Age-related pathways in cardiac regeneration : a role for lncRNAs?

F on ie s in Physiology | www. on ie sin.o g 1 Janua y 2021 | Volume 11 | A icle 583191 MINI REVIEW published: 20 Janua y 2021 doi: 10.3389/ phys.2020.583191 Edi ed by: Geo ge G an , Uni e si y o Abe deen, Uni edKingdom Re iewed by: Meijing Wang, Indiana Uni e si y Blooming on School o Medicine, Uni edS a es Riikka Ki elä, Uni e si y o Helsinki, Finland *Co espondence: Sand ina Nób ega-Pe ei a [email p o ec ed] B uno Be na des de Jesus [email p o ec ed] †These au ho s ha e con ibu ed equally o his wo k and sha e i s au ho ship Special y sec ion: This a icle was submi ed o Clinical and T ansla ional Physiology, a sec ion o he jou nal F on ie s in Physiology Recei ed: 14 July 2020 Accep ed: 16 Decembe 2020 Published: 20 Janua y 2021 Ci a ion: San os F, Co eia M, Nób ega-Pe ei a S and Be na des de Jesus B (2021) Age-Rela ed Pa hways in Ca diac Regene a ion: A Role o lncRNAs? F on . Physiol. 11:583191. doi: 10.3389/ phys.2020.583191 Age-Rela ed Pa hways in Ca diac Regene a ion: A Role o lncRNAs? F anciscoSan os 1†, MagdaCo eia 1†, Sand inaNób ega-Pe ei a 1,2* and B uno Be na des deJesus 1 * 1 Depa men o Medical Sciences and Ins i u e o Biomedicine- iBiMED, Uni e si y o A ei o, A ei o, Po ugal, 2 Faculdade de Medicina, Ins i u o de Medicina Molecula João Lobo An unes, Uni e sidade de Lisboa, Lisboa, Po ugal Aging imposes a ba ie o issue egene a ion. In he hea , aging leads o a se e e ea angemen o he ca diac s uc u e and unc ion and o a subsequen inc eased isk o hea ailu e. An in ica e ne wo k o dis inc pa hways con ibu es o age- ela ed al e a ions du ing heal hy hea aging and accoun o a highe suscep ibili y o hea disease. Ou unde s anding o he sys emic aging p ocess has al eady led o he design o an i-aging s a egies o o he adop ion o p o ec i e in e en ions. Ne e heless, ou unde s anding o he molecula de e minan s ope a ing du ing ca diac aging o epai emains limi ed. He e, wewill summa ize he molecula and physiological al e a ions ha occu du ing aging o he hea , highligh ing he po en ial ole o long non-coding RNAs (lncRNAs) as no el and aluable a ge s in ca diac egene a ion/ epai . Keywo ds: lncRNAs, hea , egene a ion, ep og amming, ansdi e en ia ion INTRODUCTION Wo ldwide, ca dio ascula diseases a e he leading cause o dea h, causing nea ly 18million dea hs in 2017. Ca dio ascula diseases comp ise se e al pa hological condi ions, including hea ailu e (Yusu e  al., 2001; Lloyd-Jones e  al., 2009, 2010; Mensah e  al., 2019). Aging is p obably he mos impo an isk ac o o hea ailu e (Li e  al., 2020a). As opposed o he neona al hea , adul mammalian hea s lose hei capaci y o ully egene a e a e an exogenous o endogenous ha m (Lam and Sadek, 2018). This may be media ed h ough se e al in e connec ed p ocesses, including cellula senescence and sec e ed ac o s, elome e a i ion, mi ochond ial damage, cell dea h, o in lamma ion ( o a comp ehensi e e iew on age- ela ed pa hways a ec ing he hea , see Li e al., 2020a). Al hough a pa ial myocy e u no e has been obse ed in adul hea a e damage (e.g., myoca dial in a c ion), i only pa ially and sligh ly es o es hea