We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in ∼30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node-like and ventricular cell-like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and α-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation.

These results suggest that the DC is a potent immunoprotective regulator during the postinfarction healing process via its control of monocyte/macrophage homeostasis.

Peripheral monocytosis is associated with LV dysfunction and LV aneurysm, suggesting a possible role of monocytes in the development of LV remodeling after reperfused AMI.

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...

Elevated serum HMGB1 levels were associated with adverse clinical outcomes in patients with MI. However, HMGB1 blockade in a rat MI model aggravated LV remodelling, possibly through impairment of the infarct-healing process. HMGB1, a novel predictor of adverse clinical outcomes after MI, may have an essential role in the appropriate healing process after MI.

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.

Summary Reasons for performing study: Overstrain injuries to the superficial digital flexor tendon (SDFT) and suspensory ligament (SL) are among the most common musculoskeletal injuries which contribute to the considerable wastage of racing Thoroughbreds. Many epidemiological studies have demonstrated the prevalence of and risk factors for tendon injury when racing but have not included those injuries sustained during training. However, since tendon injury during training is seen commonly in clinical practice, it is appropriate to determine the overall prevalence of tendon injury sustained during both training and racing. Objective: To determine the prevalence of overstrain injury to the SDFT and SL during training and racing among Thoroughbred flat racehorses in Japan in 1999. Methods: A retrospective study was performed using a sample population of 10,262 Thoroughbred racehorses. The medical information database of Thoroughbred racehorses registered by the Japan Racing Association (JRA) in 1999 was analysed for SDFT and SL overstrain injury diagnosed by a veterinarian employed by JRA during training and racing. Jump racehorses were excluded from this study. Results: The prevalence of forelimb SDFT tendonitis and SL desmitis was 11.1% (1130 cases) and 3.61% (370 cases) of the population, respectively. In the hindlimb, there were 0.06% (6 cases) and 0.14% (14 cases), respectively. Risks of SDF tendonitis in the forelimb in 3‐year‐olds or older horses were significantly higher than in 2‐year‐olds. In contrast, the risk of SL desmitis in the forelimb at age 3 and 4 years was 2.23 and 2.11 times higher, respectively, than in 2‐year‐olds, but this increased to 5.07 times in those age ≥5 years. Entire males were at greater risk in comparison to females and geldings. Conclusions: The results suggest that the prevalence of SDF tendonitis and SL desmitis in the forelimb was associated with the horse's age and sex. The prevalence of SL desmitis increased further with age compared with SDF tendonitis, possibly reflecting a more rapid accumulation of degeneration in this structure. Potential relevance: The age‐related risk demonstrated in this study provides further support that overstrain injuries are associated with accumulated degeneration. These data provide a valuable resource for further research into the aetiology of tendon injury in the racehorse.

Mechanical loading of cardiac and skeletal muscles in vivo and in vitro causes rapid activation of a number of immediate-early (IE) genes and hypertrophy of muscle cells. However, little is known as to how muscle cells sense mechanical load and transduce it into intracellular signals of gene regulation. We examined roles of putative cellular mechanotransducers, mechanosensitive ion channels, the cytoskeleton, and contractile activity in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretchactivated cation channel that was completely blocked by gadolinium (Gd3+). Inhibition of this channel by Gd3+ did not affect either the stretch-induced expression of TE genes or the increase in protein synthesis. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression and increase in protein synthesis. Arresting contractile activity of myocytes by high K+, tetrodotoxin, or Ba2+ did not affect the stretch-induced TE gene expression. Tetrodotoxinarrested myocytes could increase protein synthesis in response to stretch. These results suggest that Gd3+-sensitive ion channels, microtubules, microfflaments, and contractile activity may not be necessary for transduction of mechanical stretch into the IE gene expression and hypertrophy. The stimulus of membrane stretch may be transmitted to the cell nucleus through some mechanisms other than electrical or direct mechanical transduction in cardiac myocytes.In living animals, many types of cells are normally exposed to a variety of mechanical stimuli. Although it is a well-known fact that mechanical stimuli cause a variety of effects on the structure and function of the cell (1-3), little is known as to how cells sense the mechanical stimuli, transmit the information to second-messenger systems, and finally regulate gene expression.Cardiac and skeletal muscles rapidly change their mass and phenotype in response to the mechanical load imposed on them (4, 5), and they may be a suitable model system to study how mechanical stimuli regulate gene expression in nonsensory cells. It has been demonstrated that stretching cultured cardiac and skeletal muscle cells grown on a deformable silicone substrate increases protein synthesis (6-9); this result indicates that the ability to sense mechanical load is intrinsic to muscle cells and does not require exogenous neurohormonal factors. More recently, stretching cardiac muscle has been shown to increase inositol monophosphate and bisphosphate (10, 11) and to activate many other signal-transduction pathways (J.-i.S. and S.I., unpublished data), but how cells initially convert mechanical stimuli into biochemical signals is unknown. It has been postulated that mechanosensitive ion channels, the cytoskeleton, and contractile activity may act as mechanotransducers in muscle and other cell types (for reviews, see refs. 1-3, 12, and 13). However, experimenta...