p63 Silencing Induces Reprogramming of Cardiac Fibroblasts into Cardiomyocyte –Like Cells

Abstract

Objective

Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new heart failure therapy. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming.

Methods

p63 knockout (^−/−) and knockdown murine embryonic fibroblasts (MEFs), p63^−/− adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2 and Myocardin (HM).

Results

Flow cytometry revealed that a significantly greater number of p63^−/− MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared to wild type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, p < 0.05). HM treatment of p63^−/− MEFs increased cTnT expression to 74% ± 3% of cells, but did not induce cTnT expression in WT MEFs. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT mRNA expression compared to untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout, and expressed sarcomeric α-actinin as well as cTnT. These p63^−/− adult cardiac fibroblasts exhibited calcium transients and electrically-stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing heart failure procedures.

Conclusions

Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming.

Introduction

Nearly 5 million Americans can be expected to develop advanced congestive heart failure, typically characterized by the replacement of normal contractile cardiomyocytes with fibroblasts and scar tissue following one or more episodes of myocardial infarction.^{1, 2} Highly invasive surgical procedures such as heart transplant or mechanical circulatory support implantation represent the limited treatment options available for patients with end stage heart failure, which has traditionally been considered to be an otherwise highly lethal and irreversible condition.³ Recently, the novel strategy of in situ transdifferentiation of fibroblasts into induced cardiomyocytes (iCMs) via genetic reprogramming has offered an exciting and promising new solution for the treatment of heart failure.^{4, 5}

Inspired by the discovery that adult somatic cells could be dedifferentiated into induced pluripotent stem (iPS) cells, the concept of cellular transdifferentiation capitalized on the recognition that specific sets of transcription factors and other cellular reprogramming agents could convert one cell type into another without passing through a pluripotent, fully de-differentiated iPS stage.^4–6 More specifically, the cardiac-differentiating transcription factors Gata4, Mef2c and Tbx5 were the first to be shown to reprogram cardiac fibroblasts into cardiomyocytes, following which a wide variety of other reprogramming cocktails were shown to induce iCM generation and improvements in post-infarct ventricular function in vivo.^{5, 7–9}

Despite these encouraging findings, it was soon discovered that human cardiac fibroblasts were resistant to reprogramming compared to rodent cells, likely on the basis of more rigorous epigenetic controls imposed upon gene activation in higher order species.^{8, 10–13} Expanded cocktails of reprogramming factors were found to overcome this resistance, but the administration of complex mixtures of reprogramming factors represents a formidable barrier to human cellular reprogramming, especially in potential clinical therapeutic applications.^14–16

In the context of the intriguing observation that tumor suppressor genes such as p53, p63, p21, p19 and p16 inhibit iPS cell reprogramming,^17–21 we speculated that such genes may also impede the transdifferentiation of fibroblasts into cardiomyocytes by acting as “anti-plasticity genes”. Given the prohibitive oncogenic risk associated with downregulation of p53, we elected to narrow our focus on strategies for enhancing human cardiac cellular transdifferentiation to the downregulation of p63, a member of the p53 gene family with a reduced oncogenic profile compared to p53.^22–24

Materials and Methods

Cell culture

Previously characterized p53 knockout (^−/−) murine embryonic fibroblasts (MEFs) were gifted from Dr. Michelle Barton, MD Anderson Cancer Center.²⁵ p63 knockout (^−/−) MEFs were constructed with a deletion of the DNA binding domain for the ΔN-p63 and TA-p63 isoforms at exons 6–8 of the p63 gene.^21,26 A stable p63 knockdown MEF cell line was established after p63 lentiviral short hairpin (shRNA) transduction (GE Healthcare Dharmacon; Clone ID: V3LMM_508694), followed by puromycin selection (1μg/mL) for 5–7 days. All MEFs were cultured in DMEM supplemented with 15% fetal bovine serum (FBS), 1% non-essential amino acids (Gibco, Cat#11140050), and 1% Penicillin/Streptomycin.

