Improved Factor Combination for In Vivo Reprogramming of Cardiac Myofibroblast to Cardiomyocyte-Like Cell With Dual Recombinase Tracing

HomeCirculationVol. 148, No. 21Improved Factor Combination for In Vivo Reprogramming of Cardiac Myofibroblast to Cardiomyocyte-Like Cell With Dual Recombinase Tracing Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBImproved Factor Combination for In Vivo Reprogramming of Cardiac Myofibroblast to Cardiomyocyte-Like Cell With Dual Recombinase Tracing Jingdong Wu, Hong Zhao, Yanmeng Tao, Chunyan Yang, Yang Yang, Bin Zhou and Yang Zhao Jingdong WuJingdong Wu https://orcid.org/0000-0002-5536-7438 State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). , Hong ZhaoHong Zhao State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). , Yanmeng TaoYanmeng Tao State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). , Chunyan YangChunyan Yang https://orcid.org/0000-0003-0436-7128 State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). , Yang YangYang Yang State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). , Bin ZhouBin Zhou Correspondence to: Bin Zhou, MD, PhD, 320 Yueyang Rd, Shanghai, 200031, China, Email E-mail Address: [email protected] https://orcid.org/0000-0001-5278-5522 New Cornerstone Science Laboratory, State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China (B.Z.). and Yang ZhaoYang Zhao Yang Zhao, PhD, No 5 Yi He Yuan Rd, Haidian District, Beijing 100871, China, Email E-mail Address: [email protected] https://orcid.org/0000-0002-0856-2724 State Key Laboratory of Natural and Biomimetic Drugs, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Center for Life Sciences, Institute of Molecular Medicine, College of Future Technology, Peking University, Beijing, China (J.W., H.Z., Y.T., C.Y., Y.Y., Y.Z.). Originally published20 Nov 2023https://doi.org/10.1161/CIRCULATIONAHA.122.062810Circulation. 2023;148:1728–1731Nonrenewable cell types such as cardiomyocytes die with injury and aging, causing irreversible damage to normal heart function. In vivo direct reprogramming can promote heart regeneration by converting proliferative, extracellular matrix–secreting activated cardiac myofibroblasts into cardiomyocytes. This approach replenishes cardiomyocytes while simultaneously ameliorating cardiac fibrosis. Previous studies have reported in vivo cardiac fibroblast reprogramming into cardiomyocytes through the expression of transcription factors (such as GATA4, MEF2C, TBX5 [GMT]), microRNA silencing, or transcription factor expression in conjunction with small molecule activators.1However, our previous study showed that activated myofibroblasts, the ideal targets of in vivo cardiac reprogramming, are more difficult to reprogram than resident cardiac fibroblasts in vitro.2The efficiency remains unclear of in vivo cardiac myofibroblast–to–cardiomyocyte-like cell reprogramming using stringent lineage tracing systems. Furthermore, the discovery of genetic and chemical boosters of in vitro myofibroblast-to-cardiomyocyte reprogramming2 (Tao Y, Yang Y, Wang LP, Yang ZH, Wang SQ, Yang Z. Robust small molecule-aided cardiac reprogramming systems selective to cardiac fibroblasts. Preprint. Posted online September 21, 2023. bioRxiv 2023.09.20.558597. doi: 10.1101/2023.09.20.558597) raised the intriguing possibility of applying these boosters to further increase the efficiency of in vivo myofibroblast-to-cardiomyocyte reprogramming.Because it cannot be precluded that some resident cardiomyocytes may express myofibroblast markers, such as Col1a1 after injury,3 we adopted the IR1 dual recombinase system to stringently lineage trace myofibroblasts only.4 We generated Tnni3-Dre;IR1;Postn-MerCreMer mice to selectively label activated myofibroblasts with ZsGreen driven by the periostin (Postn) promoter3 (Figure [A]). Resident troponin I (TNNI3)+ cardiomyocytes can be selectively labeled with tdTomato by Dre during heart development with high efficiency, whereas the loxP and ZsGreen components in labeled cardiomyocytes will be removed permanently, so resident cardiomyocytes cannot express ZsGreen in response to Cre. Subsequent tamoxifen (TAM)–induced Cre-loxP recombination led to permanent labeling of POSTN+ myofibroblasts and their descendants, whereas the rox component was ablated, permanently abolishing tdTomato expression in response to Dre, even after reprogramming to induced cardiomyocytes (iCMs).Download figureDownload PowerPointFigure. In vivo cardiac MF-to-CM reprogramming with dual recombinase tracing. A, Schematic figure showing Cre-loxP recombination in CM and Dre-rox recombination