A small molecular activator of cardiac hypertrophy uncovered in a chemical screen for modifiers of the calcineurin signaling pathway

Abstract

The calcium, calmodulin-dependent phosphatase calcineurin, regulates growth and gene expression of striated muscles. The activity of calcineurin is modulated by a family of cofactors, referred to as modulatory calcineurin-interacting proteins (MCIPs). In the heart, the MCIP1 gene is activated by calcineurin and has been proposed to fulfill a negative feedback loop that restrains potentially pathological calcineurin signaling, which would otherwise lead to abnormal cardiac growth. In a high-throughput screen for small molecules capable of regulating MCIP1 expression in muscle cells, we identified a unique 4-aminopyridine derivative exhibiting an embedded partial structural motif of serotonin (5-hydroxytryptamine, 5-HT). This molecule, referred to as pyridine activator of myocyte hypertrophy, acts as a selective agonist for 5-HT2A/2B receptors and induces hypertrophy of cardiac muscle cells through a signaling pathway involving calcineurin and a kinase-dependent mechanism that inactivates class II histone deacetylases, which act as repressors of cardiac growth. 2). Activation of the calcium, calmodulin-dependent phosphatase calcineurin, is sufficient and, in many cases, necessary for pathological cardiac hypertrophy (3), a major predictor of human morbidity and mortality (4). Thus, there has been intense interest in identifying novel small molecules capable of therapeutically modulating cardiac calcineurin signaling.Many calcineurin-sensitive genes are controlled by members of the nuclear factor of activated T-cell (NFAT) family of transcription factors, which translocate to the nucleus when dephosphorylated by calcineurin (reviewed in ref. 5). The calcineurin pathway also stimulates the myocyte enhancer factor-2 (MEF2) transcription factor by multiple mechanisms (6). We have shown that calcineurin activates a kinase that phosphorylates class II histone deacetylases (HDACs), which act as MEF2 corepressors (7). Signal-dependent phosphorylation of class II HDACs triggers their export from the nucleus and activation of MEF2 target genes (8, 9). HDAC mutants lacking the signalresponsive phosphorylation sites are refractory to calcium signaling and prevent cardiomyocyte hypertrophy. Conversely, mice lacking class II HDACs are hypersensitive to the growthpromoting activity of calcineurin (7).The activity of calcineurin is influenced by cofactors known as modulatory calcineurin-interacting proteins (MCIPs) or calcipressins (reviewed in ref. 10). Recent studies in yeast (11) and mammalian cells (12-14) have revealed both positive and negative roles for these proteins in the control of calcineurin activity. Overexpression of MCIP1 (also called Down syndrome critical region 1), for example, suppresses calcineurin signaling (12). In contrast, MCIP1 also seems to potentiate calcineurin signaling, as demonstrated by the diminution of calcineurin activity in the hearts of MCIP1 knockout mice (13). Intriguingly, the MCIP1 gene is a target of NFAT and is up-regulated in response to calcineurin signaling (15), which has been propo...

Toward transcriptional therapies for the failing heart: chemical screens to modulate genes

“…Forced overexpression of class II HDACs 5 or 9 in cardiac myocytes prevents hypertrophy in response to diverse agonists (26)(27)(28). More important, mice in which the gene encoding either HDAC5 or HDAC9 has been disrupted by homologous recombination are hypersensitive to pathological signals, developing cardiomegaly and eventual cardiac failure in response to stresses such as pressure overload or introduction of the calcineurin transgene ( Figure 2B) (26)(27)(28)(29).…”

“…Instead, these HDACs are shuttled from the nucleus to the cytoplasm in response to stress (Figure 3), which provides a posttranslational mechanism to override HDACmediated repression of cardiac growth (27,28,31). This redistribution of HDACs frees MEF2 (and other transcription factors) to associate with HATs (32,33), resulting in increased local histone acetylation and activation of downstream genes that promote cell growth.…”

“…Based on the apparent cardioprotective functions of MCIP1, a high-throughput screen for small molecular activators of MCIP1 expression in cardiac myocytes was designed (27). In principle, such activators could induce MCIP1 expression through stimulation of the calcineurin/NFAT signaling pathway, in which case they would likely be prohypertrophic, or they could act through other mechanisms.…”

“…In principle, such activators could induce MCIP1 expression through stimulation of the calcineurin/NFAT signaling pathway, in which case they would likely be prohypertrophic, or they could act through other mechanisms. In a study using regulatory DNA sequences from the MCIP1 gene linked to a luciferase reporter, several novel small molecules capable of inducing luciferase expression were identified (27). One such activator, pyridine activator of myocyte hypertrophy (PAMH), dramatically stimulated MCIP1 expression.…”

“…Indeed, 5-HT receptor antagonists abolished PAMH activity on cardiomyocytes, and direct ligand binding assays showed that PAMH binds the 5-HT 2B receptor with an affinity of approximately 60 nM (27).…”