Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions.

BACKGROUND: Control of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production. METHODS: We treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production. RESULTS: In tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment. CONCLUSIONS: These data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.

Treatment with a corticotrophin releasing factor 2 receptor agonist modulates skeletal muscle mass and force production in aged and chronically ill animals.

BACKGROUND: Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting. HYPOTHESIS: We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals. METHODS: We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP. RESULTS: In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals. CONCLUSIONS: These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.

Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice.

BACKGROUND: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy. METHODS: Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression. RESULTS: We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression. CONCLUSION: Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.

Modifications of the human urocortin 2 peptide that improve pharmacological properties.

Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.

Discovery of corticotropin releasing factor 2 receptor selective sauvagine analogues for treatment of skeletal muscle atrophy.

The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.

Activation of the vasoactive intestinal peptide 2 receptor modulates normal and atrophying skeletal muscle mass and force.

Of the two known vasoactive intestinal peptide receptors (VPAC1R and VPAC2R), the VPAC2R is expressed in skeletal muscle. To evaluate the function of the VPAC2R in the physiological control of skeletal muscle mass, we utilized the VPAC1R selective agonist [K15,R16,L27]VIP(1-7) GRF(8-27)-NH2 and the VPAC2R selective agonist Ro-25-1553 to treat mice and rats undergoing either nerve damage-, corticosteroid-, or disuse-induced skeletal muscle atrophy. These analyses demonstrated that activation of VPAC2R, but not VPAC1R, reduced the loss of skeletal muscle mass and force during conditions of skeletal muscle atrophy resulting from corticosteroid administration, denervation, casting-induced disuse, increased skeletal muscle mass, and force of nonatrophying muscles. These studies indicate that VPAC2R agonists may have utility for the treatment of skeletal muscle-wasting diseases.

Urocortin II treatment reduces skeletal muscle mass and function loss during atrophy and increases nonatrophying skeletal muscle mass and function.

Two corticotropin-releasing factor 2 receptor (CRF2R)-selective peptides have been recently described, urocortin II (also known as stresscopin-related peptide) and urocortin III (stresscopin). We have used urocortin II to evaluate the effects of activation of the CRF2R on skeletal muscle-related physiological processes. Administration of urocortin II to mice prevented the loss of skeletal muscle mass resulting from disuse due to casting, corticosteroid treatment, and nerve damage. In addition, urocortin II treatment prevented the loss of skeletal muscle force and myocyte cross-sectional area that accompanied muscle mass losses resulting from disuse due to casting. Finally, we observed increased skeletal muscle mass and force in normal muscles when mice are treated with urocortin II. These results were confirmed using two additional CRF2R agonists, urocortin I and sauvagine. Thus, activation of the CRF2R modulates skeletal muscle mass in both normal and atrophying muscle. Therefore, CRF2R-selective agonists may find utility in the treatment of skeletal muscle wasting diseases including age-related muscle loss or sarcopenia.

Corticotropin-releasing factor 2 receptor localization in skeletal muscle.

Our objective in this study was to localize the corticotropin-releasing factor 2 receptor (CRF2R) in rodent and human skeletal muscle. We found CRF2R protein to be abundant in neural tissues in skeletal muscle, including large nerve fibers and bundles, neural tissue associated with mechanoreceptors, muscle spindles, and the Golgi tendon organ. CRF2R protein was also abundant in blood vessels in skeletal muscle. CRF2R protein was also observed, although with less abundance, in the endo/perimysial regions in skeletal muscle. The localization of the CRF2R to blood vessels is consistent with the CRF2R-mediated vascular phenomena observed previously, but the observation of CRF2R in neural tissue in skeletal muscle is a novel finding with an unknown function.

Phosphodiesterase 4 inhibition reduces skeletal muscle atrophy.

Several GTP-binding protein (G-protein)-coupled receptors that signal through Galphas (GTP-binding protein alpha stimulatory) and the cyclic adenosine monophosphate (cAMP) pathway increase skeletal muscle mass. In order to further evaluate the role of the cAMP pathway in the regulation of skeletal muscle mass, we utilized inhibitors of phosphodiesterase 4 (PDE 4), the major cAMP-modifying PDE found in skeletal muscle, to modulate skeletal muscle cAMP levels. We found that PDE 4 inhibitors reduced the loss of muscle mass and force resulting from denervation and casting in rats and mice. These studies indicate that PDE 4 inhibitors may have a role in the treatment of skeletal muscle-wasting diseases.

Skeletal muscle hypertrophy and anti-atrophy effects of clenbuterol are mediated by the beta2-adrenergic receptor.

Analyses were performed to evaluate the roles of the beta1- and beta2-adrenergic receptors in the skeletal muscle hypertrophy and anti-atrophy response to the beta-adrenergic agonist, clenbuterol. Treatment of wild-type mice with clenbuterol resulted in statistically significant hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles and inhibition of denervation-induced atrophy of these muscles. Treatment of beta1-adrenergic receptor knockout mice with clenbuterol also resulted in statistically significant hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles and inhibition of denervation-induced atrophy of these muscles. In contrast, in beta2-adrenergic receptor knockout mice and in mice lacking both the beta1- and beta2-adrenergic receptors, clenbuterol treatment did not result in hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles, nor did it inhibit denervation-induced atrophy in these muscles. Together these data demonstrate that the beta2-adrenergic receptor is responsible for both the skeletal muscle hypertrophy and anti-atrophy effects of the beta-adrenergic agonist clenbuterol.

Activation of the CRF 2 receptor modulates skeletal muscle mass under physiological and pathological conditions.

Two receptors activated by the corticotropin-releasing factor (CRF) family of peptides have been identified, the CRF 1 receptor (CRF1R) and the CRF 2 receptor (CRF2R). Of these, the CRF2R is expressed in skeletal muscle. To understand the role of the CRF2R in skeletal muscle, we utilized CRFR knockout mice and CRF2R-selective agonists to modulate nerve damage and corticosteroid- and disuse-induced skeletal muscle atrophy in mice. These analyses demonstrated that activation of the CRF2R decreased nerve damage and corticosteroid- and disuse-induced skeletal muscle mass and function loss. In addition, selective activation of the CRF2R increased nonatrophy skeletal muscle mass. Thus we describe for the first time a novel activity of the CRF2R, modulation of skeletal muscle mass.