MODULATION OF ESTROGEN RECEPTOR-RELATED RECEPTOR GAMMA (ERR GAMMA) AND USES THEREFOR
This application provides methods of increasing vascularization, muscle performance, muscle rehabilitation, and/or mitochondrial activity in subjects in need thereof, by administering a therapeutically effective amount of one or more agents that increases ERRγ activity to the subject. Such agents can include one or more ERRγ agonists. In some examples the method does not require that the subject exercise, and as such, the subject may be sedentary (such as bedridden or in a wheelchair).
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This application is a continuation of U.S. application Ser. No. 14/597,390 filed Jan. 15, 2015, which is a divisional of U.S. application Ser. No. 13/410,142 filed Mar. 1, 2012, now U.S. Pat. No. 8,962,546, which claims priority to U.S. Provisional Application No. 61/447,704 filed Mar. 1, 2011, herein incorporated by reference.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORTThis invention was made with government support under AR053803-03 awarded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases; under HD027183 and DK057978 awarded by the National Institutes of Health; and under U19DK62434-01 awarded by the Nuclear Receptor Signaling Atlas. The government has certain rights in the invention.
FIELDThis application relates to methods of increasing vascularization, muscle performance, and/or mitochondrial activity in subjects in need thereof, by administering a therapeutically effective amount of one or more agents that increases ERRγ activity to the subject. In some examples the subject does not receive additional exercise, and as such, the subject may be sedentary (such as bedridden or in a wheelchair).
BACKGROUNDTissue vascular supply is tightly coupled to its oxidative capacity. This is especially evident in skeletal muscle beds, each enriched in either oxidative slow-twitch or glycolytic fast-twitch myofibers (Fluck and Hoppeler, 2003; Pette and Staron, 2000). Slow-twitch muscles are characterized by high mitochondrial content, fatigue resistant (type I) fibers and dense vascularity to ensure a steady and prolonged supply of oxygen and nutrients (Annex et al., 1998; Cherwek et al., 2000; Ripoll et al., 1979). Fast-twitch (type II) muscles generally have lower oxidative capacity, a reduced blood supply and are fatigue sensitive. How the type I vs. the type II muscle vasculature is specified to match oxidative capacity is unclear.
Previous studies established that nuclear receptors such as PPARα, PPARδ and ERRα along with co-regulators PGC1α, PGC1β and Rip140 control diverse aspects of aerobic respiration including fatty acid oxidation, oxidative phosphorylation and mitochondrial biogenesis in skeletal muscle (Arany et al., 2007; Huss et al., 2004; Lin et al., 2002; Minnich et al., 2001; Muoio et al., 2002; Seth et al., 2007; Wang et al., 2004). While signaling factors such as TGFβ1, platelet-derived growth factor, fibroblast growth factor (FGF) 1 and 2, and vascular endothelial growth factor (VEGF) are known to stimulate angiogenesis (Carmeliet, 2000; Ferrara and Kerbel, 2005; Gustafsson and Kraus, 2001), whether and how these factors orchestrate dense vascularization of aerobic muscles is unclear. One possibility is vascular arborization by co-activator PGC1α that is induced by hypoxia and exercise (Arany et al., 2008). However, PGC1α knockout mice are viable, still retain oxidative muscle, and have normal vasculature (Arany et al., 2008; (Lin et al., 2004). Since the intrinsic enrichment of blood flow to aerobic muscles in the absence of exercise is unlikely to depend on PGC1α induction, we speculate the existence of a novel regulatory angiogenic pathway.
Estrogen receptor-related receptor γ (ERRγ), like other members of the ERR subfamily, is a constitutively active orphan nuclear receptor, though unlike ERRα and β, it is more selectively expressed in metabolically active and highly vascularized tissues such as heart, kidney, brain and skeletal muscles (Giguere, 2008; Heard et al., 2000; Hong et al., 1999). In vitro data indicate that ERRγ activates genes such as PDK4 and MCAD that play a regulatory role in oxidative fat metabolism (Huss et al., 2002; Zhang et al., 2006). Furthermore, a comprehensive gene expression analysis identified ERRγ as a key regulator of multiple genes linked to both fatty acid oxidation and mitochondrial biogenesis in cardiac muscles (Alaynick et al., 2007; Dufour et al., 2007). Expression of ERRγ is also induced in variety of tumors with hyper-metabolic demands and abundant vasculature (Ariazi et al., 2002; Cheung et al., 2005; Gao et al., 2006).
SUMMARYThe ability of ERRγ in controlling the intrinsic angiogenic pathway in oxidative slow-twitch muscles is demonstrated herein. It is shown herein that ERRγ is exclusively and abundantly expressed in oxidative (type I) slow-twitch muscles. Transgenic expression of ERRγ in fast-twitch type II muscle triggered aerobic transformation, mitochondrial biogenesis, VEGF induction and robust myofibrillar vascularization, all in the absence of exercise. It was observed that these intrinsic effects of ERRγ do not depend on PGC1α induction, but rather are linked to activation of the metabolic sensor AMPK. These results reveal an exercise-independent ERRγ pathway that promotes and coordinates vascular supply and metabolic demand in oxidative slow-twitch muscles.
Based on these observations, provided herein are methods for increasing vascularization in a subject. For example, such methods can include administering a therapeutically effective amount of one or more agents that increases ERRγ activity to a mammal needing increased vascularization, such as vascularization in the mammal's muscle (such as skeletal or cardiac muscle), brain, kidney, or brown adipose tissue. In some examples, the method can also include selecting a mammal in need of increased vascularization or a mammal at risk for developing a disorder that can benefit from increased vascularization.
Methods for muscle rehabilitation (such as increasing or enhancing muscle performance) in a subject are provided. For example, such methods can include administering a therapeutically effective amount of one or more agents that increases ERRγ activity to a mammal needing muscle rehabilitation (such as increased or enhanced muscle performance), such as a subject having or at risk for muscle wasting. In some examples, the method can also include selecting a mammal in need of muscle rehabilitation (such as increased muscle performance) or a mammal at risk for developing a disorder that can benefit from muscle rehabilitation (such as increased muscle performance).
In addition, provided herein are methods for increasing mitochondrial activity in a subject. For example, such methods can include administering a therapeutically effective amount of one or more agents that increases ERRγ activity to a mammal needing increased mitochondrial activity. In some examples, the method can also include selecting a mammal in need of increased mitochondrial activity or a mammal at risk for developing a disorder that can benefit from increased mitochondrial activity.
In some examples, the methods do not include exercising the mammal. As such, in some examples the subject is one who cannot exercise or is sedentary, such as a person who is bedridden or confined to a wheelchair.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOS: 1 and 2 are the nucleic acid and corresponding amino acid sequence of an exemplary ERRγ sequence.
DETAILED DESCRIPTIONUnless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. “Comprising” means “including.” Hence “comprising A or B” means “including A” or “including B” or “including A and B.”
Suitable methods and materials for the practice and/or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. All sequences associated with the GenBank® Accession numbers mentioned herein are incorporated by reference in their entirety as were present on Mar. 1, 2012. Although exemplary GenBank® numbers are listed herein, the disclosure is not limited to the use of these sequences. Many other ERRγ sequences are publicly available, and can thus be readily used in the disclosed methods. In one example, an ERRγ sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% sequence identity to any of the GenBank® numbers are listed herein.
In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:
Administration: The introduction of a composition, such as an ERRγ agonist, into a subject by a chosen route, for example topically, orally, intravascularly such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, transdermally, intrathecally, subcutaneously, via inhalation or via suppository. Administration can be local or systemic, such as intravenous or intramuscular. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. In some examples an ERRγ agonist is administered to a subject at an effective dose.
Estrogen receptor-related receptor γ (ERRγ): (OMIM 602969) A constitutively active orphan nuclear receptor of the ERR subfamily. Unlike ERRα and β, it is more selectively expressed in metabolically active and highly vascularized tissues such as heart, kidney, brain and skeletal muscles.
ERRγ sequences are publicly available. For example, GenBank® Accession Nos. NM_001134285.1, AY388461, AF058291.1 and NM_011935.2 disclose ERRγ nucleic acids, and GenBank® Accession Nos. NP_001127757.1, P62508.1, AAQ93381.1, and NP_036065.1 disclose ERRγ proteins. In certain examples, ERRγ has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to such sequences (such as SEQ ID NO: 1 or 2), and retains ERRγ activity.
