A small molecular activator of cardiac hypertrophy uncovered in a chemical screen for modifiers of the calcineurin signaling pathway Erik Bush*†, Jens Fielitz‡, Lawrence Melvin*, Michael Martinez-Arnold‡, Timothy A. McKinsey*, Ryan Plichta*, and Eric N. Olson†‡ *Myogen, Incorporated, Westminster, CO 80021; and ‡Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148 Contributed by Eric N. Olson, January 5, 2004 The calcium, calmodulin-dependent phosphatase calcineurin, regu- ative roles for these proteins in the control of calcineurin activity. lates growth and gene expression of striated muscles. The activity of Overexpression of MCIP1 (also called Down syndrome critical calcineurin is modulated by a family of cofactors, referred to as region 1), for example, suppresses calcineurin signaling (12). In modulatory calcineurin-interacting proteins (MCIPs). In the heart, the contrast, MCIP1 also seems to potentiate calcineurin signaling, MCIP1 gene is activated by calcineurin and has been proposed to as demonstrated by the diminution of calcineurin activity in the fulfill a negative feedback loop that restrains potentially pathological hearts of MCIP1 knockout mice (13). Intriguingly, the MCIP1 calcineurin signaling, which would otherwise lead to abnormal car- gene is a target of NFAT and is up-regulated in response to diac growth. In a high-throughput screen for small molecules capable calcineurin signaling (15), which has been proposed to create a of regulating MCIP1 expression in muscle cells, we identified a unique negative feedback loop that dampens calcineurin activity, which 4-aminopyridine derivative exhibiting an embedded partial structural would otherwise lead to abnormal cardiac growth. motif of serotonin (5-hydroxytryptamine, 5-HT). This molecule, re- In an effort to identify novel small molecules that might prevent ferred to as pyridine activator of myocyte hypertrophy, acts as a pathological cardiac hypertrophy by stimulating MCIP1 expression, selective agonist for 5-HT2A/2B receptors and induces hypertrophy of we performed a high-throughput screen (HTS) of a chemical cardiac muscle cells through a signaling pathway involving calcineurin library for compounds capable of activating the calcineurin͞NFAT- and a kinase-dependent mechanism that inactivates class II histone responsive promoter of the MCIP1 gene in muscle cells. We deacetylases, which act as repressors of cardiac growth. These find- describe a previously uncharacterized 4-aminopyridine that we ings identify MCIP1 as a downstream target of 5-HT2A/2B receptor refer to as pyridine activator of myocyte hypertrophy (PAMH), signaling in cardiac muscle cells and suggest possible uses for which induces MCIP1 expression and, unexpectedly, drives cardio- 5-HT2A/2B agonists and antagonists as modulators of cardiac growth myocyte hypertrophy. PAMH acts as a 5-hydroxytryptamine and gene expression. (5-HT)2A/2B receptor agonist and induces hypertrophy, at least in part, by stimulating nuclear import of NFAT and nuclear export of umerous agonists that act through G protein-coupled re- class II HDACs. These findings shed light on a powerful hypertro- Nceptors trigger calcium-dependent signal transduction path- phic signaling pathway downstream of 5-HT2A/2B receptor signaling ways that stimulate cardiac growth and gene expression (re- and suggest that chemical modulators of this pathway may be viewed in ref. 1). Postnatal cardiac myocytes respond to such efficacious in the control of cardiac growth and gene expression. signals by hypertrophic growth, characterized by an increase in Materials and Methods myocyte size and protein synthesis, assembly of sarcomeres, and activation of a fetal gene program (reviewed in ref. 2). Activation Cardiomyocyte Cultures. Neonatal rat ventricular myocytes of the calcium, calmodulin-dependent phosphatase calcineurin, (NRVMs) were cultured as described (16). For detailed proce- is sufficient and, in many cases, necessary for pathological dures, see Supporting Text, which is published as supporting cardiac hypertrophy (3), a major predictor of human morbidity information on the PNAS web site. and mortality (4). Thus, there has been intense interest in identifying novel small molecules capable of therapeutically Primary HTS. H9c2 cells (American Type Culture Collection no. ͞ modulating cardiac calcineurin signaling. CRL-1446; ref. 17) were cultured in DMEM with 10% (vol vol) ͞ ͞ Many calcineurin-sensitive genes are controlled by members FBS 4mML-glutamine 1% penicillin/streptomycin. Cells at a of the nuclear factor of activated T-cell (NFAT) family of concentration of 50,000 cells per ml were transiently transfected in transcription factors, which translocate to the nucleus when batch