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CRNK Overexpression Alters Hypertrophic Gene Expression In Cultured Neonatal Rat Ventricular Myocytes
Author(s) -
Samarel Allen Mark,
Szotek Erika,
Vijayan Kalpana,
Hart Davin,
Iyengar Rekha,
Heidkamp Maria
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1187-a
Subject(s) - myocyte , microbiology and biotechnology , gene expression , medicine , gene , endocrinology , chemistry , biology , biochemistry
We previously showed that overexpression of PYK2, a Ca 2+ ‐dependent nonreceptor protein tyrosine kinase, activated the stress‐activated protein kinases JNK1/2 and p38 MAPK , and down‐regulated SERCA2 gene transcription in neonatal rat ventricular myocytes (NRVM) (Heidkamp et al., AJP:Cell 289:C471‐C482, 2005). In the present study, we examined the effects of adenovirally (Adv)‐mediated overexpression of a C‐terminal, dominant‐negative inhibitor of PYK2, known as CRNK ( C ell adhesion kinase‐β R elated N on K inase) on cellular growth and hypertrophic gene expression in similarly cultured NRVM. NRVM were maintained in serum‐free culture medium (UI), or infected with either Adv‐CRNK or control Adv expressing green fluorescent protein (Adv‐GFP; 5 moi, 24–48h). Cell extracts were analyzed for JNK and p38 MAPK phosphorylation by Western blotting, for total protein and DNA content by spectrophotometry and fluorometry, and for SERCA2 and ANF mRNA levels by real‐time RT‐PCR. CRNK overexpression had no effect on basal JNK or p38 MAPK , but blocked the H2O2‐induced increase in JNK1/2 (but not p38 MAPK ) phosphorylation. CRNK overexpression also did not affect basal total protein/DNA ratio, and did not prevent the increase in total protein/DNA ratio produced by treatment with phorbol myristate acetate (200nM, 24h). However, CRNK overexpression significantly ( P <0.05) increased SERCA2 mRNA levels 2.1±0.5‐fold as compared to UI or GFP‐expressing NRVM. In contrast, ANF mRNA levels were significantly decreased to 36±14% of UI by CRNK overexpression. These data indicate that PYK2 is necessary to elicit specific aspects of the hypertrophic phenotype in cultured NRVM. Supported by NIH RO1 HL34328 and the Dr. Ralph and Marian Falk Medical Research Trust.