Nuclear receptors as potential therapeutic targets in peripheral arterial disease and related myopathy

Abstract

Peripheral arterial disease (PAD) is a prevalent cardiovascular complication of limb vascular insufficiency, causing ischemic injury, mitochondrial metabolic damage, and functional impairment in the skeletal muscle, and ultimately leading to immobility and mortality. While potential therapies have been mostly focused on revascularization, none of the currently available pharmacological treatments are fully effective in PAD, often leading to amputations, particularly in chronic metabolic diseases. One major limitation of focused angiogenesis and revascularization as a therapeutic strategy is limited effect on metabolic restoration and muscle regeneration in the affected limb. Therefore, additional pre-clinical investigations are needed to discover novel treatment options for PAD preferably targeting multiple aspects of muscle recovery. In this review, we propose nuclear receptors expressed in the skeletal muscle as potential candidates for ischemic muscle repair in PAD. We review classic steroid and orphan receptors that have been reported to be involved in the regulation of paracrine muscle angiogenesis, oxidative metabolism, mitochondrial biogenesis and muscle regeneration, and discuss how these receptors could be critical for recovery from ischemic muscle damage. Furthermore, we identify existing gaps in our understanding of nuclear receptor signaling in the skeletal muscle, and propose future areas of research that could be instrumental in exploring nuclear receptors as therapeutic candidates for treating PAD.

Graphical Abstract

Nuclear receptors are underwhelmingly explored as a therapeutic target in Peripheral arterial disease (PAD), a cardiovascular condition characterized by limb muscle ischemia and amputations. We review the accumulating literature on nuclear receptor signaling in skeletal muscle with an emphasis on muscle vascularization, mitochondrial metabolism, myopathy and regeneration. We underscore the untapped potential of nuclear receptors and identify research areas necessary for advancing these transcriptional factors as therapeutic targets for PAD.

Introduction

Peripheral arterial disease (PAD) is a prevalent cardiovascular complication caused by vascular occlusion of large blood vessels or regression of microvasculature resulting in decreased hemodynamic blood flow to limb musculature [1, 2]. Over the last 10 years, the number of cases of PAD have increased by 21% to approximately 200 million worldwide in both developed and underdeveloped countries [3]. In United States, over 12 million cases of PAD were reported in 2015 [4, 5]. While atherosclerosis is often the primary cause of PAD; smoking, diabetes, obesity, hypertension and aging increase the risk of PAD by over two-fold [5]. PAD is clinically categorized into asymptomatic, atypical, intermittent claudication, and critical limb ischemia [6]. In asymptomatic disease, the patient experiences no symptoms at rest, but suffers from leg pain with increased activity or mild exercise. Atypical PAD patients experience leg pain even at rest. Patients with intermittent claudication experience pain with walking or physical activity. Critical limb ischemia is the advanced form of the disease where limb ischemia leads to muscle wasting, gangrene, toe necrosis, and in advanced disease also to limb amputations. Asymptomatic PAD, intermediate claudication and critical limb ischemia (CLI) are responsible for poor mobility, and are associated with adverse cardiovascular event such as myocardial infarction, stroke or limb amputations.

Primary focus of treating PAD has been on therapeutic angiogenesis to promote revascularization of the ischemic limb by targeting angiogenic growth factors such as VEGFA and FGF1 and endothelial cell activation [1, 4]. Other interventions include anti-hypertensive agents, anti-platelet agents, and anti-diabetic drugs; however, these agents cannot be considered as first-in-line PAD treatment [1, 3, 7]. Unfortunately, none of these modalities are highly effective in ameliorating PAD, subsequently requiring surgical endovascular intervention or limb amputation [4, 8]. Extensive evidence has emerged regarding the role of mitochondrial and metabolic dysfunction as well as myopathy in the skeletal muscle in PAD pathogenesis [9-13]. These deficiencies are unlikely to be corrected by singular use of therapeutic angiogenesis strategy. Exploring molecular pathways that can effectively counter vascular and metabolic inefficiencies, and simultaneously promote regeneration in muscle ischemia is likely to lead the development of new therapeutic strategies for PAD.

Nuclear Receptors

Nuclear receptors are a super-family of transcriptional factors that respond to extracellular signals to control gene expression [14-17]. Several members are activated by physiological agents such as steroid hormones, bile acid, and metabolites or by xenobiotic chemicals. The family also consists of orphan receptors for which endogenous ligands remain unknown. Salient structural features of most nuclear receptors include N-terminal AF-1 (activation function 1) activity domain, DNA binding domain (DBD), hinge-region, a ligand binding domain (LBD) and C-terminal AF-2 activity domain [15] (Figure 1A). Activity of many receptors can be regulated via post-translational modifications such as phosphorylation, acetylation and SUMOylation [18-20] (Figure 1B). Post-translational regulation of receptor activity occurs through AF-1 and AF-2 domains. LBD’s are unique to each nuclear receptor, and has been targeted for selective pharmacological modulation of transcriptional function. DBD is the most conserved segment across different receptors, through which nuclear receptors bind to genomic sites known as hormone response elements (HRE). Nuclear receptor HRE contain half-site sequences that are arranged as direct repeats, inverted repeats or everted repeats [21]. The nuclear receptor super-family is classified into 7 classes, as shown in Table 1 [15, 16, 22]. Hormone-activated steroid receptors, such as those activated by estrogen, progesterone and testosterone, function as homodimers and bind to inverted repeat half-site sequences in the DNA. Some receptors function as obligate heterodimers with retinoid X receptors (RXR) and recognize direct repeat half-site sequences, whereas others function as homodimers binding to direct repeat half-site sequences or as monomers (Figure 1C). All nuclear receptors function as a part of a transcriptional complex, that include other regulatory factors such as co-repressors, co-activators, cAMP response element binding protein (CREB) binding protein (CBP)/p300, and histone deacetylase/acetylases (Figure 1C).

Figure 1. Basic nuclear receptor structure and regulation.

