Heart repair by reprogramming non-myocytes with cardiac transcription factors.

The adult mammalian heart possesses little regenerative potential following injury. Fibrosis due to activation of cardiac fibroblasts impedes cardiac regeneration and contributes to loss of contractile function, pathological remodelling and susceptibility to arrhythmias. Cardiac fibroblasts account for a majority of cells in the heart and represent a potential cellular source for restoration of cardiac function following injury through phenotypic reprogramming to a myocardial cell fate. Here we show that four transcription factors, GATA4, HAND2, MEF2C and TBX5, can cooperatively reprogram adult mouse tail-tip and cardiac fibroblasts into beating cardiac-like myocytes in vitro. Forced expression of these factors in dividing non-cardiomyocytes in mice reprograms these cells into functional cardiac-like myocytes, improves cardiac function and reduces adverse ventricular remodelling following myocardial infarction. Our results suggest a strategy for cardiac repair through reprogramming fibroblasts resident in the heart with cardiogenic transcription factors or other molecules.

Heart disease caused by the loss or dysfunction of cardiomyocytes is the leading cause of death worldwide[1]. Whereas the neonatal mammalian heart can regenerate following injury[2], the capacity for regeneration of adult mammalian hearts is limited[3]. A fundamental goal of regenerative cardiovascular medicine is to successfully repair injured hearts by replacing cardiomyocytes and decreasing fibrosis. Transplantation of cardiac stem cells or stem cell-derived cardiomyocytes to improve cardiac function holds clinical potential, but is relatively inefficient[4-6].

Fibroblasts can be reprogrammed into pluripotent stem cells, muscle cells, and neurons by combinations of lineage-enriched transcription factors[7-12]. The human heart is composed of ~30% cardiomyocytes and ~60%–70% cardiac fibroblasts (CFs)[13]. We sought to define the optimal combination of core cardiac transcription factors necessary and sufficient for reprogramming of adult fibroblasts into functional cardiomyocytes and to determine if these factors could improve cardiac function in injured hearts through reprogramming of endogenous cardiac fibroblasts. Here, we show that four transcription factors, GATA4, Hand2, MEF2C and Tbx5 are able to reprogram adult mouse fibroblasts into functional cardiac-like myocytes in vitro and in vivo, and expression of these factors in non-cardiomyocytes enhances function of injured hearts following myocardial infarction (MI).

Reprogramming of adult fibroblasts into functional cardiac-like myocytes

A core set of evolutionarily conserved transcription factors (GATA4, Hand2, MEF2C, MesP1, Nkx2-5, and Tbx5) controls cardiac gene expression and heart development[14,15]. Recently, GATA4, MEF2C, and Tbx5 were reported to be capable of converting neonatal fibroblasts to cardiomyocyte-like cells in vitro[16]. To search for an optimal combination of transcription factors capable of inducing cardiac lineage-reprogramming, we generated retroviruses to express each of the six core cardiac transcription factors (GATA4 (G), and Hand2 (H), MEF2C (M), MesP1 (Ms), Nkx2-5 (N), and Tbx5 (T)) in fibroblasts derived from adult mice bearing a cardiac-specific a-MHC-GFP transgene (Supplementary Figs. 1 and 2).

Potential cardiogenic activity of the above 6 factors (G, H, M, Ms, N, T) in adult tail-tip fibroblasts (TTFs) from α-MHC-GFP transgenic mice was quantified by analysis of GFP+ cells by flow cytometry 9 days after viral transduction. No GFP+ cells were observed in fibroblast cultures transduced with viral backbone or without infection. However, the 6 factors together generated a small population of cells (~7%) positive for GFP (Supplementary Fig. 3). After several rounds of withdrawing one factor, we identified multiple combinations, including GHMMsT, GHMT, HMMsT, GMT, HMT, and MT, that were capable of efficiently inducing α-MHC-GFP+ cells (Supplementary Fig. 3). The cooperativity amongst the different factors is consistent with their ability to synergistically activate cardiac gene expression and to activate each other’s expression[14, 17–20].

To determine whether the above factors could activate endogenous cardiac-specific genes in adult TTFs, we examined expression of cardiac troponin T (cTnT) and α-MHC-GFP by flow cytometry (Supplementary Fig. 4). Depending on the experiment, GHMT induced between 5.2% and 19.7% (average 9.2%) of cells to become positive for both α-MHC-GFP and cTnT. We refer to GHMT-transduced cells expressing cardiomyocyte markers as induced cardiac-like myocytes (iCLMs). By comparison, GMT induced ~2.9% (1.5% to 5.6%) of adult TTFs to adopt a cTnT+/α-MHC-GFP+ phenotype (Supplementary Fig. 4). The percentage of iCLMs generated from adult TTFs by GHMT (9.2%) was ~4-fold higher than previously reported for neonatal TTFs with GMT (2.5%)[16]. The percentage of cells expressing cTnT and α-MHC-GFP reached a peak at day 7 of transduction; the fraction of iCLMs declined thereafter due to overgrowth of non-reprogrammed fibroblasts (Supplementary Fig. 5).

