Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent.

Cardiac hypertrophy occurs as an adaptation to hypertension but a sustained hypertrophic response can ultimately lead to heart failure. Angiotensin-II (Ang II) is released following hemodynamic overload and stimulates a cardiac hypertrophic response. AngII also increases expression of the regulatory cytokine, transforming growth factor-ß1 (TGFß1), which is also implicated in the cardiac hypertrophic response and can stimulate activation of Smad2/3 as well as TGFß-activated kinase 1 (TAK1) signaling mediators. To better understand the downstream signaling events in cardiac hypertrophy, we therefore investigated activation of Smad2/3 and TAK1 signaling pathways in response to Ang II and TGFß1 using primary neonatal rat cardiomyocytes to model cardiac hypertrophic responses. Small interfering RNA (siRNA) knockdown of Smad 2/3 or TAK1 protein or addition of the TGFß type I receptor inhibitor, SB431542, were used to investigate the role of downstream mediators of TGFß signaling in the hypertrophic response. Our data revealed that TGFß1 stimulation leads to cardiomyocyte hypertrophic phenotypes that were indistinguishable from those occurring in response to Ang II. In addition, inhibition of the TGFß1 type receptor abolished Ang II-induced hypertrophic changes. Furthermore, the hypertrophic response was also prevented following siRNA knockdown of TAK1 protein, but was unaffected by knockdown of Smad2/3 proteins. We conclude that Ang II-induced cardiomyocyte hypertrophy in vitro occurs in a TAK1-dependent, but Smad-independent, manner.

The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro.

Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line, which has the advantage of being an animal-free alternative; however, the extent to which H9C2 cells can accurately mimic the hypertrophic responses of primary cardiac myocytes has not yet been fully established. To address this limitation, we have directly compared the hypertrophic responses of H9C2 cells with those of primary rat neonatal cardiomyocytes following stimulation with hypertrophic factors. Primary rat neonatal cardiomyocytes and H9C2 cells were cultured in vitro and treated with angiotensin II and endothelin-1 to promote hypertrophic responses. An increase in cellular footprint combined with rearrangement of cytoskeleton and induction of foetal heart genes were directly compared in both cell types using microscopy and real-time rtPCR. H9C2 cells showed almost identical hypertrophic responses to those observed in primary cardiomyocytes. This finding validates the importance of H9C2 cells as a model for in vitro studies of cardiac hypertrophy and supports current work with human cardiomyocyte cell lines for prospective molecular studies in heart development and disease.

Effects of enclosure size on sexual behavior of Japanese quail (Coturnix japonica).

The authors determined whether results of experiments on copulatory and affiliative behavior of pairs of Japanese quail (Coturnix japonica) conducted in a closely confining apparatus would predict behavior in a large enclosure in which female quail could avoid contact with male quail. As found previously in studies of closely confined quail, in a large enclosure containing numerous barriers, both unmated female quail and mated female quail laying unfertilized eggs were more likely to remain near a confined male quail than were mated female quail laying fertilized eggs. Furthermore, the number of copulations that a pair engaged in when closely confined predicted the number of copulations that they engaged in when they were in the large enclosure. Patterns of affiliation and of mating in a confining laboratory apparatus thus predicted behavior in a larger enclosure that provided female quail with opportunity to avoid contact with male quail.

A direct interaction between TGFbeta activated kinase 1 and the TGFbeta type II receptor: implications for TGFbeta signalling and cardiac hypertrophy.

OBJECTIVE: Transforming growth factor beta (TGFbeta)-activated kinase 1 (TAK1) is a MAP kinase kinase kinase involved in numerous signalling pathways and is strongly implicated in cardiac hypertrophy and heart failure. TGFbeta is also associated with hypertension and heart disease, and evidence suggests that TGFbeta1 and TAK1 act together in a cardiac stress signalling pathway. Canonical TGFbeta signalling is mediated through Smad transcription factors, but TGFbeta can also rapidly activate TAK1. The activation of the Smad cascade is well characterised, but little is known about how TAK1 is activated in response to TGFbeta, and no direct link between any MAPK kinase pathway and the TGFbeta receptors has yet been established. Since TAK1 is activated by TGFbeta within 1 min in cardiomyocytes, we hypothesised there might be a direct interaction between TAK1 and one of the TGFbeta receptors. METHODS: We used a combination of in vitro binding assays and co-immunoprecipitation (IP) experiments to investigate whether TAK1 interacted with the type I (ALK1 or ALK5) or type II (TBRII) TGFbeta receptors. Interactions between endogenous proteins were tested using mouse myoblast and rat cardiomyocyte cells. RESULTS: Immunoprecipitation and in vitro binding assays show that TAK1 binds directly to TBRII. Precipitation of endogenous TAK1 protein in rat cardiomyocytes shows that, in addition to a direct association with TBRII, it also interacts indirectly with ALK5. CONCLUSIONS: We describe a novel and specific interaction between TAK1 and TBRII which, for the first time, directly links TAK1 to the TGFbeta signalling cascade and potentially explains how TGFbeta signalling in cardiomyocytes mediates a hypertrophic response.