with a reporter construct (20 pg per cell) encoding firefly dephosphorylated by calcineurin (reviewed in ref. 5). The cal- luciferase under control of the exon 4 promoter from the human Ϫ ϩ cineurin pathway also stimulates the myocyte enhancer factor-2 MCIP1 gene (base pairs 874 to 30 relative to the beginning of ϫ Ϫ5 ␮ (MEF2) transcription factor by multiple mechanisms (6). We exon 4) and FuGENE transfection reagent (6 10 l per cell). have shown that calcineurin activates a kinase that phosphory- Transfected cells were plated on 96-well plates (Packard) at a lates class II histone deacetylases (HDACs), which act as MEF2 density of 5,000 cells per well. A 20,000-member library of small corepressors (7). Signal-dependent phosphorylation of class II molecule test compounds from the Myogen Library (selected for HDACs triggers their export from the nucleus and activation of molecular diversity and purchased from ChemBridge, San Diego) MEF2 target genes (8, 9). HDAC mutants lacking the signal- were then added by using a BioMEK FX robotic liquid handling responsive phosphorylation sites are refractory to calcium sig- naling and prevent cardiomyocyte hypertrophy. Conversely, Abbreviations: ANF, atrial natriuretic factor; PAMH, pyridine activator of myocyte hyper- mice lacking class II HDACs are hypersensitive to the growth- trophy; A-PAMH, antagonist of PAMH; CsA, cyclosporine A; HDAC, histone deacetylase; promoting activity of calcineurin (7). 5-HT, 5-hydroxytryptamine; HTS, high-throughput screen; NFAT, nuclear factor of acti- The activity of calcineurin is influenced by cofactors known as vated T cells; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine. modulatory calcineurin-interacting proteins (MCIPs) or calci- †To whom correspondence may be addressed. E-mail: [email protected] or pressins (reviewed in ref. 10). Recent studies in yeast (11) and [email protected]. mammalian cells (12–14) have revealed both positive and neg- © 2004 by The National Academy of Sciences of the USA 2870–2875 ͉ PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308723101 Downloaded by guest on September 28, 2021 Fig. 1. Identification of PAMH from a screen for small molecules that enhance MCIP1 expression. (A) Schematic diagram of the screen. A luciferase reporter controlled by the calcineurin-responsive exon 4 promoter of the human MCIP1 gene was transiently transfected into the H9c2 muscle cell line, which was screened in 96-well plates for luciferase expression in the presence of 20,000 individual small molecules. Our hypothesis was that activators of MCIP1 would inhibit cardiac hypertrophy by suppressing calcineurin activity. (B) Structure of PAMH and A-PAMH and their similarity to serotonin. An embedded possible structural motif shared by the three molecules is indicated in red. (C) MCIP1 protein was detected by Western blot after exposure of NRVMs to PAMH (1 ␮M) for 24 h. Calnexin was detected as a loading control. (D) MCIP1 protein was detected by Western blot in extracts derived from NRVMs infected with adenoviruses encoding lacZ (as a control), activated calcineurin (Cn), or the 28-kDa form of MCIP1 initiated from exon 4 in the presence or absence CsA for 24 h. (E) MCIP1 protein was detected by Western blot in extracts derived from NRVMs after exposure to PAMH (1 ␮M) in the presence or absence of CsA. CELL BIOLOGY system (Beckman Coulter) (one compound per well, 10 ␮M con- cardiac growth. Toward that end, we performed an HTS for centration). Sixteen control wells per plate received vehicle alone compounds able to stimulate expression of a luciferase reporter (0.1% DMSO, final). Plates were incubated for 48 h and processed gene controlled by the alternative promoter upstream of exon 4 for quantification of luciferase activity on a multiwell luminometer of the human MCIP1 gene in the H9c2 muscle cell line (Fig. 1A). (Packard Fusion). Primary HTS hits were defined as compounds This genomic region contains 15 NFAT-binding sites and confers that produced an increase in luciferase activity Ͼ3 SD from calcineurin responsiveness to MCIP1 (15). Transcripts initiated vehicle-only controls and were verified in secondary screens. from the exon 4 promoter encode a 197-aa MCIP1 protein with Ϸ Ϸ a Mr 28 kDa compared with the 38-kDa protein encoded by Western Blot Analysis and Immunostaining. A peptide correspond- transcripts containing exon 1 (20). ing to the C terminus of the murine MCIP1 protein (GenBank From a screen of 20,000 compounds, we identified 21 that accession no. AAF63486; CRPEYTPIHLS) was synthesized stimulated MCIP1-luciferase expression by 2-fold or greater. The (Sigma Genosys), incorporating an N-terminal cysteine residue strongest hit, named PAMH, is a previously uncharacterized