Cardiovascular disorders are the top cause of human death in the world, and there were around 133 deaths per 100, 000 people in 2013 based on the National Bureau of Statistics of China. This amount was 136/100, 000 in 2014. Over the past several years, therapeutic approaches, including new drug development and cellular transplantation, have had a limited effect on treating cardiovascular disorders within the clinic. Meanwhile, heart transplantation is restricted by the limited entry to donor organs and itself has considerable mortality related to immunosuppressant therapy and graft vasculopathy. Hence, it is important to discover novel therapeutic approaches for cardiac regenerative therapy.

The low regenerative capability of cardiomyocytes is the main problem for heart repair. To get new cardiomyocytes, several strategies are developed, including: induction of pluripotent stem cells (iPSCs) and differentiation directly into cardiomyocytes [ 1 , 2 ]; activation of cardiac stem cellular material or stimulation of cardiomyocytes to re-enter the cellular cycle [ 3 , 4 ]; and direct reprogramming of fibroblasts to cardiomyocytes [ 5 , 6 ]. Cardiac fibroblast cells, education for up to 50% of all cardiac cells, survive well plus couple with neighboring cells, and have been identified as a perfect cell source for direct reprogramming into cardiomyocytes [ 7 ]. After myocardial infarction, the fibroblasts expand plus constitute the majority of the cells in the infarct zone [ 8 , 9 ]. Therefore , reprogramming cardiac fibroblast cells into cardiomyocytes signifies a promising and beneficial approach for cardiovascular regeneration.

MyoD , the master regulator gene for skeletal muscle cells, was discovered many years ago, but learn regulators for other cell lineages were not been found out until 2006 [ 10 ]. What is surprising is the revolutionary discovery that the transcription factors Oct4 , Sox2 , Klf4 , and c-myc were effective at reprogramming terminally differentiated cells into iPSCs [ 11 ]. The induction of iPSCs provided new insights with regard to direct reprogramming of adult cell types into particular lineages employing a combination of transcriptional factors [ 12 ], for example Mgn3 , Pdx1 , and Mafa for pancreatic β -cells [ 13 ], and Gata4 , Hand2 , Mef2c , and Tbx5 for cardiomyocytes [ 14 , 15 ]. Therefore , induction of cardiomyocytes from endogenous fibroblasts exhibits a feasible and promising approach to restore heart function following injury.

In this article, we review previous work on direct reprogramming of fibroblasts into cardiomyocytes using mouse and individual cells, and discuss future efforts needed to apply this method to the clinic.

Direct cardiac reprogramming of murine fibroblasts

In 2010, postnatal cardiac and dermal fibroblasts were directly reprogrammed in to cardiomyocyte-like cells in vitro, with a combination of three developing transcription factors (GMT: Gata4 , Mef2c , plus Tbx5 ) from the group of Srivastava et al. [ 15 ] Afterwards, the same group demonstrated that retroviral delivery of GMT in vivo reprogrammed murine cardiac fibroblasts into caused cardiomyocytes, with decreased infarct size and modestly fallen cardiac dysfunction [ 16 ]. Meanwhile, the group of Olson et al. reported increased reprogramming efficiency in vitro and in vivo by adding Hand2 to the GMT combination, with improved heart function and reduced scar formation after myocardial infarction [ 14 ]. These studies provide a new insight into center regeneration through gene therapy. Recently, non-integrated methods had been developed to transfect mRNAs and proteins of heart transcription factors into fibroblasts to induce cardiomyocytes plus cardiac progenitor cells, respectively [ 17 , 18 ]. These reviews provided safe methods for clinical application with great possible.

Based on the compelled expression of cardiac transcription factors, many methods had been developed to enhance the reprogramming efficiency, including inhibitor/cytokine remedies and epigenetic modulation [ 14 , 16 , 19 ]. SB431542 (a changing growth factor (TGF)-β pathway inhibitor) can increase the transformation of both mouse embryonic fibroblasts and adult heart fibroblasts into cardiomyocyte-like cells up to fivefold based on the mixture of Gata4 , Hand2 , Mef2c , Tbx5 , and Nkx2. 5 [ 19 ]. Furthermore, inhibition of pro-fibrotic signaling (both TGF-β – and Rho-associated kinase pathways) reprograms embryonic fibroblasts into functional cardiomyocyte-like cells, with effectiveness up to 60% for cTnT or α -actinin [ 20 ]. Besides inhibitors, cytokines, including fibroblast growth aspect (FGF)2, FGF10, and vascular endothelial growth factor (VEGF), can increase the number of induced cardiac myocyte cells (iCMs) with spontaneous beating by 100-fold and accelerate the particular maturation of iCMs [ 21 ]. Bmi1 acts as a critical barrier in order to iCM induction through epigenetic modulation, and reduced Bmi1 expression adjustments the chromatin modification at cardiogenic loci, including improved active histone mark H3K4me3 and reduced repressive H2AK119ub. Correspondingly, cardiogenic gene expression was de-repressed during iCM conversion. These results indicate that the process of reprogramming is certainly complex and influenced by many factors. Sequential addition associated with cytokines and inhibitors holds great promise for customizing the protocol for cardiomyocyte reprogramming.

