p204 is required for the differentiation of P19 murine embryonal carcinoma cells to beating cardiac myocytes: its expression is activated by the cardiac Gata4, Nkx2.5, and Tbx5 proteins.

Among 10 adult mouse tissues tested, the p204 protein levels were highest in heart and skeletal muscle. We described previously that the MyoD-inducible p204 protein is required for the differentiation of cultured murine C2C12 skeletal muscle myoblasts to myotubes. Here we report that p204 was also required for the differentiation of cultured P19 murine embryonal carcinoma stem cells to beating cardiac myocytes. As shown by others, this process can be triggered by dimethyl sulfoxide (DMSO). We established that DMSO induced the formation of 204RNA and p204. Ectopic p204 could partially substitute for DMSO in inducing differentiation, whereas ectopic 204 antisense RNA inhibited the differentiation. Experiments with reporter constructs, including regulatory regions from the Ifi204 gene (encoding p204) in P19 cells and in cultured newborn rat cardiac myocytes, as well as chromatin coimmunoprecipitations with transcription factors, revealed that p204 expression was synergistically transactivated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. Furthermore, ectopic p204 triggered the expression of Gata4 and Nkx2.5 in P19 cells. p204 contains a nuclear export signal and was partially translocated to the cytoplasm during the differentiation. p204 from which the nuclear export signal was deleted was not translocated, and it did not induce differentiation. The various mechanisms by which p204 promoted the differentiation are reported in the accompanying article (Ding, B., Liu, C., Huang, Y., Yu, J., Kong, W., and Lengyel, P. (2006) J. Biol. Chem. 281, 14893-14906).

An engineered VEGF-activating zinc finger protein transcription factor improves blood flow and limb salvage in advanced-age mice.

Advances in understanding the relationship between protein structure and DNA binding specificity have made it possible to engineer zinc finger protein (ZFP) transcription factors to specifically activate or repress virtually any gene. To evaluate the potential clinical utility of this approach for peripheral vascular disease, we investigated the ability of an engineered vascular endothelial growth factor (VEGFa)-activating ZFP (MVZ+426b) to induce angiogenesis and rescue hindlimb ischemia in a murine model. Hindlimb ischemia was surgically induced in advanced-age C57/BL6 mice. Adenovirus (Ad) encoding either MVZ+426b or the fluorescent marker dsRed was delivered to the adducter muscle of the ischemic hindlimb, and the effects on blood flow, limb salvage, and vascularization were assessed. Ad-MVZ+426b induced expression of VEGFa at the mRNA and protein levels and stimulated a significant increase in vessel counts in the ischemic limb. This was accompanied by significantly increased blood flow and limb salvage as measured serially for 4 wk. These data demonstrate that activation of the endogenous VEGFa gene by an engineered ZFP can induce angiogenesis in a clinically relevant model and further document the feasibility of designing ZFPs to therapeutically regulate gene expression in vivo.

Loss of skeletal muscle HIF-1alpha results in altered exercise endurance.

The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). To determine the role of HIF-1alpha in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1alpha knockout mice (HIF-1alpha KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1alpha KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1alpha KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1alpha KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1alpha KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function.

Cardiac myocyte-specific HIF-1alpha deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart.

At a resting pulse rate the heart consumes almost twice-as much oxygen per gram tissue as the brain and more than 43 times more than resting skeletal muscle (1). Unlike skeletal muscle, cardiac muscle cannot sustain anaerobic metabolism. Balancing oxygen demand with availability is crucial to cardiac function and survival, and regulated gene expression is a critical element of maintaining this balance. We investigated the role of the hypoxia-inducible transcription factor HIF-1alpha in maintaining this balance under normoxic conditions. Cardiac myocyte-specific HIF-1alpha gene deletion in the hearts of genetically engineered mice caused reductions in contractility, vascularization, high-energy phosphate content, and lactate production. This was accompanied by altered calcium flux and altered expression of genes involved in calcium handling, angiogenesis, and glucose metabolism. These findings support a central role for HIF-1alpha in coordinating energy availability and utilization in the heart and have implications for disease states in which cardiac oxygen delivery is impaired. Heart muscle requires a constant supply of oxygen. When oxygen supply does not match myocardial demand cardiac contractile dysfunction occurs, and prolongation of this mismatch leads to apoptosis and necrosis. Coordination of oxygen supply and myocardial demand involves immediate adaptations, such as coronary vasodilatation, and longer-term adaptations that include altered patterns of gene expression (2-4). How the expression of multiple genes is coordinated with oxygen availability in the heart and the impact of oxygen-dependent gene expression on cardiac function are insufficiently understood. Further elucidating these relationships may help clarify the molecular pathology of various cardiovascular disease states, including ischemic cardiomyopathy and myocardial hibernation (5, 6).

Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization.

Angiogenesis is regulated by means of a balance between activators and inhibitors. However, little is known regarding the regulation of the quiescent state of adult vessels. Corticotropin-releasing factor receptor 2 (CRFR2) is found in both endothelial and smooth muscle cells (SMCs) in the vasculature, where its function has remained elusive. We have investigated the role of CRFR2 as a determinant of tissue vascularization by comparing control and CRFR2-deficient mice with immunohistological and morphometric techniques. To define the mechanisms responsible for CRFR2 inhibition of angiogenesis, we have also examined in vitro the effect of ligand activation on cell proliferation, cell cycle protein phosphorylation, and capillary tube formation. Our results demonstrate that mice deficient for CRFR2 become hypervascularized postnatally. Activation of this receptor in vitro results in reduced vascular endothelial growth factor (VEGF) release from SMCs, an inhibition of SMC proliferation, and inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibits growth factor-induced vascularization. Western blots show that cell cycle retinoblastoma protein, which is essential for cell cycle progression, is decreased by CRFR2 agonist treatment in SMCs. These results suggest that CRFR2 is a critical component of a pathway necessary for tonic inhibition of adult neovascularization. CRFR2 may be a potential target for therapeutic modulation of angiogenesis in cancer and ischemic cardiovascular disease.