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 11

Animals treated with ENPP1 inhibitor myricetin demonstrate significant improvement in heart function after injury.

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Animals treated with ENPP1 inhibitor myricetin demonstrate significant i...
(A) Strategy for using myricetin. (B) Extracellular ATP hydrolytic activity in injured and uninjured hearts of animals treated with myricetin (n = 3). (C) B and M mode echocardiogram demonstrating contractile function in diastole (green arrows) and systole (yellow arrows) in hearts of myricetin-treated animals. (D) EF, fractional shortening, and LV chamber size in systole and diastole in vehicle- or myricetin-treated animals. n = 12 (vehicle) and n = 15 (myricetin) at basal, 1 week, and 2 weeks; n = 9 (vehicle) and n = 14 (myricetin) at 3 weeks and 4 weeks. (E) Fractions of animals with mild, moderate, and severe reduction in EF at 4 weeks after injury. (F) Scar size as a fraction of LV surface area. Scale bar: 1 mm. (G) Quantitation of scar surface area. n = 9 (vehicle); n = 4 (myricetin). (H) Fractions of animals with mild, moderate, and severe fibrosis following myricetin administration. (I and J) Immunostaining demonstrating (I) p53 (Ser15 phosphorylation) expression (arrowhead) in nonmyocytes in the injured region of vehicle- versus myricetin–injected animals and under higher magnification (myocytes are stained by troponin) and quantification (n = 4). (J) pH2AX staining in nonmyocyte cells (arrowheads) in vehicle- or myricetin-injected animals at 7 days following injury, under higher magnification and quantitation (n = 4, counts normalized to number of nonmyocyte nuclei for I and J). Scale bars: 5 μm (high magnification). Low magnification, ×40. (K) Metabolomic analysis of the hearts of myricetin-injected animals. (L) Decreased adenine+adenosine/uridine ratios in myricetin-injected animals (n = 3). (M) Metabolomic analysis of serum demonstrating decreased orotate and increased deoxyuridine (day 3) and increased orotidine (day 7) in myricetin-injected versus vehicle-injected animals (n = 3). Data are represented as mean ± SEM. **P < 0.01; *P < 0.05, ordinary 2-way ANOVA with Šidák’s multiple comparisons test (D) or 2-tailed Student’s t test (B, G, and I–M).