ERRγ activity includes the ability to promote vascularization (for example in skeletal muscle), increase mitochondrial activity (such as mitochondrial respiration), promote transformation of fast- to slow-twitch type muscle, promote muscle rehabilitation, and/or enhance muscle performance.
Isolated: An “isolated” biological component (such as a nucleic acid, protein or antibody) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as, other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids and proteins which have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as those chemically synthesized.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of an ERRγ agonist or other agent that increases ERRγ activity.
In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by methods known in the art, such as chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Cells that express such molecules are referred to as recombinant or transgenic cells.
Sequence identity: The similarity between amino acid or nucleic acid sequences are expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for
Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Variants of ERRγ that retain ERRγ activity are encompassed by this disclosure typically characterized by possession of at least about 75%, for example at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity counted over the full length alignment with the amino acid or nucleic acid sequence of interest, such as any of SEQ ID NOS: 1-2. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.
Therapeutically effective amount: An amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response. A therapeutic agent, such as an ERRγ agonist, is administered in therapeutically effective amounts. In some embodiments, a therapeutically effective amount is the amount of one or more agents that increase ERRγ activity necessary to increase or more of vascularization (for example in skeletal muscle), mitochondrial activity (such as mitochondrial respiration), transformation of fast- to slow-twitch type muscle, and/or muscle performance (such as an increase of at least 20%, at least 50%, at least 60%, at least 75%, at least 80%, or at least 95% as compared to an absence of the ne or more agents that increase ERRγ activity). When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect.
Effective amounts a therapeutic agent can be determined in many different ways, such as assaying for an increase in vascularization, mitochondrial activity transformation of fast- to slow-twitch type muscle, and/or muscle performance or improvement of physiological condition of a subject having or at risk for a disease such as a mitochondrial disease, vascular disease (such as cardiovascular disease, peripheral vascular disease, ischemia), or muscular disease (such as atrophy or sarcopenia). Effective amounts also can be determined through various in vitro, in vivo or in situ assays.
Therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.
Treating a disease: “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such a sign or symptom of a mitochondrial disease, vascular disease (such as cardiovascular disease, peripheral vascular disease, ischemia), or muscular disease (such as atrophy or sarcopenia). Treatment can also induce remission or cure of a condition, such as an ischemic stroke, transient ischemia attacks (TIAs), muscle atrophy, sarcopenia, MELAS, and hearing loss. Preventing a disease refers to a therapeutic intervention to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology, such that the therapy inhibits or delays the full development of a disease, such as preventing development of a mitochondrial disease, vascular disease (such as cardiovascular disease, peripheral vascular disease, ischemia), or muscular disease (such as atrophy or sarcopenia). Treatment and prevention of a disease does not require a total absence of disease. For example, a decrease of at least 20% or at least 50% can be sufficient. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.
Upregulated or activation: When used in reference to the expression of a nucleic acid molecule, such as an ERRγ gene, refers to any process which results in an increase in production of a gene product. A gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein (such as an ERRγ protein). Therefore, gene upregulation or activation includes processes that increase transcription of a gene or translation of mRNA.
Examples of processes that increase transcription include those that facilitate formation of a transcription initiation complex, those that increase transcription initiation rate, those that increase transcription elongation rate, those that increase processivity of transcription and those that relieve transcriptional repression (for example by blocking the binding of a transcriptional repressor). Gene upregulation can include inhibition of repression as well as stimulation of expression above an existing level. Examples of processes that increase translation include those that increase translational initiation, those that increase translational elongation and those that increase mRNA stability.
Gene upregulation includes any detectable increase in the production of a gene product. In certain examples, production of a gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in an untreated cell, such as a cell not contacted with an agent that increases ERRγ activity).
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
Methods of Enhancing Vascularization, Muscle Rehabilitation, and Mitochondrial ActivityIt is shown herein that nuclear receptor ERRγ is highly expressed in type I skeletal muscle. Type I muscle is fatigue resistant, highly vascularized, aerobic, and slow-twitch. When ERRγ is transgenically expressed in anaerobic type II muscles (ERRGO mice), dually induces metabolic and vascular transformation in absence of exercise. ERRGO mice showed increased expression of genes promoting fat metabolism, mitochondrial respiration and type I fiber specification. Muscles in ERRGO mice also display an activated angiogenic program marked by myofibrillar induction and secretion of pro-angiogenic factors, neo-vascularization and a 100% increase in running endurance. At a functional level, these genetic changes impart high oxygen consuming and exercising capacity as well as resistance to diet-induced obesity to the ERRGO mice. Surprisingly, the induction of type I muscle properties by ERRγ does not involve PGC1α. Instead, ERRγ genetically activates the energy sensor AMPK, in mediating the metabo-vascular changes in the ERRGO mice. Therefore, ERRγ represents a previously unrecognized determinant that specifies intrinsic vascular and oxidative metabolic features that distinguish type I from type II muscle.
Although skeletal muscle adapts to exercise by increasing oxidative metabolism and vascular supply via induction of transcriptional regulators such as PGC1α (Arany et al., 2008; Baar et al., 2002; Huss et al., 2002; Pilegaard et al., 2003; Russell et al., 2003; Russell et al., 2005), how type I fibers achieve intrinsic vascularization even in absence of exercise is poorly understood. It is shown herein that one such molecular pathway involves nuclear receptor ERRγ, which is highly expressed in oxidative slow-twitch muscles. Targeted expression of ERRγ to quadriceps and white gastrocnemius, where the receptor is typically not expressed, morphologically endows these muscles with dense vascular supply and numerous slow-twitch characteristics.
Genome-wide expression analysis revealed that ERRγ acts by coordinately inducing gene networks promoting mitochondrial biogenesis, oxidative transformation and angiogeneis. The ERRγ program includes mobilization and oxidation of fat [e.g., Acadl, Acadm, Cpt1b, Cpt2, Lpl], electron transport [e.g., Atp5h, Cox6a2, Ndufab1, Ndufb2m Ndufv1, Sdhb], mitochondrial biogenesis [e.g., Mfn1], and formation of energy efficient slow-contractile muscle [e.g., Tnnc1, Tnni1, Tnnt1]. The observed changes constituting transformation of the contractile apparatus to a slow phenotype and increase in oxidative metabolic genes reflected in profound increase in mitochondrial (SDH) staining represents a fiber type switch. Notably, ERRγ also induces key transcriptional inducers of oxidative metabolism including Esrrb, Ppara, Ppard and Ppargc1b (Table 4) (Lin et al., 2002; Minnich et al., 2001; Muoio et al., 2002; Wang et al., 2004). Therefore, ERRγ may be an upstream genetic switch that determines metabolic fate by presiding over the expression of multiple aerobic regulators.
Without wishing to be bound by a particular model, it is proposed that the vascular program triggered by myocellular ERRγ activates a transcriptional program that directs secretion of paracrine signals into skeletal muscle microenvironment to induce angiogenesis. This model is strongly supported by the observation herein that conditioned media from ERRγ over-expressing C2C12 myotubules induces endothelial cell tube formation in culture. Indeed, ERRγ transcriptionally induced all isoforms of angiokine Vegfa in C2C12 myotubes, resulting in increased Vegfa secretion into the media. Vegfa is a key regulator of angiogenesis critical for guiding endothelial cells to their targets (Grunewald et al., 2006; Springer et al., 1998). Furthermore, ERRγ stimulates the Vegfa promoter containing putative ERR binding sites that is known to transcribe all Vegfa isoforms (Arany et al., 2008). Vegfa mRNA and protein expression is also induced in ERRGO muscle. These findings collectively indicate direct transcriptional activation of angiogenic genes by ERRγ. However, the angiogenic effects of ERRγ cannot be solely attributed to Vegfa induction and secretion. For example ERRγ additionally activates the expression of Fgf1 and Cxcl12, known to regulate endothelial cell proliferation and migration (Forough et al., 2006; Gupta et al., 1998; Partridge et al., 2000; Shao et al., 2008; Zheng et al., 2007), along with ephrin B2 proposed to recruit mural cells that are required for vessel maturation (Foo et al., 2006). Additionally, up-regulated factors such as Notch4 as well as SOX17 are transcriptional regulators of vasculogenesis (Hainaud et al., 2006; Leong et al., 2002; Matsui et al., 2006). In this aspect, ERRγ seems to serve a function similar to HIF1α, a regulator of angiogenesis during hypoxia (Pajusola et al., 2005). Interestingly, it was recently demonstrated that ERRs might physically interact with HIF1α in regulating its transcriptional activity (Ao et al., 2008). HIF1α mRNA levels—a marker for chronic hypoxia—did not change in ERRGO compared to wild type muscles indicating an absence of hypoxia or its involvement in the vascular effects of ERRγ (Hoppeler and Vogt, 2001a, b). Furthermore, HIF1α is known to negatively regulate oxidative metabolism (Mason et al., 2004; Mason et al., 2007) and is therefore unlikely to contribute to ERRγ-mediated remodeling of skeletal muscles.