unc ion. Fo ins ance, i has been ecen ly demons a ed ha manipula ion o elome e leng h h ough he exp ession o elome ase, whose exp ession is silenced in he mouse hea om day 5 o 7 (Blasco e  al., 1995; Bo ges and Liew, 1997; Richa dson e  al., 2012), may be bene icial in hea healing and heal hspan (Be na des de Jesus and Blasco, 2011; Ba e  al., 2014). Long non-coding RNAs (lncRNAs) ha e eme ged as impo an egula o s o epigene ic modula ion and gene exp ession. Al hough dep i ed om coding po en ial, lncRNAs ha e been associa ed wi h se e al biological p ocesses, including dosage compensa ion, genomic imp in ing, aging, and cell di e en ia ion (Me ce e  al., 2009; Rinn and Chang, 2012; Sousa-F anco e  al., 2019; Yao e  al., 2019). Fu he mo e, lncRNAs ha e been linked o se e al diseases including ca dio ascula diseases (Hobuß e  al., 2019; Abbas e  al., 2020). San os e al. Unde s anding Neona al Hea Regene a i e Po en ial F on ie s in Physiology | www. on ie sin.o g 2 Janua y 2021 | Volume 11 | A icle 583191 In his e iew, wewill discuss he ca diac egene a ion p ope ies o neona al and adul hea s. We will ocus on lncRNAs and hei po en ial ole in ca diac egene a ion b ie ly discussing he po en ial o ib oblas s as a sou ce o ca diomyocy es o egene a i e medicine pu poses. CARDIAC REGENERATION IN NEONATAL AND ADULT HEARTS The e is a gene al consensus on he capaci y o neona al hea s o egene a e, a e dis inc ypes o damage (Supplemen a y Figu e 1 – Po ello e  al., 2011, 2013; Haubne e  al., 2012, 2016; Jes y e  al., 2012; Mahmoud e  al., 2013, 2014, 2015; Rubin e  al., 2013; Ande sen e  al., 2014, 2016; Au o a e  al., 2014; Sadek e  al., 2014; B yan e  al., 2015; Da ehze eshki e  al., 2015; Han e  al., 2015; Jiang e al., 2015; Kon ino e  al., 2015; Aix e  al., 2016; Blom e  al., 2016; Kang e  al., 2016; Tao e  al., 2016; Valien e-Alandi e  al., 2016; Xiong and Hou, 2016; Yu e al., 2016; Ai e al., 2017; Bassa e al., 2017; Malek Mohammadi e al., 2017; Zeb owski e al., 2017; Ahmed e al., 2018; Ingason e  al., 2018; Sampaio-Pin o e  al., 2018; Se e i e  al., 2018; Cai e  al., 2019; Elhelaly e  al., 2019; Wang e  al., 2019b; Fan e  al., 2020; Pei e  al., 2020; Li e  al., 2020b, 2020c). A comp ehensi e o e iew o neona al hea egene a ion s udies has been p e iously and elegan ly de ailed by Lam and Sadek (2018). Hea egene a ion seems o be dependen on he ype o inju y ha causes loss o ca diomyocy es. Fo example, c yoinju y does no induce he same le el o egene a ion as apical esec ion o myoca dial in a c ion. Fu he mo e, i is likely ha neona al hea egene a ion is media ed by he p oli e a ion o p e-exis ing ca diomyocy es, and no by ca diac s em o p ogeni o cells. This egene a i e s a e occu s in an ex emely sho ime ame (<10days; Eschenhagen e  al., 2017) and, jus a ew days a e bi h, ca diomyocy es exi he cell cycle esul ing in a decline in hea egene a ion capaci y. This is accompanied by o he al e a ions in he ca diomyocy es, including hei me abolic needs o changes in he exp ession o bo h coding and non-coding genes. Subsequen ly, se e al s a egies ha e been designed o egene a ing he adul hea . Those app oaches may include he o