No abstract

Background Critical limb ischemia (CLI) is a manifestation of peripheral artery disease (PAD) that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered two overlapping quantitative trait loci (QTL) in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume following femoral artery or middle cerebral artery ligation, respectively. Here we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hindlimb ischemia (HLI). Methods We treated mice with either adeno-associated viruses (AAV) encoding a control (GFP), or two BAG3 variants, namely Met81 or Ile81, and subjected the mice to hindlimb ischemia. Results We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). Treating BALB/c mice with AAV encoding the BL6 BAG3 variant (Ile81) (n=25) displayed reduced limb tissue necrosis and increased limb tissue perfusion compared to Met81- (n=25) or GFP- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of AAV-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or GFP (n=5), improved ischemic limb blood flow, limb muscle histology, and restored muscle function (force production). Compared to BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Conclusions Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Presenilin-1 is required for γ-secretase activity, which participates in Notch receptor processing, the pathogenesis of Alzheimer's disease and the modulation of Ca2+ signaling. We tested the hypothesis that γ-secretase proteolytic activity modulates store-operated Ca2+ entry (SOCE) in rat dorsal root ganglion neurons. Depletion of intracellular Ca2+ stores by blocking the endoplasmic reticulum Ca2+ pump with cyclopiazonic acid (CPA) evoked a transient increase in [Ca2+]i but no sustained Ca2+ influx. However, in cells expressing a dominant negative presenilin-1 mutant (PS1-D257A), γ-secretase activity was inhibited and treatment with CPA evoked sustained Ca2+ influx. Similarly, pharmacologic inhibition of γ-secretase with DAPT for 48 hrs enhanced SOCE. SKF96365, an inhibitor of store-operated channels, blocked SOCE in cells expressing PS1-D257A. Thus, γ-secretase proteolytic activity regulates a SOCE pathway in sensory neurons.

Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.

Ferragamo MJ, Reinardy JL, Thayer SA. Ca 2ϩ -dependent, stimulus-specific modulation of the plasma membrane Ca 2ϩ pump in hippocampal neurons.