Therapeutic role of miR-19a/19b in cardiac regeneration and protection from myocardial infarction

doi:10.1038/s41467-019-09530-1

. 2019 Apr 17;10(1):1802.

doi: 10.1038/s41467-019-09530-1.

Therapeutic role of miR-19a/19b in cardiac regeneration and protection from myocardial infarction

Feng Gao¹, Masaharu Kataoka^{2

3}, Ning Liu¹, Tian Liang¹, Zhan-Peng Huang^{2

4}, Fei Gu², Jian Ding², Jianming Liu², Feng Zhang¹, Qing Ma², Yingchao Wang⁵, Mingming Zhang², Xiaoyun Hu², Jan Kyselovic⁶, Xinyang Hu⁵, William T Pu^{2

7}, Jian'an Wang⁵, Jinghai Chen⁸, Da-Zhi Wang^{9

10}

Affiliations

¹ Department of Cardiology, The Second Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, 310029, Hangzhou, China.
² Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA.
³ Department of Cardiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
⁴ Center for Translational Medicine, The First Affiliated Hospital, NHC Key Laboratory of Assisted Circulation, Sun Yat-sen University, Guangzhou, 510080, China.
⁵ Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, 310009, Hangzhou, China.
⁶ Faculty of Pharmacy, Comenius University, Bratislava, 832 32, Slovak Republic.
⁷ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, 02138, USA.
⁸ Department of Cardiology, The Second Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, 310029, Hangzhou, China. jinghaichen@zju.edu.cn.
⁹ Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA. Da-Zhi.Wang@childrens.harvard.edu.
¹⁰ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, 02138, USA. Da-Zhi.Wang@childrens.harvard.edu.

PMID: 30996254
PMCID: PMC6470165
DOI: 10.1038/s41467-019-09530-1

Therapeutic role of miR-19a/19b in cardiac regeneration and protection from myocardial infarction

Feng Gao et al. Nat Commun. 2019.

. 2019 Apr 17;10(1):1802.

doi: 10.1038/s41467-019-09530-1.

Authors

Affiliations

¹ Department of Cardiology, The Second Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, 310029, Hangzhou, China.
² Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA.
³ Department of Cardiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
⁴ Center for Translational Medicine, The First Affiliated Hospital, NHC Key Laboratory of Assisted Circulation, Sun Yat-sen University, Guangzhou, 510080, China.
⁵ Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, 310009, Hangzhou, China.
⁶ Faculty of Pharmacy, Comenius University, Bratislava, 832 32, Slovak Republic.
⁷ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, 02138, USA.
⁸ Department of Cardiology, The Second Affiliated Hospital, Institute of Translational Medicine, Zhejiang University School of Medicine, 310029, Hangzhou, China. jinghaichen@zju.edu.cn.
⁹ Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA, 02115, USA. Da-Zhi.Wang@childrens.harvard.edu.
¹⁰ Harvard Stem Cell Institute, Harvard University, Cambridge, MA, 02138, USA. Da-Zhi.Wang@childrens.harvard.edu.

