RORα nuclear receptors in protection against angiotensin II-induced cardiac hypertrophy

Nuclear hormone receptors (NRs) are a family of ligand-regulated transcription factors involved in diverse developmental and physiological functions, including the regulation of metabolism and immune and stress responses. The retinoic acid-related orphan receptors RORα (NR1F1), RORβ (NR1F2), and RORγ (NR1F3) constitute a subfamily of nuclear receptors that bind to DNA both as monomers and as dimers and display distinct patterns of tissue expression (8). RORα has been shown to play important roles in the regulation of circadian rhythm and metabolism (2) and is widely expressed in multiple tissues including liver, skeletal muscle, skin, lung, adipose tissue, kidney, thymus, brain, and, recently, also the heart (12, 18). The homozygous mutant mouse “staggerer” (RORα^sg/sg), which carries a deletion within the RORα gene (5), is characterized by severe cerebellar ataxia, staggering gait, tremor, hypotonia, and small size due to defects in the development of cerebellar granule cells and Purkinje cells (15). Importantly, engineered mice lacking RORα display the same cerebellar atrophic phenotype as the staggerer mouse (16). RORα^sg/sg mice have been widely utilized and characterized to unravel the biological relevance of RORα, leading to the identification of additional phenotypes outside the nervous system. These include, for example, a greater predisposition to atherosclerosis (11), increased resistance to diet-induced obesity (10), immunodeficiency linked to the overexpression of inflammatory cytokines (9), and abnormalities in the formation and maintenance of bone tissue (13). However, the functional relevance of RORα in the heart has not yet been fully explored. Only a few recent studies have investigated the contribution of RORα in the setting of cardiac disease. RORα receptors were shown to exert a protective role during myocardial ischemia/reperfusion (MI/R) injury (6). In that study, RORa^sg/sg mice exhibited significantly increased myocardial infarct size, myocardial apoptosis, and contractile dysfunction after MI/R compared with wild-type littermates (6). In another study, RORα deficiency resulted in worse high-fat diet-induced myocardial hypertrophy and dysfunction via mechanisms involving, at least in part, impaired mitochondrial biogenesis (18). Whether RORα plays any role in the development of pathological cardiac hypertrophy and failure has been an open question.

In an article in this issue of this journal, Beak et al. (1) provide evidence that RORα regulates angiotensin II (ANG II)-induced pathological hypertrophy by modulating proinflammatory signaling pathways and mitochondrial function (Fig. 1). In this study, to investigate the role of RORα in ANG II-induced cardiomyocyte hypertrophy, the authors used the staggerer RORα^sg/sg mouse model in combination with studies in cultured neonatal rat ventricular myocytes (NRVM). When treated for 14 days with ANG II, RORα^sg/sg mice showed an exaggerated hypertrophic response, increased fibrosis, and reduced left ventricular contractile function compared with wild-type littermates, suggesting that RORα inhibits pathological cardiac hypertrophy. Similar results were obtained in in vitro gain- and loss-of-function experiments in NRVM. Interestingly, despite showing comparable increases in heart size (heart weight-to-tibia length ratio) after ANG II treatment to those seen in wild-type animals, the RORα^sg/sg mice had a much greater increase in myocyte cross-sectional area than wild-type mice. A possible explanation for this discrepancy could be a decrease in total cardiomyocyte number in RORα^sg/sg hearts. Indeed, in line with this possibility, RORα^sg/sg hearts showed a significant increase in number of apoptotic cells upon both vehicle and ANG II treatment. In addition, loss of RORα activity per se was associated with increased oxidative stress and a decrease in mitochondrial ATP levels. It is important to note that, in contrast to previous findings (6, 18), in this study, RORα^sg/sg mice had significant baseline (vehicle treatment) differences in cardiac phenotype compared with wild-type littermates; specifically, they had a significantly lower heart weight-to-tibia length ratio, reduced left ventricular fractional shortening, and a pronounced increase in apoptosis. The reason for these differences from previous studies are not clear, and further studies are required to confirm these observations and to understand whether RORα plays a physiological role in the heart even in the absence of disease stresses.

Fig. 1.