Cardiac fibroblasts (CFs) are the most prevalent interstitial cell type in adult mammalian hearts. We transduced adult CFs with GHMT, GMT or empty viruses and analyzed expression of cardiac markers by flow cytometry one week later. GHMT induced 6.8% of CFs to become cTnT+/α-MHC-GFP+, compared with 1.4% double-positive cells with GMT (Supplementary Fig. 6a). GHMT induced both cTnT and cTnI (cardiac Troponin I) in 7.5% of cells (Supplementary Fig. 6b). Thus, GHMT represented the most optimal combination of factors for efficient initiation of cardiac gene expression in adult fibroblasts. α-MHC-GFP+ cells derived from adult TTFs and CFs by GHMT transduction showed strong immunostaining of the sarcomeric proteins α-actinin and cTnT (Fig. 1a, b, and Supplementary Fig. 7). More organized sarcomeres were observed in iCLMs at day 30 (Fig. 1b), compared to day 14 (Fig. 1a). In the presence of GHMT, three types of iCLMs, referred to as Types A, B and C, which appear to represent a spectrum of cardiac reprogramming, were induced from adult CFs and TTFs. Type A iCLMs only expressed α-MHC-GFP. Type B iCLMs expressed both α-MHC-GFP and α-actinin. Type C iCLMs not only expressed α-MHC-GFP and α-actinin, but also displayed sarcomere-like structures (Fig. 1a). GHMT induced ~15% of adult CFs and ~18% of adult TTFs to become α-MHC-GFP positive cells, and ~30% and ~10% of these became Type C iCLMs, respectively (Fig. 1c).

Figure 1

Reprogramming fibroblasts toward a cardiac phenotype in vitro by GHMT

a. and b. Immunofluorescent staining for cardiac markers, α-MHC-GFP and α-actinin. Adult CFs isolated from α-MHC-GFP reporter mice were transduced by retroviruses carrying GHMT or empty vector alone. Immunocytochemistry was performed at (a) 14 days or (b) 30 days following transduction. Three types of iCLMs were categorized based on cardiac gene expression and morphology. Type A iCLMs only express α-MHC-GFP (top panels). Type B iCLMs express both α-MHC-GFP and α-actinin, but do not display sarcomeric structures (middle panels). Type C iCLMs express both α-MHC-GFP and α-actinin, and display sarcomeric structures (bottom panels). Sarcomeres were more organized at day 30 than at day 14 post-transduction. White boxes are enlarged in insets. Scale bar, 20 μm. c. Quantification of Type A, B, and C iCLMs in adult CFs and TTFs 14 days after transduction with GHMT. Ten fields were randomly chosen from each experiment. Three independent experiments were performed. Data are presented as mean ± std. d. Representative calcium transient traces from the indicated cell types depicted as Fura-2 ratios (340/380 nm). iCLMs were derived from adult CFs and displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes. Adult CFs do not display calcium transients.

Microarray and qPCR analysis of gene expression patterns showed expression of a broad range of cardiac genes, indicative of a cardiac-like phenotype, and concomitant suppression of non-myocyte genes including Fsp1 (fibroblast-specific protein 1 or S100A4) in fibroblasts transduced with GHMT (Supplementary Figs. 8, 9). Following maintenance of GHMT-transduced adult CFs or TTFs in culture for more than 5 weeks, we observed spontaneous contractions in ~0.2% of Type C iCLMs. These beating iCLMs displayed calcium transients and action potentials (Supplementary Movies 1, 2 and 3, Fig. 1d and Supplementary Figs. 10, 11). iCLMs displayed a pattern of calcium transients most similar to neonatal ventricular cardiomyocytes (Fig. 1d). Transduction with inducible expression vectors showed that the cardiomyocyte-like phenotype was stable following termination of exogenous GHMT expression (Supplementary Fig. 12). Co-staining for cardiomyocyte markers and Myc-tagged GHMT suggests that induction of the cardiomyocyte-like phenotype by GHMT is cell-autonomous (Supplementary Fig. 13). Together, these results indicate that GHMT can activate cardiac gene expression in a sub-population of TTFs and CFs. The relatively small percentage of cells that adopts the cardiac-like phenotype perhaps indicates a precise stoichiometry of the cardiac factors required for phenotypic conversion, which is achieved in only a fraction of cells, or to differential susceptibility to reprogramming amongst heterogeneous cell types in the starting non-myocyte population. The efficiency of cellular reprogramming to iCLMs by GHMT is comparable to that of reprogramming of induced pluripotent stem (iPS) cells by pluripotency factors[7,21].

Reprogramming of Fsp1-tagged cells into iCLMs in vivo

Following MI or other forms of cardiac injury, cardiomyocytes undergo necrotic and apoptotic cell death and CFs are activated to produce collagen and other extracellular matrix components, causing fibrosis and impaired cardiac function[22]. Therefore, we sought to determine whether reprogramming CFs to a cardiomyocyte fate might blunt the decline in cardiac function post-MI. Adult mammalian cardiomyocytes do not divide and are therefore resistant to retroviral expression. We expressed the cardiogenic transcription factors in CFs and other dividing cells in vivo using a retrovirus expression system, which is specific for proliferating cells and directs expression preferentially in myofibroblasts of injured rodent hearts[23]. We confirmed the specificity of retroviral infection for replicating non-cardiomyocytes by injecting concentrated GFP retroviruses into injured hearts after ligation of the left anterior descending coronary artery (LAD), which induces MI. GFP expression was clearly detected in the ischemic area. However, none of the GFP+ cells expressed the cardiac marker, cTnT, consistent with the specificity of retroviral infection for proliferating non-cardiomyocytes (Supplementary Fig. 14).

