Estrogen‐Related Receptor Gamma Gene Therapy Promotes Therapeutic Angiogenesis and Muscle Recovery in Preclinical Model of PAD

Background Peripheral arterial disease and critical limb ischemia are cardiovascular complications associated with vascular insufficiency, oxidative metabolic dysfunction, and myopathy in the limbs. Estrogen‐related receptor gamma (ERRγ) has emerged as a dual regulator of paracrine angiogenesis and oxidative metabolism through transgenic mouse studies. Here our objective was to investigate whether postischemic intramuscular targeting of ERRγ via gene therapy promotes ischemic recovery in a preclinical model of peripheral arterial disease/critical limb ischemia. Methods and Results Adeno‐associated virus 9 (AAV9) Esrrg gene delivery vector was developed and first tested via intramuscular injection in murine skeletal muscle. AAV9‐Esrrg robustly increased ERRγ protein expression, induced angiogenic and oxidative genes, and boosted capillary density and succinate dehydrogenase oxidative metabolic activity in skeletal muscles of C57Bl/6J mice. Next, hindlimb ischemia was induced via unilateral femoral vessel ligation in mice, followed by intramuscular AAV9‐Esrrg (or AAV9‐green fluorescent protein) gene delivery 24 hours after injury. ERRγ overexpression increased ischemic neoangiogenesis and markers of endothelial activation, and significantly improved ischemic revascularization measured using laser Doppler flowmetry. Moreover, ERRγ overexpression restored succinate dehydrogenase oxidative metabolic capacity in ischemic muscle, which correlated with increased mitochondrial respiratory complex protein expression. Most importantly, myofiber size to number quantification revealed that AAV9‐Esrrg restores myofibrillar size and mitigates ischemia‐induced myopathy. Conclusions These results demonstrate that intramuscular AAV9‐Esrrg delivery rescues ischemic pathology after hindlimb ischemia, underscoring that Esrrg gene therapy or pharmacological activation could be a promising strategy for the management of peripheral arterial disease/critical limb ischemia

Muscle Arnt/Hif1β Is Dispensable in Myofiber Type Determination, Vascularization and Insulin Sensitivity

<div><p>Aryl Hydrocarbon Receptor Nuclear Translocator/ hypoxia-inducible factor 1 beta (ARNT/ HIF1β), a member of bHLH-PAS family of transcriptional factors, plays a critical role in metabolic homeostasis, insulin resistance and glucose intolerance. The contributions of ARNT in pancreas, liver and adipose tissue to energy balance through gene regulation have been described. Surprisingly, the impact of ARNT signaling in the skeletal muscles, one of the major organs involved in glucose disposal, has not been investigated, especially in type II diabetes. Here we report that ARNT is expressed in the skeletal muscles, particularly in the energy-efficient oxidative slow-twitch myofibers, which are characterized by increased oxidative capacity, mitochondrial content, vascular supply and insulin sensitivity. However, muscle-specific deletion of ARNT did not change myofiber type distribution, oxidative capacity, mitochondrial content, capillarity, or the expression of genes associated with these features. Consequently, the lack of ARNT in the skeletal muscle did not affect weight gain, lean/fat mass, insulin sensitivity and glucose tolerance in lean mice, nor did it impact insulin resistance and glucose intolerance in high fat diet-induced obesity. Therefore, skeletal muscle ARNT is dispensable for controlling muscle fiber type and metabolic regulation, as well as diet-induced weight control, insulin sensitivity and glucose tolerance.</p></div

ARNT expression in different tissues.

<p><b>(A)</b> ARNT protein expression in different organs [sub-cutaneous adipose tissue (Sc), perigonadic adipose tissue (Pg), brown adipose tissue (Bat), heart (He), liver (Li), brain (Br), kidney (Ki), pancreas (Pa), gastrocnemius muscle (Ga)] of 4 months old mice (N = 1). <b>(B)</b> Arnt gene expression in the soleus and the extensor digitorum longus (EDL) of 3 month old mice (N = 4–5). <b>(C-D)</b> ARNT expression in control and MKO muscle groups ranging from the most oxidative (soleus) to most glycolytic (EDL) (N = 3). (C) Representative images. (D) Densitometry for protein expression. (E) Arnt gene expression in EDL control and MKO muscles of 4 months old mice (N = 4–5). (*p<0.05,**p<0.01,***p<0.001, Unpaired Student’s t-test or One-way ANOVA with Tukey’s multiple comparison post-hoc test.)</p

Insulin and Glucose Tolerance.