in MF by Tnni3-Dre;IR1;Postn-MerCreMer strategy. B, Experimental strategy for MF-to-CM in vivo reprogramming study using Tnni3-Dre;IR1;Postn-MerCreMer mice. C through E, Immunostaining for tdTomato, ZsGreen, and cTnI on heart sections from MI+solvent (C), GMT+solvent (D), and GMTMS+SR (E) -treated Tnni3-Dre;IR1;Postn-MerCreMer mice. Boxed regions are magnified. White numbers in E indicate the clusters of iCMs. Scale bar, 100 μM. F, Immunostaining for tdTomato, ZsGreen, and α-actinin on heart section from GMTMS+SR-treated Tnni3-Dre;IR1;Postn-MerCreMer mouse. Boxed regions are magnified. Scale bar, 100 μM. G, Quantitative analyses for ZsGreen+tdTomato+ fused CMs in MI+solvent, GMT+solvent, and GMTMS+SR-treated Tnni3-Dre;IR1;Postn-MerCreMer mice are shown. n=5 independent biological replicates. H, Quantitative analyses for ZsGreen+tdTomato– CMs in MI+solvent, EGFP+solvent, GMT+solvent, GMTMS+solvent, GMTMS+SR, GMTS+SR, GMTM+SR, GMTMS+Ruxo, and GMTMS+SB-treated Tnni3-Dre;IR1;Postn-MerCreMer mice are shown. n=5 independent biological replicates. I and J, Left ventricular ejection fraction (LVEF) was quantified serially by echocardiography in mice treated with EGFP+solvent, GMT+solvent, and GMTMS+SR for 6 weeks. *,**GMTMS+SR vs EGFP+solvent; #,##GMTMS+SR vs GMT+solvent (I). LVEF and left ventricular fractional shortening (LVFS) were improved with GMTMS+SR after 6 weeks (J). n=6 independent biological replicates. K, Schematic figure showing Dre-rox recombination in CM and Cre-loxP recombination in nonmyocyte by Tnni3-Dre;IR1;R26-iCre strategy. L, Experimental strategy for nonmyocyte to CM in vivo reprogramming study using Tnni3-Dre;IR1;R26-iCre mice. M, Immunostaining for tdTomato, ZsGreen, and cTnI on heart sections from GMTMS+SR-treated Tnni3-Dre;IR1;R26-iCre mice. Boxed regions are magnified. Scale bar, 100 μM. N, Quantitative analyses for ZsGreen+tdTomato– CMs in MI+solvent, EGFP+solvent, GMT+solvent, and GMTMS+SR-treated Tnni3-Dre;IR1; R26- iCre mice are shown. n=5 independent biological replicates. All data are presented as mean±SEM as analyzed by 1-way ANOVA/Tukey multiple comparisons test (G, H, J, and N) or mixed-effects ANOVA/Tukey multiple comparisons test (I) using Prism 9. ns, no significance, P>0.05; *(#)P<0.05; **(##)P<0.01; ***P<0.001 vs the relevant control. CM indicates cardiomyocyte; EGFP, enhanced green gluorescent protein; GMT, GATA4, MEF2C, TBX5; GMTS, GMT with SALL4; GMTM, GMT with MYOCD; GMTMS, GMT with MYOCD and SALL4; iCM, induced cardiomyocyte; MF, myofibroblast; MI, myocardial infarction; Ruxo, ruxolitinib; SB, SB431542; SR, SB431542 and ruxolitinib; and TAM, tamoxifen.Myocardial infarction (MI) surgery was performed; meanwhile, Fu-tet-o and rtTA lentivirus were used in situ to overexpress reprogramming transcription factors or enhanced green fluorescent protein (EGFP) as control. Then, mice were administrated TAM by gavage at 0, 3, and 5 days after MI and continuously fed water with Dox at 3 days after MI to activate reprogramming factor overexpression, concurrent with daily intraperitoneal injection of small molecule boosters until euthanization (Figure [B]). By immunostaining, we did not detect any ZsGreen+tdTomato– cardiomyocytes in the MI+solvent and EGFP+solvent control group (Figure [C and H]), indicating no spontaneous myofibroblast-to-cardiomyocyte transdifferentiation after MI in untreated mice, which agreed with previous findings.4 Immunohistochemical staining showed ~335.3 ZsGreen+EGFP+ cells/section in Fu-tet-o-EGFP transduced Tnni3-Dre;IR1;Postn-MerCreMer mice (n=5, data not shown). This system was then used to investigate in vivo reprogramming by GMT. Immunohistochemical staining showed that polycistronic GMT could induce POSTN+ myofibroblast reprogramming into iCMs in vivo (Figure [D]), albeit with low efficiency of ~1.8 ZsGreen+tdTomato– cardiomyocytes/section (Figure [H]).To further investigate the effects of myofibroblast-to-cardiomyocyte reprogramming boosters in vivo, we added the MYOCD and SALL4 transcription factors to GMT (GMTMS),2 as well as a chemical combination of an ALK5 inhibitor, SB431542 (S), and a pan-Jak inhibitor, ruxolitinib (R), which were reported to improve iCM reprogramming efficiency in vitro (Tao Y, Yang Y, Wang LP, Yang ZH, Wang SQ, Yang Z. Robust small molecule-aided cardiac reprogramming systems selective to cardiac fibroblasts. Preprint. Posted online September 21, 2023. bioRxiv 2023.09.20.558597. doi: 10.1101/2023.09.20.558597). Immunohistochemistry showed markedly increased myofibroblast-to-cardiomyocyte in vivo reprogramming efficiency in GMTMS+SR treatments (Figure [E]), with a 14-fold increase in ZsGreen+tdTomato– cardiomyocyte counts, or ~25.4 ZsGreen+tdTomato– cardiomyocytes/section (Figure [H]). Furthermore, iCMs appeared more mature than those induced by GMT, with well-arranged sarcomere structures (Figure [E]) and sarcomeric α-actinin expression (Figure [F]). Comparable numbers of ZsGreen+tdTomato+ fused cardiomyocytes were detected among GMTMS+SR, GMT+solvent, and MI+solvent groups (Figure [G]).Removal of any single booster from GMTMS+SR could impede reprogramming efficiency (Figure [H]). In addition, echocardiography showed that GMTMS+SR ameliorated left ventricular ejection fraction after MI (Figure [I]). GMTMS+SR improved left ventricular ejection fraction to ~33.6% (17.4% in the GMT+solvent group) and left ventricular fractional shortening to ~16.5% (9.0% in the GMT+solvent group) at 6 weeks after MI (Figure [J]).Because POSTN+ myofibroblasts represent a subset of nonmyocytes, we assessed the overall nonmyocyte to cardiomyocyte-like cell in vivo reprogramming efficiency. Using Tnni3-Dre;IR1;R26-iCre marker-free lineage tracing to specifically label all nonmyocytes4 (Figure [K and L]), we again examined in vivo reprogramming by GMTMS+SR after MI. Confocal microscopy imaging revealed that iCMs were widely distributed in the MI border zone (Figure [M]), with 38.4 ZsGreen+tdTomato– cardiomyocytes/section (Figure [N]).Taken together, the GMTMS+SR combination can enhance myofibroblast-to-cardiomyocyte in vivo reprogramming efficiency, even within the MI microenvironment, assessed by a stringent lineage tracing system. It should be noticed that additional transcription factors and chemical compounds may lead to additional difficulties in its clinical translation, because of the limitation of delivery methods and increased incidence of side effects, which is to be solved in future investigations. Moreover, the reprogramming efficiency may be underestimated because of the limited virus infection rate, which can be optimized in future work. Cumulatively, this study provides a reliable, efficient strategy for in vivo cardiac reprogramming.All experiments involving mice were in compliance with protocols approved by the Institutional Animal Care and Use Committee of Peking University.The data and study materials that support the findings of this study, as well as experimental procedures and protocols, are available from the corresponding authors upon reasonable request.ARTICLE INFORMATIONAcknowledgmentsWe thank all members in the Y.Z. laboratory for technical assistance. We thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with the Nikon A1R confocal microscope.Sources of FundingThis work is supported by the National Key Research and Development Program of China (2018YFA0800504), the National Natural Science Foundation of China (31922020), and Plastech Pharmaceutical Technology Co, Ltd.Nonstandard Abbreviations and AcronymsGMTGATA4, MEF2C, TBX5GMTMSGMT with MYOCD and SALL4iCMinduced cardiomyocyteMImyocardial infarctionPOSTNperiostinSRSB431542 and ruxolitinibTAMtamoxifenDisclosures Y.Z. is a shareholder of Plastech Pharmaceutical Technology Ltd, but has no competing interests. The other authors report no conflicts.FootnotesFor Sources of Funding and Disclosures, see page 1731.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Bin Zhou, MD, PhD, 320 Yueyang Rd, Shanghai, 200031, China, Email zhoubin@sibs.ac.cnYang Zhao, PhD, No 5 Yi He Yuan Rd, Haidian District, Beijing 100871, China, Email yangzhao@pku.edu.cnREFERENCES1. Garry GA, Bassel-Duby R, Olson EN. Direct reprogramming as a route to cardiac repair.Semin Cell Dev Biol. 2022; 122:3–13. doi: 10.1016/j.semcdb.2021.05.019CrossrefMedlineGoogle Scholar2. Zhao H, Zhang Y, Xu XC, Sun QS, Yang CY, Wang H, Yang JB, Yang Y, Yang XC, Liu Y, et al. Sall4 and MyoCD empower direct cardiac reprogramming from adult cardiac fibroblasts after injury.Front Cell Dev Biol. 2021; 9:608367. doi: 10.3389/fcell.2021.608367CrossrefMedlineGoogle Scholar3. Kanisicak O, Khalil H, Ivey MJ, Karch J, Maliken BD, Correll RN, Brody MJ, Lin SCJ, Aronow BJ, Tallquist MD, et al. Genetic lineage tracing defines myofibroblast origin and function in the injured heart.Nat Commun. 2016; 7:12260. doi: 10.1038/ncomms12260CrossrefMedlineGoogle Scholar4. Li Y, He LJ, Huang XZ, Bhaloo SI, Zhao H, Zhang SH, Pu WJ, Tian XY, Li Y, Liu QZ, et al. Genetic lineage tracing of nonmyocyte population by dual recombinases.Circulation. 2018; 138:793–805. doi: 10.1161/CIRCULATIONAHA.118.034250LinkGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. 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