Fsp1 is expressed in non-cardiomyocytes such as fibroblasts and transitioning epithelia[24, 26]. In mouse and human hearts, expression of Fsp1 primarily colocalizes with markers of CFs and increases following MI[26]. Non-cardiomyocytes in mice carrying alleles of Fsp1-Cre and Rosa26-LacZ are specifically labeled with β-galactosidase (β-gal), providing a marker for fibroblast lineage tracing[24]. To examine whether cardiac transcription factors were able to activate cardiac genes in non-cardiomyocytes in vivo, we performed LAD ligation on Fsp1-Cre/Rosa26-LacZ mice and injected concentrated retroviruses encoding GHMT or GFP into the border zone immediately following LAD ligation. We then analyzed β-gal activity in histological sections of hearts at various times thereafter. In uninjured hearts, less than one β-gal+ cardiomyocyte per section was observed. After injury, β-galexpression was readily detected in CFs throughout the infarct zone (Fig. 2a, b). In injured hearts infected with GFP viruses, 0.05±0.13% of cardiomyocytes in the injured area were β-gal+ (Fig. 2c), which may be due to low level ectopic activation or to a basal level of new cardiomyocyte formation from a stem cell pool, as reported previously[27]. In contrast, abundant clusters of intensely stained β-gal+ cardiomyocytes were observed throughout the infarct and border zone of injured hearts infected with the GHMT retrovirus cocktail (Fig. 2b). We observed that 6.5±1.2% of cardiomyocytes in the injured area displayed β-gal activity (Fig. 2c). Generally, more β-gal+ cardiomyocytes were observed in the border zone adjacent to the infarct region, which may be due to intact vascular structures or higher viral infection in this region. Similar results were obtained upon injection with GHMMsT, whereas the inclusion of Nkx2-5 (GHMMsNT) diminished the efficacy of the other five factors (Supplementary Fig. 15), as seen in vitro. β-gal+ cardiomyocytes expressed cTnT and showed clear striations 3 weeks after viral transduction (Fig. 2b). Through quantification of β-gal+ cardiomyocytes in histological sections of infarcted hearts, we calculated that at least 10,000 new myocytes were generated in the injured area after 3 weeks of GHMT infection. This is likely an underestimate of the number of iCLMs generated in vivo since the reporter is only expressed in a subset of non-myocytes, such that unmarked cells could also be reprogrammed but go undetected.

Figure 2

Reprogramming non-cardiomyocytes toward a cardiac fate in vivo by GHMT

a. Heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. GFP-infected myocardium showed only β-gal+ non-cardiomyocytes, GHMT-infected myocardium showed extensive β-gal+ non-cardiomyocytes and cardiomyocytes. Black boxes in top panels are enlarged in lower panels. Scale bar, 40 μm. b. cTnT immunostaining and X-gal staining of heart sections of Fsp1-cre/Rosa26-LacZ mice uninjured or three weeks post-MI followed by injection of GFP or GHMT retroviruses. The same cell is marked with the same number. Several β-gal+ cells in GHMT-infected injured hearts expressed cTnT and displayed organized sarcomere structure. White boxes are enlarged in insets. Sections of injured hearts were taken at the border zone. Scale bar, 40 μm. c. Quantification of β-gal+ cardiomyocytes in border zones and infarct zones from GFP-infected (168 sections, n=3) and GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice (20 sections, n=2) post-MI. Sections were taken at 4 levels with an interval of 250 μm below LAD ligation site. Data are presented as mean ± std. d. Staining of border zone from GHMT-infected hearts of Fsp1-cre/Rosa26-LacZ mice post-MI by X-gal (blue), anti-cTnT (red) and anti-Cx43 (green). Gap junctions (green) were observed between β-gal+ and β-gal−cardiomyocytes (A and B) and between β-gal+ cardiomyocytes (C and D). The same cell is marked with the same letter. Scale bar, 20 um. e. Contractility and Ca2+ transients of β-gal+- and β-gal− -cardiomyocytes. β-gal+-cardiomyocytes (iCLMs) were labeled with a fluorogenic β-gal substrate C12FDG (green). Traces of sarcomere shortening were recorded from field-stimulatedβ-gal−-cardiomyocytes (n=15) and iCLMs (n=7) with clear striated morphology. 71.4% of iCLMs displayed a similar pattern of contractility and Ca2+ transients to β-gal−-cardiomyocytes. 28.6% of iCLMs demonstrated immature contractility.

In the heart, gap junctions composed of connexins ensure electrical and metabolic coupling between cardiomyocytes and coordinate their contractility[28]. To determine whether reprogrammed cardiomyocytes could couple with surrounding endogenous myocytes through gap junctions, we performed immunostaining for Connexin 43 (Cx43), the major connexin in functional cardiomyocytes[29]. Gap junctions were observed between β-gal+ and β-gal− cardiomyocytes and between β-gal+ cardiomyocytes (Fig. 2d), suggesting coupling of reprogrammed cardiomyocytes with surrounding myocytes. Reprogrammed β-gal+ cardiomyocytes isolated from ventricular myocardium and identified by labeling with a fluorogenic, lipophilic β-gal substrate (C12FDG) also displayed a pattern of contractility and calcium transients similar to normal β-gal− ventricular cardiomyocytes in response to electrical pacing at 1 Hz (Fig. 2e), indicating their functionality.

