BAG3 (Bcl-2–Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy

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.

Strain-Dependent Variation in Acute Ischemic Muscle Injury

“…43 We have previously described impairments in autophagic flux, mass recovery, and growth-related gene expression in myotubes differentiated from primary B/c myogenic progenitor cells, after experimental HND, that were not observed in C57 cells under the same conditions. 11,27 These findings indicate that muscle cellespecific processes differ in response to ischemia between the strains in a model that is independent of perfusion and immune response. In this study, we detected a small, but statistically significant, decrease in B/c myotube viability after 6 hours of HND compared with C57 myotubes.…”

“…9,10,41,42 Follow-up studies have used this information to assess HLI responses in B/c mice expressing C57 gene variants identified by these genome-wide association studies; they have found that expression of several C57 variants has significant effects on HLI outcomes, including enhanced number/diameter of preexisting collateral arteries in the hind limb, 28 improved postischemic perfusion recovery, 10 and augmented ischemic myoregeneration. 27 However, the aforementioned studies focused on tissue assessments that were made only during the myoregenerative phases and identified genes that are typically associated with late-stage processes in the injury recovery timeline, such as connective tissue remodeling, 10 mature muscle homeostasis, 27 and mechanotransduction. 54 Although detailed comparison of the genetic contributions to the observed sensitivity of B/c muscle to ischemic injury was outside the scope of this study, we wanted to investigate the possibility that additional inbred strains of mice that share a close common ancestor with either B/c or C57 mice would have similar phenotypes under the same conditions.…”

“…23e26 Several recent studies have indicated that ischemic outcomes in B/c mice can be enhanced by expression of exogenous genes or gene variants associated with the improved phenotypic outcomes in C57 mice. 10,27,28 These studies have focused on tissue outcomes measured days to weeks after the initial onset of ischemia, however, and have not addressed the contribution of background strain to the immediate ischemic injury sustained in these mice.…”

The Hippo network kinase STK38 contributes to protein homeostasis by inhibiting BAG3-mediated autophagy

“…In striated skeletal and heart muscle, BAG3 is localized at actin-anchoring Zdisks, which limit the contractile sarcomeric unit [67]. Loss or functional impairment of the cochaperone in animal models and patients results in a force-induced disintegration of Z-disks and aggregation of Z-disk proteins, leading to severe muscle weakness [10,19,20,22,24,68,69]. Intriguingly, knockdown of STK38 in cardiac muscle cells essentially mirrors the phenotype caused by BAG3 depletion or impairment.…”

BAG3 plays a central role in proteostasis in the heart

“…Disruption of proteostasis induces the accumulation of misfolded proteins, which then interfere with the normal function of cells through improper degradation, gain and/or loss of function, and aggregate formation. The adverse effects caused by altered proteostasis are collectively termed proteotoxicity, and pathological conditions resulting from proteotoxicity are collectively termed proteinopathies (3,4). Proteostasis is particularly important for postmitotic cells, such as neurons and adult cardiomyocytes, that have negligible regenerative potential, because in these mature cells, proteotoxicity is not readily diluted through cell division.…”