In addition to transcription factors and little molecules, microRNAs (miRNAs) have great influence on the appearance of transcription factors such as Gata4 , Hand2 , Mef2c , T-box, plus Nkx2. 5 , which usually regulate heart development. Thus, miRNAs represent an attractive plus promising direction for reprogramming. miR-1 and miR-133 are usually cardiac and skeletal muscle-specific molecules, with miR-1 marketing for ~40% of miRNAs in the mammalian heart [ 22 ]. A “ miRNA combo” (miR-1, miR-133, miR-208, and miR-499) was reported to convert cardiac fibroblasts into functional cardiomyocyte-like cells in vitro and in vivo [ 23 , 24 ]. Adeno-associated virus (AAV) vectors are appealing tools for gene therapy, but the limited cargo dimension (~4. 5  kb) of AAV restricts the manifestation of multiple transcription factors in one vector. However , thinking about the small size of miRNAs, it holds great possible to use miRNAs as gene therapy targets in vivo.

Above all, transcription factors and miRNAs perform important roles during cardiac reprogramming [

25

28

]. Their own functions in the cardiac development and direct reprogramming are usually summarized in Table 

1

.

Table 1

The particular functional mechanisms of transcription factors and microRNAs within the cardiac development and direct reprogramming

GATA4 , MEF2C , TBX5 [ 15 ]

GATA4 , MEF2C , and TBX5 would be the core components of direct reprogramming

NKX2. 5 , Mesp1 , plus Myocd : expressed in heart progenitor cells (CPCs), and induce the development of cell destiny to the mesoblastema layer

GATA4 , HAND2 , and TBX5 : induce the particular cardiac gene expression

TBX5 [ 25 ]

Stimulates the differentiation of transfected cells into beating cardiomyocytes

NKX2. 5

Induces Ryr2 gene expression

Hand2

Induces tropomyosin and cTnT in human dermal fibroblasts

Mesp1 [ 26 ]

Expressed in CPCs plus programs nascent mesoderm toward a cardiovascular cell destiny

Myocd

Manages the development of cardiomyocytes and smooth muscle cells, and improved the expression of cardiac sarcomeric proteins

miR-1, miR-133, miR-208, miR-499 [ 24 ]

Alters H3K27 methyltransferase and demethylase expression

Promotes cardiomyocyte proliferation and suppresses apoptosis; raises expression of contractile proteins (MHC); influences the development of ventricular septum

miR-1 [ 27 ]

Promotes cardiomyocyte proliferation and suppresses apoptosis

Encourages cardiomyocyte proliferation and suppresses apoptosis

miR-133 [ 28 ]

miR-133-mediated Snai1 repression

Promotes cardiomyocyte expansion

During the iPSC induction process there is a pluripotent intermediate condition, showing plastic developmental potential. After transfection of 4 Yamanaka factors and manipulating pathways for cardiogenesis, computer mouse embryonic fibroblasts (MEFs) can be reprogrammed into cardiomyocytes along with spontaneous contraction [

29

]. Contracting cells resembling cardiomyocytes were also observed during iPSC induction via the chemical substance combination CRFVPTZ (CHIR99021, RepSox, Forskolin, VPA, Parnate, TTNPB, and DZnep). Furthermore, these chemically induced cardiomyocytes (CiCMs) are not generated through the iPSC stage, but via a heart precursor-like stage. These results indicate that the intermediate condition is plastic and provides a new reprogramming strategy to generate cardiomyocytes [

30

]. Rgarding the safety problem of hereditary manipulation, it is promising that the chemical cocktails could reprogram fibroblast cells into induced cardiomyocyte-like cells. However , thinking of reprogramming myocardial fibroblasts in situ, how to release these types of small molecules into the infarct region and reprogram myofibroblasts successfully into cardiomyocytes in vivo is still challenging. Brand new materials that control drug release may overcome this issue. Meanwhile, it is worth noting that the strategy may have the chance of tumorigenicity since the specificity of small molecules cannot be assured, and this procedure can also induce iPSCs. Typical methods for murine fibroblast reprogramming are summarized in Table 

2

.