Reprogramming of inducible Tcf21-tagged cells into iCLMs in vivo

To rule out the possibility that injury or viral infection might somehow activate the Fsp1 reporter in cardiomyocytes, we generated a strain of mice harboring an inducible MerCreMer expression cassette inserted by homologous recombination into the Tcf21 (capsulin/epicardin) locus (Tcf21iCre), which is expressed specifically in non-cardiomyocytes in the heart (Supplementary Fig. 16)[30]. Intercrossing of these mice with mice bearing the Cre-inducible R26RtdTomato reporter showed specific expression of Tomato predominantly in cardiac fibroblasts with less expression in endothelial cells (Supplementary Fig. 16). However, we only observed one Tomato+ cardiomyocyte in over 40 histological sections of normal hearts. Tcf21iCre/R26RtdT mice were treated with tamoxifen for three days to mark Tcf21-expressing cells. Eight days after the last tamoxifen treatment, LAD ligation was performed and animals were injected with empty vector, GFP or GHMT retroviruses and analyzed 3–4 weeks later. Numerous Tomato+ cardiomyocytes were observed in GHMT-injected hearts compared to GFP-injected ones (Fig. 3a).

Figure 3

Lineage tracing of GHMT-induced iCLMs in vivo

a. Border zone of heart sections isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice that were subjected to LAD ligation followed by injection of GFP or GHMT retroviruses. Three weeks post-MI, hearts were fixed, sectioned and stained for cTnT (green) and visualized for tomato (red). Upper panels show GFP-injected heart in which tomato is seen only in fibroblasts. Lower panels show GHMT-injected heart in which tomato is seen in both fibroblasts and cardiomyocytes. Three types of iCLMs (positive for cTnT and tomato) were observed in injured hearts injected with GHMT-virus: (cell A) small cells without sarcomeres; cell B displays sarcomeres and expresses equivalent cTnT to neighboring tomato-negative cardiomyocytes; cell C displays sarcomeres and expresses less cTnT than neighboring tomato-negative cardiomyocytes. Scale bars, 20 μm. b. Tomato+ iCLMs isolated from tamoxifen treated Tcf21iCre/Rosa26RtdT mice one month post-MI followed by injection of GHMT retroviruses. Scale bar, 20 μm. c. Quantification of tomato+ cardiomyocytes isolated from uninjured hearts (n=2), injured hearts treated with empty vector retroviruses (n=3), or GHMT retroviruses (n=3). Data are presented as mean ± std. d. Recording of action potential of tomato+ iCLMs and endogenous cardiomyocytes by patch-clamping. Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz by whole-cell current patch-clamping. (n=5 for each group).

To quantify Tomato+ cardiomyocytes, we isolated cardiomyocytes from intact hearts using the Langendorff perfusion method. In GHMT-injected hearts, 2.4±1.5% of cardiomyocytes in the injured area were Tomato+, and these Tomato+ cardiomyocytes displayed clear striations (Fig. 3b and c). However, in empty vector-injected injured hearts, only 0.004±0.005% of cardiomyocytes were Tomato+ (Fig. 3c). Tcf21iCre marks a smaller, more restricted fraction of non-myocytes than Fsp1-Cre. Hence the number of new myocytes marked with this reporter is lower than with Fsp1-Cre. These findings suggest that forced expression of GHMT in non-myocytes of the heart is sufficient to induce the expression of cardiac markers. The Tomato+ cardiomyocytes induced by GHMT in vivo were able to spontaneously contract in vitro (Supplementary Fig. 17 and Supplementary Movie 4). Recording of action potentials of reprogrammed Tomato+ cardiomyocytes and endogenous cardiomyocytes by whole-cell patch clamping suggested functionality of cardiomyocytes induced by GHMT in vivo (Fig. 3d).

To further test whether GHMT indeed promoted the formation of new cardiomyocytes following MI and to rule out the possibility that labeled cardiomyocytes obtained from Fsp1-Cre or Tcf21iCre/Rosa marker mice injected with GHMT might arise from fusion of native cardiomyocytes with non-myocytes, we utilized mice with an inducible α-MHC-MerCreMer transgene and Rosa26-LacZ reporter. Gavage of these mice with tamoxifen for seven consecutive days resulted in labeling of 87.7±1.9% of cardiomyocytes in the left ventricular myocardium. Following LAD ligation and injection of GFP retroviruses, we observed a reduction in β-gal-labeled cardiomyocytes (83.2±4.6%) in the border zone adjacent to the infarct, suggesting replenishment of cardiomyocytes following injury, as described previously[27, 31]. Injection of GHMT retroviruses further reduced the percentage of β-gal+ cardiomyocytes in the border zone (76.0±5.2%) (Supplementary Fig. 18). These findings suggest that GHMT promotes the formation of new cardiomyocytes from a non-α-MHC lineage in vivo following injury.

