Severity of stroke varies widely among individuals. Whether differences in the extent of the native (preexisting) pial collateral circulation exist and contribute to this variability is unknown. We addressed these questions and probed for potential genetic contributions using morphometric analysis of the collateral circulation in 15 inbred mouse strains recently shown to exhibit wide differences in infarct volume. Morphometrics were determined in the unligated left hemisphere (for native collaterals) and ligated right hemisphere (for remodeled collaterals) 6 days after permanent middle cerebral artery (MCA) occlusion. Variation among strains in native collateral number, diameter, MCA, anterior cerebral artery (ACA), and posterior cerebral artery (PCA) tree territories were, respectively: 56-fold, 3-fold, 42%, 56%, and 61%. Collateral length (P < 0.001) and the number of penetrating arterioles branching from them also varied (P < 0.05). Infarct volume correlated inversely with collateral number (P < 0.0001), diameter (P < 0.0001), and penetrating arteriole number (P < 0.05) and directly with MCA territory (P < 0.05). Relative collateral conductance and MCA territory, when factored together, strongly predicted infarct volume (P < 0.0001). Outward remodeling of collaterals in the ligated hemisphere varied B3-fold. These data show that the extent of the native pial collateral circulation and collateral remodeling after obstruction vary widely with genetic background, and suggest that this variability, due to natural polymorphisms, is a major contributor to variability in infarct volume.

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, ␣1, ␤1, and ␤2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergentsolubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from ␣1-, ␤1-, and ␤2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated α-syntrophin null mice by targeted gene disruption to test the function of this association. The α-Syn−/− mice show no evidence of myopathy, despite reduced levels of α-dystrobrevin–2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the α-Syn−/− mice, even where other syntrophin isoforms are present. α-Syn−/− neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, α-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.

The molecular machinery underlying neurotransmitter receptor immobilization at postsynaptic sites is poorly understood. The NMDA receptor subunit NR1 can form clusters in heterologous cells via a mechanism dependent on the alternatively spliced C1 exon cassette in its intracellular C-terminal tail, suggesting a functional interaction between NR1 and the cytoskeleton. The yeast two-hybrid screen was used here to identify yotiao, a novel coiled coil protein that interacts with NR1 in a C1 exon-dependent manner. Yotiao mRNA (11 kb) is present modestly in brain and abundantly in skeletal muscle and pancreas. On Western blots, yotiao appears as an approximately 230 kDa band that is present in cerebral cortex, hippocampus, and cerebellum. Biochemical studies reveal that yotiao fractionates with cytoskeleton-associated proteins and with the postsynaptic density. With regard to immunohistochemistry, two anti-yotiao antibodies display a somatodendritic staining pattern similar to each other and to the staining pattern of NR1. Yotiao was colocalized by double-label immunocytochemistry with NR1 in rat brain and could be coimmunoprecipitated with NR1 from heterologous cells. Thus yotiao is an NR1-binding protein potentially involved in cytoskeletal attachment of NMDA receptors. Consistent with a general involvement in postsynaptic structure, yotiao was also found to be specifically concentrated at the neuromuscular junction in skeletal muscle.

Rationale: Collaterals are arteriole-to-arteriole anastomoses that connect adjacent arterial trees. They lessen ischemic tissue injury by serving as endogenous bypass vessels when the trunk of 1 tree becomes narrowed by vascular disease. The number and diameter ("extent") of native (preexisting) collaterals, plus their amount of lumen enlargement (growth/remodeling) in occlusive disease, show remarkably wide variation among inbred mouse strains (eg, C57BL/6 and BALB/c), resulting in large differences in tissue injury in models of occlusive disease. Evidence suggests similar large differences exist among healthy humans. Objective: To identify candidate loci responsible for genetic-dependent collateral variation. Methods and Results: Cerebral collateral number and diameter were determined in 221 C57BL/6؋BALB/c F2 progeny, followed by linkage analysis to identify quantitative trait loci (QTL) for collateral number and diameter. Four QTL were obtained for collateral number, including epistasis between 2 loci. A QTL that was identical to the strongest QTL for collateral number on chromosome 7 (logarithm of the odds [LOD]‫,92؍‬ effect size‫)%73؍‬ was also mapped for collateral diameter (LOD‫,71؍‬ effect size‫.)%03؍‬ Chromosome substitution strain analysis confirmed this locus. We also obtained a unique QTL on chromosome 11 for collateral remodeling after middle cerebral artery occlusion. Association mapping within the chromosome 7 QTL interval using collateral traits measured for 15 inbred strains delineated 172-kbp (P‫)20000.0؍‬ and 290-kbp (P‫)4000.0؍‬ regions on chromosome 7 containing 2 and 7 candidate genes, respectively. Conclusions: We conclude that collateral extent and remodeling are unique, highly heritable complex traits, with 1 QTL predominantly affecting native collateral number and diameter. (Circ Res. 2010;107:558-568.)Key Words: collateral vessels Ⅲ genetics Ⅲ quantitative trait loci Ⅲ cerebral circulation Ⅲ arteriogenesis A therosclerotic, atherothrombotic, and thromboembolic occlusive vascular diseases constitute the primary cause of morbidity and mortality in developed countries. Many physiological systems are concomitantly recruited, albeit with significant interindividual variation, which lessen the accompanying ischemic tissue injury. Among these, 3 vascular protective mechanisms are paramount: (1) the number and diameter of arteriole-to-arteriole anastomoses present in the tissue before the onset of disease that cross-connect occasional distal-most arterioles of adjacent trees (ie, the "native collateral extent"); (2) an anatomic increase in lumen diameter and wall thickness of these vessels caused by obstruction of flow to one of the trees, a process termed collateral remodeling, collateral growth, or arteriogenesis; and (3) ischemic angiogenesis, ie, the sprouting of additional capillaries. [1][2][3][4][5][6] Arteriogenesis, which requires days to weeks to achieve up to an approximately 10-fold increase in diameter depending on tissue and species, occurs when perfusion of an adjacent tree is c...