Table 2

Factors and results in mouse direct heart reprogramming

GMT [ 15 ]

CF, TTDF

cTnT + : 30% of α -MHC cells; α -actinin + : the majority of cTnT + cells

+

+

+

OSKM; JI1, BMP4 [ 29 ]

MEF

cTnT + : 40%

+

+

+

GMT [ 16 ]

CF

α -MHC-EYFP + : ~40% at border zone

+

+

+

miR-1 , 133 , 208 , 499 ; JAK inhibitor I [ 23 ]

CF

α -MHC-GFP + : ~28%

+

+

+

GMT, Myocd , Srf , Mesp1 , Smarcd3 [ 42 ]

MEF

Myh6. Egfp + : 2 . 4%

+

Hand2 , Nkx2. 5 , Gata4 , Mef2c , Tbx5 [ 43 ]

MEF, CF

Troponin T-GCaMP5 + activity: 1 . 6%

ND

+

+

OSKM; PEG hydrogel [ 44 ]

MEF, TTF

Defeat patch per cm 2 : nine. 4%

α -actinin + : 1 . 72 fold/control

ND

+

+

GHMT, MyoD area [ 45 ]

HF, LBF, TTF

cTnT + : 4. 9%

ND

+

+

GHMT and SB431542 [ 19 ]

CF

Troponin T-GCaMP5 + activity: 9. 27%

ND

+

GHMT, Myod domain [ 46 ]

HF

cTnT + : 19%

ND

+

+

GMT, Mesp1 , Myocd and miR-133 [ 28 ]

MEF, CF

α -MHC-GFP & cTnT + : 8. 1%; α -actinin + : 19. 9%

ND

 

OCT4 , SCPF [ 35 ]

MEF, TTF

beating clusters: ~40/well associated with 24-well plate

+

+

+

GHMT [ 47 ]

MEF,

Sarcomere + : ~32%; NPPA + : 35% of sarcomere + ; MYL2 + : 22% of sarcomere +

+

ND

+

GMT mRNA, C_lipo [ 17 ]

CF

α -MHC-GFP + : 0. 5% of transfected CF

ND

ND

miR-1 , miR-133 , miR-208 , miR-499 [ 24 ]

CF

tdTomato + Troponin T + : 12%

+

ND

+

OSKM, Ascorbic acid [ 48 ]

MEF

GATA4 + : ~40%; MHC + : ~24%

+

ND

+

CHIR99021, RepSox, Forskolin, VPA [ 30 ]

MEF, TTF

α -actinin + : 14. 5%; α -MHC + : 9%

+

+

+

GHMT, miR-1 , miR-133 , Y-27632, A83-01 [ 20 ]

MEF, AF

cTnT + : ~60% with A83-01; α -actinin + : ~60% with A83-01

+

+

+

Direct heart reprogramming of human fibroblasts

Compared to murine fibroblasts, it takes a long time in order to reprogram human fibroblasts into cardiomyocytes and it is more difficult to acquire mature cardiomyocytes from human somatic cells. After reprogramming in mice, Nam et al. [ 31 ] reported in 2013 that the combination of GATA4 , HAND2 , TBX5 , MYOCD (myocardin), miR-1, and miR-133 activated cardiac marker expression, but that many induced cardiomyocytes were in a partially reprogrammed state. Within the same year, Wada et al. [ 32 ] discovered that reprogramming fibroblasts with the transcription factors GATA4 , MEF2C , TBX5 , MESP1 , and MYOCD (referred in order to as GMTMM) changed the cell morphology from a spindle shape to a rod-like shape, and exhibited spontaneous California 2+ oscillations. Srivastava and co-workers discovered that GMT ( GATA4 , MEF2C , and TBX5 ) was inadequate for reprogramming of human fibroblasts into cardiomyocytes, as well as the addition of ESRRG and MESP1 to GMT could induce cardiomyocyte-like cells along with cardiac-specific gene expression and sarcomere formation. Furthermore, digging in MYOCD plus ZFPM2 led to more features of cardiomyocytes, including global cardiac gene appearance and a phenotypic shift to a cardiac state [ 6 ]. Although the reprogramming efficiency in human cells is very reduced, these reports represent a great step towards therapeutic app in the clinic.