ced e-en e ing in he cell cycle o he p e-exis ing ca diomyocy es, o may include cell ansdi e en ia ion s a egies, in which soma ic cells can be con e ed in o unc ional ca diomyocy es o cell eplacemen he apy (Qian e  al., 2012; Addis and Eps ein, 2013; Nam e  al., 2013; Wada e  al., 2013; Ghi oldi e  al., 2017; Amin e  al., 2018; Engel and A dehali, 2018a). A ROLE FOR lncRNAs IN HEART REGENERATION The impo ance o lncRNAs in hea egene a ion has been b ough o ligh ecen ly (Ba e  al., 2016; Abbas e al., 2020). LncRNAs a e a as ca ego y o non-coding, poo ly conse ed, and issue- and de elopmen al s age-speci ic ansc ip s wi h dis inc unc ions in se e al biological p ocesses, including epigene ic, ansc ip ional, and pos - ansc ip ional egula ion. Rega ding he ole o lncRNAs in hea egene a ion, we will discuss some ecen s udies desc ibing lncRNAs di ec ly ac ing (p omo ing o inhibi ing) on hea egene a ion (Table 1). LncRNAs Tha P omo e Ca diomyocy e P oli e a ion and Ca diac Regene a ion P7 mice subjec ed o LAD liga ion and injec ed wi h adeno i us con aining NR_045363 exhibi ed imp o ed le en icula ejec ion ac ion and educed in a c size compa ed o he con ol-injec ed g oup (Wang e  al., 2019a). Mice o e exp essing NR_045363 showed highe exp ession o ca diomyocy e mi o ic ma ke s, such as Ki67 and phospho yla ed his one H3 (pH3), sugges ing ha imp o ed hea unc ion a e MI was due o ca diomyocy e p oli e a ion. The au ho s epo ed ha NR_045363 ac ed as a compe ing endogenous RNA (ceRNA), binding o miR-216a (Wang e al., 2019a). miR-216a is known o ep ess JAK2, leading o dec eased le els o phospho yla ion o STAT3 (Hou e  al., 2015). Fu he mo e, dele ion o STAT3 was shown o impai ca diomyocy e p oli e a ion a e apical esec ion (Ku di e  al., 2018), sugges ing ha NR_045363 p omo ed ca diomyocy e p oli e a ion by modula ing he JAK2-STAT3 pa hway. So, he absence o NR_045363 (which esul s in an up egula ion o miR216a) led o educed ac i i y o he JAK2-STAT3, whils NR_045363 o e exp ession (which leads o a down egula ion o miR-216a) esul ed in an inc ease o he phospho yla ion TABLE1 | LncRNAs wi h epo ed oles in ca diac egene a ion.. LncRNA Repo ed ole in ca diac egene a ion Re e ence Nega i e egula o s AZIN2-s ↓ ca diomyocy e p oli e a ion by seques e ing miR-214 and leading o a dec ease in he phospho yla ion o Ak and Cyclin D Li e al., 2018b CAREL ↓ ca diomyocy e p oli e a ion by seques e ing miR-296 and ac i a ing T p53inp1 and I m2a Cai e al., 2018 CPR ↓ ca diomyocy e p oli e a ion by he ec ui men o DNMT3A, leading o inc eased le els o me hyla ion o he MCM3 p omo e Ponnusamy e al., 2019 CRRL ↓ ca diomyocy e p oli e a ion by seques e ing miR-199a-3p, leading o an inc eased exp ession o Hopx Chen e al., 2018 LncDACH1 ↓ ca diomyocy e p oli e a ion by egula ing PP1A/YAP1 signaling Cai e al., 2020a SARRAH ↑ ca diomyocy e apop osis by inc easing caspase ac i i y T embinski e al., 2020 Posi i e egula o s NR_045363 ↑ ca diomyocy e p oli e a ion ia he miR- 216a/JAK-STAT3 pa hway Wang e al., 2019a; Chen e al., 2020↓ ca diomyocy e apop osis by blocking p53 ac i a ion ECRAR ↑ ca diomyocy e p oli e a ion by p omo ing phospho yla ion o