BAG3 Attenuates Ischemia-Induced Skeletal Muscle Necroptosis in Diabetic Experimental Peripheral Artery Disease.

Peripheral artery disease (PAD) is characterized by impaired blood flow to the lower extremities, resulting in ischemic limb injuries. Individuals with diabetes and PAD typically have more severe ischemic limb injuries and limb amputations, but the mechanisms involved are poorly understood. Previously, we identified BAG3 as a gene within a mouse genetic locus termed limb salvage QTL1 on mouse chromosome 7 that determined the extent of limb necrosis following ischemic injury in C57Bl/6 mice. Whether BAG3 deficiency plays a role in the severe ischemic injury observed in diabetic PAD is not known. In vitro, we found simulated ischemia enhanced BAG3 expression in primary human skeletal muscle cells, whereas BAG3 knockdown increased necroptosis markers and decreased cell viability. In vivo, ischemic skeletal muscles from hind limbs of high-fat diet (HFD)-fed mice showed poor BAG3 expression compared to normal chow diet (NCD)-fed mice, and this was associated with increased limb amputations. BAG3 overexpression in ischemic skeletal muscles from hind limbs of HFD mice rescued limb amputation and improved autophagy, necroptosis, skeletal muscle function and regeneration. Therefore, BAG3 deficiency in ischemic skeletal muscles contributes to the severity of ischemic limb injury in diabetic PAD, likely through autophagy and necroptosis pathways.

MicroRNA regulation of BAG3.

B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that modulates major biological processes, including apoptosis, autophagy, and development to promote cellular adaptive responses to stress stimuli. Although BAG3 is constitutively expressed in several cell types, its expression is also inducible and is regulated by microRNAs (miRNAs). miRNAs are small non-coding RNAs that mostly bind to the 3'-UTR (untranslated region) of mRNAs to inhibit their translation or to promote their degradation. miRNAs can potentially regulate over 50% of the protein-coding genes in a cell and therefore are involved in the regulation of all major functions, including cell differentiation, growth, proliferation, apoptosis, and autophagy. Dysregulation of miRNA expression is associated with pathogenesis of numerous diseases, including peripheral artery disease (PAD). BAG3 plays a critical role in regulating the response of skeletal muscle cells to ischemia by its ability to regulate autophagy. However, the biological role of miRNAs in the regulation of BAG3 in biological processes has only been elucidated recently. In this review, we discuss how miRNA may play a key role in regulating BAG3 expression under normal and pathological conditions.

Mitochondrial Oxidative Phosphorylation Function and Mitophagy in Ischaemic/Reperfused Hearts from Control and High-Fat Diet Rats: Effects of Long-Term Melatonin Treatment.

PURPOSE: Oxidative stress causes mitochondrial dysfunction in myocardial ischaemia/reperfusion (I/R) as well as in obesity. Mitochondrial depolarization triggers mitophagy to degrade damaged mitochondria, a process important for quality control. The aims of this study were to evaluate (i) the effect of I/R on mitochondrial oxidative phosphorylation and its temporal relationship with mitophagy in hearts from obese rats and their age-matched controls, and (ii) the role of oxidative stress in these processes using melatonin, a free radical scavenger. METHODS: Male Wistar rats were divided into 4 groups: control (normal diet ± melatonin) and high-fat sucrose diet (HFSD ± melatonin). Rats received melatonin orally (10 mg/kg/day). After 16 weeks, hearts were removed and subjected to 40-min stabilization, and 25-min global ischaemia/10-min reperfusion for preparation of mitochondria. Mitochondrial oxidative phosphorylation was measured polarographically. Western blotting was used for evaluation of PINK1, Parkin, p62/SQSTM1 (p62) and TOM 70. Infarct size was measured using tetrazolium staining. RESULTS: Ischaemia and reperfusion respectively reduced and increased mitochondrial QO2 (state 3) and the ox-phos rate in both control and HFSD mitochondria, showing no major changes between the groups, while melatonin pretreatment had little effect. p62 as indicator of mitophagic flux showed up- and downregulation of mitophagy by ischaemia and reperfusion respectively, with melatonin having no significant effect. Melatonin treatment caused a significant reduction in infarct size in hearts from both control and diet groups. CONCLUSIONS: The results suggest that I/R (i) affects mitochondria from control and HFSD hearts similarly and (ii) melatonin-induced cardioprotection is not associated with reversal of mitochondrial dysfunction or changes in the PINK1/Parkin pathway.