The abovementioned three reviews in human cells all concern transcription factors shipped through a virus until Ding et al. reported upon small molecules [

36

]. Small molecules have results in the reprogramming of human pancreatic lineages and nerve organs stem cells from somatic cells [

33

,

34

], and have enormous influence in the process of transdifferentiation of fibroblasts towards cardiomyocytes with reduced transcription factor numbers [

35

]. Thereafter, Ding’ s group found that human somatic cells could be transdifferentiated to cardiomyocyte-like cells which was similar to naive human cardiomyocytes with regards to the properties of transcriptome, epigenetics, and electrophysiology, with nine small molecules (9C: CHIR99021, A83-01, BIX01294, AS8351, SC1, Y27632, OAC2, SU16F, plus JNJ10198409) in 2016 [

36

]. Moreover, human fibroblasts treated with 9C could be converted into cardiomyocytes in the infarcted computer mouse heart, and enhanced the function of infarcted cardiovascular [

36

]. To understand cardiac reprogramming further, we described typical methods for human fibroblast reprogramming (Table 

3

).

Table 3

Factors and results in direct heart reprogramming of human cells

ETS2 , MESP1 [ 49 ]

DF

NKX2. 5-tdTomato + : 30 colonies/plate (cardiac progenitor)

GATA4 , MEF2C , TBX5 , MESP1 , MYOCD [ 32 ]

HCF

cTnT + : 5. 9%

α -actinin + : 5. 5%

+

+

+

GATA4 , MEF2C , TBX5 , ESRRG , MESP1 , MYOCD , ZFPM2 [ 6 ]

ESC, FH, neonatal skin

α -MHC-mCherry + : 15. 8%

α -MHC-mCherry + & cTnT + : 13%

+

+

ND

GATA4 , MEF2C , TBX5 , MESP1 , MYOCD , miR-133 [ 28 ]

HCF

cTnT + : 27. 8%

α -actinin + : 8%

ND

+

+

GATA4 , HAND2 , MYOCD , TBX5 , miR-1 , miR-133 [ 31 ]

HFF

cTnT + : 34. 1%

ND

+

+

CHIR99021, A83-01, BIXO1294, AS8351, SC1, Y27632, OAC2, SU16F, JNJ [ 36 ]

HFF

cTnT + : 6. 6%

+

+

+

Direct cardiac reprogramming in vivo

Reprogramming fibroblasts into cardiomyocytes in vivo is required just for heart regeneration. Transplanting reprogrammed cells and transdifferentiation elements into the infarcted heart represent two strategies towards this particular purpose. Firstly, cardiac fibroblasts were transduced with GMT for 1  day and were transplanted into computer mouse hearts. These cells were reprogrammed to cardiomyocytes within vivo [ 15 ]. Thereafter, in situ repair from the heart was performed by targeting endogenous cardiac fibroblasts through virus transfection. After coronary ligation, resident non-myocytes in the infarct zone can be reprogrammed into cardiomyocyte-like tissues by local delivery of GMT through a virus. Additionally , thymosin β 4 can improve the migration ability associated with fibroblasts. Co-injection of thymosin β 4 and GMT further improved the ejection fraction and reduced scar tissue formation [ 16 ]. Using a retrovirus expression system, pressured expression of GHMT (GATA4, HAND2, MEF2C, and TBX5) can also reprogram cardiac fibroblasts into beating cardiomyocytes within vivo [ 14 ]. In fact , the cardiac niche within vivo improves the efficiency of transdifferentiation. This gives a lot more hope to increasing the reprogramming efficiency and maturity [ 37 ]. These results suggest the possibility for repairing the center through gene therapy by targeting myofibroblasts. However , a comparatively safe gene delivery method needs to be developed. AAV vectors show great potential for gene therapy, but limited capability restricts their application for multiple genes. Reprogramming along with miRNAs may solve the problem; furthermore, cell-penetrating proteins furthermore hold great promise.

Transplanting of human fibroblasts treated with 9C may efficiently get chemically induced cardiomyocytes in vivo plus enhance the function of the infarcted heart [ 36 ]. When compared with transcription factors and miRNAs, small molecules have many benefits in vitro, such as better temporal control, more effective cellular delivery, and they are non-immune, less expensive, and safer. Moreover, it really is more convenient to control the process of programming through varying small chemical concentrations and combinations. However , there are still some questions in regards to the use of small molecules for reprogramming in situ. Little molecules can enter the blood and spread to other internal organs with ambiguous influence, and the impact time should be purely controlled to convert fibroblasts into target cells. Consequently , novel biomaterials should be developed to help local delivery associated with multiple drugs in a controllable manner.