PMID: 30996254
PMCID: PMC6470165
DOI: 10.1038/s41467-019-09530-1

Abstract

The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart. Previously, we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation. Here, we report that expression of miR-19a/19b, members of the miR-17-92 cluster, is induced in heart failure patients. We show that intra-cardiac injection of miR-19a/19b mimics enhances cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction (MI) injury. miR-19a/19b protected the adult heart in two distinctive phases: an early phase immediately after MI and long-term protection. Genome-wide transcriptome analysis demonstrates that genes related to the immune response are repressed by miR-19a/19b. Using an adeno-associated virus approach, we validate that miR-19a/19b reduces MI-induced cardiac damage and protects cardiac function. Finally, we confirm the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI. Our study establishes miR-19a/19b as potential therapeutic targets to treat heart failure.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
Expression of mir-19a/miR-19b in the heart and cardiomyocytes. a qRT-PCR of miR-19a and miR-19b at 2 and 12 days postnatal mouse hearts. n = 3 mice. b In isolated adult cardiomyocytes and non-cardiomyocytes, qRT-PCR detection of the expression of miR-19a. n = 3 mice. c, d In mouse myocardial infarction (MI) model, qRT-PCR detection of the expression of miR-19a and miR-19b (c) at 3 days post-MI (n = 6 mice) and (d) at 2 weeks and 4 weeks post-MI. n = 3–6 mice. e In mouse transverse aortic constriction (TAC) induced cardiac hypertrophy model, qRT-PCR detection of the expression of miR-19a, miR-19b, and miR-21 at 3 days and 2 weeks post-TAC. Expression of miR-21 was considered as positive control. n = 2–3 mice. f In human heart disease samples, qRT-PCR detection of the expression of pri-mir19a in hearts with dilated cardiomyopathy (DCM) and coronary artery disease (CAD). The expression of ANF (encoded by *Nppa*) was considered as positive control. All panels, statistical significance was calculated using Student’s t-test and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 2**
Direct injection of miR-19a/19b mimics protects the heart from myocardial infarction. a Gene structure of the *miR-17–92* cluster and conserved sequences of miR-19a and miR-19b across species. Seed sequences are highlighted. b Echocardiography analyses of mice with intra-cardiac injection of individual miR-19a, miR-19b or control mimics at 5 days, 2, 7, and 9 weeks post MI. n = 3–6 mice. c Transverse sections of miR-19a, miR-19b or control mimic injected hearts at 2 months post MI. Sirius red/fast green marks myocardium (green) and scar (red). Scale bar = 2 mm. d Quantification of the size of scar. n = 3–4 mice. e qRT-PCR of miR-19a in heart at 4 days, 2 months, and 1 year after intra-cardiac injection of miR-19a/19b mimics. n = 3–5 mice. f Experimental design. Mice receiving miR-19a/19b mimic post-MI were assessed for cardiac function short term and long term, as well as morphological assessment. g Left ventricular fractional shortening (FS%) and (h) LV internal dimension at end-systole (LVID;s). n = 5 mice in control mimic group, n = 10 mice in miR-19a/19b mimic group. i Representative images of series of transverse sections after injection of miR-19a/19b mimics compared to control group at 2 months after MI. Sirius red/fast green collagen staining marks myocardium (green) and scar (red). Scale bar = 2 mm. j Quantification of the size of scar in the hearts after injection of miR-19a/19b mimics. n = 5 control mice; n = 4 miR-19a/19b mimic-treated mice. k qRT-PCR detection of expression of pathological remodeling marker genes BNP (encoded by *Nppb*) and β-MHC (encoded by *Myh7*). n = 5 control mice; n = 6 miR-19a/19b mimic-treated mice. Statistical significance was calculated using Student’s t-test in b, d, e, g, h, j, and k and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 3**
Short-term and long-term cardiac protection after myocardial infarction and injection of miR-19a/19b mimics. a Kaplan–Meier survival curves after injection of miR-19a/19b mimics compared to injection of control mimic after MI injury. n = 11 mice mimic control; n = 13 mice, miR-19a/19b mimics. b, c Echocardiography analyses of cardiac function after miR-19a/19b mimic injection at both short term of 2–4 weeks and 4 months (b) and long term of 12 months (c) after MI injury compared to their control group. FS% left ventricular fractional shortening. LVIDs LV internal dimension at end-systole. n = 3–12 mice. d Representative images of series of transverse sections after injection of miR-19a/19b mimics compared to control mimic at long term of 12 months after MI injury. Sirius red/fast green collagen staining marks myocardium (green) and scar (red). Scale bar = 2 mm. e Quantification of the size of scar after injection of miR-19a/19b mimics compared to control mice at long term of 12 months after MI injury. n = 3 mice. f qRT-PCR detection of expression of fibrotic remodeling marker genes 4 days after mimic injection and MI injury. n = 3–7 mice. g qRT-PCR detection of expression of pathological remodeling marker genes 12 months after mimic injection and MI injury. n = 4–6 mice. Statistical significance was calculated using Student’s t-test in b, c, e, f, and g and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 4**
