The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.

Granulocyte colony-stimulating factor (G-CSF) was reported to induce myocardial regeneration by promoting mobilization of bone marrow stem cells to the injured heart after myocardial infarction, but the precise mechanisms of the beneficial effects of G-CSF are not fully understood. Here we show that G-CSF acts directly on cardiomyocytes and promotes their survival after myocardial infarction. G-CSF receptor was expressed on cardiomyocytes and G-CSF activated the Jak/Stat pathway in cardiomyocytes. The G-CSF treatment did not affect initial infarct size at 3 d but improved cardiac function as early as 1 week after myocardial infarction. Moreover, the beneficial effects of G-CSF on cardiac function were reduced by delayed start of the treatment. G-CSF induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes in the infarcted hearts. G-CSF also reduced apoptosis of endothelial cells and increased vascularization in the infarcted hearts, further protecting against ischemic injury. All these effects of G-CSF on infarcted hearts were abolished by overexpression of a dominant-negative mutant Stat3 protein in cardiomyocytes. These results suggest that G-CSF promotes survival of cardiac myocytes and prevents left ventricular remodeling after myocardial infarction through the functional communication between cardiomyocytes and noncardiomyocytes.

Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) after depolarization of transverse tubules. The ryanodine receptor exists as a 'foot' protein in the junctional gap between the sarcoplasmic reticulum and the transverse tubule in skeletal muscle, and is proposed to function as a calcium-release channel during excitation-contraction (E-C) coupling. Previous complementary DNA-cloning studies have defined three distinct subtypes of the ryanodine receptor in mammalian tissues, namely skeletal muscle, cardiac and brain types. We report here mice with a targeted mutation in the skeletal muscle ryanodine receptor gene. Mice homozygous for the mutation die perinatally with gross abnormalities of the skeletal muscle. The contractile response to electrical stimulation under physiological conditions is totally abolished in the mutant muscle, although ryanodine receptors other than the skeletal-muscle type seem to exist because the response to caffeine is retained. Our results show that the skeletal muscle ryanodine receptor is essential for both muscular maturation and E-C coupling, and also imply that the function of the skeletal muscle ryanodine receptor during E-C coupling cannot be substituted by other subtypes of the receptor.

We have recently shown that mechanical stress induces cardiomyocyte hypertrophy partly through the enhanced secretion of angiotensin II (ATII). Endothelin-1 (ET-1) has been reported to be a potent growth factor for a variety of cells, including cardiomyocytes. In this study, we examined the role of ET-1 in mechanical stress-induced cardiac hypertrophy by using cultured cardiomyocytes of neonatal rats. ET-1 (10maximally induced the activation of both Raf-1 kinase and mitogen-activated protein (MAP) kinases at 4 and 8 min, respectively, followed by an increase in protein synthesis at 24 h. All of these hypertrophic responses were completely blocked by pretreatment with BQ123, an antagonist selective for the ET-1 type A receptor subtype, but not by BQ788, an ET-1 type B receptorspecific antagonist. BQ123 also suppressed stretch-induced activation of MAP kinases and an increase in phenylalanine uptake by approximately 60 and 50%, respectively, but BQ788 did not. ET-1 was constitutively secreted from cultured cardiomyocytes, and a significant increase in ET-1 concentration was observed in the culture medium of cardiomyocytes after stretching for 10 min. After 24 h, an ϳ3-fold increase in ET-1 concentration was observed in the conditioned medium of stretched cardiomyocytes compared with that of unstretched cardiomyocytes. ET-1 mRNA levels were also increased at 30 min after stretching. Moreover, ET-1 and ATII synergistically activated Raf-1 kinase and MAP kinases in cultured cardiomyocytes. In conclusion, mechanical stretching stimulates secretion and production of ET-1 in cultured cardiomyocytes, and vasoconstrictive peptides such as ATII and ET-1 may play an important role in mechanical stress-induced cardiac hypertrophy.Cardiac hypertrophy, a major underlying cause of heart diseases such as myocardial infarction and cardiac arrhythmias (1), is formed when increased external stimuli such as hemodynamic overload and neurohumoral factors are continuously imposed on cardiac myocytes (2, 3). These external stimuli are generally transduced into the nucleus through protein kinase cascades of phosphorylation (4), and Raf-1 kinase (Raf-1) 1 (5) and mitogen-activated protein (MAP) kinases (6, 7) are important components in these cascades. We have recently reported that stretching of cardiomyocytes sequentially activates Raf-1 and MAP kinases, followed by an increase in protein synthesis (8). Interestingly, all of these events were partially suppressed by a specific antagonist of the angiotensin II (ATII) type 1 receptor, CV11974 (9). These results suggest that mechanical stress exemplified by stretching might stimulate the secretion of ATII from cardiomyocytes and that ATII participates in the activation of the protein kinase cascades and the production of cardiomyocyte hypertrophy through the ATII type 1 receptor. However, because the inhibition of these hypertrophic events by CV11974 is incomplete, factors other than ATII should also be involved in cardiomyocyte hypertrophy induced by mechanical stress.Endothelin-1 (ET-1) ...

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.