Primary adult mouse cardiac fibroblasts were generated from inducible knockout mice (p63^fl/fl, strain C57BL/6, age 8–10 weeks), which have Cre-activated loxP sequences flanking the ΔNp63 gene (Figure S1).^{21, 27} These fibroblasts were isolated using a Miltenyibiotec GentleMACS dissociator under a protocol approved by the Baylor College of Medicine (BCM) Institutional Animal Care and Use Committee, following which they were cultured on 0.1% gelatin coated flasks in IMDM culture medium supplemented with 20% FBS, 1% Pen/Strep.

Adult human cardiac fibroblasts were harvested from patients undergoing heart transplant or mechanical circulatory support procedures for heart failure (BCM Institutional Review Board approval H-33421). The same p63 shRNA lentivirus described above was used to induce p63 knockdown in these cells. These cells were maintained in FGM^™ Fibroblast Growth Media (Lonza, CC-4526) after infection as previously described.¹²

Vectors

Lentiviral vectors were prepared from a doublet plasmid encoding Hand2-Myocardin ([HM]; SystemBio).²⁸ Adenovirus (Ad) control vectors encoding green fluorescent protein (GFP), or Ad vectors encoding Cre and GFP, were obtained from the BCM Vector Development Lab. Viral transduction was performed respectively with multiplicity of infection (MOI) of 20 (for lentiviruses) or 200 (for Ad) using transfection reagent polybrene (8 μg/mL, Millipore), based on our experience that these dosages provide the greatest level of transgene expression. After 24–48 hours transfection, media was replaced with reprogramming media consisting of DMEM and M199 supplemented with 10% FBS, 1% NEAA, 1% Pen/Strep. All cells were maintained for up to 3 weeks prior to analysis by flow cytometry, qRT-PCR and immunofluorescence.

Cell characterization assays

Flow cytometry

Identification of cardiomyocyte markers via flow cytometry was performed following trypsin detachment of cells from culture plates, after which cells were fixed in 4% paraformaldehyde (PFA) and permeabilized in 0.02% Tween-20, 0.5% DMSO in PBS. Blocking was then performed with 5% bovine serum album in permeabilization buffer. The cells were stained with cardiac troponin T antibody (Abcam, Cat#Ab8295) and goat anti-mouse Alexa 647 secondary antibody (Abcam, Cat#Ab150115). The cells were analyzed in a BD LSR Fortessa cell sorter using FlowJo software.

qRT-PCR

Relevant gene expression was detected via qRT-PCR from total RNA purified from cultured cells using TRIzol reagent (Invitrogen, Cat#10296010), following which cDNA was synthesized using Bio-Rad iScript Reverse Transcription Supermix (Cat#1708841) on an Eppendorf Mastercycler (Model 6321), following the manufacturer’s instruction. qRT-PCR was performed with SYBR Green PCR Master Mix (Cat#4309155) on a ViiA 7 Real-Time PCR System. Results were normalized to GAPDH. All primer sequences are listed in Table S1.

Immunofluorescence (IF) studies

After fixation of cells grown on IF-qualified cover slips (VWR, Cat#89015-724) with 4 % PFA, cells were permeabilized with 0.3% Triton and blocked in 10% goat serum. IF was performed using antibodies against cTnT (ThermoFisher, Cat#MS295-P0), α-actinin (Sigma Aldrich, Cat#A7811), and cardiac myosin heavy chain (Abcam, Cat#ab15). Goat anti-mouse Alexa 568 was used as the secondary antibody (ThermoFisher, Cat#A11004). Images were captured with a BioTek Cytation 5 Imaging Multi-Mode Reader at 40× magnification.

Co-culture and measurements of contractility and calcium transient

Adult murine p63^fl/fl cardiac fibroblasts were seeded onto a 6-well plate coated with 0.1% gelatin at a density of 150,000/well. Two days later, the cells were infected with Ad-GFP control or Ad-Cre-GFP, with or without lentivirus encoding Hand2 and Myocardin (HM), and iCM media was applied the next day.¹² One week later, the cells were harvested and re-plated at a ratio of 1:4 onto cultures of primary neonatal rat cardiomyocytes.¹² These had been isolated using the Miltenyibiotec GentleMACS dissociator and seeded at a density of 50,000 cells/well onto 0.1% gelatin coated 12-well plates in DMEM, M199, 4% horse serum, and pre-conditioned medium obtained from neonatal rat cardiomyocytes.⁷ Co-cultured cells were incubated for a total of 7 weeks.