<p>Following parameters were measured in control and MKO mice on HFD. (A) Weight gain (N = 6–12). (B) Fat mass in 4 month old mice (N = 6–12). (C) Lean mass in 4 month old mice (N = 6–12). (D) Insulin tolerance test (ITT) in 4 months old mice (N = 6–11). (E) Area Above the Curve (AAC) for ITT. (F) Glucose tolerance test (GTT) in 4 months old mice (N = 6–11). (G) Area Under the Curve (AUC) for GTT. (H) Ex vivo p-AKT ser473/panAKT stimulation by insulin measured in the gastrocnemius muscles of the 5 months old mice (N = 3–5). $indicates the treatment effect; * indicate the genotype effect.$ p<0.05; $$ $/*** p<0.001 (Unpaired Student’s t-test or Two-way ANOVA with a Bonferroni’s repeated measure test).</p

Metabolic dysfunction and altered mitochondrial dynamics in the utrophin-dystrophin deficient mouse model of duchenne muscular dystrophy.

The utrophin-dystrophin deficient (DKO) mouse model has been widely used to understand the progression of Duchenne muscular dystrophy (DMD). However, it is unclear as to what extent muscle pathology affects metabolism. Therefore, the present study was focused on understanding energy expenditure in the whole animal and in isolated extensor digitorum longus (EDL) muscle and to determine changes in metabolic enzymes. Our results show that the 8 week-old DKO mice consume higher oxygen relative to activity levels. Interestingly the EDL muscle from DKO mouse consumes higher oxygen per unit integral force, generates less force and performs better in the presence of pyruvate thus mimicking a slow twitch muscle. We also found that the expression of hexokinase 1 and pyruvate kinase M2 was upregulated several fold suggesting increased glycolytic flux. Additionally, there is a dramatic increase in dynamin-related protein 1 (Drp 1) and mitofusin 2 protein levels suggesting increased mitochondrial fission and fusion, a feature associated with increased energy demand and altered mitochondrial dynamics. Collectively our studies point out that the dystrophic disease has caused significant changes in muscle metabolism. To meet the increased energetic demand, upregulation of metabolic enzymes and regulators of mitochondrial fusion and fission is observed in the dystrophic muscle. A better understanding of the metabolic demands and the accompanied alterations in the dystrophic muscle can help us design improved intervention therapies along with existing drug treatments for the DMD patients

Increased oxygen consumption relative to integral force.

<p>A) The fatigue profile of WT and DKO EDL during the 10 minutes fatigue shows that DKO EDL generates lesser force and fatigues less. B) The % of initial force after the 10 minute fatigue is higher in DKO EDL indicating less fatigue (p = 0.0019). C) The quantified force time integral over the entire 10 minutes fatigue protocol is significantly reduced in the DKO EDL compared to WT (p = 0.0097). D) Oxygen consumption over 10 minutes fatigue is not significantly different in WT and DKO EDL muscle. E). Oxygen consumed per unit integral force produced is significantly higher in DKO EDL compared to WT (p = 0.0110). p< 0.05 is significant. * = p<0.05, ** = p<0.01.</p

Whole body energy expenditure of WT and DKO mice.

<p>A) Rate of oxygen consumption in DKO mice is not significantly different from WT at both night and B) day. Respiratory exchange ratio (F) is similar in WT and DKO mice both at C) night and D) day. Activity counts measured in WT and DKO mice during E) night and F) day. Activity counts are significantly reduced (p = 0.0060) in DKO mice at night compared to WT. Oxygen consumption per unit activity is significantly higher in the DKO mice both during G) night (p = 0.0023) and H) day (p = 0.0463) p<0.05 = significant. * = p<0.05, ** = p<.01.</p

Transmission electron microscopic images show mitochondrial localization is altered in DKO EDL muscle.

<p>A) WT EDL B) DKO EDL at 14000X magnification. C) WT and D) DKO at 34000x magnification. The arrows point to the localization of mitochondria (M) which are at the I band on either side of the Z disc in WT, but this tight localization is reduced in the DKO EDL.</p

Increased expression of mitochondrial fusion and fission regulators in DKO muscles.

<p>A) Western blots depicting mitochondria fission (Drp 1) and fusion (Mfn 2) regulators in WT and DKO EDL. B) Mfn 2 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0073). C) Drp 1 protein level normalized to GAPDH is significantly higher in DKO EDL compared to WT (p = 0.0089). D) Western blots depicting mitochondria fission (Drp 1) and fusion (Mfn 2) regulators in WT and DKO diaphragm. E) Mfn 2 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0074). F) Drp 1 protein level normalized to GAPDH is significantly higher in DKO diaphragm compared to WT (p = 0.0007). Western blots depicting similar mitochondrial electron transport chain complex protein levels in G) EDL and H) diaphragm of WT and DKO mice. p< 0.05 is significant. * = p<0.05, ** = p<0.01. Mfn 2- Mitofusin 2, Drp 1—Dynamin related protein 1, CV ATP5A- Complex V F1-F0 ATP synthase subunit, CIII UQCRC2- Complex III ubiquinol-cytochrome c reductase subunit, CIV MTCO1- Complex IV Cytochrome C Oxidase core subunit, CII SDHB- complex II succinate dehydrogenase subunit.</p