ERRGO mice exhibited increased oxygen consumption, decreased respiratory exchange ratio, high running endurance and resistance to diet-induced weight gain. These changes are physiological hallmarks of increased aerobic capacity in mice, and are a direct consequence of engineering highly oxidative and vascularized muscle by ERRγ. While similar remodeling of skeletal muscle and aerobic physiology are triggered by exercise, the data herein demonstrate that generation of a fully functional “endurance vasculature” is not exercise dependent (Bloor, 2005; Egginton, 2008; Gavin et al., 2007; Gustafsson and Kraus, 2001; Jensen et al., 2004; Waters et al., 2004).
A surprising finding was lack of change in the expression of PGC1α, a known and inducible regulator of aerobic muscles, in the ERRγ-transformed muscle. One alternative possibility is post-translational activation of PGC1α without change in its expression (Jager et al., 2007; Puigserver et al., 2001; Rodgers et al., 2005). De-acetylation of PGC1α is critical for its activation in the skeletal muscle (Canto et al., 2010; Gerhart-Hines et al., 2007; Lagouge et al., 2006). However, ERRγ over-expression did not lead to de-acetylation of PGC1α, which remained comparably acetylated in both the wild type and ERRGO muscles. The lack of post-translational activation of the co-factor in ERRGO mice is further underscored by a previous report that non-genomic activation of PGC1α typically leads to its transcriptional induction, which we did not observe in these studies (Jager et al., 2007). Along the same lines, it was recently shown that both PGC1α and β are dispensable for fiber type specification in the skeletal muscle (Zechner et al., 2010). In contrast, an alternative aerobic master regulator, AMPK, was found to be activated by ERRγ in the skeletal muscles. AMPK is typically activated by exercise (Fujii et al., 2000; Winder and Hardie, 1996; Wojtaszewski et al., 2000) and is essential for exercise-mediated switch to aerobic myofibers in the skeletal muscle (Rockl et al., 2007). Indeed, transgenic activation of AMPK in the skeletal muscle increases the proportions of oxidative myofibers in absence of any exercise (Rockl et al., 2007). It is shown herein that chemical activation of AMPK by AICAR triggers aerobic transformation of type II muscle. However, AMPK alone is unlikely to mediate all the ERRγ effects, and contribution by additional metabolic regulators (e.g., calcineurin, SIRT1, etc.) in ERRGO mice cannot be ruled out. This is possible because, unlike ERRγ, AMPK activation apparently does not lead to a complete transformation to a type I phenotype, but to a more intermediate type IIa and IIx oxidative myofibers (Rockl et al., 2007). In this context, it is peculiar that AMPK was naturally and selectively active in soleus (pre-dominantly type I myofibers) compared to quadriceps (pre-dominantly type II myofibers). Previous studies have suggested AMPK activity to be similar between soleus and EDL (also pre-dominantly made up of type II myofibers) (Dzamko et al., 2008; Jensen et al., 2007; Jorgensen et al., 2004). Speculatively, this discrepancy may have technical attributes or may even be linked to possible differences in recruitment of EDL and quadriceps for postural activity that might affect basal AMPK activation. Nevertheless, the results herein demonstrate that in the context of over-expression, ERRγ is sufficient to initiate both metabolic and vascular pathways to drive aerobic remodeling of sedentary muscle independent of PGC1α by recruiting alternative regulators such as AMPK (see
Multiple diseases including obesity and diabetes are commonly linked to deregulation of both oxidative metabolism and vascularity. A shared therapeutic approach to these conditions includes exercise that activates a plethora of transcriptional pathways to increase aerobic metabolism and vascularization to ultimately enhance performance (Bloor, 2005; Egginton, 2008; Gavin et al., 2007; Gustafsson and Kraus, 2001; Jensen et al., 2004; Waters et al., 2004). These findings indicate that regulation of ERRγ activity can be used to simultaneously regulate oxidative capacity and vascularity. High expression levels of this receptor in tissues most prone to metabolic and vascular diseases (e.g., heart, skeletal muscle, brain and kidney) further potentiates its value as a pharmacologic target (Ariazi et al., 2002; Cheung et al., 2005; Gao et al., 2006; Giguere, 2008; Heard et al., 2000; Hong et al., 1999). In summary, it is shown herein that ERRγ controls mitochondrial function and metabolism, together with angiogenesis that anatomically synchronizes vascular arborization to oxidative metabolism.
Based on this observation, provided herein are methods of increasing vascularization, mitochondrial activity, muscle rehabilitation and/or muscle endurance by increasing ERRγ activity, for example by use of ERRγ nucleic acids, proteins, agonists, or combinations thereof. Such methods can be used to treat or prevent a disorder associated with defects in vascularization or mitochondrial activity, as well as disorders that may result from being sedentary or being unable to exercise. The agent(s) which enhance ERRγ activity can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intracerebral, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the dosing is given by injections, such as intravenous or intramuscular injections, depending in part on whether the administration is brief or chronic.
In some examples, the methods do not include providing additional exercise to the subject being treated with the agents that increase ERRγ activity. For example, the subject may undergo some moderate activity during such treatment (such as that needed to perform menial tasks), the subject does not undergo any strenuous activity (such as running, biking, swimming, or walking for more than 5 or 10 minutes at a time). In some examples, the subject treated is one who cannot exercise, such as one confined to a bed or wheelchair. In another example, the subject treated is one who is sedentary, such as one with no or irregular physical activity, for example one who sits or remains inactive for most of the day with little or no exercise.
In one example, methods are provided for increasing vascularization. Such methods can include administering a therapeutically effective amount of one or more agents that increase ERRγ activity to a mammal needing increased vascularization. In some examples, the method increases vascularization in the desired tissue by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to an amount of vascularization in the absence of administration of the one or more agents that increases ERRγ activity. Methods of measuring vascularization are provided herein and are known in the art, and can include measuring expression of one or more VEGF molecules, detection of vasculature (for example using angiography), and the like. In some examples, the method also includes selecting a mammal in need of increased vascularization or a mammal at risk for developing a disorder that can benefit from increased vascularization. In one example, vascularization is needed or occurs in the mammal's muscle, brain, kidney, or brown adipose tissue. Thus, the agents that increase ERRγ activity can be used for the prophylaxis or treatment of a mammal, for instance, a human subject who has been diagnosed with a vascular disorder, such as ischemia or peripheral vascular disease (PVD), or a person at risk for developing a vascular disorder, such as one who is sedentary or is unable to exercise.
In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a vascular disorder in skeletal muscle. For example, the subject may have or be at risk for muscle atrophy, sarcopenia, or peripheral vascular disease (such as peripheral arterial disease, PAD). In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a vascular disorder in cardiac muscle. For example, the subject may have or be at risk for a heart attack or ischemia in a cardiac muscle. In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a vascular disorder in the brain. For example, the subject may have or be at risk for a cerebrovascular disease, such as ischemic stroke, migraines, transient ischemic attacks (TIAs), dementia and the like. In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a vascular disorder in the kidney. For example, the subject may have or be at risk for a kidney failure. In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a vascular disorder in brown adipose tissue. For example, the subject may have or be at risk for obesity (such as diet-induced obesity). One skilled in the art will appreciate that complete elimination of symptoms associated with the vascular disorder is not required for the method to be effective, as long as at least some symptoms are eased.
In one example, methods are provided for increasing mitochondrial activity. Such methods can include administering a therapeutically effective amount of one or more agents that increase ERRγ activity to a mammal needing increased mitochondrial activity. In some examples, the method increases mitochondrial activity by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to an amount of mitochondrial activity in the absence of administration of the one or more agents that increases ERRγ activity. Methods of measuring mitochondrial activity are provided herein and are known in the art, and can include measuring mitochondrial respiration, expression of mitochondrial genes, and the like. In some examples, the method also includes selecting a mammal in need of increased mitochondrial activity or a mammal at risk for developing a disorder that can benefit from increased mitochondrial activity. In one example, increased mitochondrial activity is needed or occurs in the mammal's muscle (e.g., skeletal or cardiac), brain, or ear. Thus, the agents that increase ERRγ activity can be used for the prophylaxis or treatment of a mammal, for instance, a human subject who has been diagnosed with a mitochondrial disorder, such as mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), or a person at risk for developing a mitochondrial disorder, such as one who is sedentary or is unable to exercise.