Cell shortening and Ca²⁺ transients

Myocytes were field-stimulated at room temperature to contract by using a Grass S5 stimulator through platinum electrodes placed alongside the bath (1 Hz, bipolar pulses with voltages 50% above myocyte voltage threshold). ²⁹ Cells from random fields were videotaped and motion was digitized on a computer to assess contractility. For Ca²⁺ signal measurements, cells were loaded with 1.8 mM Ca²⁺ and 2 μmol/L Fura-2/AM (Life Technologies) for 1 h at room temperature, washed, and incubated for an additional 1 h to allow de-esterification of the dye, and then alternately excited at 340 and 380 nm using a Delta Scan dual-beam spectrophotofluorometer (Photon Technology International). Ca²⁺ transients were expressed as the 340/380 nm ratios of the resulting 510-nm emissions. Data were analyzed with Felix software (Photon Technology International).

Cell proliferation and soft agar oncogenicity assays

To assess cell proliferation, 1×10⁴ cells were seeded onto a 24-well plate and total number of viable cells was counted daily for 1 week. Adherent cells were detached with Trypsin 0.05%, stained with Trypan Blue and counted using a Bio-rad Automated Cell Counter. Soft agar assays were performed as previously described.³⁰ Briefly, 10,000 cells were seeded in a 6-well plate containing soft agar, and images were taken 2 weeks after culture (400×).

Statistical analysis

Data are represented as mean values ± standard deviation (SD), unless otherwise indicated. Differences between groups were examined for statistical significance using the Student t-test or ANOVA. We tested the assumption of normality in each group using the Shapiro-Wilk test and Kolmogrov-Smirnov test. We determined that our data was normal distributed in all groups. Then, we applied post-hoc testing after ANOVA and adjusted our p-values with the Bonferroni method. A p value < 0.05 was considered to indicate significance.

Results

Cardiomyocyte marker expression induced by downregulation of p63

To determine if the p53 gene family could be modulated to enhance cellular reprogramming, we first analyzed cardiac troponin T (cTnT) expression in cultures of p53^−/− and WT MEFs. Flow cytometry demonstrated that cTnT was expressed in 12% ± 0.3% vs 1.2% ± 0.6% of these cells, respectively (p < 0.05). We next demonstrated that significantly increased proportions of p63^−/− MEFs also expressed cTnT vs WT controls (38% ± 11% vs 0.9% ± 0.9% of cells, p < 0.05; Figure 1A, 1B).

Figure 1. Cardiomyocyte marker expression in MEFs induced by knockout of p63.

Wild type (WT) and p63^−/− MEFs were treated in vitro with lentiviruses expressing GFP or HM, and analyzed 3 weeks later (n=2). (A) Representative flow cytometry plots demonstrating that cTNT expression is increased in p63^−/− and HM-treated p63^−/− MEFs. (B) Percentage of (WT) and p63^−/− MEFs cells expressing cTnT⁺ after treatment with lentiviruses expressing GFP or HM. *p<0.05 vs WT (control). (C) qRT-PCR analysis demonstrating increased relative mRNA expression levels of cTnT and the indicated cardiac-differentiating factors in p63^−/− versus WT MEFs. Gene expression was normalized to GAPDH. *p<0.05 vs WT (control).

When p63^−/− MEFs were treated with Hand2 and Myocardin (HM), the percentage of cells expressing cTnT increased to 74% ± 3%, whereas HM treatment did not induce cTnT expression in WT MEFs. p63^−/− MEFs also demonstrated increased expression of the cardiac-transdifferentiation genes Gata4, Mef2c and Tbx5, (Figure 1C), suggesting a potential mechanism of action underlying the cardiodifferentiation of cells with p63 deletion.

To assess if (shRNA-mediated) p63 knockdown could also induce cTnT⁺ cardiomyocyte-like cells, we first confirmed that p63 shRNA-treated MEFs demonstrated an appropriate level of reduction of p63 mRNA expression (Figure 2A). Following this validation, we observed that p63 knockdown in MEFs induced approximately a 20-fold increase in cTnT mRNA expression, and a 3–5-fold increase in the mRNA levels of Gata4, Mef2c, and Tbx5 (Figure 2B), consistent with our observations in p63^−/− MEFs.