ERK1/2 o ac i a e Cyclins D1 and E1 Chen e al., 2019 Si 1 an isense lncRNA ↑ ca diomyocy e p oli e a ion and ↓ ca diomyocy e apop osis by s abilizing Si 1 Li e al., 2018a San os e al. Unde s anding Neona al Hea Regene a i e Po en ial F on ie s in Physiology | www. on ie sin.o g 3 Janua y 2021 | Volume 11 | A icle 583191 le els o JAK2 and STAT3, hus p omo ing ca diomyocy e p oli e a ion (Wang e  al., 2019a). Mo e ecen ly, NR_045363 was associa ed wi h ca diomyocy e apop osis. Chen e al. (2020) epo ed ha loss o NR_045363 led o he ac i a ion o he p53 signaling pa hway, p omo ing apop osis. On he o he hand, o e exp essing NR_045363 inhibi ed apop osis and imp o ed ca diac unc ion a e MI, hus po en ially media ing he ca diac unc ions obse ed a e NR_045363 modula ion. Long non-coding RNA endogenous ca diac egene a ion- associa ed egula o (ECRAR) was ound o be up egula ed in he e al hea , and i s exp ession g adually dec eased in pos na al hea s. O e exp ession o ECRAR in pos na al a ca diomyocy es, bo h in i o and in i o, esul ed in an inc ease o DNA syn hesis, and an inc ease o cy okinesis (pH3 and au o a B kinase), sugges ing a di ec in ol emen in ca diomyocy es p oli e a ion (Chen e  al., 2019). O e exp ession o ECRAR esul ed in he phospho yla ion o ERK1/2, hei subsequen ansloca ion o he nucleus and he ansc ip ion o cell p oli e a ion and cell cycle- ela ed genes (Chen e  al., 2019). Li e  al. (2018a) iden i ied Si 1 an isense lncRNA (Si 1-as), whose exp ession was high du ing hea de elopmen . O e exp ession o his lncRNA esul ed in an inc ease o Ki67- and pH3-posi i e ca diomyocy es. On he o he hand, silencing o Si 1-as, bo h in i o and in i o, led o a dec ease o Ki67- and pH3-posi i e ca diomyocy es, indica ing a po en ial decline in cell di ision (Li e  al., 2018a). Fu he mo e, o e exp ession o Si 1-as a e MI in adul mice esul ed in an inc eased exp ession o cell-cycle speci ic ac o s Ki67 and pH3, hus sugges ing a po en ial implica ion in ca diac heal h (Li e  al., 2018a). Mo e ecen ly, Wilson e  al. (2020) desc ibed BANCR, a lncRNA exclusi ely exp essed in p ima e e al ca diomyocy es. BANCR p omo es ca diomyocy e mig a ion in i o and en icula enla gemen in i o. To elucida e he egula ion o BANCR in ca diomyocy es, he au ho s sugges ed ha TBX5 binding was esponsible o he e al hea -speci ic exp ession o BANCR. Addi ionally, he au ho s iden i ied TEAD4 and YAP1 ( wo ac o s in ol ed in he HIPPO pa hway) in he same enhance , p omo ing BANCR exp ession. Finally, he au ho s iden i ied a ole o BANCR in hea disease, demons a ing highe exp ession in pedia ic bu no adul dila ed ca diomyopa hy (Nelakan i and Xiao, 2020; Wilson e al., 2020). O he lncRNAs associa ed wi h aged hea s include lncRNA H19 (down egula ed in aged o ischemic hea ; Ho mann e al., 2019), and MALAT1 a lncRNA which, i sel , is egula ed by an an isense lncRNA ansc ip (TALAM1; Zong e  al., 2016; Gomes e  al., 2019), was also shown o be dec eased in aged hea s (Bink e  al., 2019; Gomes e  al., 2019), and his dec ease was shown o bein ol ed in ca diac dys unc ion (Zhu e  al., 2019; Li e  al., 2020a). LncRNAs Tha Inhibi Ca diomyocy e P oli e a ion and Ca