FRI625 Diabetic Ischemic Skeletal Muscle Injury Occurs Through Pkc-beta/sp1 Regulation Of Bag3 Expression

Abstract

Abstract Disclosure: A. Mani: None. V. Lamin: None. T. Wong: None. M.V. Singh: None. A.O. Dokun: None. Peripheral arterial disease (PAD) affects more than 200 million people worldwide and is characterized by impaired blood flow to the lower extremities. Individuals with diabetes (DM) and PAD have more severe ischemic limb injuries and increased limb amputations, but the mechanisms involved are poorly understood. Following experimental PAD, C57Bl/6 mice develop limited ischemic limb injury largely due to protective effect of genes within the limb salvage QTL-1 (LSq-1) a genetic locus on chromosome 7. However, when diabetes is induced in C57Bl/6 mice they develop severe ischemic limb injuries following experimental PAD, akin to the findings in humans. Previously, we identified BAG3 as a gene within LSq-1 that is protective against ischemic limb injuries. BAG3 is upregulated in ischemic limbs from non-DM C57Bl/6 mice but poorly expressed in ischemic limbs of DM mice. The mechanism of BAG3 upregulation in ischemia is not known however the BAG3 promoter has a Specificity protein 1 (Sp1) transcription factor binding site suggesting its possible role. Moreover, how DM impairs BAG3 expression in ischemic skeletal muscles in not known. PKCβ regulates expression of transcription factors, and its phosphorylation has been implicated in the vascular pathology in DM but a role in ischemic diabetic skeletal muscle injury has not been established. Here we assessed the role of Sp1 in BAG3 upregulation in ischemic skeletal muscle cells (HSMC), we found Sp1 is upregulated in ischemic HSMCs and overexpression of Sp1 enhanced BAG3 promoter luciferase activity while knock down of Sp1 impaired BAG3 upregulation in ischemia. However, in vitro, ischemic HSMCs under DM conditions (High glucose or HG, 25mM + Palmitic acid or PA, 200µM) express lower BAG3 (fold change: Vehicle, 1.0±0.02 vs HG+PA, 0.57±0.04; n=4; p<0.05) and Sp1 (fold change: Vehicle, 1.0±0.15 vs HG+PA, 0.22±0.03; n=4; p<0.05). Overexpression of BAG3 improved survival of ischemic HSMCs in DM conditions in vitro (fold change: Vehicle, 1.0±0.02 vs HG+PA, 1.3±0.02 vs BAG3+HG+PA, 1.03±0.03; n=4-5; p<0.05) and improved extent of limb injury in vivo (Necrosis Score; DM mice without BAG3 2.67±0.29 vs DM mice + BAG3, 0.87±0.29; n=8-9; p<0.05). Additionally, under DM conditions ischemic HSMCs showed increased phosphorylation of PKCβ on the Ser661. Blocking PKCβ activation with Ruboxistaurin (Rbx), a specific inhibitor of PKCβ phosphorylation prevented the DM induced phosphorylation of PKCβ on the Ser661 (p-PKCβ/PKCβ ratio: Vehicle, 0.08±0.006 vs HG±PA, 0.19±0.03 vs Rbx+HG+PA, 0.10±0.006; n=4; p<0.05) and rescued BAG3 and Sp1 expression (BAG3, fold change: Vehicle, 1.0±0.06 vs HG±PA, 0.50±0.02 vs Rbx+HG+PA, 0.76±0.03; SP1, fold change: Vehicle, 1.0±0.02 vs HG±PA, 0.57±0.03 vs Rbx+HG+PA, 0.85±0.02; n=4; p<0.05). We conclude that impaired BAG3 expression in ischemic HSMC is mediated by impaired Sp1 expression due to increased PKCβ phosphorylation in DM. Presentation: Friday, June 16, 2023