Figure 2. Cardiomyocyte marker expression induced by p63 knockdown.

(A) qRT-PCR analysis demonstrating decreased p63 mRNA expression levels in MEFs following p63 shRNA (shp63) treatment, as described in Methods, that is equivalent to p63 mRNA levels found in p63^−/− MEFs (n=2). Scr: scrambled shRNA control. (B) qRT-PCR analysis demonstrating approximately 20-fold increase in cTnT expression vs Scr control cells, and increased expression of cardiac-transdifferentiating factors in (shp63-treated) p63 knockdown MEFs after 3 weeks of culture. Gene expression was normalized to GAPDH..

Adult p63^−/− cardiac fibroblasts demonstrated cardiomyocyte-like features

Having determined that p63 silencing could enhance the transdifferentiation of embryonic cells into cardiomyocyte-like cells, we next asked if p63 downregulation would induce similar changes in adult cells. Adult murine p63^fl/fl cardiac fibroblasts were accordingly treated with adenovirus encoding Cre recombinase (Ad-Cre) or a negative control vector (Ad-GFP). As expected, Ad-Cre administration substantially decreased p63 expression, as verified by qRT-PCR (Figure 3A). Depletion of p63 in these cells resulted in a 200-fold increase in the expression levels of cTnT (Figure 3B). Immunofluorescence staining likewise revealed intracellular expression of the cardiac-specific protein markers sarcomeric α-actinin and cTnT in these p63 ^−/− cells, but not in cells treated HM alone or in Ad-GFP treated control cells (Figures 3C, 3D).

Figure 3. Cardiomyocyte marker expression in adult murine p63^−/− cardiac fibroblasts.

(A) qRT-PCR analysis showing decreased p63 mRNA expression levels in adult murine cardiac fibroblasts 5 days following Ad-Cre (to induce p63 knockout) vs Ad-GFP administration (vector control) in vitro (n=2). (B) qRT-PCR analysis showing an approximately 200-fold increase in cTnT gene expression 3 weeks after Ad-Cre vs Ad-GFP treatment (p63^−/− vs “WT” cells, respectively [n=2]). (C, D) Representative immunofluorescence images of adult murine cardiac fibroblasts treated as indicated on the left, and stained with cardiomyocyte-specific markers cTnT (Figure 3C) or sarcomeric α-actinin (Figure 3D). Images were captured 3 weeks after Ad-GFP or Ad-Cre treatment, with or without HM co-transduction. The first column depicts the nucleic stain 4′, 6′-diamidino-2-phenylindole (DAPI); second column, GFP fluorescence to identify infected cells; third column, cTnT (Figure 3C)) or α-actinin (Figure 3D); fourth column, merged image. Scale bars represent 30 μM. NRVM, neonatal rat ventricular cardiomyocytes (positive control). Gene expression was normalized to GAPDH Quantitative data are presented as mean ± SD from two independent repeats.

To demonstrate that these changes in molecular phenotype corresponded to relevant functional changes in cells with induced p63 silencing, we performed calcium transient and cellular contraction assays on p63^−/− adult murine cardiac fibroblasts co-cultured with neonatal rat cardiomyocytes, as recommended by previous investigators as a means of inducing iCM contractility in vitro.^{10, 12} In this analysis, approximately 2% of the p63^−/− cardiac fibroblasts treated with HM demonstrated contractions in response to electrical field stimulation, whereas no contractions were observed in cells treated with Ad-GFP or Ad-Cre alone (Figure 4A, Supplemental Videos). HM-treated p63 ^−/− cardiac fibroblasts likewise demonstrated calcium transients and cell shortening corresponding to their visual contraction (Figure 4B).