In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of a mitochondrial disorder in skeletal muscle. For example, the subject may have or be at risk for muscle atrophy, sarcopenia, or muscle wasting. In some embodiments, the agents that increase ERRγ activity is administered to treat or prevent the development of hearing loss, Leber's hereditary optic neuropathy (LHON), or mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode syndrome (MELAS). One skilled in the art will appreciate that complete elimination of symptoms associated with the mitochondrial disorder is not required for the method to be effective, as long as at least some symptoms are eased.
In one example, methods are provided for muscle rehabilitation (such as increasing muscle performance). Such methods can include administering a therapeutically effective amount of one or more agents that increase ERRγ activity to a mammal needing muscle rehabilitation (such as increased muscle performance). In some examples, the method increases muscle rehabilitation or performance by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to an amount of muscle performance in the absence of administration of the one or more agents that increases ERRγ activity. In some examples, the method increases aerobic transformation of a type II muscle by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to an amount of aerobic transformation of a type II muscle (e.g., quadriceps or white gastrocnemius) in the absence of administration of the one or more agents that increases ERRγ activity. In some examples, the method increases an amount of type I fibers in a muscle by at least 25%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, as compared to an amount of type I fibers in a muscle in the absence of administration of the one or more agents that increases ERRγ activity. Methods of measuring muscle performance are provided herein and are known in the art, and can include measuring the strength and endurance of the muscle, testing creatine kinase levels in the blood, electromyography (measuring electrical activity in muscles), elastography, and the like. In some examples, the method also includes selecting a mammal in need of muscle rehabilitation (such as increased muscle performance) or a mammal at risk for developing a disorder that can benefit from muscle rehabilitation (such as increased muscle performance). In one example, the muscle rehabilitated is skeletal muscle or cardiac muscle. Thus, the agents that increase ERRγ activity can be used for the prophylaxis or treatment of a mammal, for instance, a human subject who has been diagnosed with a muscle performance disorder, muscle atrophy or sarcopenia, or a person at risk for developing a muscle performance disorder, such as one who is sedentary or is unable to exercise.
Thus, increasing ERRγ activity, for example without the addition of exercise, can result in one or more of the following in skeletal or cardiac muscle: increased oxidative metabolism (such as one or more of aerobic transformation of fast-twitch muscles, increased mitrochondrial biogenesis and respiration, increased lipid metabolism, increased fatigue-resistant type I fibers, increase running endurance), increased vascularization (such as one or more of increased synthesis and release of pro-angiogenic factors by myotubes, increased vascularization of skeletal muscle (e.g., hyper-vascularization). Thus, increasing ERRγ activity can enhance muscle performance, such as performance of type II muscles, such as quadriceps and white gastrocnemius. In one example, increasing ERRγ activity increases expression of or more of myoglobin, cytochrome c and UCP3, and has increased oxidative myofibers, relative to untreated muscle. In some examples such increases are an at least 1.5-fold or an at least 2-fold increase.
Increasing ERRγ ActivityThe present disclosure provides methods and pharmaceutical compositions for increasing vascularization, mitochondrial activity, and/or muscle performance by increasing ERRγ activity and thereby treating or preventing disorders associated with decreased vascularization, muscle performance, or mitochondrial activity. ERRγ activity may be increased by increasing the amount of ERRγ protein being produced or by enhancing the activity of ERRγ protein. This can be achieved, for example, by administering a nucleotide sequence encoding for an ERRγ protein, an agent which enhances ERRγ expression, a substantially purified ERRγ protein, or an ERRγ agonist. An ERRγ agonist includes compounds which increase the ERRγ activity in a cell or tissue.
Administration of ERRγ Proteins
In one example, ERRγ activity is increased by administering to the subject an ERRγ protein, such as a pharmaceutical composition containing such a protein. ERRγ protein sequences are known. For example, GenBank® Accession Nos. NP_001127757.1, P62508.1, AAQ93381.1, and NP_036065.1 disclose exemplary ERRγ protein sequences. However, one skilled in the art will appreciate that variations of such proteins can also retain ERRγ activity. For example such variants may include one or more deletions, substitutions, or additions (or combinations thereof), such as 1-50 of such changes (such as 1-40, 1-30, 1-20, or 1-10 of such changes). In certain examples, ERRγ has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to such sequences (such as SEQ ID NO: 2), and retains ERRγ activity. In some examples, changes are not made to the ERRγ ligand binding domain (LBD). In some examples, residues Asp328, Arg316 and/or Asp275 are not changed.
One of skill will realize that variants of ERRγ proteins can be used, such as a variant containing conservative amino acid substitutions. Such conservative variants will retain critical amino acid residues necessary for ERRγ activity, and will retain the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, at most five, or at most 10 amino acid substitutions, such as 1 to 10 or 1 to 5 conservative substitutions) can be made in an ERRγ protein sequence to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
An ERRγ protein can be derivatized or linked to another molecule (such as another peptide or protein). For example, the ERRγ protein can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as an antibody, a detection agent, or a pharmaceutical agent.
Methods of making proteins are routine in the art, for example by recombinant molecular biology methods or by chemical peptide synthesis. In one example, an ERRγ protein is expressed in a cell from a vector encoding the protein. In some examples, the expression vector encoding ERRγ also encodes a selectable marker. In some examples, the sequence encoding ERRγ also encodes a purification tag sequence (such as a His-tag, β-globin-tag or glutathione S-transferase-(GST) tag) at the N- or C-terminus of ERRγ, to assist in purification of the protein.
For example, expression of nucleic acids encoding ERRγ proteins can be achieved by operably linking the ERRγ DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter, including a cytomegalovirus promoter and a human T cell lymphotrophic virus promoter (HTLV)-1. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes (such as E. coli) or eukaryotes (such as yeast or a mammalian cell). Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
To obtain high level expression of ERRγ, expression cassettes can include a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription/translation terminator. Exemplary control sequences include the T7, trp, lac, tac, trc, or lambda promoters, the control region of fd coat protein, a ribosome binding site, and can include a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40, polyoma, adenovirus, retrovirus, baculovirus, simian virus, promoters derived from the promoter for 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, the promoter of the yeast alpha-mating factors or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding EERγ, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40), retrovirus, adenovirus, adeno-associated virus, Herpes virus, or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One can readily use an expression system, such as plasmids and vectors, to produce proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa, fibroblast cell lines, lymphoblast cell lines, and myeloma cell lines.
Once expressed, the recombinant ERRγ protein can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, PROTEIN PURIFICATION, Springer-Verlag, N.Y., 1982). The recovered ERRγ protein need not be 100% pure. Once purified, partially or to homogeneity as desired, the ERRγ protein can be used therapeutically.
Modifications can be made to a nucleic acid encoding ERRγ without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of ERRγ into a fusion protein. Such modifications are well known and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.
In one example, ERRγ protein is synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N,N′-dicylohexylcarbodimide) are well known.
Expression of ERR' in a Subject
In one example, ERRγ activity is increased by administering to the subject a nucleic acid molecule encoding an ERRγ protein. ERRγ coding sequences are known. For example, GenBank® Accession Nos. NM_001134285.1, AY388461, AF058291.1 and NM_011935.2 disclose exemplary ERRγ nucleic acid sequences. However, one skilled in the art will appreciate that variations of such sequences can also encode a protein with ERRγ activity. For example such variants may include encode a protein with one or more deletions, substitutions, or additions (or combinations thereof), such as 1-50 of such changes (such as 1-40, 1-30, 1-20, or 1-10 of such changes). In certain examples, an ERRγ coding sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to such sequences (such as SEQ ID NO: 1), and encodes a protein having ERRγ activity. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same ERRγ protein sequence.
Nucleic acid sequences encoding a ERRγ protein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase phosphoramidite triester method described by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that longer sequences may be obtained by the ligation of shorter sequences.
Exemplary ERRγ nucleic acids can be prepared by routine cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), and Applied Biosystems (Foster City, Calif.), as well as many other commercial sources. Nucleic acids can also be prepared by amplification methods. Amplification methods include but are not limited to polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known.