Background-Peroxisome proliferator-activated receptors (PPARs) are transcription factors of the nuclear receptor superfamily. It has been reported that the thiazolidinediones, which are antidiabetic agents and high-affinity ligands for PPAR␥, regulate growth of vascular cells. In the present study, we examined the role of PPAR␥ in angiotensin II (Ang II)-induced hypertrophy of neonatal rat cardiac myocytes and in pressure overload-induced cardiac hypertrophy of mice. Methods and Results-Treatment of cultured cardiac myocytes with PPAR␥ ligands such as troglitazone, pioglitazone, and rosiglitazone inhibited Ang II-induced upregulation of skeletal ␣-actin and atrial natriuretic peptide genes and an increase in cell surface area. Treatment of mice with a PPAR␥ ligand, pioglitazone, inhibited pressure overload-induced increases in the heart weight-to-body weight ratio, wall thickness, and myocyte diameter in wild-type mice and an increase in the heart weight-to-body weight ratio in heterozygous PPAR␥-deficient mice. In contrast, pressure overload-induced increases in the heart weight-to-body weight ratio and wall thickness were more prominent in heterozygous PPAR␥-deficient mice than in wild-type mice. Conclusions-These results suggest that the PPAR␥-dependent pathway is critically involved in the inhibition of cardiac hypertrophy.

Pretreatment with a combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) has been reported to attenuate left ventricular (LV) remodeling after acute myocardial infarction (MI). We here examined whether the cytokine treatment started after MI has also beneficial effects. Anterior MI was created in the recipient mice whose bone marrow had been replaced with that of transgenic mice expressing enhanced green fluorescent protein (GFP). We categorized mice into five groups according to the following treatment: 1) saline; 2) administration of G-CSF and SCF from 5 days before MI through 3 days after; 3) administration of G-CSF and SCF for 5 days after MI; 4) administration of G-CSF alone for 5 days after MI; 5) administration of SCF alone for 5 days after MI. All the three treatment groups with G-CSF showed less LV remodeling and improved cardiac function and survival rate after MI. The number of capillaries, which express GFP, was increased and the number of apoptotic cells was decreased in the border area of all the treatment groups with G-CSF. Even if the cytokine treatment is started after MI, it could prevent LV remodeling and dysfunction after MI--at least in part--through an increase in neovascularization and a decrease in apoptosis in the border area.

Abstract-Mechanical stress activates various hypertrophic responses, including activation of mitogen-activated protein kinases (MAPKs) in cardiac myocytes. Stretch activated extracellular signal-regulated kinases partly through secreted humoral growth factors, including angiotensin II, whereas stretch-induced activation of c-Jun NH 2 -terminal kinases and p38 MAPK was independent of angiotensin II. In this study, we examined the role of integrin signaling in stretch-induced activation of p38 MAPK in cardiomyocytes of neonatal rats. Overexpression of the tumor suppressor PTEN, which inhibits outside-in integrin signaling, strongly suppressed stretch-induced activation of p38 MAPK. Overexpression of focal adhesion kinase (FAK) antagonized the effects of PTEN, and both tyrosine residues at 397 and 925 of FAK were necessary for its effects. Stretch induced tyrosine phosphorylation and activation of FAK and Src. Stretch-induced activation of p38 MAPK was abolished by overexpression of FAT and CSK, which are inhibitors of the FAK and Src families, respectively, and was suppressed by overexpression of a dominant-negative mutant of Ras. Mechanical stretch-induced increase in protein synthesis was suppressed by SB202190, a p38 MAPK inhibitor. These results suggest that mechanical stress activates p38 MAPK and induces cardiac hypertrophy through the integrin-FAKSrc-Ras pathway in cardiac myocytes. Key Words: hypertrophy, cardiac Ⅲ myocytes Ⅲ stress, mechanical Ⅲ integrins Ⅲ protein kinases M echanical stress plays a crucial role in tissue morphogenesis and remodeling. Changes of cell morphology, which rely on the organization of cytoskeletal components and the modulation of cell adhesion, evoke specific intracellular signals from cell adhesion receptors and induce the expression of specific genes. 1,2 We and others have developed an in vitro system by which cultured cardiac myocytes are subjected to mechanical stress, and have demonstrated that mechanical stress induces a variety of hypertrophic responses such as activation of mitogen-activated protein kinases (MAPKs), 3-5 reprogramming of gene expressions, 4,6 and an increase in protein synthesis. 4,7,8 MAPKs-including extracellular signal-regulated kinases (ERKs), c-Jun NH 2 -terminal kinase (JNK), and p38 MAPK-play pivotal roles in a variety of cell functions in many cell types. 9,10 Although ERKs were activated by mechanical stretch partially through enhanced secretion of angiotensin (Ang) II and endothelin-1, 11-13 JNK was strongly activated by stretch independently of Ang II, 3 suggesting that JNK might be directly activated by mechanical stress. p38 MAPK-which consists of ␣-, ␤-, ␥-, and ␦-isoforms-was first isolated as a mammalian homolog of yeast HOG1, 14 which is activated by mechanical stresses, including osmotic stress. 15 The ␣-and ␤-isoforms of p38 MAPK induce apoptosis and hypertrophy, respectively, in cardiac myocytes. 16 In addition, it has recently been reported that cardiac hypertrophy is induced by p38 MAPK but is suppressed by JNK, 17 and that SB20358...