Figure 4. Cell contractility and calcium transients induced by p63 knockout and HM treatment in adult murine cardiac fibroblasts, when co-cultured with primary neonatal rat cardiomyocytes.

p63^fl/fl adult murine cardiac fibroblast treated with Ad-GFP, Ad-Cre, or Ad-Cre and Hand2 and Myocardin (Ad Cre + HM) and were co-cultured with neonatal rat cardiomyocytes 1 week after viral transduction for a total of 7 weeks. (A) Representative immunofluorescence staining for GFP. White arrow indicates non-beating GFP⁺ cell, yellow arrow indicates beating GFP⁺ cells, which correspond to Supplemental Videos S1 & S2. (B) Representative contraction and transient peaks corresponding to cells denoted by arrows in same column in Figure 4A, reflecting cell shortening (contraction) and Ca²⁺ transients under 1 Hz frequency and 1.8 mM calcium stimulation.

Several observations support the conclusion that the cells observed to be contracting in these functional studies were the (HM-treated) p63^−/− cardiac fibroblasts, and rule out the possibility that these findings could represent the artifact of neonatal cardiomyocyte contractions in this preparation. First, cell contractility was not observed when neonatal cardiomyocytes were cultured without the addition of p63^fl/fl fibroblasts, consistent with the known loss of this native cardiomyocyte phenotype after the 7 weeks in culture at which time point our contractility studies were undertaken.³¹ Second, contractility was also not seen in wells containing cardiomyocytes co-cultured with Ad-GFP-treated p63^fl/fl cardiac fibroblasts (i.e., WT control cells), or with (Ad-Cre-treated) p63^−/− cells without HM treatment (Figure 4A, Supplemental Videos). Third, given that contractions were detected only in GFP⁺ (i.e., Ad-infected) cells, and that (GFP-labeling) adenovirus was only administered to p63^fl/fl fibroblasts prior to their introduction into co-culture, it is unlikely that untreated neonatal cardiomyocytes could be mistakenly analyzed as (GFP-labeled) p63^−/− cardiac fibroblasts.

Cardiomyocyte marker expression in adult human cells with p63 knockdown

On the basis of our analysis of the effects of p63 silencing in embryonic and adult rodent fibroblasts, we analyzed the effects of p63 knockdown in (primary culture) adult human cardiac fibroblasts obtained from patients with heart failure. In these studies, p63 knockdown human cardiac fibroblasts demonstrated up to a 30-fold increase in cTnT mRNA expression levels compared to untreated human cardiac fibroblasts (Figure 5).

Figure 5. Cardiomyocyte marker expression in adult human cells treated with shp63.

(A) qRT-PCR analysis showing decreased p63 mRNA expression levels in shp63-treated vs scrambled shRNA treated human cardiac fibroblasts, obtained from patients undergoing heart transplant or circulatory support implants, as detailed in Methods (n=2). (B) Human cardiac fibroblasts procured as described in Methods demonstrating increased cTnT mRNA expression levels seven days after treatment with shp63 as compared cells treated with a scrambled shRNA control (Scr.). Gene expression was normalized to GAPDH (n=2).

Minimal neoplastic transformation with p63 knockout

We also evaluated whether p63 silencing could potentially induce neoplastic transformation, given its homology to p53, using two standard assays for this parameter: colony formation in soft agar and in vitro proliferation assays.^22–24 p63^−/− MEFs demonstrated negligibly increased replication rates compared to WT MEFs, while p53^−/− MEFs, used as a control comparator, demonstrated significantly increased replication rates (Figure 6A). Likewise, while p53^−/− MEFs grew into large colonies in soft agar cultures – a marker of oncogenicity, the p63^−/− MEFs produced smaller colonies that were comparable with those of wild type cells (Figure 6B).

Figure 6. Neoplastic transformation assessment assays.

(A) WT, p53^−/−, and p63^−/− MEFs (1×10⁴ cells) were seeded into a 24 well plate, and the total number of cells was counted daily for a total of 7 days. An increased proliferation rate was observed in p53^−/− but not in p63^−/− MEFs compared to WT cells (n=3). (B) Representative images of soft agar assays, performed as described in Methods, comparing anchorage-independent growth of WT, p53^−/−, and p63^−/− MEFs, demonstrating markedly larger p53^−/− colonies compared to WT and p63^−/− cells.

