The contribution of the angiotensin (Ang) II type 2 receptor (AT2R) to cardiac hypertrophy is still controversial. Here we examined the effect of overexpressing the human AT2R in cultured porcine cardiac fibroblasts (pFib) on proliferation, procollagen I mRNA expression, and - as putatively underlying signal-transduction pathways - on mitogen-activated protein kinase ERK1/ERK2 and phosphotyrosine phosphatase activities. As quantitated by 125I-(Sar1,Ile8)-Ang II binding, transduction of cardiac fibroblasts with the adenoviral AT2R expression vector led to a six- to tenfold higher AT2 than endogenous Ang II type 1 receptor (AT1R) expression. The overexpressed AT2R had the same apparent molecular mass as the endogenous AT2R in rat PC12W cells. Proliferation was not significantly lower in AT2R expressing pFib than in antisense-transduced controls (TA2) upon stimulation with Ang II (AT2R 110.5+/-4.8% vs. TA2 110.2+/-5.5%), Ang II plus the AT1R blocker Irbesartan (97.1+/-1.4% vs. 108.0+/-5.0; P=0.052) and the partial AT2R antagonist CGP42112 at the agonistic concentration of 50 nM (92.1+/-2.7% vs. 99.8+/-3.1%; P=0.053). Procollagen Ialpha2 (COL1A2) mRNA levels were quantitated by (a) northern blot analysis and (b) reverse transcriptase polymerase chain reaction. COL1A2/GAPDH (a) and COL1A2/beta-actin (b) ratios revealed no differences between AT2R-transduced fibroblasts and antisense controls when stimulated with Ang II (1 microM, 24 h) plus Irbesartan and 10 ng/ml transforming growth factor beta1. Ang II stimulation of the endogenous AT1R increased extracellular signal regulated kinase 1/2 activities. This response was reduced by Irbesartan, but PD123319 had no effect. Time course and magnitude of Ang II stimulated ERK1/ERK2 activation was identical in AT2R-transduced and control cells. Also, neither simultaneous nor Ang II pre-stimulation, suggested to induce gene expression of the MAP kinase phosphatase 1, modulated phorbol myristate acetate-stimulated ERK1/ERK2 activation in AT2R-transduced pFib, in AT2R-transduced human umbilical vein endothelial cells, and in PC12W cells. By the use of a tyrosine phosphatase assay we observed an inhibition of phosphotyrosine phosphatase activity by 30.8% (P=0.009, n=5) after 5 min Ang II stimulation of AT2R-expressing pFib. Immunoprecipitation-tyrosine phosphatase assays revealed that inhibition of phosphotyrosine phosphatase 1B, which regulates insulin signaling, contributed to this effect. In conclusion, stimulation of the overexpressed human AT2R in porcine cardiac fibroblasts inhibited tyrosine phosphatase activity but had no significant effect on fibroblast functions related to cardiac fibrosis. It is conceivable that possible antifibrotic AT2R effects are species specific and/or require the interaction between fibroblasts and cardiomyocytes, probably via paracrine factors, or mechanical load.

BackgroundRecently, supplementation of L-carnitine to obese rats was found to improve the carnitine status and to counteract an obesity-induced muscle fiber transition from type I to type II. However, it has not been resolved if the change of muscle fiber distribution induced in obese rats and the restoration of the “normal” muscle fiber distribution, which is found in lean rats, in obese rats by supplemental L-carnitine is causally linked with the carnitine status. In the present study we hypothesized that fiber type distribution in skeletal muscle is dependent on carnitine status.MethodsTo test this, an experiment with 48 piglets which were randomly allocated to four groups (n = 12) was performed. All piglets were given orally either 60 mg sodium bicarbonate/kg body weight (group CON), 20 mg L-carnitine and 60 mg sodium bicarbonate/kg body weight (group CARN), 30 mg pivalate (dissolved in sodium bicarbonate)/kg body weight (group PIV) or 20 mg L-carnitine and 30 mg pivalate/kg body weight (group CARN + PIV) each day for a period of 4 weeks.ResultsConcentrations of total carnitine in plasma, liver and longissimus dorsi and biceps femoris muscles were 2.0–2.7 fold higher in group CARN than in group CON, whereas these concentrations were 1.9–2.5-fold lower in group PIV than in group CON. The concentrations of total carnitine in these tissues did not statistically differ between group CARN + PIV and group CON. Fiber type distribution of longissimus dorsi and biceps femoris muscles, mRNA and protein levels of molecular regulators of fiber distribution in longissimus dorsi and biceps femoris muscles and mRNA levels of genes reflecting the metabolic phenotype of longissimus dorsi and biceps femoris muscles did not differ between groups.ConclusionChanges in the systemic carnitine status and the muscle carnitine concentration induced by either supplementing L-carnitine or administering pivalate have no impact on the contractile and metabolic phenotype of skeletal muscles in pigs.

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