diac Regene a ion Cai e  al. (2018) explo ed he ole o lncRNAs du ing hea egene a ion a e ischemic inju y, in bo h neona al and adul mice. CAREL, a lncRNA whose exp ession g adually inc eased in he neona al hea s om P1 o P10 mice, wi h P7 co esponding o he ime poin a which he hea egene a i e capaci y is los in mice (Cai e  al., 2018). Ca diac-speci ic o e exp ession o CAREL led o a dec ease o ca diomyocy e p oli e a ion and educed hea egene a ion in neona al mice a e inju y. On he con a y, silencing CAREL p omo ed ca diac egene a ion and imp o ed hea unc ional pa ame e s a e myoca dial in a c ion in neona al and adul mice (Cai e al., 2018). CAREL was ound o bea ceRNA, seques e ing miR-296. I was sugges ed ha he CAREL-miR-296 in e ac ion led o he ac i a ion o T p53inp1 and I m2a, leading o a dec ease in ca diomyocy e p oli e a ion, hus esul ing in a educ ion o egene a ion. In amyoca dial adminis a ion o CAREL o p1 neona al mice inhibi ed ca diomyocy e mi osis and inc eased he o ma ion o ca diac sca and, on he o he hand, o e exp ession o miR-256 p omo ed ca diomyocy e p oli e a ion and ca diac egene a ion a e inju y. Simila ly, lncRNA ca diomyocy e p oli e a ion egula o (CPR) was shown o be a nega i e egula o o ca diomyocy e p oli e a ion and ca diac epai . Ponnusamy e al. (2019) obse ed ha highe le els o CPR hampe ed ca diomyocy e p oli e a ion, whils silencing CPR esul ed in ca diomyocy e p oli e a ion in pos na al and adul hea s. CPR exp ession le els we e ound o be highe in he adul hea , which is consis en wi h hei lack o egene a ion. The au ho s epo ed ha CPR ec ui s DNMT3A o se e al locus leading, in pa icula , o inc eased le els o me hyla ion in he MCM3 p omo e (Ponnusamy e  al., 2019). In di iding issues, MCM3 p omo es he ini ia ion o DNA eplica ion and cell cycle p og ession (Lin e  al., 2008), some hing hal ed by CPR in he hea and leading o he inhibi ion o ca diomyocy es p oli e a ion. Ano he lncRNA ha nega i ely egula es ca diac egene a ion is LncDACH1. This lncRNA was ound o beg adually up egula ed in pos na al hea s, which is in acco dance wi h he loss o myoca dial egene a i e capaci y soon a e bi h (Cai e  al., 2020). The au ho s sugges ha LncDACH1 binds p o ein phospha ase 1 ca aly ic subuni alpha (PP1A), educing i s dephospho yla ion capaci y, and inc eases he phospho yla ion o yes-associa ed p o ein 1 (YAP1), p e en ing i s ansloca ion o he nucleus and, hus, he ac i a ion o cell p oli e a ion- ela ed genes ( on Gise e  al., 2012; Cai e  al., 2020). Ca diac- speci ic o e exp ession o LncDACH1 esul ed in he supp ession o neona al hea egene a ion and agg a a ion o ca diac unc ion a e apical esec ion. These pheno ypes we e accompanied by a dec ease in he numbe o ca diac-cells exp essing p oli e a i e ma ke s (Cai e  al., 2020). Ca diomyocy e egene a ion- ela ed lncRNA (CRRL) was also ound o be in ol ed in hea egene a ion. CRRL silencing was associa ed wi h an inc eased exp ession o EdU, Ki67, and pH3in P1 and P7 a ca diomyocy es (Chen e  al., 2018). Simila esul s we e ob ained in neona al a s pos -MI, concomi an ly wi h be e p ognosis such as educ ion o he ib o ic leng h o he in a c wall and ib