Characterization of the regulatory mechanisms of activating transcription factor 3 by hypertrophic stimuli in rat cardiomyocytes.

AIMS: Activating transcription factor 3 (ATF3) is a stress-activated immediate early gene suggested to have both detrimental and cardioprotective role in the heart. Here we studied the mechanisms of ATF3 activation by hypertrophic stimuli and ATF3 downstream targets in rat cardiomyocytes. METHODS AND RESULTS: When neonatal rat cardiomyocytes were exposed to endothelin-1 (ET-1, 100 nM) and mechanical stretching in vitro, maximal increase in ATF3 expression occurred at 1 hour. Inhibition of extracellular signal-regulated kinase (ERK) by PD98059 decreased ET-1- and stretch-induced increase of ATF3 protein but not ATF3 mRNA levels, whereas protein kinase A (PKA) inhibitor H89 attenuated both ATF3 mRNA transcription and protein expression in response to ET-1 and stretch. To characterize further the regulatory mechanisms upstream of ATF3, p38 mitogen-activated protein kinase (MAPK) signaling was investigated using a gain-of-function approach. Adenoviral overexpression of p38α, but not p38ß, increased ATF3 mRNA and protein levels as well as DNA binding activity. To investigate the role of ATF3 in hypertrophic process, we overexpressed ATF3 by adenovirus-mediated gene transfer. In vitro, ATF3 gene delivery attenuated the mRNA transcription of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1), and enhanced nuclear factor-κB (NF-κB) and Nkx-2.5 DNA binding activities. Reduced PAI-1 expression was also detected in vivo in adult rat heart by direct intramyocardial adenovirus-mediated ATF3 gene delivery. CONCLUSIONS: These data demonstrate that ATF3 activation by ET-1 and mechanical stretch is partly mediated through ERK and cAMP-PKA pathways, whereas p38 MAPK pathway is involved in ATF3 activation exclusively through p38α isoform. ATF3 activation caused induction of modulators of the inflammatory response NF-κB and Nkx-2.5, as well as attenuation of pro-fibrotic and pro-inflammatory proteins IL-6 and PAI-1, suggesting cardioprotective role for ATF3 in the heart.

Distinct regulation of B-type natriuretic peptide transcription by p38 MAPK isoforms.

Persistent controversy underlies the functional roles of specific p38 MAPK isoforms in cardiac biology and regulation of hypertrophy-associated genes. Here we show that adenoviral gene transfer of p38ß but not p38α increased B-type natriuretic peptide (BNP) mRNA levels in vitro as well as atrial natriuretic peptide mRNA levels both in vitro and in vivo. Overexpression of p38α, in turn, augmented the expression fibrosis-related genes connective tissue growth factor, basic fibroblast growth factor and matrix metalloproteinase-9 both in vitro and in vivo. p38ß-induced BNP transcription was diminished by mutation of GATA-4 binding site, whereas overexpression of MKK6b, an upstream regulator of p38α and p38ß, activated BNP transcription through both GATA-4 and AP-1. Overexpression of MKK3, upstream regulator of p38α, induced BNP transcription independently from AP-1 and GATA-4. These data provide new evidence for diversity in downstream targets and functional roles of p38 pathway kinases in regulation of hypertrophy-associated cardiac genes.

p38 Kinase rescues failing myocardium after myocardial infarction: evidence for angiogenic and anti-apoptotic mechanisms.

As a leading cause of heart failure, postinfarction left ventricular remodeling represents an important target for therapeutic interventions. Mitogen-activated protein kinases regulate critical cellular processes including stress response and survival, but their role in left ventricular remodeling is unknown. In the present study, rats were subjected to myocardial infarction by ligating the left anterior descending coronary artery. Western blot and kinase assay analysis revealed an inactivation of p38 kinase after myocardial infarction. Local adenovirus-mediated cotransfection of wild-type (WT) p38 kinase and constitutively active MKK3b reduced infarct size (26+/-3% vs. 47+/-4%, P<0.05 vs. LacZ-treated control) associated with improved ejection fraction (66.9+/-5.5% vs. 44.4+/-4.0%, P<0.001), fractional shortening (30.2+/-2.1% vs. 19.7+/-2.2%, P<0.001), and decreased left ventricular diastolic diameter (8.5+/-0.4 mm vs. 9.5+/-0.2 mm, P<0.01). p38 kinase gene transfer increased capillary density (2423+/-107/mm(2) vs. 1934+/-86/mm(2), P<0.001) and resulted in microvessel enlargement in the ischemic border zone. Apoptosis (35+/-7 vs. 69+/-13 cells, P<0.01) and fibrosis (16+/-3% vs. 34+/-8%, P<0.05) were reduced, while the number of c-kit positive cardiac stem-like cells remained unchanged. These results indicate that reduced p38 signaling predisposes to adverse postinfarction remodeling. The rescue of failing myocardium with p38 kinase may be a potential new therapy for heart failure after myocardial infarction.