Discussion

Given the now well-documented barriers to the reprogramming of human cells compared to that of lower order species, we sought to enhance the susceptibility of cells to reprogramming as an alternative to the use of expanded reprogramming cocktail mixtures used by others to overcome this challenge.^{8, 10–16} We were able to demonstrate that the transdifferentiation of both rodent and human fibroblasts into cardiomyocyte-like cells was enhanced by the downregulation of p63, a member of the cell senescence family of genes of which p53 is the prototype. This approach was stimulated by the observation that downregulation of either of these genes enhance iPS reprogramming, suggesting they serve as “anti-plasticity” genes that may normally suppress excessive or promiscuous gene activation.^18–22

Given the large number of human neoplasms associated with p53 mutations, we elected to target p63, which has been associated with a far smaller human oncogenic profile than has p53.^{23, 24} We did not explore the reprogramming effects of other p53 family members such as p73, which have not been described to have anti-plasticity effects like p53 and p63.²⁴ Encouragingly, p63 downregulation appears to be even more effective than p53 downregulation in enhancing cellular reprogramming (Figure 1), consistent with observations that p63 is a potent anti-plasticity factor.²¹ Likewise, p63 downregulation resulted in cTnT expression levels four-fold greater than that which we have observed using optimized reprogramming cocktails on wild type cells.^{9, 12, 32}

Given the apparent potency of p63 silencing, it is prudent to first ask whether p63 downregulation incurs the risk of neoplastic transformation, despite the lack of evidence of this risk in the clinical literature.^{23, 24} Consistent with prior studies, encouragement can be derived from our observations of a lack of such oncogenicity effects with p63 versus p53 downregulation in soft agar and cell replication assays - the “gold standards” for in vitro neoplastic transformation. ^{23, 24, 33} Additional relevant safeguards may be found in the limited duration of p63 downregulation that is apparently needed to enhance reprogramming (as can be provided with adenovirus vectors),²⁸ and the ability to deliver p63 shRNA locally to cardiac tissues, which naturally have a very low baseline predilection to malignancy. It may also be possible to narrow therapy to a single isoform of p63 or to a downstream effector of p63, such as DCGR8.²¹ Notwithstanding these assurances, more rigorous in vivo validation of these safety data will certainly be needed before clinical applications are considered.

Our findings of the potency of p63 silencing on cardiac fibroblast transdifferentiation is surprising given that p63 has been previously associated primarily with epithelial development.^{21, 23, 24} In this context, it is interesting to consider that the spontaneous differentiation of p63^−/− MEFs into cardiomyocyte-like cells observed in our studies may represent a “default” differentiation pattern, consistent with the heart being the first organ formed in embryonic development.³⁴ In this paradigm, once p63 gene repression is released, cells revert to a predominant cardiomyocyte gene expression pathway, and the addition of additional cardio-differentiating genes like HM simply enhance this endogenous pathway. ^{7, 8, 10–12}

The molecular mechanism underlying cellular cardiodifferentiation mediated by p63-silencing may be related to our observation of the upregulation of the cardiac-reprogramming genes Gata4, Mef2c and Tbx5 in p63 deficient cells (Figs. 1, 2); specifically, that p63 repression allows for the increased expression of these otherwise repressed cardio-differentiating genes. The observation that exogenous GMT together with HM can substitute for p63 downregulation in inducing human cellular reprogramming is consistent with these data.^10–12 This premise is supported by our findings of effective cardiac reprogramming with p63 downregulation in adult human cardiac fibroblasts as well as embryonic and adult rodent cells.

On the other hand, it is possible that p63 downregulation also activates genes, such as the Wnt and B-catenin signaling pathways, which have been shown to be modulated by p63 expression and facilitate cardiac cellular reprogramming.¹³

While we have not yet identified the specific epigenetic processes by which p63 downregulation leads to enhanced gene activation and cardio-differentiation, one recently discovered pathway involves the complexing of p63 with histone deacetylases (HDACs) to induce chromatin condensation and the rendering of various gene promoter sites inaccessible to activator complexes.³⁵ Conceivably, release of p63 repression may thus allow access to these otherwise excessively repressed promoter sites in human cells.