osis a ea in he non-in a c zone. Ins ead, o e exp ession o CRRL leads o a dec ease in pH3-posi i e ca diomyocy es and inhibi ion o unc ional eco e y pos -MI. CRRL unc ion seemed o be media ed h ough he binding o miR-199a-3p, esul ing in an inc eased exp ession o Hopx, which is a nega i e egula o o ca diomyocy e p oli e a ion (T i edi e  al., 2010). LncRNA AZIN2-s , a splice a ian o he AZIN2 gene, was ound o be up egula ed in human adul hea s. AZIN2-s was San os e al. Unde s anding Neona al Hea Regene a i e Po en ial F on ie s in Physiology | www. on ie sin.o g 4 Janua y 2021 | Volume 11 | A icle 583191 epo ed o nega i ely egula e ca diomyocy e p oli e a ion, bo h in i o and in i o (Li e al., 2018b). O e exp ession o AZIN2-s led o an an i-p oli e a i e pheno ype, ma ked by dec eased le els o EdU-, Ki67-, pH3-, and Au o a-B. On he o he hand, silencing AZIN2-s p omo ed ca diomyocy e p oli e a ion and imp o ed ca diac unc ion a e MI. AZIN2-s seques e s miR-214, leading o he elease o i s a ge PTEN, esul ing in a dec ease in he phospho yla ion o Ak and Cyclin-D, he e o e inhibi ing ca diomyocy e p oli e a ion. Reduced le els o AZIN2-s allow miR-214 o ep ess PTEN, leading o inc eased le els o phospho yla ed Ak and Cyclin-D1, hus p omo ing ca diomyocy e p oli e a ion. Mo e ecen ly, T embinski e  al. (2020) iden i ied lncRNA SCOT1-an isense RNA egula ed du ing aging in he hea (SARRAH), whose exp ession declines du ing aging. Inhibi ion o Sa ah induces caspase ac i i y in mouse and human ca diomyocy es, p omo ing apop osis. Gene se en ichmen analysis a e SARRAH silencing showed en ichmen o apop osis- ela ed pa hways, co obo a ing p e ious obse a ions (T embinski e al., 2020). SARRAH was also ound o di ec ly bind o he p omo e s h ough RNA-DNA iplex helix s uc u es, sugges ing ha i s binding may ac i a e gene exp ession. Indeed, i was epo ed ha SARRAH in e ac ed wi h ca diac ansc ip ion ac o cys eine- ich p o ein 2 (CRIP2) and p300, which ace yla es his one H3 lysine 27 o ac i a e ansc ip ion (T embinski e  al., 2020). On he o he hand, o e exp ession o SARRAH led o a dec ease in caspase ac i i y. In adul mice a decline in apop osis was obse ed a e o e exp essing SARRAH, sugges ing ha educed exp ession le els o his lncRNA in aged mice migh con ibu e o ca diomyocy e cell dea h in i o. Fu he mo e, educed le els o Sa ah we e obse ed in he in a c ed and bo de egions a e acu e MI (T embinski e  al., 2020). Fu he mo e, se e al lncRNAs ha e been iden i ied as p omo e s o ca diac ib osis (Liang e  al., 2018; Wang e  al., 2018; Hao e  al., 2019; Zhang e  al., 2019a). Aged issues accumula e signals ha p omo e he epi helial-mesenchymal ansi ion (EMT), inducing he ansdi e en ia ion o epi helial cells o mesenchymal cells, such as ib oblas s, which a e he main media o s o ib osis h ough he deposi ion o ex acellula ma ix. In he hea , many ib oblas s de i e om endo helial cells, leading o excessi e deposi ion o ex acellula ma ix and causing ca diac ib osis, which is common in pa ien s wi h hea ailu e ( e iewed by San os e  al., 2019). Thus, a ge ing hese lncRNAs may also beconside ed o imp o ing hea unc ion. REPROGRAMMING OF FIBROBLASTS