In some examples, it may only be necessary to introduce the ERRγ genetic or protein elements into certain cells or tissues. For example, introducing ERRγ into only the muscle, such as skeletal or cardiac muscle (or even a particular muscle), may be sufficient. However, in some instances, it may be more therapeutically effective and simple to treat all of the patient's cells, or more broadly disseminate the ERRγ nucleic acid or protein, for example by intravascular administration.
Nucleic acids encoding ERRγ can be introduced into the cells of a subject using routine methods, such as by using recombinant viruses (e.g., viral vectors) or by using naked DNA or DNA complexes (non-viral methods). Thus, in some embodiments, a method of increasing ERRγ activity in persons suffering from, or at risk for, a vascular disease, mitochondrial disease, and/or muscle performance disease, is achieved by introducing a nucleic acid molecule coding for ERRγ into the person. A general strategy for transferring genes into donor cells is disclosed in U.S. Pat. No. 5,529,774. The nucleic acid encoding ERRγ can be administered to the subject by any method which allows the recombinant nucleic acid to reach the appropriate cells. Exemplary methods include injection, infusion, deposition, implantation, and topical administration. Injections can be intradermal, intramuscular, iv, or subcutaneous.
In one example, an ERRγ coding sequence is introduced into a subject in a non-infectious form, such as naked DNA or liposome encapsulated DNA. Such molecules can be introduced by injection (such as intramuscular, iv, ip, pneumatic injection, or a gene gun), or other routine methods (such as oral or nasal). In one example, ERRγ coding sequence is part of a lipoplex, dendrimer, or inorganic nanoparticle to assist in its delivery.
In one example, viral vectors are used. Generally, such methods include cloning an ERRγ coding sequence into a viral expression vector, and that vector is then introduced into the subject to be treated. The virus infects the cells, and produces the ERRγ protein sequence in vivo, where it has its desired therapeutic effect. The nucleic acid sequence encoding ERRγ can be placed under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the gene's native promoter; retroviral LTR promoter; adenoviral promoters, such as the adenoviral major late promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMTV promoter; the metallothionein promoter; heat shock promoters; the albumin promoter; the histone promoter; the β-actin promoter; TK promoters; B19 parvovirus promoters; and the ApoAI promoter.
Exemplary viral vectors include, but are not limited to: pox viruses, recombinant vacciniavirus, retroviruses (such as lentivirus), replication-deficient adenovirus strains, adeno-associated virus, herpes simplex virus, or poliovirus.
Adenoviral vectors may include essentially the complete adenoviral genome. Alternatively, the adenoviral vector may be a modified adenoviral vector in which at least a portion of the adenoviral genome has been deleted. In one embodiment, the vector includes an adenoviral 5′ ITR; an adenoviral 3′ ITR; an adenoviral encapsidation signal; a DNA sequence encoding a therapeutic agent such as EDA1-II, dl or DL; and a promoter for expressing the DNA sequence encoding a therapeutic agent. The vector is free of at least the majority of adenoviral E1 and E3 DNA sequences, but is not necessarily free of all of the E2 and E4 DNA sequences, and DNA sequences encoding adenoviral proteins transcribed by the adenoviral major late promoter. Such a vector may be constructed according to standard techniques, using a shuttle plasmid which contains, beginning at the 5′ end, an adenoviral 5′ ITR, an adenoviral encapsidation signal, and an E1a enhancer sequence; a promoter (which may be an adenoviral promoter or a foreign promoter); a tripartite leader sequence, a multiple cloning site (which may be as herein described); a poly A signal; and a DNA segment which corresponds to a segment of the adenoviral genome. The DNA segment serves as a substrate for homologous recombination with a modified or mutated adenovirus, and may encompass, for example, a segment of the adenovirus 5′ genome no longer than from base 3329 to base 6246. The plasmid may also include a selectable marker and an origin of replication. The origin of replication may be a bacterial origin of replication. A desired DNA sequence encoding a therapeutic agent may be inserted into the multiple cloning site of the plasmid. The plasmid may be used to produce an adenoviral vector by homologous recombination with a modified or mutated adenovirus in which at least the majority of the E1 and E3 adenoviral DNA sequences have been deleted. Homologous recombination may be effected through co-transfection of the plasmid vector and the modified adenovirus into a helper cell line, such as 293 cells, by CaPO4 precipitation. The homologous recombination produces a recombinant adenoviral vector which includes DNA sequences derived from the shuttle plasmid between the Not I site and the homologous recombination fragment, and DNA derived from the E1 and E3 deleted adenovirus between the homologous recombination fragment and the 3′ ITR.
In one embodiment, the viral vector is a retroviral vector. Examples of retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, lentivirus, myeloproliferative sarcoma virus, and mammary tumor virus. The vector can be a replication defective retrovirus particle. Retroviral vectors are useful as agents to effect retroviral-mediated gene transfer into eukaryotic cells. Retroviral vectors are generally constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (e.g., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. An ERRγ coding sequence can be incorporated into a proviral backbone using routine methods. In the most straightforward constructions, the structural genes of the retrovirus are replaced by a ERRγ gene which then is transcribed under the control of the viral regulatory sequences within the long terminal repeat (LTR). Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter. Alternatively, two genes may be expressed from a single promoter by the use of an Internal Ribosome Entry Site.
In one example, the viral vector is an adeno-associated virus (AAV). Gene therapy vectors using AAV can infect both dividing and non-dividing cells and persist in an extrachromosomal state without integrating into the genome of the host cell. In some examples, the rep and cap are removed from the DNA of the AAV. The ERRγ coding sequence together with a promoter to drive transcription is inserted between the inverted terminal repeats (ITR) that aid in concatamer formation in the nucleus after the single-stranded vector DNA is converted by host cell DNA polymerase complexes into double-stranded DNA.
The viral particles are administered in an amount effective to produce a therapeutic effect in a host. The exact dosage of viral particles to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient to be treated, and the nature and extent of the disease or disorder to be treated. The viral particles may be administered as part of a preparation having a titer of viral particles of at least 1×105 pfu/ml, at least 1×106 pfu/ml, at least 1×107 pfu/ml, at least 1×108 pfu/ml, at least 1×109 pfu/ml, or at least 1×1010 pfu/ml, and in some examples not exceeding 2×1011 pfu/ml. The viral particles can be administered in combination with a pharmaceutically acceptable carrier, for example in a volume up to 10 ml. The pharmaceutically acceptable carrier may be, for example, a liquid carrier such as a saline solution, protamine sulfate or Polybrene.
Agonists
An ERRγ agonist is an agent that induces or increases ERRγ activity or expression. Agonists of ERRγ are commercially available, and can be generated using routine methods. In some examples, the agonist is an agonist of ERRγ, but not ERRα or ERRβ. In some examples, the agonist is an agonist of ERRγ, as well as of ERRα and/or ERRβ.
ERRγ agonists are known in the art, and additional ERRγ agonists can be identified using known methods (e.g., see Zuercher et al., 2005, J. Med. Chem. 48(9):3107-9; Coward et al. 2001, Proc Natl Acad Sci USA. 8(15):8880-4; and Zhou et al., 1998, Mol. Endocrin. 12:1594-1604).
For example, phenolic acyl hydrazones GSK4716 (e.g., Santa Cruz Catalog #sc-203986) and GSK9089 (also known as DY131, see for example, U.S. Pat. No. 7,544,838) (N-[(E)-[4-(diethylamino)phenyl]methylideneamino]-4-hydroxybenzamide; e.g., Tocris Bioscience Catalog #2266 or Santa Cruz Catalog #sc203571) are agonists of ERRβ and ERRγ.
Kim et al. (J. Comb. Chem. 11:928-37, 2009) disclose a screening assay for agonists of ERRγ derived from GSK4716. Such a screening method can also be used to identify other agonists of ERRγ. E6 was discovered as being selective for ERRγ but not ERRα and β.
U.S. Pat. Nos. 7,544,838 and 8,044,241 also provide ERRγ agonists that can be used with the disclosed methods, such as DY131. In addition, DY159, DY162, DY163 and DY164 were also observed to activate ERRγ (and ERRα and β), for example in the presence of PGC-1a.
US Patent Application Publication Nos. 2011/0218196 and 2009/0281191 also provide ERRγ agonists that can be used with the disclosed methods.