Enhancement of cardiac regeneration of injured hearts

We examined whether forced expression of GHMT in non-cardiomyocytes could lead to measurable improvement in function of ischemic hearts. Cardiac function following MI was assessed in a blinded fashion by fractional shortening (FS), ejection fraction (EF), and stroke volume using echocardiography and magnetic resonance imaging (MRI). Twenty-four hours after LAD ligation, FS and EF assessed by echocardiography of all mice decreased relative to sham-operated mice. Thereafter, cardiac function of GFP-injected mice continued to decline, reaching a stable value 2 weeks post-MI; FS ~13% and EF ~ 30%. In contrast, infection of injured myocardium with GHMT retroviruses blunted further worsening of cardiac function 3 weeks post-MI; FS ~26% and EF ~51% (Fig. 4a). By comparison, functional improvement was delayed and less complete with GMT, consistent with the reduced efficiency of this transcription factor combination in reprogramming in vitro.

Figure 4

Attenuation of cardiac function of injured hearts by GHMT

a. Cardiac function of mice subjected to LAD ligation followed by intramyocardial injection of GFP or GHMT retroviruses was evaluated at various time points by echocardiography. Data are presented as mean ± std. *: p<0.05, **: p<0.005, ns: not statistically significant. b. Cardiac function at 6 and 12 weeks post-MI was assessed by MRI. Data are presented as mean ± std. *: p<0.05, **: p<0.005.

To determine whether functional improvement was sustained, we assessed cardiac function at 6 weeks and 12 weeks by EF and stroke volume using cardiac MRI. EF of GFP-injected mice decreased to reach a stable value of ~28% 6 weeks post-MI (Fig. 4b). In contrast, infection of injured myocardium with GHMT blunted worsening of EF 6 weeks post-MI (~49%) with further significant improvement at 12 weeks post-MI (~57%) (Fig. 4b). This long-term effect on EF by GHMT was accompanied by significant increases in stroke volume at 12 weeks compared to 6 weeks (Fig. 4b). Individual mice in each group demonstrated similar functional changes in both cardiac parameters, indicating the reliability of cardiac MRI to assess cardiac function (Supplementary Fig. 19). These data suggest that expression of GHMT in non-cardiomyocytes in injured hearts can sustain cardiac function. Moreover, GHMT- and GHMMsT-infected hearts showed a pronounced reduction in fibrosis and increased muscle tissue, compared with GFP-infected hearts after MI (Fig. 5a, b and Supplementary Fig. 20).

Figure 5

Attenuation of fibrosis in response to MI by GHMT

a. Comparison of cardiac fibrosis and scar formation between GFP and GHMT infected myocardium 4 weeks after LAD ligation. Cardiac fibrosis was evaluated at 5 levels (L1–L5) by trichrome staining 4 weeks post-MI. The ligation site is marked as X. Severity of cardiac fibrosis was classified as mild, moderate or severe (fibrotic area <20%, 20–40% or >40%, respectively). Numbers indicate the various type of severity/total number of hearts examined in each group. A graph shows animal distribution in each category. Scale bar, 1mm. b. Quantification of fibrotic area in heart sections displayed in (a). Fibrotic area (%) = (the sum of fibrotic area at levels 3 and 4/the sum of myocardial area in the LV at levels 3 and 4) × 100. Data are presented as mean ± std. *: p<0.05, **: p<0.005.

Discussion

Our results demonstrate that GATA4, Hand2, MEF2C, and Tbx5 (GHMT) can reprogram CFs into functional cardiac-like myocytes in vivo and in vitro, confirming and extending prior studies[16]. Moreover, exogenous GHMT expression in non-cardiomyocytes of the heart post-MI also reduces fibrosis and improves cardiac function.

Improvement of cardiac function post-MI by different transcription factor combinations correlated with their ability to convert fibroblasts into iCLMs in vitro (Supplementary Fig. 21), suggesting that cardiac repair results, at least in part, from reprogramming of non-cardiomyocytes toward a cardiomyocyte fate. We feel it is important to emphasize, however, that the dramatic functional improvement of GHMT-injected hearts seems greater than might be predicted from the relative inefficiency of reprogramming of adult CFs to iCLMs in vitro. We speculate that the native milieu of the intact heart, containing extracellular matrix, growth factors, persistent contractility, surrounding contractile cells and other cell types, is more permissive than plastic tissue culture dishes for functional reprogramming. In this regard, reprogramming of pancreatic beta-cells has been observed in vivo but not in vitro[32]. It is also conceivable that other mechanisms, such as a blockade to the activation of CFs, enhanced survival of cardiomyocytes, facilitated differentiation of activated cardiac progenitors into cardiomyocytes, as described previously[27, 31], or improved angiogenesis contribute to the benefits observed upon expression of GHMT in the heart post-MI. Irrespective of these uncertainties, the strategy presented here, while still requiring optimization, provides a potential means of improving cardiac function in vivo, bypassing many of the obstacles associated with cellular transplantation.

Methods Summary

All experimental procedures with animals were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center.

Adult TTFs and CFs were isolated by an explanting method in which fibroblasts migrate from minced tissue and grow in fibroblast growth medium. Fibroblasts were transduced with a mixture of polybrene (Sigma; 6μg/ml) and freshly made retroviruses expressing transcription factors generated in Platinum E cells (Cell Biolabs). Twenty-four hours after viral transduction, the viral medium was changed to a cardiac induction medium. Medium was changed every two days. Expression of cardiac genes was analyzed by flow cytometry, microarray, and immunocytochemistry.

