GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

We have previously shown that mesenchymal stem cells (MSC) improve function upon integration in ischemic myocardium. We examined whether specific cytokines and growth factors produced by MSCs are able to affect angiogenesis, cellular migration and apoptosis. Conditioned media (CM) was prepared by culturing MSC for 48 hours. CM displayed significantly elevated levels of VEGF, Monocyte Chemoattractant Protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), MIP-1β and monokine induced by IFN-γ (MIG) compared to control media. MSC contained RNA for these factors as detected by RT-PCR. CM was able to induce angiogenesis in canine vascular endothelial cells. MCP-1 and MIP-1α increased cell migration of MSC while VEGF reduced it. H9c2 cells treated with CM under hypoxic conditions for 24 hours displayed a 16% reduction in caspase-3 activity compared to controls. PI 3-kinase γ inhibitor had no effect on controls but reversed the effect of CM on caspase-3 activity. MCP-1 alone mimicked the protective effect of CM while the PI 3-Kγ inhibitor did not reverse the effect of MCP-1. CM reduced phospho-BAD (Ser112) and phospho-Akt (Ser473) while increasing phospho-Akt (Thr308). MCP-1 reduced the level of phospho-Akt (Ser473) while having no effect on the other two; the PI 3-Kγ inhibitor did not alter the MCP-1 effect. ERK 1/2 phosphorylation was reduced in CM treated H9c2 cells, and inhibition of ERK 1/2 reduced the phosphorylation of Akt (Ser473), Akt (Thr308) and Bad (Ser112). In conclusion, MSC synthesize and secrete multiple paracrine factors that are able to affect MSC migration, promote angiogenesis and reduce apoptosis. While both MCP-1 and PI3-kinase are involved in the protective effect, they are independent of each other. It is likely that multiple pro-survival factors in addition to MCP-1 are secreted by MSC which act on divergent intracellular signaling pathways.

Significant numbers of myocytes die by apoptosis during myocardial infarction. The molecular mechanism of this process, however, remains largely unexplored. To facilitate a molecular genetic analysis, we have developed a model of ischemia-induced cardiac myocyte apoptosis in the mouse. Surgical occlusion of the left coronary artery results in apoptosis, as indicated by the presence of nucleosome ladders and in situ DNA strand breaks. Apoptosis occurs mainly in cardiac myocytes, and is shown for the first time to be limited to hypoxic regions during acute infarction. Since hypoxia-induced apoptosis in other cell types is dependent on p53, and p53 is induced by hypoxia in cardiac myocytes, we investigated the necessity of p53 for myocyte apoptosis during myocardial infarction. Myocyte apoptosis occurs as readily, however, in the hearts of mice nullizygous for p53 as in wild-type littermates. These data demonstrate the existence of a p53-independent pathway that mediates myocyte apoptosis during myocardial infarction. ( J.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKC ␤ was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Cysteine-rich protein 3 or muscle LIM protein (MLP) is thought to be a mechanosensor in cardiac myocytes. Therefore, the subcellular location of MLP may have functional implications in health and disease. Our hypothesis is that MLP becomes mislocalized after prolonged overload, resulting in impaired mechanosensing in cardiac myocytes. Using the techniques of biochemical subcellular fractionation and immunocytochemistry, we found MLP exhibits oligomerization in the membrane and cytoskeleton of cultured cardiac rat neonatal myocytes. Nuclear MLP was always monomeric. MLP translocated to the nucleolus in response to 10% cyclic stretch at 1 Hz for 48 h. This was associated with a threefold increase in S6 ribosomal protein (P Ͻ 0.01; n ϭ 3 cultures). Adenoviral overexpression of MLP also resulted in a twofold increase in S6 protein, suggesting that MLP can activate ribosomal protein synthesis in the nucleolus. In ventricles from aortic-banded and myocardially infarcted rat hearts, nuclear MLP increased by twofold (P Ͻ 0.01; n ϭ 7) along with a significant decrease in the nonnuclear oligomeric fraction. The ratio of nuclear to nonnuclear MLP increased threefold in both groups (P Ͻ 0.01; n ϭ 7). In failing human hearts, there was almost a complete loss of oligomeric MLP. Using a flag-tagged adenoviral MLP, we demonstrate that the COOH terminus is required for oligomerization and that this is a precursor to stretch sensing and subsequent nuclear translocation. Therefore, reduced oligomeric MLP in the costamere and cytoskeleton may contribute to impaired mechanosensing in heart failure.hypertrophy; mechanosensing; cytoskeleton; nucleocytoplasmic shuttling PROLONGED HEMODYNAMIC OVERLOAD results in cardiac hypertrophy with detrimental changes in myocardial gene expression and morphology (7,8,19,20,24,32) that predict morbidity and mortality (9, 28). Stretch sensors, necessary for myocyte enlargement, are thought to become impaired as a result of prolonged overload (11). However, the mechanisms by which chronic overload leads to decompensation and failure are poorly understood.Many proteins shuttle from subcellular compartments to the nucleus and may act as molecular sensors (4). In myocytes, some of these nucleocytoplasmic proteins are located in costameric focal adhesions and the cytoskeleton where mechanical forces are transmitted. One such protein, muscle LIM protein (MLP) or cysteine-rich protein 3, contains two zinc finger LIM domains. MLP is known to interact with many proteins of the cytoskeleton, including ␣-actinin and the titin-binding protein telethonin (34). MLP is thought to be part of a mechanosensing mechanism in myocytes, and this is likely to occur through two main mechanisms. First, the LIM domains enable the protein to interact with a variety of other cellular proteins, many of which are associated with known signaling pathways. The potential role of MLP as a sensor of mechanical activity through its interactions with other proteins has been thoroughly reviewed recently (18). Second, the protein contains...