(A) Different conserved regions within the nuclear receptor (NR) structure including AF-1, DNA binding domain (DBD), hinge region, ligand binding domain (LBD), and AF-2. (B) Nuclear receptors can function as monomers, homodimers or heterodimers. Nuclear receptor transcriptional complexes contain various regulatory co-factors that orchestrate NR-dependent gene expression via hormone response elements (HRE) in the promoters. (C) Nuclear receptor transcriptional activity is regulated via different mechanisms including ligand binding, post-translational modification, trafficking and protein degradation.

Table 1. Nuclear Receptor Classification.

There are 48 mammalian nuclear receptors including sub-types, which are classified into 7 groups, as shown below. TRα/β, thyroid receptors; RAR, retinoic acid receptors; PPAR, peroxisome proliferator activated receptors; Rev-erbs, reverse strand ERBA oncogene; ROR, RAR-related orphan receptor; LXR, liver X receptor; FXR, farnesoid X receptor, VDR, vitamin D receptor; PXR, photocell receptor specific nuclear receptor; CAR, constitutive androstane receptor; HNF, hepatocyte nuclear factor; RXR, retinoid X receptor; TR2/4, testicular receptors ; TLX, Tailless gene homology; COUPTF, chicken ovalbumin upstream promoter transcription factor; EAR, erbA-related gene; ER, estrogen receptors, ERR, estrogen-related receptors; GR, glucocorticoid receptor; MR, mineralocorticoid receptor; PR, progesterone receptor; AR, androgen receptor; NGF1B, nerve growth factor-induced gene; NURR, nuclear receptor related 1; NOR, neuron-derived orphan receptor; SF-1, steroidogenic factor 1; LRH-1, liver receptor homolog 1; GCNF, germ cell nuclear factor; DAX1, dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1; SHP, short heterodimer partner.

CLASS I	CLASS II	CLASS III	CLASS IV	CLASS V	CLASS VI	CLASS 0
TRα/β	HNF4s	ERs	NGF1B	SF-1	GCNF	DAX1
RARs	RXRs	ERRs	NURR1	LRH-1		SHP
PPARs	TR2/4	GR	NOR1
REV-ERBs	TLX COUPTFs	MR PR
RORs	EAR2	AR
LXRs
FXRs
VDR
PXR
CAR

While many more nuclear receptors are found across different species, there are 48 mammalian nuclear receptors which are ubiquitously or specifically expressed in different organs or cell types [23-27]. These receptors exert transcriptional control over multiple key aspects of human physiology including reproduction, metabolism and immunity. Perturbations in nuclear receptor signaling underlie major health problems including post-menopause complications, metabolic syndrome, cardiovascular dysfunction and cancer. Due to their characteristic ligand binding domain for which synthetic modulators can be potentially designed, nuclear receptors remain an attractive pharmacological target in diverse therapeutic disciplines.

The role of nuclear receptors in heart and vascular physiology as well as cardiac myopathies and atherosclerosis has been well-studied and extensively reviewed [28-30]. Surprisingly, nuclear receptors have not been extensively explored as a therapeutic target in PAD. While vascular insufficiency and ischemia are triggers of PAD, this initial insult is often followed by inflammation, metabolic and mitochondrial dysfunction, and myofibrillar damage in the limb musculature distal to the occlusion site [9-13]. Here, we describe the importance of investigating nuclear receptors as potential therapeutic targets in PAD.

Skeletal muscle is the major tissue affected by PAD, and is subjected to chronic vascular insufficiency/ischemia leading to tissue damage, mitochondrial damage, metabolic dysfunction, muscle wasting, and functional impairment (Figure 2A). Therefore, the central emphasis of this review is on nuclear receptor signaling and effects within the skeletal muscle that might be beneficial in combating the common PAD pathology. We highlight nuclear receptors that are involved in orchestration of paracrine angiogenesis, mitochondrial function and myocellular regeneration in the skeletal muscle, which could contribute to the resolution of ischemic muscle damage in PAD (Figure 2B). Furthermore, we identify gaps and potential new directions for nuclear receptor signaling that need further exploration for advancing therapeutics in PAD and related myopathies.

Figure 2. Potential therapeutic effects of nuclear receptor activation in PAD-related myopathy.

(A) Limb pathology in PAD resulting from vascular occlusion and insufficient perfusion to the limb musculature. (B) Summary of potential mechanisms though which NR signaling can stimulate recovery in PAD-related myopathy. In addition to myocellular signaling, nuclear receptors could orchestrate ischemic muscle repair through non-muscle cells such as endothelial progenitor cells (EPCs), endothelial cells (ECs), macrophages and fibro-adipogenic progenitors (FAPs).

Paracrine Angiogenesis.

Skeletal muscle controls its own vascularization in a paracrine fashion by secreting angiogenic factors [31, 32]. Under physiological conditions, oxidative mitochondria-rich muscle fibers are densely vascularized due to higher expression and secretion of angiogenic factors by these myofibers [33]. Exercise can also promote myofiber secretion of angiogenic factors leading to paracrine muscle angiogenesis and vascularization [31, 32]. While much remains to be decoded regarding the transcriptional regulation of paracrine angiogenic programming in the skeletal muscle, several nuclear receptors have been identified that regulate muscle neo-angiogenesis.

Estrogen-related receptors (ERRs):

Direct evidence for paracrine regulation of angiogenesis via muscle nuclear receptor signaling arises from studies on two subtypes of orphan estrogen-related receptors (ERR) – ERRα and ERRγ. In in vitro co-culture studies, ERRs induce the mRNA and protein expression of Vascular endothelial growth factor A (VEGFA) in myotubes, which is a secretory growth factor that activates angiogenesis in endothelial cells [34, 35]. Muscle-specific overexpression in mice of either receptor increases baseline vascularization, as well as enhances ischemic neo-angiogenesis in a murine model of hindlimb ischemia [36]. In addition, muscle-specific ERRγ overexpression prevents diabetes-induced capillary regression in dB/dB mice [37]. Genome-wide expression studies suggest that ERRs induce a plethora of pro-angiogenic genes, activating a comprehensive angiogenic program in the skeletal muscle that may facilitate formation of functional blood vessels via paracrine regulation [36, 38]. While, the interpretation of these studies is based on ectopic overexpression/transgenic models, further elucidation of the role of endogenous ERRs in angiogenic gene program and muscle paracrine angiogenesis is needed, and will require use of muscle-specific single and/or compound ERRα/γ knockout mice. Notably, global ERRα knockout mice fail to undergo exercise-induced muscle angiogenesis based on CD31 capillary staining [39]. Lastly, the effect of activating ERR program in post-ischemic fashion either through pharmacology or intramuscular gene therapy needs to be explored for therapeutic consideration.