Mitochondrial oxidative phosphorylation and mitophagy in myocardial ischaemia/reperfusion: effects of chloroquine.

AIM: The aim of this study was to evaluate the temporal relationship between mitochondrial oxidative phosphorylation and mitophagy in rat hearts subjected to ischaemia/reperfusion. Measurements were made at specific points during the experimental protocol (snapshot approach) and by assessments of mitophagic flux, using chloroquine pre-treatment. METHODS: Isolated working rat hearts were subjected to 25 or 30 minutes of global ischaemia/10 minutes of reperfusion. Half of each group received chloroquine (10 mg/kg, intraperitoneally) one hour before experimentation. Mitochondria were isolated after stabilisation, ischaemia and reperfusion, and oxidative phosphorylation was measured polarographically. Mitochondrial mitophagy markers were detected by Western blot analysis. RESULTS: Mitochondrial oxygen uptake (state 3) and oxidative phosphorylation rate were reduced by ischaemia and increased by reperfusion. Chloroquine pre-treatment increased both parameters. Using a snapshot approach, exposure to ischaemia ± reperfusion had little effect on mitochondrial PINK1, Parkin and p62/SQSTM1 expression. Ischaemia reduced Rab9 expression, and reperfusion upregulated the phosphor DRP1, phosphor/total DRP1 ratio and Rab9 levels. Chloroquine significantly reduced PINK1, p62/SQSTM1, Rab9 and particularly Parkin expression during reperfusion, without an effect on mitochondrial total and phospho DRP1 levels. CONCLUSIONS: Ischaemia/reperfusion-induced changes in mitochondrial oxidative phosphorylation function occurred concomitantly with changes in mitophagic flux. Pre-treatment with chloroquine profoundly affected mitochondrial function as well as the pattern of mitophagy during ischaemia/reperfusion.

In experimental peripheral arterial disease, type 2 diabetes alters post-ischemic gene expression.

Peripheral arterial disease is characterized by impaired blood flow to tissues outside the heart due to atherosclerosis and it most frequently occurs in the lower extremities. Type 2 diabetes (T2D) is a well-known risk factor that accelerate the course and contributes to poor clinical outcomes of PAD. While there is some evidence that T2D is associated with altered expression of genes involved in regulating PAD severity, our knowledge about the specific genes and pathways involved remains incomplete. We induced experimental PAD or hind limb ischemia in T2D and non-diabetic mice and subjected the ischemic gastrocnemius muscle tissues to genome-wide mRNA transcriptome analysis. We subsequently performed pathway analysis on the top 500 genes that showed the most significant expression differences between the ischemic diabetic and ischemic non-diabetic muscle tissues. Pathway analysis of the differentially expressed genes identified pathways involved in essential biological processes such as "metabolic pathways," "phagosomes," "lysosomes," and "regulation of actin cytoskeleton". Overall, our data provides the opportunity to test hypotheses on the potential role of the altered genes/molecular pathways in poor PAD outcomes in diabetes.

Revisiting the Cardiotoxic Effect of Chloroquine.

PURPOSE: Cardiotoxicity is a well-known side effect of chloroquine. Several studies have proposed chloroquine as a potential anti-diabetic treatment but do not address this problem. The current study investigated the effect of ex vivo chloroquine treatment on (1) heart function and glucose uptake, (2) mitochondrial function and (3) in vivo treatment on heart function. METHODS: Control or obese male Wistar rats were used throughout. Dose responses of increasing chloroquine concentrations versus vehicle on cardiac function were measured using isolated, Langendorff-perfused hearts whilst glucose uptake and cell viability were determined in ventricular cardiomyocytes. Mitochondrial function was assessed with a Clark-type oxygraph (Hansatech) after ex vivo perfusion with 30 µM chloroquine versus vehicle. Animals were treated orally with 5 mg/kg/day chloroquine for 6 weeks. RESULTS: Acute chloroquine treatment of 10 µM was sufficient to significantly decrease heart function (p < 0.05) whilst 30 µM significantly reduced heart rate (p < 0.05). Chloroquine became toxic to isolated cardiomyocytes at high concentrations (100 µM), and had no effect on cardiomyocyte glucose uptake. Ex vivo treatment did not affect mitochondrial function, but chronic low-dose in vivo chloroquine treatment significantly decreased aortic output and total work in hearts (p < 0.005). CONCLUSION: Low and intermediate chloroquine doses administered either chronically or acutely are sufficient to result in myocardial dysfunction.