Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair
Shen Li, Tomohiro Yokota, Ping Wang, Johanna ten Hoeve, Feiyang Ma, Thuc M. Le, Evan R. Abt, Yonggang Zhou, Rimao Wu, Maxine Nanthavongdouangsy, Abraham Rodriguez, Yijie Wang, Yen-Ju Lin, Hayato Muranaka, Mark Sharpley, Demetrios T. Braddock, Vicky E. MacRae, Utpal Banerjee, Pei-Yu Chiou, Marcus Seldin, Dian Huang, Michael Teitell, Ilya Gertsman, Michael Jung, Steven J. Bensinger, Robert Damoiseaux, Kym Faull, Matteo Pellegrini, Aldons J. Lusis, Thomas G. Graeber, Caius G. Radu, Arjun Deb
Shen Li, Tomohiro Yokota, Ping Wang, Johanna ten Hoeve, Feiyang Ma, Thuc M. Le, Evan R. Abt, Yonggang Zhou, Rimao Wu, Maxine Nanthavongdouangsy, Abraham Rodriguez, Yijie Wang, Yen-Ju Lin, Hayato Muranaka, Mark Sharpley, Demetrios T. Braddock, Vicky E. MacRae, Utpal Banerjee, Pei-Yu Chiou, Marcus Seldin, Dian Huang, Michael Teitell, Ilya Gertsman, Michael Jung, Steven J. Bensinger, Robert Damoiseaux, Kym Faull, Matteo Pellegrini, Aldons J. Lusis, Thomas G. Graeber, Caius G. Radu, Arjun Deb
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Research Article Cardiology

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair

Abstract

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Authors

Shen Li, Tomohiro Yokota, Ping Wang, Johanna ten Hoeve, Feiyang Ma, Thuc M. Le, Evan R. Abt, Yonggang Zhou, Rimao Wu, Maxine Nanthavongdouangsy, Abraham Rodriguez, Yijie Wang, Yen-Ju Lin, Hayato Muranaka, Mark Sharpley, Demetrios T. Braddock, Vicky E. MacRae, Utpal Banerjee, Pei-Yu Chiou, Marcus Seldin, Dian Huang, Michael Teitell, Ilya Gertsman, Michael Jung, Steven J. Bensinger, Robert Damoiseaux, Kym Faull, Matteo Pellegrini, Aldons J. Lusis, Thomas G. Graeber, Caius G. Radu, Arjun Deb

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Figure 2

ENPP1 in the presence of ATP induces cardiomyocytes to release proapoptotic molecules.

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ENPP1 in the presence of ATP induces cardiomyocytes to release proapopto...
(A) Coculture of rat ventricular cardiomyocytes (CMs, red) with control CF or ENPP1-CF (green) with or without ATP (arrowheads show decrease in ENPP1 CF). Scale bar: 100 μm. (B) Number of CMs and CFs following 48 hours of coculture (n = 4). (C) Control CFs or ENPP1 CFs (green) in the presence or absence of added ATP but without any cardiomyocytes, and quantitation of cell numbers after 48 hours of ATP/vehicle addition (n = 3). Scale bar: 100 μm. (D) Transfer of control or ENPP1+ATP MCndM to CFs and images 48 hours later demonstrating decreased numbers of CFs treated with ENPP1+ATP MCndM (arrowheads). Scale bar: 50 μm. (E) Flow cytometry to demonstrate the fraction of dead (PI+) or apoptotic cells (annexin V+, PI–) following treatment with ENPP1+ATP MCndM or control MCndM (n = 7). (F) TUNEL and caspase staining (arrowheads) of CFs treated with vehicle MCndM or ENPP1+ATP MCndM and (G) quantitation (n = 3). Scale bar: 50 μm. (H) CFs treated with vehicle MCndM, PPi MCndM, or AMP MCndM for 48 hours showing loss of cells with treatment with AMP MCndM (arrowheads) and (I) quantitation of dead cells (n = 7). Scale bar: 50 μm. (J) Treatment of macrophages, HUVECs, and human vascular smooth muscle cells (hVSMCs) with vehicle MCndM or ENPP1+ATP MCndM and (K) corresponding flow cytometry to determine cell death (arrowheads). (n = 6, BM macrophages; n = 6, HUVECs, n = 3 hVSMCs). Scale bars: 50 μm. (L) ENPP1+ATP MCndM does not cause cell death when added to myocytes (n = 4). Scale bar: 100 μm. Data are represented as mean ± SEM. **P < 0.01; *P < 0.05, ordinary 1-way ANOVA with Tukey’s multiple comparison test (B, C, E, and I) or 2-tailed Student’s t test (G, K, and L).