Peroxisome proliferator activator receptors (PPARs):

PPARs first emerged in prominence due to their multi-tissue role in energy homeostasis, via regulation of fatty acid synthesis, storage and metabolism [40]. Studies using pharmacological agonists suggest that PPAR’s can regulate angiogenic gene expression and vascularization through local effects in the skeletal muscle. PPAR agonists including cilostazol, GW0742, pioglitazone, pemafibrate and fenofibrate induce ischemic angiogenesis and/or prevent capillary regression in rodent models of hindlimb ischemia and diabetes, co-relating with induction of VEGFA and endothelial nitric oxide synthase (eNOS) in the ischemic muscle tissue [41-47]. However, the extent to which skeletal muscle PPARs contribute to the pharmacological actions of these agents remains to be determined, as studies have demonstrated the role of endothelial and endothelial progenitor PPARs in the regulation of angiogenesis [48-50]. New studies using muscle-specific PPAR transgenic mice [51-53] are necessary to understand the precise molecular mechanisms involving PPAR agonist effects in the skeletal muscle.

Steroid receptors:

Steroid hormones and their receptors have also been implicated in the regulation of skeletal muscle angiogenesis. 5α-dihydroxytestosterone promotes ischemic revascularization in the ischemic hindlimb tissue in castrated wild type mice, an effect which is blunted in androgen receptor (AR) global knockout (KO) mice [54]. The beneficial effect of 5α-dihydrotestosterone is linked to activation of hypoxia inducible factor 1A (HIF1A) in the ischemic muscle, as well as mobilization of bone marrow derived progenitors. Another study demonstrated decreased baseline capillarity in AR KO mice, as well as impaired revascularization in hindlimb ischemia leading to auto-amputation in both male and female mice [55]. In this study however, AKT signaling and not HIF1A was found to be the mediator of AR effects. A part of the androgen action on ischemic revascularization may also be dependent on recruitment of bone marrow-derived progenitor cells to the site of ischemia and stimulation of vasculogenesis [56]. In addition to androgens, estrogen or estrogen mimetic agents have also been shown to promote ischemic angiogenesis in the skeletal muscle. Studies using ovariectomized female mice or rabbits show that ischemic revascularization is impaired by estrogen deficiency, and can be restored by supplementation with estrogen or other compounds such as Aucubin with estrogenic properties [57-59]. Notably, in the Aucubin study, its effects were blunted in estrogen receptor β (ERβ) knockout mice [60]. Ischemic neo-angiogenesis by estrogen activation correlated with the activation of AKT, eNOS and/or VEGFA pathway in the skeletal muscle. As with PPARs, the precise contribution of skeletal muscle AR and ER via transcriptional signaling remains to be determined. Furthermore, the detailed molecular and transcriptional mechanisms through which muscle steroid receptor signaling control functional angiogenesis, beyond activation of VEGFA and eNOS also remain unclear. Generally, the possibility of aging and/or menopause-related decline in steroid hormones as a contributing factor to development of PAD-related pathology in the skeletal muscle remains to be definitively explored.

In summary, several nuclear receptors and their ligands support neo-angiogenesis in the ischemic skeletal muscle. However, a direct evidence for paracrine angiogenesis via myocellular regulation of angiogenic factor expression has been reported only for ERRα and ERRγ, and for their coregulators peroxisome proliferator activator receptor co-activator α and β (PGC1α and PGC1β) [34, 61]. In-depth studies are warranted using conditional genetic mouse models in conjunction with transcriptome analysis to fully define skeletal muscle receptor circuitry involved in the regulation of myocellular angiogenic factors and paracrine angiogenesis.

Mitochondrial dysfunction.

Clinical as well as pre-clinical studies have identified impairment in mitochondrial content and function following vascular insufficiency/ischemia as key contributing factors to PAD-associated myopathy [62-70]. In PAD patients, severity of the disease is linked to decreased mitochondrial copy number, suppressed mitochondrial respiratory capacity, increased reactive oxygen species (ROS) production, as well as decreased calcium retention capacity (which is associated with apoptotic cell death) in skeletal muscle biopsies. The mitochondrial defects are recapitulated in pre-clinical mouse models of ischemia or ischemia/reperfusion injury [71, 72]. In addition, perturbations in the proportions of oxidative (mitochondria-rich) vs. glycolytic (sparse mitochondria) myofibers have been reported in PAD skeletal muscles [73], potentially contributing to overall decrease in oxidative capacity and muscle function. Nuclear receptors have been extensively studied in the regulation of skeletal muscle fiber type, mitochondrial function and endurance in context of sedentary and exercise-activated states. However, not many studies have examined nuclear receptor-mediated restoration of mitochondrial health and oxidative function in models of skeletal muscle ischemic injury. We highlight key nuclear receptor sub-groups and their metabolic effects in the skeletal muscle, that are likely to be beneficial in PAD-related ischemia, and warrant further investigation in ischemic injury.

ERRs.