Taken together, the findings of the present study suggest that downregulation of p63 may be a safe and effective strategy for inducing human cardiac cellular reprogramming as a potential therapeutic strategy for the treatment of heart failure. This strategy seeks to overcome barriers to human cellular reprogramming by increasing target cell susceptibility to this process, as compared to present strategies that largely rely upon adding a greater number of reprogramming factors to treatment cocktails. The latter may prove to be unduly complex and cumbersome for potential clinical application.

Study limitations

Limitations of the current study include our lack of a more expansive analysis of factors other than Hand2 and Myocardin that might optimally enhance cardiac reprogramming, an expanded analysis of additional potential mechanisms of action such as HDAC repression or cellular apoptosis, and our utilization of a relatively limited, albeit previously validated panel of cardiomyocyte markers (i.e.; cTnT, α-actinin, and myosin heavy chain) to define a cardiomyocyte-like phenotype.

Further, while we did demonstrate critically important physiologic cardiomyocyte markers (i.e., calcium transients and cell contraction) in p63-downregulated cells, co-culture of these cells with neonatal cardiomyocytes was needed to induce this phenotype. This is consistent with the use of this requisite reprogramming methodology in other in vitro reprogramming studies,¹² presumably because of the need for an appropriate cardiac “milieu” to induce reprogramming, as suggested by greater efficacy of reprogramming effects observed in in vivo vs in vitro studies.^7–12 The above considerations also apply to the relatively low efficiency of reprogrammed cells demonstrating a “full” (contractile) phenotype, which prior studies suggest might be more robust in the in vivo cardiac milieu.^7–12

Finally, while our use of human cardiac fibroblasts is encouraging in terms of the ultimate clinical applicability of this proposed human cardiac reprogramming strategy, it must be recognized that the cell culture process inherently selects out the “healthier” cells that can survive the explantation and in vitro culture propagation process. It is therefore not possible to definitively extrapolate these in vitro findings to potential results of in situ cell reprogramming that will ultimately require in vivo testing data that we now plan to perform.

Supplementary Material

1. Video S1. p63 ^−/− adult murine cardiac fibroblasts treated with HM.

Contraction was monitored after 7 weeks of co-culture with neonatal rat cardiomyocytes. Contractions are observed in response to external stimulation. Video images correspond to immunofluorescence image of Cell 1 in Figure 4A.

2. Video S2. p63 ^−/− adult murine cardiac fibroblasts treated with HM.

Contraction was monitored after 7 weeks of co-culture with neonatal rat cardiomyocytes. Contractions are observed in response to external stimulation. Video images correspond to immunofluorescence image of Cell 2 in Figure 4A.

3. Video S3. p63^fl/fl cells treated with Ad-GFP (wild type control).

Evidence of cell contraction was not detected in response to external stimulation after 7 weeks of co-culture with neonatal rat cardiomyocytes.

4

S1. Schematic diagram depicting p63^fl/fl inducible knockout mouse model. p63^fl/fl inducible knockout mice were generated using the Cre-loxP system, as described previously.²⁶ LoxP sequences, which recognize Cre, flank the 3′ exon of the p63 gene. Addition of Cre to p63^fl/fl removes the targeted region (highlighted in yellow) through recombination, rendering a p63 ^−/− phenotype.

Table S1. qRT-PCR Primer Sequences for Murine and Human Samples

Figure 7. Central Picture.

Silencing p63 in cardiac fibroblasts transdifferentiates cardiomyocyte–like cells.

Perspective.

Recent success reprogramming rodent cardiac cells has been tempered by the relative human cell resistance to reprogramming with currently defined transdifferentiation factors, perhaps due to more rigorous epigenetic constraints in higher order species. Downregulation of anti-plasticity genes such as p63 may enhance cellular plasticity and thereby improve reprogramming efficiency in human cells.

Glossary of Abbreviations

ICM

induced cardiomyocyte

KNOCKDOWN

knockdown

KO

knockout

MEFs

mouse embryonic fibroblasts

GFP

green fluorescent protein

HM

Hand2, Myocardin

cTnT

cardiac Troponin T

mRNA

micro RNA

qRT-PCR

quantitative reverse transcriptase polymerase chain reaction

shRNA

short hairpin RNA

IF

immunofluorescence

FACS

fluorescence activated cell sorting

MOI

multiplicity of infection