Abstract-This study was designed to determine whether mechanical stretch activates the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in cardiomyocytes and, if so, by what mechanism. Neonatal rat/murine cardiomyocytes were cultured on malleable silicone dishes and were stretched by 20%. Mechanical stretch induced rapid phosphorylation of JAK1, JAK2, Tyk2, STAT1, STAT3, and glycoprotein 130 as early as 2 minutes and peaked at 5 to 15 minutes. It also caused gel mobility shift of sis-inducing element, which was supershifted by preincubation with anti-STAT3 antibody. Preincubation with CV11974 (AT 1 blocker) partially inhibited the phosphorylation of STAT1, but not that of STAT3. Preincubation with TAK044 (endothelin-1-type A/B-receptor blocker) did not attenuate this pathway. RX435 (anti-glycoprotein 130 blocking antibody) inhibited the phosphorylation of STAT3 and partially inhibited that of STAT1. Phosphorylation of STAT1 and STAT3 was strongly inhibited by HOE642 (Na ϩ /H ϩ exchanger inhibitor) and BAPTA-AM (intracellular calcium chelator), but not by gadolinium (stretch-activated ion channel inhibitor), EGTA (extracellular Ca 2ϩ chelator), or KN62 (Ca 2ϩ /calmodulin kinase II inhibitor). Chelerythrine (protein kinase C inhibitor) partially inhibited the phosphorylation of STAT1 and STAT3. Mechanical stretch also augmented the mRNA expression of cardiotrophin-1, interleukin-6, and leukemia inhibitory factor at 60 to 120 minutes. These results indicated that the JAK/STAT pathway was activated by mechanical stretch, and that this activation was partially dependent on autocrine/ paracrine-secreted angiotensin II and was mainly dependent on the interleukin-6 family of cytokines but was independent of endothelin-1. Moreover, certain levels of intracellular Ca 2ϩ were necessary for stretch-induced activation of this pathway, and protein kinase C was also partially involved in this activation. C ardiac hypertrophy is a compensatory response that allows the heart to cope with the pathogenic stimuli found with many cardiovascular diseases. 1 Cardiac hypertrophy is induced by mechanical load and humoral factors, such as angiotensin II (Ang II), 2 endothelin-1 (ET-1), 3 and norepinephrine. 4 Mechanical stretch is one of the most important stimuli of cardiac hypertrophy. 5,6 Mechanical stretch-induced signal transduction is characterized by simultaneous activation of multiple second messenger systems. Many studies have demonstrated that mechanical stretch caused activation of multiple intracellular signal transduction pathways in cultured neonatal cardiomyocytes, such as phospholipases (C, D, and A 2 ), tyrosine kinases, p21 ras , Raf-1, mitogen-activated protein kinases, c-jun N-terminal protein kinases, and protein kinase C (PKC). 7-9 Autocrine/paracrine-secreted growth factors such as Ang II and ET-1 play an important role in the stretch-induced hypertrophic response. 10,11 Although mechanical stretch activates multiple second messenger systems, it remains unclear which molecules ar...

This study was designed to investigate whether angiotensin II induces the interleukin (IL)-6 family of cytokines in cardiac fibroblasts and, if so, whether these cytokines can augment cardiac hypertrophy. Angiotensin II increased IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 mRNA by 6.5-, 10.2-, and 2.0-fold, respectively, but did not affect IL-11, ciliary neurotrophic factor, or oncostatin M in cardiac fibroblasts. Enzyme-linked immunosorbent assay revealed that angiotensin II-stimulated conditioned medium from cardiac fibroblasts contained 9.3 ng/ml IL-6 at 24 h, which was 24-fold higher than the control. It phosphorylated gp130 and STAT3 in cardiomyocytes, which was reduced with RX435 (anti-gp130 blocking antibody). It increased [ 3 H]phenylalanine uptake and cell area by 44% and 86% in cardiomyocytes compared with mock medium. RX435 suppressed these increases by 26% and 38%, while TAK044 (endothelin-A/B-R blocker) suppressed them by 52% and 52%, respectively. Antisense oligonucleotides against LIF and cardiotrophin-1 blocked their up-regulation, and attenuated the conditioned medium-induced increase in [ 3 H]phenylalanine uptake by 21% and 13%, respectively. The combination of antisense oligonucleotides to LIF and cardiotrophin-1 decreased their uptake by 33%. These results indicated that angiotensin II induced IL-6, LIF, and cardiotrophin-1 in cardiac fibroblasts, and that these cytokines, particularly LIF and cardiotrophin-1, activated gp130-linked signaling and contributed to angiotensin II-induced cardiomyocyte hypertrophy.

This study delineates a mechanism for antiapoptotic signaling initiated by atrial natriuretic peptide (ANP) stimulation leading to elevation of cGMP levels and subsequent nuclear accumulation of Akt kinase associated with zyxin, a cytoskeletal LIM-domain protein. Nuclear targeting of zyxin induces resistance to cell death coincident with nuclear accumulation of activated Akt. Nuclear translocation of zyxin triggered by cGMP also promotes nuclear Akt accumulation. Additional supportive evidence for nuclear accumulation of zyxin-enhancing cardiomyocyte survival includes the following: (a) promotion of zyxin nuclear localization by cardioprotective stimuli; (b) zyxin association with phospho-Akt 473 induced by cardioprotective stimuli; and (c) recruitment of zyxin to the nucleus by activated nuclear-targeted Akt as well as recruitment of Akt by nuclear-targeted zyxin. Nuclear accumulation of zyxin requires both Akt activation and nuclear localization. Potentiation of cell survival is sensitive to stimulation intensity with high-level induction by ANP or cGMP signaling leading to apoptotic cell death rather than enhancing resistance to apoptotic stimuli. Myocardial nuclear accumulation of zyxin and Akt responds similarly in vivo following treatment of mice with ANP or cGMP. Thus, zyxin and activated Akt participate in a cGMP-dependent signaling cascade leading from ANP receptors to nuclear accumulation of both molecules. Nuclear accumulation of zyxin and activated Akt may represent a fundamental mechanism that facilitates nuclear-signal transduction and potentiates cell survival.

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