ERRα is expressed uniformly and abundant in all the skeletal muscle. ERRγ is also expressed in all muscles, but its expression is much higher in oxidative slow-twitch than glycolytic fast-switch myofibers [74, 75]. ERRα global KO mice have a decreased gene expression of components of oxidative phosphorylation, tricarboxylic acid cycle (TCA), pyruvate dehydrogenase complex (PDH), and fatty acid oxidation (FAO) in the skeletal muscle. ERRα KO also show an increase in respiratory exchange ratio (RER), impaired TCA cycle, increased exercise-induced lactate accumulation, and decreased spontaneous physical activity and capacity for exercise [76]. While these studies support a role for ERRα in muscle oxidative capacity, the universal loss of ERRα in all tissues confounds interpretation regarding muscle-specific role, and therefore studies using conditional deletion of ERRα are warranted. Nevertheless, gain-of-function muscle-specific overexpression studies support the role of ERRα in the regulation of oxidative type IIA and IIX myofibers, as well as gene expression of molecules linked with oxidative metabolism of fatty acids [38]. Whether overexpression of ERRα in skeletal muscle improves endurance, exercise tolerance and energy expenditure at the level of myofibers and whole-body remains to be investigated. Likewise, evidence for the regulation of mitochondrial size and number by ERRα in the skeletal muscle is still lacking, despite indications from gene expression analysis.

Insights into muscle ERRγ function were gained from transgenic mice that either over-express VP16-ERRγ or apo-ERRγ, or lack ERRβ and ERRγ specifically in skeletal muscle [74, 75, 77]. Overexpression of ERRγ increases mitochondrial size and content, triggers a fiber type shift towards oxidative fatigue resistant myofibers (with prominent increases in oxidative/glycolytic fast type IIA and oxidative/glycolytic fast type IIX, and decreases in glycolytic fast type IIB myofibers), and improves exercise tolerance [36, 74, 75, 78]. ERRγ transgenic mice also show increased whole-body oxygen consumption, particularly a higher peak VO2 during running, and decreased RER, consistent with increased energy expenditure and a shift from carbohydrate to lipid oxidative metabolism. Conversely, mice lacking ERRγ (along with ERRβ) in skeletal muscle show some of the opposite effects, such as loss of a type I fibers and type I contractile gene expression, and decreased endurance capacity [77]. These effects may be predominantly due to loss of ERRγ because ERRβ expression is undetectable in the skeletal muscle. At the gene expression level, ERRγ transgenic mice have higher levels of genes linked to oxidative phosphorylation, FAO, TCA cycle, and type I contractile program [74, 75]. Many of these genes are direct ERRγ targets, based on genomic binding data of ERRγ in heart [79]. However, genome-wide binding studies have yet to be performed for ERRs in the skeletal muscle. ERRs can also affect oxidative metabolic gene program indirectly, via the induction of microRNAs (miR-499 and miR-208b) and Perm1 (a regulator of mitochondrial biogenesis and oxidative capacity in skeletal muscle), and the activation of AMPK [75, 77, 80, 81].

PPARs.

PPARβ/δ is the highly expressed PPAR isoform in the skeletal muscle [53]. PPARβ/δ is preferentially expressed in the oxidative myofibers, and is induced by exercise [51, 53]. Muscle-specific over-expression of either apo-PPARβ/δ or VP16-PPARβ/δ (constitutively active receptor variant that is tagged with the VP16 viral transactivation domain) induces genes linked to glucose utilization (e.g. Glut4, Ldhb), fatty acid oxidation (Cpt1b) and oxidative phosphorylation (Cycs, CoxII, Cox IV), and increases mitochondrial content [51, 53, 82]. PPARβ/δ also drives fiber type switching to the more oxidative, mitochondria-rich and fatigue-resistant type I myofibers [51, 53]. These PPARβ/δ-driven adaptations impact both muscle performance and energy balance. Muscle-specific PPARβ/δ transgenic mice show increased tolerance for endurance as well as sprint exercise [53, 82]. Activation of endogenous PPARβ/δ by its pharmacological agonist GW1516 synergizes with exercise training to enhance responses to exercise (i.e. increases in oxidative metabolism genes and in endurance running capacity), via interactions between PPARβ/δ and exercise-activated AMPK signaling [83]. PPARβ/δ muscle transgenic mice also show increased energy expenditure, resistance to obesity, and insulin sensitization [51, 53, 82]. Conversely, muscle-specific deletion of PPARβ/δ in skeletal muscle decreases the oxidative myofiber proportion, suppresses the expression of fatty acid transport proteins (e.g. Lpl, Cd36, Fabp3), fatty acid oxidation enzymes (e.g. Lcad, Mcad), TCA cycle genes, uncoupling proteins (e.g. Ucp3), as well as nuclear and mitochondria-encoded respiratory chain complex proteins. Muscle PPARβ/δ deficiency also impairs exercise performance, insulin sensitivity, glucose homeostasis and promotes obesity [52, 53]. Some of the effects of PPARβ/δ are via direct activation of target genes with characterized PPAR response elements (PPREs) [84]. At least part of the effects of PPARβ/δ are mediated via other transcription factors, such as PGC1α, which is known to activate mitochondrial biogenesis; myocyte enhancing factor 2C (MEF2C), which drives Glut4 expression; and ERRγ, which enhances mitochondrial oxidative phosphorylation capacity and induces miR-208b and miR-499 (regulators of the type I contractile program) expression [52, 77, 82].

Similar to PPARβ/δ, PPARα overexpression in skeletal muscle activates genes involved in fatty acid import, fatty acid binding and FAO, as well as genes linked with mitochondrial TCA cycle, oxidative phosphorylation and uncoupling, and protects from high fat diet induced obesity [85]. In contrast to PPARβ/δ, PPARα represses glucose uptake (Glut4) and glycolysis (Pfk) genes, thus leading to a selective increase in lipid oxidation. PPARα transgenic mice also exhibit repression of type I contractile gene program, diminished capacity for high intensity sprint running, and high fat diet-induced glucose intolerance [77, 82, 85]. These studies suggest that while activation of PPARα in skeletal muscle may protect from obesity, the net effect on overall health maybe harmful, compromising exercise capacity and glucose homeostasis. These observations regarding PPARα are important particularly in the context of aforementioned studies where PPARα agonist improved ischemic angiogenesis in the skeletal muscle.

