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).
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.
These data suggest that vascular smooth muscle contractions can occur under conditions where the [Ca2+]i is low and fixed and that these contractions may be mediated by PKC.
The type-II cAMP-dependent protein kinase (A-Kinase) partitions primarily into the particulate fraction in gastric parietal cells. Localization of this kinase to particular subcellular domains is mediated through the binding of the regulatory subunit (RII) dimer to A-Kinase-anchoring proteins (AKAPs). Using a [32P]RII overlay assay, we have screened a rabbit gastric parietal cell cDNA library and have isolated a single RII-binding protein clone. Sequence analysis revealed an open reading frame coding for 1022 amino acids (AKAP120). Recombinant fragments of the full-length clone were prepared and the RII-binding region mapped to an area between amino acids 489 and 549. This area contained a putative alpha-helical RII-binding region between amino acids 503 and 516. Incubation of [32P]RII with a synthetic peptide of AKAP120-(489-522) completely inhibited the binding of [32P]RII to the recombinant AKAP120 fragments that demonstrated RII binding. In vitro RII-binding affinity studies indicated a high-affinity interaction between AKAP120 and RII with a Kapp between 50 and 120 nM for the three recombinant fragments that bound [32P]RII. RNase-protection analysis revealed that AKAP120 is a widely distributed protein, with the highest levels of mRNA observed in gastric fundus. The presence of this novel high-affinity AKAP in gastric parietal cells suggests that it may regulate RII subcellular sequestration in this cell type.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.