Adult mice, 8–10 weeks old, underwent either a sham operation or ligation of the LAD. Concentrated retroviruses (~108~9 pfu viruses) were injected into the border zone using a gastight 1710 syringe (Hamilton). Cardiac function was assessed using echocardiography and MRI. Hearts were harvested from euthanized animals for histological studies.

Isolation of cardiomyocytes, electrophysiological measurements, histology and immunohistochemistry were performed as described previously[2, 33–37].

Methods

Isolation of primary fibroblasts

Tail-tip fibroblasts (TTFs)

Adult mouse tails were skinned and cut into small pieces which were plated on tissue culture dishes and cultured in DMEM supplemented with 15% FBS and antibiotics. The medium was changed every 2 to 3 days. TTFs migrated out from the explants after 2 or 3 days. One week later, TTFs were frozen or replated for viral transduction.

Cardiac fibroblasts (CFs)

Hearts from adult mice (older than 4 weeks of age) were minced into small pieces and plated on tissue culture dishes. Three minutes later, culture medium (DMEM:199 (4:1), 15% FBS and antibiotics) was gently added to the dishes. CFs started to migrate out of the minced heart tissue after two days. The medium was replaced every two days. Ten days later, CFs were frozen or replated for viral transduction.

Generation of retroviruses

Retroviral plasmid DNA was generated by subcloning EGFP, Myc-tagged Nkx2-5, GATA4, Tbx5, Hand2, MEF2C, and FLAG-tagged MesP1 cDNAs into the retroviral vector pBabe-X[38].

Ten micrograms of retroviral plasmid DNA was transfected using Fugene 6 (Roche) into Platinum E cells (Cell Biolabs) which were plated on a 10-cm tissue culture dish at a density of 3×106 cells per dish, 24 hours prior to transfection. Twelve hours after transfection, medium was changed to 12 ml of fresh medium (DMEM supplemented with 10% FBS and antibiotics). After 36 hours of transfection, viral medium was harvested and filtered through a 0.45 μm cellulose filter. The viral supernatant was mixed with polybrene (Sigma) to a final concentration of 6 μg/ml.

Viral transduction

TTFs and CFs were plated on tissue culture dishes pre-coated with SureCoat (Cellutron) at a density of 0.8×104/cm2. After 24 hours, the fibroblast growth medium was replaced with freshly made viral mixture containing polybrene. Twenty four hours later, viral medium was replaced with induction medium, composed of 10% conditioned medium obtained from neonatal rat/mouse cardiomyocyte culture, DMEM/199 (4:1), 10% FBS, 5% horse serum, antibiotics, non-essential amino acids, essential amino acids, B-27, insulin-selinum-transferin, vitamin mixture, and sodium pyruvate (Invitrogen). Conditioned medium was filtered through a 0.22 μm pore size cellulose filter. Medium was changed every two days until cells were harvested.

qPCR, Western blot analysis and immunocytochemistry

Total RNA was extracted from cultured cells and cDNA was synthesized by reverse transcription. All qPCR probes were obtained from Applied Biosystems. Western blots were performed with anti-Myc (Santa Cruz, clone A-14 1:1000) and anti-FLAG (Sigma, 1:2000) antibody. For immunocytochemistry, cells were fixed in 4% paraformaldehyde and incubated with primary antibodies: anti-GFP (Torrey Pines Biolabs 1:400), anti-cTnT (Thermo Scientific 1:400), anti-Myc (Santa Cruze, clone A-14 1:200), and anti-α-actinin (Sigma, 1:400). After washing with PBS, Alexa fluorogenic secondary antibodies (Invitrogen) were used to detect the signal.

DNA microarray

Total RNA was isolated from uninfected CFs, CFs transduced with either empty vector or GHMT retroviruses and adult heart. Microarray analysis was performed on the platform of Illumina Mouse-6 Beadchip by the DNA Microarray Core Facility at the University of Texas Southwestern Medical Center. Data were analyzed using GeneSpring GX software (Agilent).

Flow cytometry

For the initial assay to detect αMHC-GFP expression, adherent fibroblasts were washed with PBS and detached from culture dish by treatment with accutase (Millipore) for 10 min at 37°C. Cells were then washed with 2% FBS in PBS and filtered through a cell strainer. Cells were incubated with propidium iodide (1:1000 dilution in 1% FBS in PBS) for 15 min at room temperature. Dead cells were excluded by propidium iodide staining and live cells were analyzed for GFP expression using FACS Caliber (BD Sciences) and FlowJo software.

For intracellular staining of cardiac-specific markers, cells were fixed with 4% paraformaldehyde for 15 min after being harvested, as described above. Fixed cells were washed with PBS and permeabilized with saponin for 10 min at room temperature. After being washed with PBS, cells were incubated with 5% goat and donkey serum in PBS at room temperature for 30 min, followed by incubation with primary antibodies (rabbit polyclonal anti-GFP antibody (Invitrogen) at a 1:100 dilution and mouse monoclonal anti-cTnT antibody (Thermo Scientific) at a 1:400 dilution in 0.2% goat and donkey serum in PBS for 30 min at room temperature. After washing twice with PBS, cells were incubated with secondary antibodies for 30 min at room temperature. Secondary antibodies were goat anti-rabbit Alexa fluor 488 (Invitrogen) at a 1:200 dilution and donkey anti-mouse Cy5 (Jackson Laboratory) at a 1:400 dilution in PBS containing 0.2% goat and donkey serum. Cells were washed with PBS three times, and then analyzed for GFP and cTnT expression using FACS Caliber (BD Sciences) and FlowJo software.