Compared to PPARα and PPARβ/δ, the regulatory control of metabolism by PPARγ in the skeletal muscle, where the receptor is sparsely expressed, is incompletely understood. Consistent with its role in adipose tissue, PPARγ activation in muscle may increase gene expression of molecules involved in fatty acid synthesis, enabling storage and sequestration of lipids [86, 87]. PPARγ may also promote glycogen storage [88]. Muscle-specific deletion of PPARγ results in increased adiposity, whole body triglyceride accumulation, and glucose intolerance, via poorly understood mechanisms [86, 87]; but showing that sparsely expressed PPARγ may still be important for muscle homeostasis.

Rev-erbs:

Rev-erbα is highly expressed in oxidative compared to glycolytic muscle, and is induced by exercise [89]. Rev-erbα knockout mice show decreased skeletal muscle mitochondrial content and oxidative function, decreased capacity for exercise, and decreased maximal oxidative capacity while running. The defects are partly attributed to the reduced expression of mitochondrial biogenesis factors [Ppargc1a, Ppara, Tfam and Nrf1] and partly to the stimulation of expression of autophagy and mitophagy genes [Ulk1, Beclin 1, Atg5 and Bnip3, Ctsl and Park2]. The mechanism by which Rev-erbα, a constitutive repressor, enhances the expression of mitochondrial biogenesis factors is not clear, but seems to be indirect, via modulation of the LKB1-AMPK-SIRT1-PGC1α pathway [89]. In contrast, the effects of Rev-erbα on the autophagy genes seem to be direct, as Rev-erbα binds regulatory sites in several of them, and represses their expression [89]. Notably, activation of Rev-erbα [via overexpression in C2C12 cells, AAV (adeno-associated virus)-mediated expression in skeletal muscle, or treatment with Rev-erbα/β agonists] leads to effects opposite from those seen in Rev-erbα KO mice, such as increased oxidative and exercise capacity [89]. The Rev-erbα/β agonists (SR9009, SR9011) administered in vivo also leads to increased whole-body energy expenditure, protection from diet-induced obesity, and decreased plasma lipids [90]. The extent to which the Rev-erbα/β agonist acts via muscle Rev-erbs or in other tissues is currently unclear. It is also unclear whether SR9009 acts through on-target mechanisms, as its specificity has been called into question [91]. The physiological role of muscle Rev-erbβ, which controls gene expression of molecules linked to fatty acid uptake (Fabp3 and 4, Fat/Cd36) and FAO (Cpt1, Mcad1, Acs4, Ucp3) [92, 93], remains to be systematically studied.

Nuclear Receptor 4A sub-types (NR4As):

All three NR4A sub-types are expressed in skeletal muscle and induced by either beta-adrenergic stimulation or exercise [94-98]. Nur77 is preferentially expressed in the glycolytic fast-twitch muscles; and its expression is dependent on intact muscle innervation [99]. Nur77 is necessary for mediating neuronal stimulation of glucose utilization genes in the skeletal muscle, including genes involved in glucose uptake (Glut4), glycolysis (Pfkm, Pgam2, Bpgm), glycerophosphate shuttle (Gpd1) and glycogenolysis (Phkg1, Pygm) – which is the primary fuel source used by the glycolytic fast-twitch muscles [99]. Global Nur77 null mice are susceptible to diet-induced weight gain, insulin resistance and glucose intolerance, which is partly linked to impaired glucose utilization in the skeletal muscle of the mutant mice [100]. Interestingly, in a subsequent study Chao et al. found that muscle-specific overexpression of Nur77 can induce an oxidative slow-twitch program, involving induction of slow-twitch myofiber type markers (Myh7, TnI1), mitochondrial content, mitochondrial respiration and oxidative metabolism [succinate dehydrogenase (SDH), nicotine adenine dinucleotide dehydrogenase (NADH), cytochrome c oxidase], and repression of pyruvate dehydrogenase (PDH) complex (limiting glucose oxidation) (79). In this model, glucose utilization genes were only moderately stimulated by Nur77 overexpression. Muscle-specific Nur77 overexpression improved muscle fatigue resistance in muscle contractility assays as well as grip strength; however, it failed to protect against diet-induced glucose intolerance, suggesting that muscle Nur77 signaling may not be sufficient for maintaining whole-body insulin and glucose homeostasis in the scenario of high fat consumption.

Similar to Nur77, Nor1 positively regulates gene expression of molecules involved in both glucose and fatty acid metabolism, and enhances mitochondrial biogenesis and oxidative respiratory capacity in the skeletal muscle [101]. In terms of skeletal muscle myofiber composition, Nor1 induces type IIA and IIX myofibers (representing myofibers with both glycolytic and oxidative capacities), suppresses the glycolytic type IIB myofibers, without changing the oxidative type I myofiber, perhaps explaining the dual regulation of glycolytic and oxidative genes by this receptor. Nor1-mediated remodeling increases glucose tolerance, oxygen consumption and exercise endurance, as well as decreases adiposity, and protects against diet-induced obesity and hyperglycemia in mice [102].

A recent study has examined the muscle-specific role of Nurr1 in metabolic homeostasis. Genetic or pharmacological activation of Nurr1 induces type I myofiber and glucose metabolism genes, mildly increased mitochondria copy number, and leads to an increase in exercise tolerance and protects against diet-induced obesity and weight gain [103]. Overall, NR4A’s exert overlapping regulation of various aspects of muscle energy expenditure, including glucose and fat metabolism, as well as mitochondrial biogenesis and oxidative capacity. NR4As are potentially mediating the effects of neuronal/beta-adrenergic stimulation or exercise on muscle energy expenditure; however, the extent to which these receptors act as effectors to these stimuli will require the development of single or compound muscle-specific knockout mice.

Steroid Receptors:

Steroid receptors have been extensively studied in the skeletal muscle metabolic homeostasis, and consideration of all the receptors and associated studies is beyond the scope of this review. Therefore, we discuss key recent studies to highlight the role of muscle steroid receptors as it may relate to muscle ischemic recovery. Pharmacological administration of testosterone elevates heat production, increases oxidative biomarker expression and mitochondrial biogenesis in the skeletal muscle. Converse effects are observed in the skeletal muscle from AR KO mice [104]. Skeletal muscles from AR KO mice also exhibit fiber type switch from type II to I, as well as decreased muscle mass and decreased force production. These effects seem to be selective in the male mice. In another study, mice with orchidectomy-induced androgen deficiency as well as AR KO mice exhibit metabolic dysfunction and susceptibility to diet-induced obesity [104], suggesting that androgen deficiency could potentially contribute to the development of diabetes-associated PAD.