SubjectsExemplary subjects that can benefit from the disclose therapies include human and veterinary mammalian subjects, such as cats, dogs, horses, rodents, and the like. In one example, the subject treated has, or is at risk for developing, a vascular disease, mitochondrial disease, and/or muscle performance disease. Thus, such therapies can be used to prevent or treat the disease provided herein. In some examples the patient is at risk for such diseases due to smoking, alcoholism, diabetes (e.g., diabetes mellitus), obesity, head trauma, hypertension, stroke, heart attack, dyslipidemia, atherosclerosis, sedentary lifestyle, inability to exercise, and the like, and thus such patients can be treated using the methods provided herein.
In one example, the subject has or is at risk to develop a vascular disease that can be treated or prevented by increased blood flow. In one example, the vascular disease is a cerebrovascular disease, such as a vascular disease of the brain. Exemplary diseases that can be treated or prevented by increased blood flow in or to the brain include migraines, dementia, and ischemia, such as ischemia resulting from or due to ischemic stroke, transient ischemic attacks (TIAs), and carotid stenosis. In some examples such patients are diabetic, smokers, have hypertension, sedentary, or have suffered head trauma.
In one example, the vascular disease is a cardiovascular disease, such as a vascular disease of the heart or blood vessels. Exemplary diseases that can be treated or prevented by increased blood flow in the heart or vessels include ischemic heart disease, coronary heart disease, and cardiomyopathy. In some examples such patients are diabetic, smokers, have hypertension, have suffered a heart attack, or are sedentary.
In one example, the vascular disease is a vascular disease of the kidney. Exemplary diseases that can be treated or prevented by increased blood flow in the kidney include kidney failure. In some examples such patients are diabetic, smokers, have hypertension, or are sedentary.
In one example, the vascular disease is a vascular disease of the brown adipose tissue. Exemplary diseases that can be treated or prevented by increased blood flow in the brown adipose tissue include obesity. In some examples such patients are diabetic, smokers, have hypertension, or are sedentary.
In one example, the vascular disease is a vascular disease of the skeletal muscle. Exemplary diseases that can be treated or prevented by increased blood flow in the skeletal muscle include peripheral vascular or artery disease, muscle atrophy, muscle wasting and sarcopenia. In some examples such patients are diabetic, smokers, have hypertension, or are sedentary.
In one example, the subject has or is at risk to develop a mitochondrial disease that can be treated or prevented by increased mitochondrial activity. Exemplary mitochondrial diseases that can be treated or prevented by increased mitochondrial activity include muscle disease (such as muscle atrophy, muscle wasting and sarcopenia), diabetes mellitus and deafness (DAD), hearing loss, Leber's hereditary optic neuropathy (LHON), Leigh syndrome, neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP), myoneurogenic gastrointestinal encephalopathy (MNGIE), myoclonic epilepsy with ragged red fibers (MERRF) progressive myoclonic epilepsy, mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In some examples such patients are sedentary.
In one example, the subject has or is at risk to develop a skeletal or cardiac muscle disease that can be treated or prevented by increased muscle performance. Exemplary muscle diseases that can be treated or prevented by muscle rehabilitation, such as increased muscle performance, include muscle disease (such as muscle atrophy, muscle wasting and sarcopenia, and heart disease). In some examples such patients are diabetic, smokers, have hypertension, or are sedentary.
Muscle atrophy, or disuse atrophy, is a decrease in the mass of the muscle; it can be a partial or complete wasting away of muscle. When a muscle atrophies, this leads to muscle weakness. Muscle atrophy can result from cancer, AIDS, congestive heart failure, COPD (chronic obstructive pulmonary disease), renal failure, severe burns, Dejerine Sottas syndrome (HSMN Type III), inactivity (e.g., when a cast is put on a limb), weightlessness (e.g., due to spaceflight), extended bedrest (e.g., during a prolonged illness), cachexia, liver failure, starvation, and disuse. Thus, the disclosed methods can be used to treat or prevent atrophy resulting from such conditions.
Sarcopenia refers to the process during aging, where there is a gradual decrease in the ability to maintain skeletal muscle function and mass.
In addition to the simple loss of muscle mass (atrophy), or the age-related decrease in muscle function (sarcopenia), other muscle diseases which may be caused by structural defects in the muscle (muscular dystrophy), or by inflammatory reactions in the body directed against muscle (the myopathies) can be treated using the disclosed methods.
Administration of Agents that Increase ERRγ ActivityCompositions that include one or more agents that increase ERRγ activity, such as ERRγ nucleic acids, ERRγ proteins, and ERRγ agonists that can be used to increase vascularization, mitochondrial activity, and/or muscle performance, are suited for the preparation of pharmaceutical compositions.
Pharmaceutical compositions that include one or more agents that increase ERRγ activity are provided. These pharmaceutical compositions can be used in methods of treatment/prevention of vascular, mitochondrial, and muscular disorders, and can be formulated with an appropriate physiologically acceptable solid or liquid carrier, depending upon the particular mode of administration chosen. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well known sterilization techniques. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs. Compositions including one or more agents that increase ERRγ activity are of use, for example, for the treatment of a vascular, mitochondrial, or muscular disorders, such as those resulting from inactivity.
The pharmaceutically acceptable carriers and excipients useful in this disclosure, for either therapeutic or diagnostic methods, are conventional. The one or more agents that increase ERRγ activity can be formulated for systemic or local (such as inhalational) administration. In one example, the one or more agents that increase ERRγ activity is formulated for parenteral administration, such as intravenous or intramuscular administration. For instance, parenteral formulations usually include injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
The compositions can be prepared in unit dosage forms for administration to a subject. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral and suppository formulations can be employed. Topical preparations can include ointments, sprays and the like. Inhalation preparations can be liquid (such as solutions or suspensions) and include mists, sprays and the like. Oral formulations can be liquid (for example, syrups, solutions or suspensions), or solid (such as powders, pills, tablets, or capsules). Suppository preparations can also be solid, gel, or in a suspension form. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
The pharmaceutical compositions that include one or more agents that increase ERRγ activity can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the pharmaceutical compositions may be administered in a single dose or as in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions. Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated, the severity of the affliction, whether the therapeutic agent is administered for preventive or therapeutic purposes, previous prophylaxis and therapy, the subject's clinical history and response to the therapeutic agent, and the manner of administration, and can be left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. A therapeutically effective amount of one or more agents that increase ERRγ activity is one that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. These compositions can be administered in conjunction with another agent, such as angiogenic therapy (e.g., those that include a VEGF, such as VEGF-A), either simultaneously or sequentially. The one or more agents that increase ERRγ activity also can be used or administered as a mixture, for example in equal amounts, or individually, provided in sequence, or administered all at once.
Single or multiple administrations of the compositions can be administered depending on the dosage and frequency as required and tolerated by the subject. The composition should provide a sufficient quantity of one or more agents that increase ERRγ activity to effectively treat the subject or inhibit the development of the desired disease. The dosage can be administered once but can be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy. In one example, a dose of the one or more agents that increase ERRγ activity is infused for thirty minutes every other day. In this example, about one to about ten doses can be administered, such as three or six doses can be administered every other day. In a further example, a continuous infusion is administered for about five to about ten days. The subject can be treated at regular intervals, such as monthly, until a desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of a disease without producing unacceptable toxicity to the patient.
In one specific, non-limiting example, a unit dosage for intravenous or intramuscular administration of an ERRγ agonist includes at least 0.5 μg agonist per dose, such as at least 5 μg agonist per dose, at least 50 μg agonist per dose, or at least 500 μg agonist per dose. In some examples, doses are administered three-times in one week.
In one specific, non-limiting example, an ERRγ agonist daily dosage is from about 0.01 milligram to about 500 milligram per kilogram of animal body weight, for example given as a single daily dose or in divided doses two to four times a day, or in sustained release form. For most large mammals, the total daily dosage is from about 0.01 milligrams to about 100 milligrams per kilogram of body weight, such as from about 0.5 milligram to about 100 milligrams per kilogram of body weight, which can be administered in divided doses 2 to 4 times a day in unit dosage form containing for example from about 10 to about 100 mg of the compound in sustained release form. In one example, the daily oral dosage in humans is between 1 mg and 1 g, such as between 10 mg and 500 mg, 10 mg and 200 mg, such as 10 mg. The dosage regimen may be adjusted within this range or even outside of this range to provide the optimal therapeutic response. Oral administration of an ERRγ agonist can be carried out using tablets or capsules, such as about 10 mg to about 500 mg of the ERRγ agonist. Exemplary doses in tablets include 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, and 500 mg of the ERRγ agonist. Other oral forms can also have the same dosages (e.g., capsules). In one example, a dose of an ERRγ agonist administered parenterally is at least 10 mg, such as 10 to 500 mg or 10 to 200 mg of the ERRγ agonist.
