A simple and convenient procedure for chemoselectively protecting the amino groups of chitosan has been developed to provide N-phthaloyl-chitosan that is indispensable as a soluble N-protected precursor for further controlled modification reactions of chitosan. Although the conventional N-phthaloylation of chitosan in N,N-dimethylformamide was accompanied by partial phthaloylation of the hydroxy groups, the addition of a small amount of hydroxy-containing compounds effectively suppressed the O-phthaloylation. Of some compounds examined, water proved particularly suitable, resulting in the formation of chemoselectively N-phthaloylated chitosan without any appreciable O-phthaloyl groups. The resulting N-phthaloyl-chitosan was found to be crystalline despite the presence of a bulky substituent. A solubility test indicated that N-phthaloyl-chitosan exhibited considerable affinity for organic solvents.

Although many animal studies indicate insulin has cardioprotective effects, clinical studies suggest a link between insulin resistance (hyperinsulinemia) and heart failure (HF). Here we have demonstrated that excessive cardiac insulin signaling exacerbates systolic dysfunction induced by pressure overload in rodents. Chronic pressure overload induced hepatic insulin resistance and plasma insulin level elevation. In contrast, cardiac insulin signaling was upregulated by chronic pressure overload because of mechanical stretch-induced activation of cardiomyocyte insulin receptors and upregulation of insulin receptor and Irs1 expression. Chronic pressure overload increased the mismatch between cardiomyocyte size and vascularity, thereby inducing myocardial hypoxia and cardiomyocyte death. Inhibition of hyperinsulinemia substantially improved pressure overloadinduced cardiac dysfunction, improving myocardial hypoxia and decreasing cardiomyocyte death. Likewise, the cardiomyocyte-specific reduction of insulin receptor expression prevented cardiac ischemia and hypertrophy and attenuated systolic dysfunction due to pressure overload. Conversely, treatment of type 1 diabetic mice with insulin improved hyperglycemia during pressure overload, but increased myocardial ischemia and cardiomyocyte death, thereby inducing HF. Promoting angiogenesis restored the cardiac dysfunction induced by insulin treatment. We therefore suggest that the use of insulin to control hyperglycemia could be harmful in the setting of pressure overload and that modulation of insulin signaling is crucial for the treatment of HF.

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs) 1 . Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown 1,2 . Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, lowdensity lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.The heart is the first organ to form during embryogenesis, and abnormalities in this process result in congenital heart diseases, the most common cause of birth defects in humans 3 . Molecules that mediate cardiogenesis are of particular interest because of their potential use for cardiac regeneration 4,5 . Previous studies have shown that soluble growth factors such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), Wnts and Wnt inhibitors mediate the tissue interactions that are crucial for cardiomyocyte specification 3,4 . We proposed that there might be additional soluble factors that modulate cardiac development and/or cardiomyocyte differentiation.P19CL6 cells differentiate into cardiomyocytes with high efficiency in the presence of 1% dimethylsulphoxide (DMSO) 6 . We cultured P19CL6 cells with culture media conditioned by various cell types in the absence of DMSO, and screened the cardiogenic activity of the conditioned media. The extent of cardiomyocyte differentiation was assessed by the immunostaining with MF20 monoclonal antibody that recognizes sarcomeric myosin heavy chain (MHC). Among the several cell types tested, culture media conditioned by a murine stromal cell line OP9 induced cardiomyocyte differentiation of P19CL6 cells without DMSO treatment (Fig. 1a, left and middle panels). Increased MF20-positive area was accompanied by the induction of cardiac marker genes such as aMHC, Nkx2.5 and GATA-4, and by the increased protein levels of cardiac troponin T (cTnT) (Fig. 1a, right panel). In contrast, culture media conditioned by COS7 cells, mouse embryonic fibroblasts, NIH3T3 cells, HeLa cells, END2 cells (visceral endoderm-like cells), neonatal rat cardiomyocytes and neonatal rat cardiac fibroblasts did not induce cardiomyocyte differentiation of P19CL6 cells in the absence of DMSO (Fig. 1a and data not shown). From these observations, we p...

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