Studies using short-term ovariectomy along with estrogen replacement therapy showed that estrogen signaling is involved in mitochondrial respiratory function, through the regulation of mitochondrial membrane microviscocity and H2O2 emission potential, as well as complex I and I + III activities [105]. ERα global knockout female mice exhibit reduced energy expenditure and glucose intolerance, along with insulin resistance, lipid accumulation and decreased biomarkers of oxidative metabolism in the skeletal muscle [106]. Muscle-specific ERα knockout female mice exhibited decreased oxidative metabolism and lipid accumulation, associated with abnormal mitochondrial morphology, impaired mitochondrial fission dynamics, impaired turnover of mitochondrial DNA abundance, and increased production of reactive oxygen species [107]. In another study, muscle-specific deletion of ERα in female mice resulted in age-dependent decline in muscle mass, impairment in muscle strength and various contractile properties such as eccentric, sub-maximal and maximal isometric force, as well as fatigability and force recovery [108]. In contrast, ERα seems dispensable in post-natal high fat diet studies. Inducible deletion of ERα in adult mice on high fat diet did not affect body composition, VO2, VCO2, RER and energy expenditure. Mitochondrial respiratory capacity and oxidative phosphorylation was not affected by inducible ERα deletion in the skeletal muscle [109]. Nevertheless, the overall importance of estrogen signaling in regulation of mitochondrial metabolism and energy homeostasis in part through effects in the skeletal muscle are established.

In summary, steroid and orphan nuclear receptors control multiple aspects of metabolism including glucose and fatty acid metabolism, along with the regulation of mitochondrial homeostasis in the skeletal muscle. While discussion of all steroid receptor pathways is beyond the scope of this review; several other steroid hormones (e.g. thyroid, vitamin A, vitamin D) have direct or indirect effects on skeletal muscle metabolic homeostasis. Even though enhanced anaerobic glucose metabolism is critical in early stages of muscle ischemic damage for energy production, eventual recovery from ischemic muscle damage would require restoration of mitochondria and oxidative capacity. Therefore, it will be interesting to explore the temporal role of different nuclear receptors in the post-ischemic metabolic recovery of the skeletal muscle.

Regulation of skeletal muscle mass and regeneration.

Skeletal muscle regeneration after ischemic injury is the most understudied aspect of PAD. Most studies that examine therapeutic angiogenesis have typically measured vascular regeneration, but not necessarily muscle repair, which would be a critical aspect for functional recovery in PAD. Conversely, the vast majority of the mechanistic pathway studies on muscle regeneration have been performed using cardiotoxin or BaCl₂-mediated muscle injury in mice or pre-clinical muscular dystrophy models. Using these models, several nuclear receptors have been identified to play a role in muscle regeneration. However, the orchestration of muscle regeneration by nuclear receptors (through effects in different cell types including muscle stem cells, myofibers and immune cells) after ischemic injury have not been investigated. We have summarized below the current understanding of how nuclear receptors may regulate muscle regeneration, which could be extrapolated to ischemic muscle injury.

ERRs:

Experiments in C2C12 myoblasts or primary myocytes using overexpression, knockdown, or knockout strategies show that ERRα/γ are involved in myocyte differentiation [110, 111]. In case of ERRα, a similar inhibitory effect on myogenesis is observed with an inverse agonist/inhibitor [111], suggestive of a potential for pharmacological regulation of ERRs. Whereas impaired regeneration in ERRα-deficient myocytes is linked to decreased ERK signaling, ERRγ-deficient myocytes have reduced mitochondrial content, an imbalance in glucose vs. fat metabolism, increasing ROS production, and activation of catabolic factors, such as Foxhead Box O (FoxO) and nuclear factor-kappa B (NF-κB). Loss-of-function studies using muscle-specific ERRα knockout mice have demonstrated impaired muscle regeneration in response to cardiotoxin-induced muscle injury [112]. While loss of function studies in mice have not been performed for ERRγ, muscle-specific transgenic overexpression studies suggest that ERRγ can promote muscle recovery in models of muscular dystrophy and ischemia [36, 78]. Although these studies indicate a role for ERRs in muscle regeneration, additional in-depth studies are warranted to examine the expression as well as role of ERRs in satellite cell regulation, and the mechanisms through which ERRs promote myocellular regeneration in ischemia.

PPARs:

Amongst the three PPARs, only PPARβ/δ has so far been associated with the regulation of skeletal muscle mass and regeneration. Global PPARβ/δ knockout mice exhibit reduced muscle mass, myofiber atrophy, co-relating with decreased satellite cell number. These knockout mice also exhibit impaired skeletal muscle regeneration in response to myotoxin-induced injury. These deficiencies in the PPARβ/δ KO mice were associated with increased expression of myostatin, a muscle atrophy factor [113]. Observations in the global knockout mice were further corroborated by findings in mice with Myf5+ muscle progenitor specific PPARβ/δ knockout mice. These mice not only developed metabolic syndrome, but exhibit impaired satellite cell function and muscle regeneration [114]. Pharmacological activation of PPARβ/δ stimulates myofiber repair and restoration of mitochondrial integrity in mdx mice [115]. In fact, the potential role of mitochondrial metabolism in PPARβ/δ-mediated repair in dystrophic muscle has been supported by other studies [116, 117].

NR4As:

NR4A1 is reported to be involved in the regulation of skeletal muscle size. NR4A1 mRNA, as well as myonuclear protein expression is induced upon differentiation of C2C12 myoblasts into myotubes. While NR4A1 is undetectable in quiescent satellite cells, it is induced upon stem cell activation and differentiation, correlating with the expression of classical myogenic markers [118]. In concert with this report, another study found that global or skeletal muscle-specific inducible NR4A1 knockout mice exhibit reduced muscle mass as well as myofiber size, suggesting a role for NR4A1 in the regulation of muscle mass at developmental and post-natal levels. The positive regulation of muscle mass by NR4A1 is linked to repression of muscle atrophy genes (e.g. Smad2 and FoxO3) [119]. NR4A2 and NR4A3 are expressed in the skeletal muscle; however, their effect on muscle regeneration has not been yet examined using genetic mouse models.

