Angiotensin II type 1 receptor blockade attenuates TGF-beta-induced failure of muscle regeneration in multiple myopathic states.

Skeletal muscle has the ability to achieve rapid repair in response to injury or disease. Many individuals with Marfan syndrome (MFS), caused by a deficiency of extracellular fibrillin-1, exhibit myopathy and often are unable to increase muscle mass despite physical exercise. Evidence suggests that selected manifestations of MFS reflect excessive signaling by transforming growth factor (TGF)-beta (refs. 2,3). TGF-beta is a known inhibitor of terminal differentiation of cultured myoblasts; however, the functional contribution of TGF-beta signaling to disease pathogenesis in various inherited myopathic states in vivo remains unknown. Here we show that increased TGF-beta activity leads to failed muscle regeneration in fibrillin-1-deficient mice. Systemic antagonism of TGF-beta through administration of TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor blocker losartan normalizes muscle architecture, repair and function in vivo. Moreover, we show TGF-beta-induced failure of muscle regeneration and a similar therapeutic response in a dystrophin-deficient mouse model of Duchenne muscular dystrophy.

Circulating transforming growth factor-beta in Marfan syndrome.

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-beta (TGF-beta). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-beta activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-beta might be mirrored in circulating TGF-beta concentrations. METHODS AND RESULTS: Serum obtained from MFS mutant mice (Fbn1(C1039G/+)) treated with losartan was analyzed for circulating TGF-beta1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-beta1 concentrations increased with age and were elevated in older untreated Fbn1(C1039G/+) mice compared with wild-type mice (P=0.01; n=16; mean+/-SEM, 115+/-8 ng/mL versus n=17; mean+/-SEM, 92+/-4 ng/mL). Losartan-treated Fbn1(C1039G/+) mice had lower total TGF-beta1 concentrations compared with age-matched Fbn1(C1039G/+) mice treated with placebo (P=0.01; n=18; 90+/-5 ng/mL), and circulating total TGF-beta1 levels were indistinguishable from those of age-matched wild-type mice (P=0.8). Correlation was observed between circulating TGF-beta1 levels and aortic root diameters in Fbn1(C1039G/+) and wild-type mice (P=0.002). In humans, circulating total TGF-beta1 concentrations were elevated in patients with MFS compared with control individuals (P<0.0001; n=53; 15+/-1.7 ng/mL versus n=74; 2.5+/-0.4 ng/mL). MFS patients treated with losartan (n=55) or beta-blocker (n=80) showed significantly lower total TGF-beta1 concentrations compared with untreated MFS patients (P< or =0.05). CONCLUSIONS: Circulating TGF-beta1 concentrations are elevated in MFS and decrease after administration of losartan, beta-blocker therapy, or both and therefore might serve as a prognostic and therapeutic marker in MFS.

Timeline of cardiac dystrophy in 3-18-month-old MDX mice.

The dystrophin-deficient (mdx) mouse remains the most commonly used model for Duchenne muscular dystrophy (DMD). Mdx mice show a predominantly covert cardiomyopathy, the hallmark of which is fibrosis. We compared mdx and normal mice at six ages (3, 6, 9, 12, 15, and 18 months) using in vivo assessment of cardiac function, selective collagen staining, and measures of TGF-ß mRNA, Evans blue dye infiltration, macrophage infiltration, and aortic wall thickness. Clear temporal progression was demonstrated, including early fragility of cardiomyocyte membranes, which has an unrelated impact on cardiac function but is associated with macrophage infiltration and fibrosis. Aortic wall thickness is less in older mdx mice. Mdx mice display impaired responses to inotropic challenge from a young age; this is indicative of altered adrenoreceptor function. We draw attention to the paradox of ongoing fibrosis in mdx hearts without a strong molecular signature (in the form of TGF-ß mRNA expression).

Angiotensin II-dependent TGF-ß signaling contributes to Loeys-Dietz syndrome vascular pathogenesis.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-ß receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-ß signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-ß signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-ß in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-ß signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-ß target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-ß1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-ß1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-ß signaling contributes to postnatal aneurysm progression in LDS.

Losartan restores skeletal muscle remodeling and protects against disuse atrophy in sarcopenia.

Sarcopenia, a critical loss of muscle mass and function because of the physiological process of aging, contributes to disability and mortality in older adults. It increases the incidence of pathologic fractures, causing prolonged periods of hospitalization and rehabilitation. The molecular mechanisms underlying sarcopenia are poorly understood, but recent evidence suggests that increased transforming growth factor-ß (TGF-ß) signaling contributes to impaired satellite cell function and muscle repair in aged skeletal muscle. We therefore evaluated whether antagonism of TGF-ß signaling via losartan, an angiotensin II receptor antagonist commonly used to treat high blood pressure, had a beneficial impact on the muscle remodeling process of sarcopenic mice. We demonstrated that mice treated with losartan developed significantly less fibrosis and exhibited improved in vivo muscle function after cardiotoxin-induced injury. We found that losartan not only blunted the canonical TGF-ß signaling cascade but also modulated the noncanonical TGF-ß mitogen-activated protein kinase pathway. We next assessed whether losartan was able to combat disuse atrophy in aged mice that were subjected to hindlimb immobilization. We showed that immobilized mice treated with losartan were protected against loss of muscle mass. Unexpectedly, this protective mechanism was not mediated by TGF-ß signaling but was due to an increased activation of the insulin-like growth factor 1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. Thus, blockade of the AT1 (angiotensin II type I) receptor improved muscle remodeling and protected against disuse atrophy by differentially regulating the TGF-ß and IGF-1/Akt/mTOR signaling cascades, two pathways critical for skeletal muscle homeostasis. Thus, losartan, a Food and Drug Administration-approved drug, may prove to have clinical benefits to combat injury-related muscle remodeling and provide protection against disuse atrophy in humans with sarcopenia.

Noncanonical TGFß signaling contributes to aortic aneurysm progression in Marfan syndrome mice.

Transforming growth factor-ß (TGFß) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFß can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal-regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFß. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase-1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFß signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Long-term administration of pirfenidone improves cardiac function in mdx mice.

Duchenne muscular dystrophy, an X-linked recessive neuromuscular disorder due to lack of the protein dystrophin, manifests as progressive muscle degeneration and cardiomyopathy with increased fibrosis. The exact mechanisms involved in fibrosis are unknown, but a cytokine, transforming growth factor-beta (TGF-beta), is a likely mediator. This study tested whether the TGF-beta antagonist, pirfenidone, could reduce cardiac fibrosis. Eight-month-old mdx mice were treated for 7 months with 0.4%, 0.8%, and 1.2% pirfenidone in drinking water; untreated water was given to control mdx and C57 mice. Mice treated with 0.8% and 1.2% pirfendone had lowered cardiac TGF-beta mRNA and improved in vitro cardiac contractility (P < 0.05) to levels consistent with C57 mice, yet without a change in cardiac stiffness or fibrosis. These results show that the TGF-beta antagonist, pirfenidone, can improve cardiac function in mdx mice, potentially providing a new avenue for developing cardiac therapies for patients with Duchenne muscular dystrophy.