INTO CARDIOMYOCYTES AS A POTENTIAL CELL REPLACEMENT THERAPY – A ROLE FOR lncRNA Cell ep og amming has eme ged as a no el s a egy o egene a i e medicine and cell-based he apy. The ep og amming o mouse and human ib oblas s in o induced plu ipo en s em cells (iPSCs) using ansc ip ion ac o s (TFs) known o play key oles in he main enance o emb yonic s em cell iden i y sugges ed ha pa ien -de i ed iPSCs could be p oduced om soma ic cells. This s a egy allowed he con e sion o ully di e en ia ed cells in o cells wi h he po ency o bedi e en ia ed in issues om di e en de elopmen lineages (Takahashi and Yamanaka, 2006; Takahashi e al., 2007; Yamanaka, 2009; Abad e al., 2013). Addi ionally, many o he ep og amming ba ie s, such as he obs acles imposed by aging, ha e been add essed h ough he di ec manipula ion o umo supp esso genes, p53 o Ink4a/ARF (Li e  al., 2009; Ma ion e  al., 2009), o he EMT-p omo ing ac o ZEB2 (Be na des de Jesus e  al., 2018; San os e  al., 2019). The ep og amming o ib oblas s in o iPSCs opened doo s o di ec cell ep og amming. Di ec ca diac ep og amming o ib oblas s in o ca diomyocy es [usually e med induced ca diomyocy es (iCMs)] has eme ged as an a ac i e s a egy o eplacing los o damaged cells in he hea . Mouse pos na al ca diac and de mal ib oblas s ha e been ansdi e en ia ed in o iCMs h ough he combined exp ession o h ee di e en ca diac- speci ic TFs: Ga a4, Me 2c, and Tbx5 (GMT). The ec opic exp ession o GMT ac i a es a ca diac-like gene exp ession p og am and p omo es he con e sion o ib oblas s in o iCMs (Ieda e al., 2010; Qian e al., 2012). Compa a i e gene exp ession analyses epo ed ha iCMs gene a ed in i o exhibi ed bona ide adul ca diomyocy e-like ea u es, such as a y acid oxida ion o cell cycle exi (Mu aoka e al., 2019). Rema kably, his app oach has been adap ed in i o, whe e ca diac ib oblas s ha e been ansdi e en ia ed in o iCMs (Song e  al., 2012; Zhang e  al., 2019b, 2019c), bypassing he need o e e ib oblas s o a plu ipo en s a e (Liu e al., 2017; Mu aoka e al., 2019). Endogenous ca diac ib oblas s comp ise abou 50% o all he cells in he hea , making hem a po en ial sou ce o ca diomyocy es o egene a i e he apy (Ieda e al., 2010). In ac , iCMs ep og ammed om endogenous ca diac ib oblas s enhanced ca diac unc ion a e myoca dial in a c ion, ully demons a ing he po en ial o his s a egy o ca diac epai (Miyamo o e  al., 2018; Bek ik and Fu, 2019; Lee e  al., 2020). Despi e se e al encou aging esul s, cu en ep og amming me hodologies emain somewha ine icien , as e y ew ib oblas s a e ully con e ed in o unc ional iCMs. Di e en ial exp ession pa e ns o lncRNAs ha e been obse ed in se e al de elopmen al s ages, including ca diogenesis, and in ol e he exp ession o lncRNAs B a ehea , Fend , and Ca men. In ac , lncRNA ZEB2-NAT has been modula ed in o de o imp o e he ep og amming o ib oblas s in o iPSCs (Be na des de Jesus e al., 2018). Ha ing hese concep s in mind, i seems easonable o expec ha modula ing lncRNAs migh imp o e he e iciency o di ec ca diac ep og amming. In i o The apeu ic Deli e y – Cu en Issues Rega ding phenocon e sion o ca diac cells, i is impo an o men ion, howe e , ha many