Rev-Erbs:

Multiple studies suggest that Rev-erbα is a negative regulator of muscle regeneration. Initial studies suggested that Rev-erbα represses the proliferation and differentiation of cultured myogenic precursor cells [120, 121]. Animal studies using Rev-erbα agonist and antagonist as well as muscle-specific knockout demonstrated that Rev-erbα likely plays a role in suppression of satellite cell proliferation and expansion, as well as regeneration. Mechanistically, Rev-erbα may target proliferative pathways, as well as Wnt signaling in the skeletal muscle [122]. In another study, Rev-erbα antagonist SR8278 stimulated muscle regeneration in an acute injury model, increasing muscle mass and muscle function, as well as decreasing muscle fibrosis and muscle protein degradation. Indeed, Rev-erbα antagonism works through increasing satellite cell pool via Notch and Wnt signaling. Moreover, Rev-erbα functions as a transcriptional repressor at cell fate regulatory sites (NF-Y binding sites). Differentiation cue results in the release of Rev-erbα mediated repression at promoters and enhancers of a number of myogenic genes [123]. Subsequent studies have shown that Rev-erb antagonist SR8278 mitigates dystrophic damage by increasing lean mass and muscle function, as well as by decreasing fibrosis and protein degradation [124]. These pharmacology studies are supported by genetic deletion of Rev-erbα in dystrophic mice, where loss of Rev-erbα results in the activation of proliferative pathways and Wnt signaling, increased myogenesis, and decreased inflammation resulting in overall mitigating dystrophic muscle damage [125].

Steroid receptors.

Steroid receptors including androgen, estrogen and thyroid hormone receptors are involved in the regulation of skeletal muscle mass or regeneration. Satellite cell-specific deletion of androgen receptor resulted in the loss of muscle mass mainly associated with decrease in type IIA myofibers, leading to a decrease in grip strength. Curiously, androgen induces myostatin in the skeletal muscle and consequently the anabolic effect of androgen is even more pronounced in myostatin knockout mice [126]. Additional studies using androgen receptor agonist or androgen deficient mice support the role of androgen signaling in muscle regeneration via satellite cell activation. Molecular gene expression studies suggest that androgen effect on satellite cell activation is mediated through induction of follistatin (FST), Insulin-like growth factor 1 (IGF-1), C-X-C chemokine receptor 4 (CXCR4), hepatocyte growth factor (HGF), and glucocorticoid receptor (GR) [127].

Studies using 17β-estradiol, ERα/ERβ selective agonist, ovariectomized mice, as well as ERα and ERβ knockout mice have identified a role for ERβ in the regulation of skeletal muscle growth, particularly through the induction of IGF-1, potentially through satellite cell activation, and suppression of immune response. In ovariectomized mice, 17β-estradiol can also restore muscle contractile function, as well as increase satellite cell number in female mice [128]. These studies are further supported by the findings using muscle-specific and satellite cell-specific ERβ knockout mice [129]. Female mice displayed loss of fast-type muscle mass. These mice also exhibited decrease in regenerative capacity resulting from proliferation defect and apoptosis in satellite cells. RNA-sequencing revealed that loss of ERβ alters gene expression of extracellular matrix components, includes laminin and collagen. This study suggests a sex specific regulation of muscle growth associated with ERβ [130]. 17β-estradiol also prevented muscle loss in tumor bearing female mice, through AMPK-mTORC1 pathway. Thyroid hormone and its receptors are also involved in skeletal muscle homeostasis. Thyroid hormone resistant mice exhibit a decrease in muscle mass, along with depletion of satellite cells. Satellite cells from these mice were also defective in proliferation and differentiation, and these mice have impaired regenerative capacity in response to chemical injury [131]. Pharmacological studies using T3, along with TRα and TRβ knockout mice demonstrate that TRα but not TRβ are involved in muscle precursor cell proliferation and differentiation leading to stimulation of muscle regeneration [132]. Glucocorticoids are typically associated with muscle wasting, however a recent study showed that an intermittent treatment regimen with glucocorticoids is devoid of muscle wasting effects, and instead results in a pro-regenerative response through a direct activation of muscle repair genes such as Annexins 1 and 2 [133].

These studies show that in addition to regulation of muscle angiogenesis and metabolism, nuclear receptors expressed in the skeletal muscle could be instrumental in driving muscle regeneration after ischemic injury. However, potential role of the majority of these receptors in promoting ischemic regeneration through signaling in the muscle cells remain to be explored and will be an important area for research in the future.

Extra-muscular effects of nuclear receptors

Nuclear receptors in non-muscle cells are also likely to contribute to orchestration of ischemic muscle recovery in PAD. Briefly, nuclear receptors expressed in endothelial cells (e.g. PPARs) can regulate angiogenesis [134], and therefore promote revascularization of ischemic muscle. Furthermore, nuclear receptor regulation of immune cells (e.g. macrophages) contributes to inflammatory remodeling and muscle regeneration, and are likely to play a role in ischemic resolution [135-137]. Other cell types (e.g. pericytes, FAPs) may also express nuclear receptors and contribute to regulating muscle repair.

