1- and PE-induced activation of ERK in cardiomyocytes was inhibited by U0126 with IC50 values of 1.3 M (Fig. 1A) and 1.4 M (Fig. 1B), respectively (n ϭ 4)
Our results demonstrate that ET-1- or PE-induced myofibrillar organization in cardiomyocytes was remarkably inhibited by U0126, consistent with previous studies that demonstrated a positive link between the ERK activation and the enhanced myofibrillar reorganization [12] and the inhibitory effect of PD98059 (50 M) on sarcomeric reorganization [16]
Summary
Primary Neonatal Rat Cardiac Myocyte Cultures—Primary cultures of neonatal rat cardiomyocytes from 1- to 2-day-old Harlan SpragueDawley rats were prepared by the method reported previously [21]. Measurement of Cell Surface Area—Cardiac myocyte cultured on chamber slides (2.5 105 cells/well) were fixed and immunostained with a monoclonal antibody against sarcomeric ␣-actinin (Sigma) [23] as described below. Northern Analysis of ANF Expression in Cardiomyocytes—The probe for ANF spanned a 216-base pair fragment of the rat ANF cDNA (accession number M27498) It was generated by reverse transcriptase polymerase chain reaction from total RNA obtained from rat heart tissue. ANF Enzyme-linked Immunosorbent Assay (ELISA)—Myocytes cultured in 24-well plates at 2 ϫ 105 cells/well were treated with vehicle or U0126 for 20 min prior to the addition of ET-1 or PE in serum-free medium for 24 h. The cells were exposed to ET-1 (100 nM) or PE (50 M) in serum-free medium for an additional 24 – 48 h before being fixed and stained with anti-actinin antibody (cell size measurement) or Oregon Green 514 phalloidin (sarcomere organization). Differences with a value of p Ͻ 0.05 were considered significant
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