Generation of α-MHC-GFP transgenic mice

A plasmid containing a 5.5 kb genomic fragment upstream of the mouse α-MHC gene plus exons 1–3 and intronic sequences[39], inserted upstream of a neomycin-resistance cassette followed by an Internal Ribosomal Entry Sequence (IRES) and a GFP reporter was linearized and micro-injected into the pronucleus of zygotes. The founders were crossed to C57BL/6 mice to get stable lines.

Induction of Cre by tamoxifen administration

Tamoxifen (Sigma) was dissolved in sesame oil (90%) and ethanol (10%) at a concentration of 50 mg/ml. To induce Cre activity, tamoxifen (0.2 mg/g body weight) was administered by gavage with a 22-gauge feeding needle into mice bearing Tcf21iCre/+/R26RtdT or α-MHC-MerCreMer[40]/Rosa26-LacZ for three to five or seven consecutive days, respectively. Mice were analyzed or subjected to MI surgery at day 8 post-oral gavage of the last dosage.

Sorting of Tcf21-expressing cells and gene expression

Three-month old Tcf21iCre/+/R26RtdT mice were induced with 0.2mg/g tamoxifen for 3 consecutive days by gavage, and a week later hearts were isolated and processed (atria and aorto-pulmonary trunk were removed) to generate single cell suspensions for FACS sorting, as described previously[41]. The suspension was filtered through tissue strainers, centrifuged at 400 × g for 5 minutes and resuspended in 10% CM media (10% Hyclone FBS, 3:1 DMEM/M-199, 10 mM HEPES, 1.2% antibiotic/antimycotic) before sorting with a MoFlo flow cytometer (Cytomation Inc) using Summit software. For transcript analysis, sorted cells were collected into lysis buffer for RNA extraction (RNAqueous Micro kit from Ambion). A fraction of each sample was also collected into PBS for post-sort assessment of purity. Complimentary DNA was synthesized using Superscript III reverse transcriptase (Invitrogen) and random hexamers (Roche). Gene expression profiles were generated using standard qPCR methods with iTAQ SYBR Green master mix (Bio-Rad) on a CFX96 instrument (Bio-Rad). Samples were run in triplicate and normalized to cyclophilin expression. Fold enrichment was determined with respect to the unsorted population.

MI surgery and intramyocardial injection of retroviruses

Mice were anesthetized with isoflurane, intubated with a polyethylene tube (size 60), and then ventilated with a volume-cycled rodent respirator with a 2–3 ml/cycle at a respiratory rate of 120 cycles/min. Thoracotomy was performed at the third intercostal space and self-retaining microretractors were placed to separate the third and fourth rib to visualize the LAD. A 7.0 prolene suture (Ethicon, Johnson & Johnson, Brussels, Belgium) was then passed under the LAD at 1.5 mm distal to the left atrial appendage, immediately after the bifurcation of the left main coronary artery. The LAD was doubly ligated. The occlusion was confirmed by the change of color (becoming paler) of the anterior wall of the left ventricle. Sham-operated mice underwent the same procedure without ligation. Immediately after ligation of the LAD, 50 μl of concentrated retrovirus was injected into the border zone of the infarct at 5 different areas using a gastight 1710 syringe (Hamilton). The chest wall was then closed with a 5.0 Dexon absorbable suture (Tyco Healthcare, United States Surgical, USA), and the skin was closed with Topical Tissue Adhesive (Abbott Laboratory, IL, USA). Mice were extubated and allowed to recover from surgery under a heating lamp. The mouse surgeon was blinded to the study. Mice with FS>30% at day 1 post-MI were removed from the study.

Transthoracic echocardiography

Cardiac function was evaluated by two-dimensional transthoracic echocardiography on conscious mice using a VisualSonics Vevo2100 imaging system. Fractional shortening (FS) and ejection fraction (EF) were used as indices of cardiac contractile function. M-mode tracings were used to measure LV internal diameter at end diastole (LVIDd) and end systole (LVIDs). FS was calculated according to the following formula: FS (%) = [(LVIDd − LVIDs)/LVIDd] × 100. EF is estimated from (LVEDV-LVESV)/LVEDV 100%. Left ventriclular end systolic volume, LVESV; end diastolic volume, LVEDV. All measurements were performed by an experienced operator blinded to the study.

Cardiac MRI

Six and twelve weeks after MI, the cardiac function of mice was re-evaluated by cardiac MRI using a 7T small animal MR scanner (Varian, Inc, Palo Alto, CA) with a 38 mm birdcage RF coil. Under anesthesia by inhalation of 1.5 – 2% isoflurane mixed with medical-grade air via nose-cone, the animals were placed prone on a mouse sled, (Dazai Research Instruments) equipped with a pneumatic respiratory sensor and ECG electrodes for cardiac sensing, head first with the heart centered with respect to the center of the RF coil. The mouse chests were shaved and a conducting gel was applied to optimize ECG contact between electrodes and mouse. All MRI acquisitions were gated using both cardiac and respiratory triggering. The bore temperature was kept at 35°C to assure adequate and constant heart rate.