Unexplored areas for NR signaling in skeletal muscle biology

Substantial research has been conducted on nuclear receptor function in skeletal muscle homeostasis using natural hormone ligands, pharmacological agents as well as global and muscle-specific transgenic mice in combination with classical muscle biology to measure histological changes, gene expression changes, as well as impact on muscle fitness and metabolism. While steroid hormones and their receptors have been established as major regulators in the skeletal muscle, orphan nuclear receptors are emerging as equally important modulators of muscle function. However, several areas of research are wide open that need to be undertaken to not only advance our understanding of nuclear receptor signaling in the skeletal muscle, but also to appraise the therapeutic potential of these receptors in PAD as well as other myopathies. First, several of the aforementioned studies have employed microarray and recently RNA sequencing analysis to measure global impact of nuclear receptor modulation on myocellular gene expression. However, much remains unknown about transcriptional mechanisms through which nuclear receptors directly or indirectly regulate gene expression at genome-wide level in myocytes. High-throughput transcriptomics (e.g. ChIP-seq, ATAC-seq) and epigenetics [138] in skeletal muscles for candidate nuclear receptors at physiological and pathophysiological ischemic conditions will provide insights into nuclear receptor transcriptional regulation in the skeletal muscle. Furthermore, single cell sequencing approach to measure the impact of nuclear receptor modulation on gene expression simultaneously in muscle and resident non-muscle cells (e.g. endothelial cells, macrophages, pericytes, faps) will also provide important insights into the impact of nuclear receptors on muscle function and recovery from injury. Second, nuclear receptor transcriptional regulation is controlled by a plethora of transcriptional coregulators. While some of these cofactors such as PGCs, Nuclear receptor co-repressors (NCoRs) and RIP140 have been individually studied in the skeletal muscle [138-142], their interaction with different nuclear receptors, as well as genome-wide interaction at conserved nuclear receptor response elements in muscle cells remain unexplored. The composition of major nuclear receptor transcriptional complexes in the skeletal muscle and their impact on myocellular function are also unknown. Investigations in these directions, particularly in the context of hypoxia, nutrient deprivation or ischemia will provide important insights into the transcriptional regulation by nuclear receptors in the skeletal muscle in normal and pathological conditions related to PAD.

At a more fundamental level, the role of nuclear receptors in several skeletal muscle-linked cellular processes remains less well-defined. Such cellular processes include mitochondrial dynamics (fission, fusion, autophagy), protein homeostasis (protein synthesis and degradation), calcium homeostasis, redox regulation, as well as contractile apparatus regulation. Similarly, as mentioned above regulation of muscle regeneration by nuclear receptor signaling in important non-muscle cell types such as resident immune cells, endothelial cells, fibro-adipogenic progenitors, as well as in muscle stem cells would be another important area of inquiry. Each of these processes or cell types are likely to be perturbed by ischemic muscle injury in PAD. Study of the regulation of aforementioned processes and cell types by nuclear receptors will provide a comprehensive picture of nuclear hormone receptor function in skeletal muscle homeostasis and in resolution of ischemia in PAD.

Summary and Scope in PAD

The potential ameliorative function of nuclear receptor signaling in skeletal muscle ischemia remains understudied, thus presenting opportunity to explore these receptors as potential therapeutic candidates in PAD. Receptors belonging to PPAR, ERR, Rev-erb and NR4A sub-families are particularly well-poised for in-depth exploration in models of PAD for their effects on muscle revascularization, metabolism and regeneration. Furthermore, given the ubiquitous nature of nuclear receptor expression, signaling through other cell types such as macrophages, endothelial cells, and muscle stem cells are likely to contribute to ischemic muscle recovery, and therefore critical for PAD-related studies. The relative contribution from each of these cell types will require use of conditional overexpression or knockout strategies along with available pharmacological agents in models of skeletal muscle ischemia. For promising candidates, the studies need to be extended to ischemic injury models with chronic disease such as atherosclerosis and diabetes. Potential association between nuclear receptor expression or polymorphisms and PAD is poorly explored. We can across a single study associating PPARγ2 (Pro12Ala) polymorphism with PAD [143], suggesting that receptor expression or gene perturbation may be linked with PAD. Lastly, as nuclear receptors research in ischemia advances, particular focus is needed on delivering nuclear receptor therapeutics in PAD. While pharmacological agents for orphans as well as steroid receptors continue to be developed [144, 145] and nuclear receptors remain favorable pharmaceutical targets due to their unique ligand binding pockets, our current understanding of whether these agents are suitable therapeutics outweighing adverse effects in PAD is still in infancy. Alternatively, direct intramuscular delivery of candidate nuclear receptor genes at injury site in PAD can be envisioned to have lesser side effects. Notably, AAV delivery vectors have been extensively studied and optimized for gene delivery in muscular dystrophies [146, 147]. AAV9 strategies can be easily adapted for nuclear receptors in PAD. In this regard, we recently showed that AAV9-mediated ERRα delivery to murine skeletal muscle facilitates angiogenic gene expression and vascularization [148]. Intramuscular AAV9 gene delivery for ERRα and other nuclear receptors need to be further optimized in pre-clinical models of PAD. Lipid nanoparticle delivery platform has been developed [149, 150], which have potential in cardiovascular diseases and can be adapted for nuclear receptor gene delivery in PAD. To our knowledge, neither pharmacological nor gene delivery approaches have been extensively studied or advanced for nuclear receptors in ischemia and PAD, and is likely to become a major area of clinical exploration as nuclear receptors emerge as potential targets in PAD. In conclusion, our review highlights that comprehensive studies on nuclear receptors and ischemic muscle recovery are clearly warranted and should be undertaken to provide valuable insights into development of new pharmacological therapies for PAD.

ABBREVIATIONS

AR

Androgen receptor

AMPK

AMP-activated protein kinase

eNOS

Endothelial nitric oxide synthase

ER

Estrogen receptor

ERR

Estrogen-related receptors

FGF1

Fibroblast growth factor 1

FOXO

Foxhead box O

FAPS

Fibro-adipogenic progenitors

HIF1A

Hypoxia-inducible factor 1A

LKB1

Liver Kinase B1

MEF2C

Myocyte Enhancer Factor 2C

NR

Nuclear receptors

NF-κB

Nuclear factor-kappa B

NCoR

Nuclear receptor co-repressor

PAD

Peripheral arterial disease

PPAR

Peroxisome proliferator activator receptor

PGC

Peroxisome proliferator activated receptor co-activator

PERM1

PGC-1 And ERR-Induced Regulator In Muscle Protein 1

RER

Respiratory exchange ratio

SIRT1

Sirtuin 1

VEGF-A

Vascular endothelial growth factor-A