Model of the protective role of retinoic acid-related orphan receptor-α (RORα) in angiotensin (ANG) II-induced cardiomyocyte hypertrophy. AT1/2R, ANG II receptor 1/2. A: RORα modulates proinflammatory responses by negatively regulating the nuclear factor (NF)-κB and interleukin-6-signal transducer and activator of transcription 3 (IL-6-STAT3) (pSTAT3-Tyr⁷⁰⁵) signaling pathways. Activation and nuclear translocation of NF-κB induces expression of NF-κB transcriptional targets, including IL-6, leading to enhanced STAT3-mediated inflammatory signaling. RORα regulates NF-κB via the NF-κB inhibitory protein IκBα, and it may also negatively regulate IL-6 levels at a transcriptional level. RORα improves mitochondrial function by promoting STAT3 phosphorylation at Ser⁷²⁷ via an unknown mechanism. B: in the absence of RORα, there is enhanced NF-κB nuclear translocation and expression of IL-6 with increased downstream STAT3 signaling (pSTAT3-Tyr⁷⁰⁵), while phosphorylation of STAT3 at Ser⁷²⁷ decreases, thus leading to exaggerated cardiac hypertrophy, increased inflammation, oxidative stress, and cell death.

Studies in astrocytes (7) and smooth muscle cells (3) have shown that RORα acts as a negative regulator of the inflammatory response by inhibiting tumor necrosis factor-α (TNF-α)-induced IL-6 expression and negatively regulating the nuclear factor (NF)-κB signaling pathway via induction of the NF-κB inhibitory protein IκBα. Given that IL-6 and NF-κB are known mediators of ANG II-induced cardiomyocyte hypertrophy (14) and pressure overload (17), the authors explored the possibility that the antihypertrophic effects of RORα might be mediated by repression of IL-6 and NF-κB signaling. Indeed, Beak et al. (1) showed that the absence of RORα boosts the expression of cardiac inflammatory mediators after ANG II treatment. In fact, enhanced upregulation of IL-6 and of downstream signal transducer and activator of transcription 3 (STAT3) signaling (pSTAT3-Tyr⁷⁰⁵) was found in RORα^sg/sg hearts compared with wild-type hearts after ANG II treatment. In line with previous findings (3), the authors also demonstrated that total and phosphorylated levels of IκBα were increased in the absence of RORα and that, as a consequence, NF-κB translocation into the nucleus also increased. Additionally, they were able to demonstrate a direct transcriptional regulation of IL-6 by RORα in vitro, with RORα found to bind to a ROR response element (RORE) in the IL6 promoter region. Taken together, these findings suggest that RORα may regulate IL-6 levels both directly and indirectly via an increase in NF-κB nuclear translocation and subsequent expression of NF-κB transcriptional targets, including IL-6. Further studies are required to confirm these interesting findings in the in vivo setting. When phosphorylated at Tyr⁷⁰⁵, STAT3 translocates into the nucleus, where it acts as a transcriptional activator. However, when phosphorylated at Ser⁷²⁷, STAT3 has been shown to translocate into the mitochondria and to increase mitochondrial respiration (4). Here, the authors showed that, in the absence of RORα, STAT3 phosphorylation at Tyr⁷⁰⁵ and the induction of proinflammatory and stress responses were enhanced, while there was a concomitant decrease in STAT3 phosphorylation at Ser⁷²⁷ along with decreased mitochondrial function and increased oxidative stress. It remains to be determined what RORα-dependent signaling pathways control the specific phosphorylation status of STAT3 at different residues and thereby the balance between inflammatory signaling and mitochondrial effects.

Interestingly, RORα levels seem significantly decreased both in a mouse model of heart failure and in failing human heart tissue, suggesting that downregulation of RORα may contribute to the pathophysiology of cardiac hypertrophy and failure. However, the mechanisms by which RORα abundance is regulated in the setting of pathological hypertrophy and heart failure remains an open question.

In conclusion, the current study by Beak et al. (1) reveals novel functions for RORα in the heart, as RORα emerges as a potential cardioprotective nuclear receptor. A limitation of this work is the global disruption of RORα in RORα^sg/sg mice, meaning that the contribution of different cell types to the observed exaggerated hypertrophic phenotype is unclear. Future studies in cell-specific and inducible RORα knockout mice would be informative in further delineating the roles of RORα in the heart.

GRANTS

This work is supported by the British Heart Foundation (RG/13/11/30384), a Fondation Leducq Transatlantic Network of Excellence award, and in part by the Department of Health via a National Institute for Health Research (NIHR) Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.Z. and A.M.S. drafted manuscript; A.Z. and A.M.S. edited and revised manuscript; A.Z. and A.M.S. approved final version of manuscript.