Protein kinase A activation inhibits DUX4 gene expression in myotubes from patients with facioscapulohumeral muscular dystrophy

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator double homeobox 4 (DUX4) is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing DUX4 expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce DUX4 gene expression and hence DUX4 activity. Among those, β₂-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased DUX4 expression by >75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces DUX4 expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how DUX4 expression is regulated in FSHD and point to potential areas of therapeutic intervention.

Keywords: D4Z4; DUX4; FSHD; adrenergic receptor; cyclic AMP (cAMP); drug discovery; facioscapulohumeral muscular dystrophy; muscle atrophy; muscular dystrophy; protein kinase A (PKA).

Figure 1.

DUX4- and Dux4-dependent gene expression is induced in FSHD but not healthy myoblasts upon differentiation. A, immunofluorescence of MYHC (red) and nuclei (blue) in healthy (Skmdc) and FSHD1 (FSHD940 and FSHD869) human primary myotubes during 7 days of differentiation. Each individual image is nine 10× fields. B, quantification of MYHC-positive nuclei by high-content imaging (percentage of total nuclei). C, qPCR for MYOG (left) and CKM (right). D, immunofluorescence of Dux4 (green), MYHC (red), and nuclei (blue) in FSHD940 and FSHD869 myotubes after 5 and 7 days of differentiation, respectively. E, qPCR for ZSCAN4 (left) and TRIM43 (right). ND, not detected. Error bars, S.E.

Figure 2.

ZSCAN4 promoter-based reporter gene assay detects endogenous Dux4 activity in FSHD1 myotubes. A, luciferase activity measured in ZSCAN4 RGA–transduced healthy (Skmdc) and FSHD1 (FSHD940) myoblasts and myotubes. B, qPCR for DUX4 in Skmdc and FSHD940 myoblasts and myotubes. C, luciferase activity measured in ZSCAN4 RGA–transduced FSHD940 myotubes transfected with a nontargeting siRNA (CON) or siRNA against Dux4 (DUX4). D, qPCR for DUX4 in FSHD940 myotubes transfected with a nontargeting siRNA (CON) or siRNA against Dux4 (DUX4); **, p < 0.01. Error bars, S.E.

Figure 3.

β2AR agonists reduce DUX4 expression in FSHD myotubes. A, luciferase activity measured in ZSCAN4 RGA–transduced FSHD1 (FSHD940) myotubes treated with formoterol (FOR) and albuterol (ALB). B, qPCR for ZSCAN4 (top) and TRIM43 (bottom) in FSHD940 myotubes treated with varying concentrations of formoterol (FOR) and albuterol (ALB). C, qPCR for DUX4 in FSHD940 myotubes treated with 100 nm formoterol (FOR) and albuterol (ALB); *, p < 0.05. D, quantification of MYHC-positive nuclei by high content imaging (percentage of DMSO control). Myotubes were treated with 100 nm formoterol (FOR) or albuterol (ALB) for 48 h. Error bars, S.E.

Figure 4.

Formoterol induces cAMP production in FSHD myotubes through the β2AR. A, measurement of cAMP produced in FSHD1 (FSHD940) myotubes after formoterol treatment. B, cAMP produced in FSHD940 myotubes treated with varying concentrations of formoterol in the presence of the β2AR antagonist ICI118551, the β₁-adrenergic receptor antagonist CGP20712, or DMSO (CON). Error bars, S.E.

Figure 5.

PDE inhibitors reduce DUX4 expression in FSHD myotubes. A, qPCR for ZSCAN4 (top) and TRIM43 (bottom) in FSHD1 (FSHD940) myotubes treated with varying concentrations of crisaborole and ibudilast. B, qPCR for DUX4 in FSHD940 myotubes treated with 10 μm crisaborole and ibudilast; *, p < 0.05. C, quantification of MYHC-positive nuclei by high content imaging (percentage of DMSO control). Myotubes were treated with 10 μm crisaborole or ibudilast for 48 h. Error bars, S.E.

Figure 6.

The cAMP analog 8-Br-cAMP reduces DUX4 expression in FSHD myotubes. A, luciferase activity measured in ZSCAN4 RGA–transduced FSHD1 (FSHD940) myotubes treated with 8-Br-cAMP. B, qPCR for ZSCAN4 (top) and TRIM43 (bottom) in FSHD940 myotubes treated with varying concentrations of 8-Br-cAMP. C, qPCR for DUX4 in FSHD940 myotubes treated with 500 μm 8-Br-cAMP; *, p < 0.05. Error bars, S.E.

Figure 7.

Active PKA is sufficient to reduce DUX4 activity in FSHD myotubes. FSHD940 myotubes were treated at day 5 for 48 h followed by protein extraction, SDS-PAGE, and Western blotting. A, immunoblot analysis of phosphorylated PKA substrates (p-PKA), phosphorylated CREB (p-CREB), total CREB, and HA-tagged (HA) caPKA from FSHD940 myotubes transduced with varying concentrations of caPKA and empty (CON) adenovirus. vp, viral particle. B, qPCR for ZSCAN4 (top) and TRIM43 (bottom) in FSHD940 myotubes transduced with varying concentrations of caPKA or CON adenovirus or treated with 100 nm formoterol (FOR) for 48 h; **, p < 0.01; *, p < 0.05. Error bars, S.E.

Figure 8.

Reduction of Dux4 activity by formoterol in FSHD myotubes requires PKA activity. A, FSHD1 (FSHD940) myotubes were transduced with adenovirus at day 5 for 48 h, followed by treatment with formoterol for 60 min. Protein was extracted, and SDS-PAGE/Western blotting was performed. Shown is immunoblot analysis of phosphorylated PKA substrates (p-PKA), phosphorylated CREB (p-CREB), total CREB, and HA-tagged (HA) dnPKA from FSHD940 myotubes transduced with varying concentrations of dnPKA and empty (CON) adenovirus. vp, viral particle. B, qPCR for ZSCAN4 (left) and TRIM43 (right) in FSHD940 myotubes transduced with 0.3 × 10⁹ viral particle/ml dnPKA or CON adenovirus and treated with 100 nm formoterol (FOR) or DMSO (VEH) for 48 h; **, p < 0.01; *, p < 0.05; ns, not significant. Error bars, S.E.