miR-19a/19b promotes cardiomyocyte proliferation after myocardial infarction. a Immunofluorescence staining of EdU incorporation on transverse sections of adult hearts injected with miR-19a/19b or control mimic 2 months post MI injury. EdU labels proliferating cells (green); cardiac troponin T (cTNT) marks cardiomyocytes (red); wheat germ agglutinin (WGA) marks cell surfaces (white) and DAPI labels nuclei (blue). Arrows point to EdU-positive signal in cardiomyocytes and stars mark EdU-positive signal in non-cardiomyocytes. Scale bar = 48 μm. b, c Quantification of percentages of EdU-positive cardiomyocytes. n = 5 hearts for control group, n = 3 hearts for miR-19a/19b mimics group, 25–30 fields per heart for each group. d Immunofluorescence staining of pH3 on transverse sections of adult hearts injected with miR-19a/19b or control mimic 4 days post MI injury. pH3 (green); α-actinin (red); WGA (white) and DAPI (blue). pH3-positive signal in cardiomyocytes (arrows) and non-cardiomyocytes (asterisk) are marked. Scale bar = 20 μm. e Quantification of pH3-positive cardiomyocytes. n = 3 hearts for each group, 6–8 fields per heart for each group. f Immunofluorescence staining of Aurora B adult hearts. Aurora B (red); α-ACTININ (green); WGA (white); and DAPI (blue). Arrows point to Aurora B-positive signals in cardiomyocytes. Scale bar = 20 μm. g Quantification of Aurora B-positive cardiomyocytes. n = 4 hearts for each group, five sections for each heart. h qRT-PCR of cell cycle marker genes. n = 4–5 hearts. i Representative images of cardiomyocytes isolated from adult hearts injected with control or miR-19a/19b mimic 3 weeks post MI injury. Scale bars = 100 μm. j Quantification of the number of cardiomyocytes. Approximately 4 × 10³ cardiomyocytes were counted per group, using eight independent heart samples. k Immunostaining of isolated cardiomyocytes with Connexin 43 (red), α-actinin (green), and DAPI (blue). Scale bars = 10 μm. l For nucleation, ~1 × 10³ cardiomyocytes were counted per sample, using nine control hearts and six miR-19a/19b mimic-treated hearts. Statistical significance was calculated using Student’s t-test in b, c, e, g, h, j, and l and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 5**
miR-19a/19b reduces myocardial infarction-induced inflammation and cell death. a TUNEL staining (red) on transverse sections of adult hearts injected with miR-19a/19b or control mimic 4 days after MI. cTNT marks cardiomyocytes (green) and DAPI marks nuclei (blue). Scale bars = 48 μm. b, c Quantification of TUNEL-positive cardiomyocytes (b) and non-cardiomyocytes (c). n = 4 hearts, 6–14 fields per heart. d qRT-PCR detection of apoptosis gene expression. n = 3–7 hearts. e Western blot analysis of protein levels of PTEN, BIM, cleaved Caspase 3, and BAX in adult hearts. f Quantification of Western blot band density. n = 4 hearts. g Hierarchical clustering of 544 differentially expressed genes between miR-19a/19b and control mimic injected hearts 4 days after MI. Red and blue colors indicate up-regulated or down-regulated genes. h Gene ontology (GO) analysis of 393 down-regulated genes. (i) qRT-PCR analysis of expression of down-regulated genes related to immune response, cell death, and fibrosis. Up-regulated genes were also examined. NPPA serves as a control for cardiomyopathy. n = 3 hearts. **j–l** Immunohistochemistry analysis of M1 markers, (j) CD80, (k) iNOS, and M2 marker (l) Arg-1 in adult hearts injected with miR-19a/19b or control mimics at 48 and 72 h post MI. n = 4–8 hearts. Scale bars= 50 μm. m qRT-PCR analysis of expression of M1 and M2 marker genes and potential target genes in adult hearts. n = 3–6 hearts. n Western blot analysis of protein levels of SOCS and STAT3 pathways in adult hearts. o Quantification of Western blot band density. n = 3–4 hearts. Statistical significance was calculated using Student’s t-test in b, c, d, f, i, m, and o and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 6**
AAV9-mediated overexpression of miR-19a/19b protects the heart from myocardial infarction. a Schematic of the AAV9-miR-19a/19b and AAV9-Control constructs and the experimental procedure of the AAV9-miR-19a/19b therapeutic trial in adult mice after myocardial infarction injury. ITR inverted terminal repeat. b qRT-PCR detection of the level of miR-19a in heart after intra-cardiac injection of AAV9-miR-19a/19b 3 months post-MI. n = 2–3 hearts. c, d Echocardiography analyses of cardiac function after intra-cardiac injection of AAV9-miR-19a/19b versus control 3 months post-MI. AAV9-miR-19a/19b injection (c) significantly increased left ventricular fractional shortening (FS%) and (d) decreased LV internal dimension at end-systole LVID;s. n = 4–7 hearts. e Representative images of series of transverse sections after intra-cardiac injection of AAV9-miR-19a/19b versus control at 3 months post-MI. Sirius red/fast green collagen staining marks myocardium (green) and scar (red). Scale bar = 2 mm. f Quantification of the scar size of the heart sections. n = 4 hearts. **g–i** qRT-PCR detection of expression of (g) cardiomyopathy marker genes, (h) fibrosis marker genes and (i) miR-19 target gene PTEN in hearts after intra-cardiac AAV9-miR-19a/19b injection 3 months post-MI. n = 4–6 hearts. j Western blot analysis of protein level of PTEN in hearts after intra-cardiac AAV9-miR-19a/19b injection 7 days post-MI. k Quantification of Western blot band density. n = 3 hearts. Statistical significance was calculated using Student’s t-test in b, c, d, f, g, h, i, and k and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

