Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Mar 2;101(9):2870-5.
doi: 10.1073/pnas.0308723101. Epub 2004 Feb 19.

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

Erik Bush  1 , Jens Fielitz, Lawrence Melvin, Michael Martinez-Arnold, Timothy A McKinsey, Ryan Plichta, Eric N Olson
Affiliations

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

Erik Bush et al. Proc Natl Acad Sci U S A. .

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-HT(2A/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. These findings identify MCIP1 as a downstream target of 5-HT(2A/2B) receptor signaling in cardiac muscle cells and suggest possible uses for 5-HT(2A/2B) agonists and antagonists as modulators of cardiac growth and gene expression.

PubMed Disclaimer

Figures

Fig. 1.
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 μM) 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 μM) in the presence or absence of CsA.
Fig. 2.
Fig. 2.
Stimulation of cardiac hypertrophy by PAMH. (A) α-Actinin and perinuclear ANF staining of NRVMs in the absence or presence of PAMH (1 μM) for 24 h. PAMH induces hypertrophy, ANF, and sarcomere assembly. (B–E) Cell size, protein content, ANF secretion, and β-myosin heavy chain expression were detected in NRVMs in the absence or presence of PAMH (1 μM) or PE (10 μM) for 24 h. (F) Venn diagram representing array elements regulated by PAMH and PE. Array elements that were regulated at least 2-fold by either PAMH or PE were scored for statistically significant regulation by either compound by using the Affymetrix statistical parameters. The areas of the Venn diagram represent the number of array elements that were significantly regulated by only one drug or by both drugs.
Fig. 3.
Fig. 3.
Effects of receptor antagonists on the activity of PAMH. (A) NRVMs were pretreated for 1 h with the indicated compound followed by PAMH (1 μM) for 24 h. The ratio of ANF/GAPDH mRNA expression was determined. Values are expressed relative to the ANF/GAPDH ratio in cells treated with PAMH alone, which was assigned a value of 100%. Compounds were used at the concentrations described in Supporting Text. (B) Competition curve showing the percent inhibition of binding of radiolabeled lysergic acid diethylamide to the 5-HT2B receptor by the indicated concentrations of unlabeled PAMH. (C) NRVMs were treated with PAMH (1 μM) and the indicated concentrations of cyproheptadine and ketanserin for 24 h and MCIP1 was detected by Western blot. (D) NRVMs were treated with PAMH (1 μM) in the presence or absence of A-PAMH at the indicated concentrations and secreted ANF was detected. Values are expressed relative to the maximal level of ANF expression in the presence of PAMH. (E) NRVMs were treated with PAMH (1 μM) and A-PAMH at the indicated concentrations, and MCIP1 was detected by Western blot.
Fig. 4.
Fig. 4.
Effects of inhibitors on the activity of PAMH. NRVMs were pretreated with the indicated inhibitors for 1 h followed by PAMH (1 μM) for 24 h, and ANF transcripts were detected, as in Fig. 3A. Compounds were used at the concentrations described in Supporting Text.
Fig. 5.
Fig. 5.
Signaling to NFAT and class II HDACs by PAMH. (A) Stimulation of NFAT nuclear import by PAMH. NRVMs transfected with a GFP-NFATc expression plasmid were exposed to PAMH (1 μM) or PE (10 μM) for 24 h, as indicated, and GFP was detected. In unstimulated cells, GFP-NFATc is localized to the cytoplasm. Both agonists drive GFP-NFATc to the nucleus. (B) Stimulation of GFP-HDAC5 nuclear export by PAMH. NRVMs infected with an adenovirus encoding GFP-HDAC5 were exposed to PAMH (1 μM) or PE (10 μM), as indicated, for 24 h, and GFP was detected. In unstimulated cells, GFP-HDAC5 is localized to the nucleus. Both agonists promote translocation to the cytoplasm, and A-PAMH blocks this effect. (C) Blockade to PAMH activity by signal-resistant HDAC. NRVMs infected with an adenovirus encoding a signal-resistant HDAC5 mutant were exposed to PAMH, as indicated, and stained for α-actinin. The HDAC5 mutant prevents hypertrophy and sarcomere assembly in response to PAMH.
Fig. 6.
Fig. 6.
Possible signaling pathways controlled by PAMH. The activity of PAMH is blocked by staurosporin and the tyrosine kinase inhibitor AG490, implicating serine, threonine kinases, and tyrosine kinases, respectively, in the signaling pathway controlled by PAMH. PAMH drives nuclear export of HDAC5 and nuclear import of NFATc and stimulates the expression of MCIP1 and other genes involved in cardiac growth and remodeling. Signaling events that appear to play dominant roles in PAMH signaling are shown in bold. Those that are documented, but for which the relative importance remains to be established, are shown by thin lines, and those that remain hypothetical are shown by dashed lines. Prohypertrophic effectors are in green, and antihypertrophic effectors are in red.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Frey, N. & Olson, E. N. (2003) Annu. Rev. Physiol. 65, 45-79. - PubMed
    1. MacLellan, W. R. & Schneider, M. D. (2000) Annu. Rev. Physiol. 62, 289-319. - PubMed
    1. Olson, E. N. & Williams, R. S. (2000) Cell 101, 689-692. - PubMed
    1. Kannel, W. B. & Cobb, J. (1992) Cardiology 81, 291-298. - PubMed
    1. Hogan, P. G., Chen, L., Nardone, J. & Rao, A. (2003) Genes Dev. 17, 2205-2232. - PubMed

Publication types

MeSH terms

LinkOut - more resources