Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS), but does not have the ocular and cardiovascular complications that characterize MFS. CCA and MFS result from mutations in highly similar genes, FBN2 and FBN1, respectively. All the identified CCA mutations in FBN2 cluster in a limited region similar to where severe MFS mutations cluster in FBN1, specifically between exons 23 and 34. We screened exons 22 through 36 of FBN2 for mutations in 13 patients with classic CCA by single stranded conformational polymorphism analysis (SSCP) and then by direct sequencing. We successfully identified 10 novel mutations in this critical region of FBN2 in these patients, indicating a mutation detection rate of 75% in this limited region. Interestingly, none of these identified FBN2 mutations alter amino acids in the calcium binding consensus sequence in the EGF-like domains, whereas many of the FBN1 mutations alter the consensus sequence. Furthermore, analysis of the clinical data of the CCA patients with characterized FBN2 mutation indicate that CCA patients have aortic root dilatation and the vast majority lack evidence of congenital heart disease. These studies have implications for our understanding of the molecular basis of CCA, along with the diagnosis and genetic counseling of CCA patients.

The acute adaptation of myocardial glucose metabolism in response to low-flow ischemia and reperfusion was investigated in isolated working rat hearts perfused with bicarbonate saline containing glucose (10 mM) and insulin (40 microU/ml). Reversible low-flow ischemia was induced by reducing coronary perfusion pressure from 100 to 35 cmH2O. Tritiated glucose was used to assess rates of glucose transport and phosphorylation, flux from glucose to pyruvate, and oxidation of exogenous glucose. Rates of glycogen synthesis and glycolysis were also assessed. With ischemia, cardiac power decreased by more than two-thirds. Rates of glucose uptake and flux from glucose to pyruvate remained unchanged, while glucose oxidation declined by 61%. Rates of lactate release more than doubled, and fractional enrichment of glycogen remained the same. During reperfusion, glucose oxidation returned to the preischemic values. When isoproterenol was added during ischemia, glucose uptake increased, glycogen decreased, and lactate release increased. No effect was seen with pacing. We conclude that during low-flow ischemia and with glucose as the only exogenous substrate, net glucose uptake remains unchanged. There is a reversible redirection between glycolysis and glucose oxidation, while glycogen synthesis continues during ischemia and is enhanced with reperfusion.

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.