**Fig. 7**
Therapeutic potential of miR-19a/19b in treating infarcted hearts. a Schematic of tail-vein injection of RNALancerII-delivered miRNA mimics post myocardial infarction (MI). b qRT-PCR detection of miR-19a and miR-19b expression in the heart 6 h after injection post MI injury. n = 1 heart each group. c qRT-PCR detection of miR-19a and miR-19b expression in the heart at 24 h after injection with or without MI injury. n = 2 hearts each group. d, e Echocardiography analyses of cardiac function after tail-vein injection of miR-19a/19b and control mimic at different time points post MI. d FS%, left ventricular fractional shortening. e LVIDs LV internal dimension at end-systole. n = 3–10 mice. f Representative images of series of transverse heart sections after tail-vein injection of miR-19a/19b or control mimics post MI. Sirius red/Fast green collagen staining marks myocardium (green) and scar (red). g Quantification of the size of scar. n = 3 hearts. h Western blot analysis of protein levels of SOCS and STAT3 in the heart 24 h after tail-vein injection of miR-19a/19b or control mimics post MI. Statistical significance was calculated using Student’s t-test in d, f, and g and data are presented as means ± s.e.m. *p < 0.05, **p < 0.01 vs. control. Source data are provided as a Source Data file

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References

1. Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 2013;14:529–541. doi: 10.1038/nrm3619. - DOI - PMC - PubMed
1. van Rooij E. Cardiac repair after myocardial infarction. N. Engl. J. Med. 2016;374:85–87. doi: 10.1056/NEJMcibr1512011. - DOI - PubMed
1. Porrello ER, et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080. doi: 10.1126/science.1200708. - DOI - PMC - PubMed
1. Nakada Y, et al. Hypoxia induces heart regeneration in adult mice. Nature. 2017;541:222–227. doi: 10.1038/nature20173. - DOI - PubMed
1. Kennedy-Lydon, T. & Rosenthal, N. Cardiac regeneration: all work and no repair? Sci. Transl. Med. 9, eaad9019 (2017). - PubMed

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[1] Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 2013;14:529–541. doi: 10.1038/nrm3619. - DOI - PMC - PubMed

[2] Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat. Rev. Mol. Cell Biol. 2013;14:529–541. doi: 10.1038/nrm3619. - DOI - PMC - PubMed

[3] van Rooij E. Cardiac repair after myocardial infarction. N. Engl. J. Med. 2016;374:85–87. doi: 10.1056/NEJMcibr1512011. - DOI - PubMed

[4] van Rooij E. Cardiac repair after myocardial infarction. N. Engl. J. Med. 2016;374:85–87. doi: 10.1056/NEJMcibr1512011. - DOI - PubMed

[5] Porrello ER, et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080. doi: 10.1126/science.1200708. - DOI - PMC - PubMed

[6] Porrello ER, et al. Transient regenerative potential of the neonatal mouse heart. Science. 2011;331:1078–1080. doi: 10.1126/science.1200708. - DOI - PMC - PubMed

[7] Nakada Y, et al. Hypoxia induces heart regeneration in adult mice. Nature. 2017;541:222–227. doi: 10.1038/nature20173. - DOI - PubMed

[8] Nakada Y, et al. Hypoxia induces heart regeneration in adult mice. Nature. 2017;541:222–227. doi: 10.1038/nature20173. - DOI - PubMed

[9] Kennedy-Lydon, T. & Rosenthal, N. Cardiac regeneration: all work and no repair? Sci. Transl. Med. 9, eaad9019 (2017). - PubMed

[10] Kennedy-Lydon, T. & Rosenthal, N. Cardiac regeneration: all work and no repair? Sci. Transl. Med. 9, eaad9019 (2017). - PubMed

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Therapeutic role of miR-19a/19b in cardiac regeneration and protection from myocardial infarction

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Therapeutic role of miR-19a/19b in cardiac regeneration and protection from myocardial infarction

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