In one specific, non-limiting example, a unit dosage for oral administration (such as a table or capsule), or for oral intravenous or intramuscular administration, of an ERRγ protein includes about 1 μg to 1000 mg of ERRγ protein per dose, such as 1 μg to 100 μg ERRγ protein per dose, 1 μg to 500 μg ERRγ protein per dose, 1 μg to 1 mg ERRγ protein per dose, 1 mg to 1000 mg ERRγ protein per dose, or 10 mg to 100 mg ERRγ protein per dose. In some examples, doses are administered at least three-times in one week.
In one specific, non-limiting example, a unit dosage for administration of an ERRγ nucleic acid (such as injection, gene gun, pneumatic injection, or topical) includes at least 10 ng, at least 100 ng, at least 1 μg, at least 10 μg, at least 100 μg, or at least 500 μg nucleic acid per dose. Saline injections can use amounts of DNA, such as from 10 μg-1 mg, whereas gene gun deliveries can require 100 to 1000 times less DNA than intramuscular saline injection (such as 0.2 μg-20 μg). These amounts can vary from species to species, with mice, for example, requiring approximately 10 times less DNA than primates. Saline injections may require more DNA because the DNA is delivered to the extracellular spaces of the target tissue (e.g., muscle), where it has to overcome physical barriers before it is taken up by the cells, while gene gun deliveries bombard DNA directly into the cells.
In one specific, non-limiting example, a unit dosage for intravenous or intramuscular administration of a viral vector that encodes ERRγ includes at least 1×108 viral particles per dose, such as at least 1×109 viral particles per dose, at least 1×1010 viral particles per dose, or at least 1×1011 viral particles per dose.
Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
Agents that increase ERRγ activity (such as a ERRγ protein or agonist) can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The resulting solution can then added to an infusion bag containing 0.9% sodium chloride, USP, and can be administered in some examples at a dosage of from 1 to 300 mg/kg of body weight. Considerable experience is available in the art in the administration of proteins or nucleic acids. Such molecules can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
The agents that increase ERRγ can be administered to humans or other mammal using routine modes of administration, such as topically, orally, intravascularly such as intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, intracraneally, via inhalation or via suppository. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic).
Controlled release parenteral formulations of agents that increase ERRγ activity can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems (see Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly (see Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).
Polymers can be used for ion-controlled release. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, R., Accounts Chem. Res. 26:537, 1993). For example, the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425, 1992; and Pec et al., J. Parent. Sci. Tech. 44:58, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri, et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systems for controlled delivery of therapeutic proteins are known (see, for example, U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and 5,534,496).
Site-specific administration of the agents that increase ERRγ activity can be used, for instance by applying the agent to a region of the body in need of treatment, such as the brain, particular muscle, or kidney. In some embodiments, sustained release of the pharmaceutical preparation that includes a therapeutically effective amount of the one or more agents that increase ERRγ activity may be beneficial.
The present disclosure also includes combinations of one or more agents that increase ERRγ activity with one or more other agents useful in the treatment of a vascular, mitochondria, or muscular disorder. For example, the compounds of this disclosure can be administered in combination with effective doses of other angiogenic agents, such as vascular endothelial growth factor (VEGF), or fibroblast growth factor (FGF). The term “administration in combination” or “co-administration” refers to both concurrent and sequential administration of the active agents.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
EXAMPLE 1 Materials and MethodsThis example describes the materials and methods used for Examples 2-8.
Animals. Mouse ERRγ cDNA was placed downstream to the human a-skeletal actin promoter and upstream of the SV40 intron/poly (A) sequence. The purified transgene was injected into C57BL/6J x CBA F1 zygotes. Two transgenic founders (TG 425 and 421) were obtained that were backcrossed for 5 generations with C57BL/6J. All experiments used age (2-3 months) and sex (male) matched transgenic and wild type (WT) littermates. Mice were maintained on a normal chow diet. ERRγ+/−mice and tissue β-galactosidase staining has been described previously (Alaynick et al., 2007).
Drug treatment. Male C57Bl/6J mice (8 weeks old) were intra-peritoneally injected with vehicle or AICAR (500 mg/kg/day), as previously described (Narkar et al., 2008).
Gene and protein expression analysis. RNA was extracted using the Trizol extraction method from quadriceps or soleus isolated from WT and transgenic mice. Additionally, protein lysates were prepared from quadriceps and analyzed by western blotting with myoglobin (Dako), CYCS (Santacruz), UCP3 (Affinity Bioreagents), phospho-AMPK alpha (Cell Signaling, Cat no #2535) and total-AMPK alpha (Cell Signaling, Cat no #2532) antibodies. The AMPK antibodies detect both the alpha 1 and 2 catalytic subunits of AMPK (Narkar et al., 2008).
Microarray Analysis. Global gene expression analysis was performed in quadriceps from WT and transgenic mice, as previously described (Narkar et al., 2008).
Fluorescence Micro-angiography. Blood vessel mapping was performed as previously described (Johnson et al., 2004; Springer et al., 2000). Briefly, a red fluorescent microsphere (0.1 μM) suspension was intra-ventricularly perfused (10 ml, 1 ml/min) followed by euthanasia and tissue collection. Longitudinal cryo-sections (10 μM) of frozen gastrocnemius were processed and subjected to confocal microscopy to image skeletal muscle vasculature.
Cell culture, in vitro angiogenesis and Vegfa ELISA. C2C12 myoblasts were grown in 20% FBS-DMEM and differentiated in 2% horse serum-DMEM [with penicillin/streptomycin]. Conditioned media from two day differentiated WT and ERR/over-expressing C2C12 myotubules were used in the in vitro angiogenesis assay. Murine endothelial SVEC4-10 cells were cultured and maintained in DMEM containing 10% fetal bovine serum and penicillin/streptomycin. On the day of the experiment 4×105 cells/500 μl/well were plated in matrigel-coated 12-well plates. The cells were immediately treated for 7 hr with C2C12 cell conditioned media (2500), followed by evaluation of tube formation. Vegfa concentration in the conditioned media was measured using commercial Elisa kit according manufacturer's instructions [Research & Diagnostics].
Oxymetery and treadmill assays. Oxygen consumption, respiratory exchange ratio and ambulatory activity were measured in 3 month old, WT and transgenic male mice (N=6-7/group) of comparable weight using Comprehensive Lab Animal Monitoring System to obtain oxymetric measurements (Columbus Instruments). These mice were first acclimated in the monitoring system for 1 day, followed by data collection for 24 hr to include a 12 hr light and dark cycle. For each animal, the average of all the data points within the light or dark phase was used as a representative value of the respective cycle. Diurnal differences between the light and dark cycles were detectable in all animals, validating the method of data collection.
Endurance was determined in WT and transgenic (N=6 mice/group), as previously described (Narkar et al., 2008). The treadmill endurance test was performed as follows. WT and transgenic mice were acclimated to treadmill running (8 meters/min for 15 min) every other day for 1 week before the test. For the endurance testing, the mice were run on a treadmill at 5° inclination as the speed was gradually increased to 14 meters/min. After reaching 14 m/min, mice were run to exhaustion at constant speed. Endurance was measured as the function of time and distance ran.
Succinate Dehydrogenase (SDH) Staining. SDH staining was performed on 6 μM cryo-sections of gastrocnemius. Briefly, WT and transgenic sections were incubated at 37° C. for 10 min in substrate buffer [0.2M Phosphate buffer containing sodium succinate (250 mg/10 ml) and NBT (10 mg/10 ml)]. Following incubation, sections were washed three times with water following by two washes each with increasing and decreasing concentrations of acetone (30%, 60%, 90%). Finally, the sections were washed three times with water and mounted in an aqueous mounting media.
Immunohistochemistry. Gastrocnemius muscles isolated from WT and transgenic mice were equilibrated in 30% sucrose (in PBS) for 2-3 hr and frozen in OCT. Cryo-sections (10[1M) were fixed (4% paraformaldehyde-PBS), permeabilized (0.3% Triton X-PBS) and blocked (normal goat serum-PBS) before antibody treatment. Further, the sections were incubated overnight at 4° C. with anti-PECAM 1 antibody (1:25 in PBS, SEROTEC), washed three times with PBS, incubated with anti-rat secondary antibody (1:250, ALEXA FLOR 344), washed three times with PBS and mounted in VECTASHIELD. For negative controls, primary antibody was replaced with normal goat serum-PBS for overnight incubation.