o he cu en p o ocols depend on i al ec o s o gene deli e y. The e a e a ew sa e y issues associa ed wi h he use o len i i al and e o i al ec o s, as hey in eg a e hei genome in he hos cell. They could po en ially dis u b endogenous gene exp ession and a e associa ed San os e al. Unde s anding Neona al Hea Regene a i e Po en ial F on ie s in Physiology | www. on ie sin.o g 5 Janua y 2021 | Volume 11 | A icle 583191 wi h he isk o inse ional mu agenesis, hampe ing he clinical applica ion o his me hod. Howe e , non-in eg a i e i uses, such as Sendai i us, and non- i al ep og amming sys ems ha e eme ged as sa e al e na i es o clinical applica ion (Engel and A dehali, 2018b; Miyamo o e  al., 2018; Tani e  al., 2018; Chang e  al., 2019). CONCLUSION As p e iously discussed, se e al lncRNAs a e exp essed du ing he de elopmen o he hea and du ing hea pa hologies. Ta ge ing lncRNAs may be a no el s a egy agains hea diseases (Ba e  al., 2016). Technically, he de elopmen o speci ic and deli e able an isense ansc ip s (e.g., LNA-GapmeRs) has been p o ed powe ul and e icien ca ie s o in i o a ge ing and RNase H-media ed deg ada ion o speci ic a ge s (Be na des de Jesus e  al., 2018). Simila app oaches may bedesigned o exp ession o selec ed lncRNAs, down egula ed in ca diac diseases. We ha e o ace, howe e , ha mos human lncRNAs a e non-conse ed be ween species, making i ex emely challenging o iden i y he unc ional lncRNAs in i o. The lack o sequence conse a ion poses a challenge o he ansla ional applica ion o human lncRNAs. Since lncRNAs a e species-speci ic, weo en can only isualize hei impac when s udied in hei speci ic sys em. This challenge may only be add essed h ough a humanized expe imen al model whe e he de ailed unc ion o non-conse ed lncRNAs may be es ed. In conclusion, unde s and lncRNAs speci ic p o iles in di iding s. non-di iding ca diomyocy es may allow he de ec ion o po en ially d uggable a ge s o adul hea epai . AUTHOR CONTRIBUTIONS FS, MC, SN-P, and BB planned, w o e, and discussed he pape . SN-P and BB e ised he pape . All au ho s con ibu ed o he a icle and app o ed he submi ed e sion. FUNDING This wo k was suppo ed by Fundação pa a a Ciência e Tecnologia (FCT; ERA-CVD 2018 / 3599-PPCDT - ERA-CVD/0001/ 2018 - INNOVATION). SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ phys.2020.583191/ ull#supplemen a y-ma e ial Supplemen a y Figu e 1 | S udies on neona al hea egene a ion depic ing di e en me hodologies. The s udies wi h blue backg ound obse ed hea egene a ion whe he he s udies in ed could no de ec hea healing. REFERENCES Abad, M., Mos ei o, L., Pan oja, C., Caname o, M., Rayon, T., O s, I., e al. (2013). Rep og amming in i o p oduces e a omas and iPS cells wi h o ipo ency ea u es. Na u e 502, 340–345. doi: 10.1038/na u e12586 Abbas, N., Pe bellini, F., and Thum, T. (2020). Non-coding RNAs: eme ging playe s in ca diomyocy e p oli e a ion and ca diac egene a ion. Basic Res. Ca diol. 115:52. doi: 10.1007/s00395-020-0816-0 Addis, R. C., and Eps ein, J. A. (2013). Induced egene a ion-- he p og ess and p omise o di ec ep og amming o hea epai . Na . 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