Two-dimensional (2D) gradient echo images on three orthogonal planes (transverse, coronal and sagittal) were acquired to determine the long-axis of the heart in each mouse. Axial images perpendicular to the long axis of the heart were chosen for cine-imaging. Cine images at 12 phases per cardiac cycle were obtained with an echo time of 2.75 ms, repetition time = EKG R-R interval/12, flip angle of 45°, and NEX= 4. Each scan consisted of seven to ten contiguous slices from apex to LV outflow with 1 mm thickness, a matrix size of 128 × 128, and a field of view of 30 × 30 mm.

Epicardial and endocardial borders were manually traced for calculation of LVESV, LVEDV using NIH ImageJ software. Total LV volumes were calculated as the sum of all slice volumes. Stroke volume was calculated by the equation, LVEDV-LVESV. EF was calculated by the equation, (LVEDV-LVESV)/LVEDV 100%. Investigators performing MRI acquisition and analysis were blinded to the assignment of mice group.

Isolation of adult mouse cardiomyocytes, measurement of cardiomyocyte sarcomere shortening and Ca2+ transients

Mouse cardiomyocytes were isolated using enzymatic digestion and mechanical dispersion methods previously described[33]. In brief, after retrograde perfusion with Ca2+ -free Krebs-Ringer buffer (KR, 35 mM NaCl, 4.75 mM KCl, 1.19 mM KH2P04, 16 mM Na2HPO4, 134 mM sucrose, 25 mM NaCO3, 10 mM glucose, 10 mM HEPES, pH 7.4, with NaOH) and digestion with collagenase solution (Collagenase II, 8 mg/mL), the LV myocytes were separated using a fine scalpel and scissors. After gentle trituration, cells were kept in KB solution (10 mM taurine, 70 mM glutamic acid, 25 mM KCl, 10 mM KH2PO4, 22 mM glucose, 0.5 mM EGTA,pH 7.2 with KOH) and studied within 6 hours at room temperature.

To examine myocyte contractile capacity, isolated cardiomyocytes were incubated with 33 μm C12FDG for 30 min. The green C12FDG+ cardiomyocytes were identified by a fluorescence microscope. Adult cardiomyocytes from wild-type mice incubated with C12FDG were used to determine autofluorescence of cardiomyocytes. C12FDG+ cardiomyocytes and C12FDG− cardiomyocytes were field stimulated at 1 Hz while being superfused with extracellular buffer at room temperature. Images were acquired at 240 Hz through a 60xmicroscope objective using a variable field rate CCD camera (IonOptix, Milton, MA). Cell length was measured by a video edge-detection system, using an IonOptix interface system. Intracellular Ca2+ transients were measured as previously described[33-36]. Analyses were carried out with the software (IonWizard, IonOptix Corp.). Onlyrod-shaped, clearly striated cardiomyocytes that were Ca2+ tolerant were used in the experiments.

Calcium transient measurements in spontaneous beating cardiomyocytes

Calcium imaging of beating iCLMs, cultured neonatal mouse ventricular cardiomyocytes was performed using the PTI (Photon Technology International) Ca2+ Imaging System (Birmingham, NJ) with an automated fluorescence microscope and a CCD camera, as described previously[35]. Calcium transients in individual spontaneous beating cell were calculated by measurement of Ca2+-induced fluorescence at both 340 and 380 nm.

Membrane potential recordings of iCLMs in vitro

Action potentials of beating iCLMs with di-4-ANEPPS (Invitrogen) were assessed, as described previously[37].

Action potential recordings of iCLMs isolated from adult mouse hearts

Tomato+ cardiomyocytes were identified under a Nikon inverted fluorescence microscope with a red filter. Whole-cell current clamp experiments (Axopatch 200B, Molecular Devices) were conducted to measure cardiomyocyte membrane action potentials (AP). Cells were plated in a chamber that was superfused at 1 to 2mL/min with standard solution A containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES (pH 7.4 with NaOH), and 10 mM glucose (310 mOsm). Recording pipettes were prepared with resistances of 2–3 mΩ when filled with internal solution containing 135 mM KCl, 10 mM EGTA, 10 mM HEPES, 5 mM glucose (pH7.2 with KOH; 300 mOsm). Action potentials were recorded in response to brief (1–2 ms) depolarizing current (1–2 nA) injections delivered at 1 Hz. All data were filtered at 5 kHz and analyzed by pCLAMP 9 software (Molecular Devices).

Histology and immunohistochemistry

Hematoxylin and eosin (H&E) and LacZ staining were performed as described[37]. Frozen sections from ischemic hearts were stained with anti-GFP (Aves Labs 1:800) antibody according to manufacturer’s instructions. Paraffin-embedded sections were stained with anti-cTnT (1:400) and anti-Cx43 (Cell Signaling, 1:50). Frozen sections from tamoxifen-treated Tcf21iCre/+/R26RtdT mice (~5 week old) were stained with anti-P4HB (ProteinTech, 1:200), anti-cTnT (Thermo Scientific, 1:400), anti-isolectin B4 (Vector Labs, 1:100), and anti-SM22α (Abcam, 1:200). Signals were detected with Alexa fluorogenic secondary antibodies (Invitrogen).