Alkaline phosphatase (AP) staining. For AP staining, 10 μM muscle sections were fixed in ice-cold acetone (5 min, −20° C.), incubated in Tris-buffered Naphthol AS-MX phosphate/N,N Dimethylformamide solution (30 min, 37° C.), rinsed with distilled water (3×2 min) and mounted with aqueous media.
Data Analysis. Data was analyzed using either one way ANOVA with an appropriate post hoc test, or unpaired student's t-test, as indicated.
The global gene expression data has been deposited in the NCBI Gene Expression Omnibus under the GEO series accession number (pending).
EXAMPLE 2 Skeletal Muscle ERRγ ExpressionThis example describes methods used to examine ERRγ expression in skeletal muscle.
Because skeletal muscle is a functionally heterogeneous tissue containing both aerobic slow-twitch and glycolytic fast-twitch muscles, ERRγ expression was evaluated in the context of different myofibrillar beds. The ERRγ transcript was highly expressed in oxidative muscles such as soleus and red gastrocnemius, with minimal expression in glycolytic quadriceps and white gastrocnemius (
Viable ERRγ+/−mice are available in which a β-galactosidase protein-coding region without the promoter was introduced in-frame with the initiation site of the Esrrg gene (Alaynick et al., 2007) such that the enzyme mimics the expression of endogenous ERRγ. β-Galactosidase staining of different muscle beds from ERRγ+/−adult mice confirmed that the receptor is highly expressed in oxidative (e.g., soleus and red gastrocnemius) compared to the minimal levels in glycolytic muscles (e.g., quadriceps, white gastrocnemius) (
This example describes methods used to demonstrate the role of ERRγ in oxidative and slow-twitch muscle biology.
Transgenic mice were generated that selectively expressed ERRγ in skeletal muscles under the control of the human alpha-skeletal actin promoter (Muscat and Kedes, 1987; Wang et al., 2004). Two ERRγ over-expressing (ERRGO) transgenic lines were obtained (TG 421 and 425) showing both transcript (lower panel) and protein (upper panel) in fast-twitch quadriceps (
This example describes methods used to determine the transcriptional effect of ERRγ by measuring muscle gene expression in quadriceps from wild type and ERRGO mice.
In gene array analysis, it was observed that ERRγ regulated 1123 genes in skeletal muscles, of which 623 genes were induced. Gene ontology-based classification of these genes is presented in
Furthermore, contractile genes, especially ones associated with slow myofibers, were also activated raising the possibility of fast-to-slow transformation linked to the metabolic switch (Table 2).
Key biomarker genes associated with oxidative metabolism [Ucp3, Pdk4, Cycs, Cox5a, Lpl] and oxidative myofibers [Mhc Ia, Mhc IIa], but not glycolytic myofibers [Mhc IIb] were induced by ERRγ in quadriceps of transgenic mice (
To access the metabolic effects of ERRγ at the cellular level, the mitochondrial bioenergetics were measured in wild type and ERRγ over-expressing C2C12 cells using an extracellular flux analyzer. Specifically, the oxygen consumption rate (OCR) (an indicator of mitochondrial respiration) along with the extracellular acidification rate (ECAR) (a measure of glycolysis) was determined in these cells (
These results show that ERRγ promotes an overt conversion of glycolytic fast-twitch muscles, such as quadriceps, to an oxidative slow-twitch phenotype.
EXAMPLE 5 ERRγ Promotes Skeletal Muscle VascularizationIntrinsic vascularization of slow-twitch myofibers enables a baseline of exercise independent fatigue resistance. This example provides results demonstrating that ERRγ, by virtue of its restricted expression to type I fibers can, in addition to promoting oxidative metabolism, simultaneously induce vessel formation to match the increased oxidative demand.
Muscle cryo-sections were stained for PECAM 1 (CD31), an endothelial cell marker that is routinely used to detect angiogenesis and changes in tissue vasculature. Transgenic muscles showed increased PECAM 1 (
To show that ERRγ supports formation of functional non-leaky blood vessels micro-angiography following intra-ventricular perfusion of a fluorescent microspheres (0.1 μM) was used. The impermeability of the microspheres allows their vascular retention, enabling confocal angiographic “vascular mapping” of intact and mature blood vessels. Examination of perfused microspheres in wild type and transgenic gastrocnemius revealed an increase in muscle vascularity by ERRγ (
This example provides results showing how ERRγ expressed in myofibers can regulate proximal vascular development.
Gene expression studies (
As shown in Table 4, ERRγ also induces key transcriptional inducers of oxidative metabolism including Esrrb, Ppara, Ppard and Ppargc1b.
To directly test whether ERRγ triggers paracrine angiogenesis an SVEC4-10 (murine endothelial cells) tube formation assay was employed. It was reasoned that conditioned media from ERRγ over-expressing muscle cells would contain the appropriate signals to induce tube formation in endothelial cells. Indeed, treatment of SVEC4-10 cells with conditioned media from ERRγ over-expressing C2C12 myotubes stimulated tube formation in 7-8 hr (
Aerobic exercise-induced remodeling of skeletal muscles depends on both an increase in oxidative capacity and new blood vessel formation; changes that are a critical part of the physiologic adaptation to training (Bloor, 2005; Egginton, 2008; Gavin et al., 2007; Gustafsson and Kraus, 2001; Jensen et al., 2004; Waters et al., 2004). This example describes results showing the ability of ERRγ to promote physiological re-modeling.
First, in metabolic cage oxymetric studies, the transgenic mice exhibited an increase in oxygen consumption (during both the light and dark cycles) in concert with the observed increased oxidative metabolism and blood supply to skeletal muscles (
Based on these combined changes the ability of ERRGO mice to acquire enhanced running endurance was determined. ERRγ transgenic mice were able to run longer and further compared to the wild type littermates (
These findings demonstrate that targeting of ERRγ increases oxidative metabolism and blood supply to skeletal muscle leading to increased oxygen consumption, better endurance and resistance to weight gain.
EXAMPLE 8 PGC1α-Independent Regulation of Aerobic Muscle by ERRγPGC1α is induced by hypoxia and exercise to promote HIF1α-independent vascularization of type II muscle (Arany et al., 2008) and further activated by post-translational modifications such as deacetylation (Jager et al., 2007; Puigserver et al., 2001; Rodgers et al., 2005). This example describes results showing that the ERRγ-induced changes in the muscle were due to the induction and/or activation of PGC1α.
The levels of PGC1α mRNA, protein and acetylation remained unchanged in the ERRγ-transformed skeletal muscle (
To determine if AMPK can ERRγ control metabolism, VEGF induction and vasculature remodeling in ERRGO mice in absence of enhanced PGC1α signaling, the following methods were used. AMPK is an alternative aerobic master-regulator, having a role in metabolic (Fujii et al., 2008; Fujii et al., 2007) and vascular adaptation (Zwetsloot et al., 2008). While AMPK is normally induced by exercise or hypoxia, it was surprisingly found to be constitutively activated in ERRGO muscle (
These observations indicate a convergence between ERRγ and AMPK pathways that comprise an exercise-independent mechanism to direct intrinsic vascularization and oxidative metabolism in type I muscle, as depicted in
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In view of the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.
Claims
1. A method of increasing mitochondrial activity, comprising:
- administering a therapeutically effective amount of one or more agents that increases ERRγ activity to a mammal needing increased mitochondrial activity; and
- not exercising the mammal.
2. The method of claim 1, wherein the method further comprises:
- selecting a mammal in need of increased mitochondrial activity or a mammal at risk for developing a disorder that can benefit from increased mitochondrial activity.
3. The method of claim 1, wherein increased mitochondrial activity is needed in the mammal's muscle.
4. The method of claim 1, wherein the mammal has or is at risk for muscle atrophy, muscle wasting, sarcopenia, hearing loss, or mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode syndrome (MELAS).
5. The method of claim 1, wherein the one or more agents that increases ERRγ activity comprises:
- a nucleic acid molecule encoding ERRγ;
- one or more ERRγ agonists;
- an ERRγ protein; or
- combinations thereof.
Type: Application
Filed: Jan 17, 2017
Publication Date: Jul 6, 2017
Applicant: Salk Institute for Biological Studies (La Jolla, CA)
Inventors: Vihang A. Narkar (Houston, TX), Michael Downes (San Diego, CA), Ruth T. Yu (La Jolla, CA), Ronald M. Evans (La Jolla, CA)
Application Number: 15/407,940
