Improving heart muscle cell reprogramming by targeting cell aging through the Rb1 gene

Mouse embryonic fibroblasts (MEF) were obtained from the α-MHC/GFP transgenic mouse on embryonic day 13.5, as previously described [2, 5]. In short, hearts were digested with 0.1% trypsin and plated on plastic dishes. Attached fibroblasts were cultured for 7 days and α-MHC-GFP⁺ cells were sorted and cultured in DMEM/M199 medium containing 10% FBS at a density of 10⁴/cm². To generate a MEF GMT cell line, MEFs were transfected with a PiggyBac vector [16] carrying an inducible TetOn3G promotor, the mouse GMT reprogramming factors, and mCherry. The PiggyBac transposon system, which integrates randomly into the genome, as described in Yusa et al. [17]. PiggyBac system's random integration ensures that the GMT cassette is expressed independently of the endogenous GMT genes, which remain intact. After co-transfection by using Lipofectamine 3000 (Invitrogen, Catalog number: L3000001), the cells were selected with puromycin at a final concentration of 2 µg/ml for 3 days (Gibco, A11138-03) to obtain a stable cell line.

Human fetal cardiac fibroblasts (hFCFs) cell line carrying an inducible cardiac reprogramming cassette was created with the PiggyBac (PB) transposon system as described above. hFCFs were transfected with either a GMT vector or an empty vector (EV) together with a transposase plasmid. As a control, the EV-hFCFs cell line was created containing the same TetOn 3G promotor and mCherry tag, but not containing the reprogramming cassette. After co-transfection by using Lipofectamine 3000 (Invitrogen, Catalog number: L3000001), the cells were selected with puromycin at a final concentration of 2 µg/ml (Gibco, A11138-03) to obtain a stable cell line. Due to the tetOn3G promotor, it is possible to activate and inactivate the reprogramming cassette on every preferred time point by adding or removing doxycycline (Dox) [18].

Cells were seeded at a density of 5 × 10⁴ cells per well in a 6-well plate coated with 0.1% gelatin and cultured in the reprogramming medium (DMEM with 10% FBS and 1% P/S). Reprogramming medium was changed every other day. Dox (1 µg/ml, Clontech, 631311) was added one day after the cells were plated to start reprogramming and maintained during cardiac reprogramming. The day of Dox administration was considered as day 0.

All shRNAs constructed in the pLKO.1 HIV-based lentiviral vector were purchased from Sigma-Aldrich. The lentivirus packaging and envelop vectors, pCMVR8.74 (#22036) and pCMV-VSV-G (#8454) were obtained from Addgene. The coding sequences of RB1, RBBP4, RBBP7, CBX8 and CDKN1B were PCR amplified from MEF cDNA and cloned into pMXs-puro vector with compatible restriction enzyme sites. Each gene was designed with two pairs of coding sequences. pLKO.1 vector itself was used as the control shRNA. The primers required in this study are listed in supplementary table S1 and purchased from Integrated DNA Technologies.

Lentivirus packaging was conducted in 293 T cell line. This cell lines were maintained in DMEM growth media containing DMEM with 10% FBS, 1% PS (Penicillin/Streptomycin). One day before transfection, 293 T cells were plated at a density of 6 × 10⁶ cells per T175 flask. The next day, 10 µg shRNA viral vector, 5 µg pCMVR8.74 and 5 µg pCMV-VSV-G were co-transfected to 293 T cells. Supernatant was collected 72 h post-transfection and filtered through 0.45 μm syringe filter (CORNING, 516–1954) and incubated with PEG6000 solution (8% final concentration) overnight at 4°C. The next day, mixtures were centrifuged at 6000 rpm for 30 min at 4 °C, and the pellets were resuspended in 100 µl 50mM Tris-HCl per T175 flask virus. Lentiviruses were freshly added to target cells with 4 µg/ml polybrene (sigma, TR-1003-G) or frozen at −80 °C freezer for future usage. For transfection, MEFs/Hfcfs were plated into gelatin-coated 24 well plates at a density of 2 × 10⁴ cells per well in DMEM medium. The next day, 5–10 µl of lentiviruses expressing shRNAs were added in iCM media (10% FBS of DMEM/M199 (4:1)) for 16 h and changed every 2–3 days followed by selection with 2 µg/ml puromycin for 48 h to obtain stable cell line. At the indicated time points, reprogramming cells were harvested for analyses.

Total RNA was isolated at the indicated time points using the Nucleospin RNA isolation kit from Macherey-Nagel (740.955.250). The qScript cDNA synthesis kit from Quanta BioSciences (95047-100) was used to synthesize cDNA from the isolated RNA samples (300 ng per sample). The quantitative real-time polymerase chain reaction (qRT-PCR) was performed with iQ SYBR Green Supermix (Bio-Rad). β-actin was selected as the housekeeping gene and was used for the calculation of normalized gene expression levels (ΔCt). The primers required in this study are listed in supplementary table S2 and purchased from Integrated DNA Technologies.

RNA concentration and integrity were analyzed with the Agilent 2100 Bio analyzer prior to proceeding with sequencing (Agilent RNA 6000 Nano Kit). RNA concentration for each single sample must be at least more than 15ng/ul and the total RNA amount is no less than 0.3ug. The library construction kit is MGIEasy RNA Directional Library Prep Set (1000006385), made in MGI. Sequence on the DNBSEQ-G400 platform with PE150. The sequencing data was filtered with SOAPnuke by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2. After that, Ericscript (v0.5.5) and rMATS (V3.2.5) were used to detect fusion genes and differential splicing genes (DSGs), respectively. Bowtie2 was applied to align the clean reads to the gene set, a database built by BGI (Beijing Genomic Institute in ShenZhen), in which known and novel, coding and noncoding transcripts were included. Expression level of gene was calculated by RSEM (v1.3.1). Essentially, differential expression analysis was performed using the DESeq2(v1.4.5). These RNA sequencing and data mapping is conducted in BGI Hongkong Tech Solution NGS Lab. Gene expression profiles were analyzed using Omics Explorer 3.2 (Qlucore) and principal component analysis (PCA) was used to visualize the data. Gene set enrichment analysis (GSEA) was also performed with Qlucore to determine if a gene set of interest was significantly enriched in one condition compared to another. Gene sets with (False Discovery Rate [FDR] < 0.1) were considered significantly enriched in the comparison made.

For MEFs, cells from different groups were first washed with PBS, detached by trypsin and collected to 15 ml Eppendorf tube, filtered with Flowmi™ Cell Strainers, 40 μm (southern labware, BAH136800040) and were analyzed by Cytoflex flow cytometer (Beckman Coulter, #A00-1-1102). For hFCFs, cells were washed and fixated in 70% cold ethanol for 30 min. Later, cells were washed twice with PBS and blocked with 5% goat serum for 30 min, washed twice with PBS and stained with primary antibody α-Actinin (Sigma, A7811, 22ug/ml) overnight at 4℃, washed with PBS twice and stained with goat anti-mouse IgG Alexa Fluor 488 s antibody (Invitrogen, A11001, in 1:400 dilution) for one hour at room temperature, and then washed with PBS twice, filtered with Flowmi™ Cell Strainers, 40 μm and analyzed by Cytoflex flow cytometer in the similar setting.

Cell apoptosis was determined by Annexin V/PI staining. The cells were stained with Annexin V/PI for 15 min in the dark and then analyzed using a flow cytometer (Accuri C6, USA). Annexin V⁺/PI⁺ cells were regarded as apoptotic cells in the late stage. A minimum of 10,000 cells within the gated region was measured by flow cytometry.

To visually compare the senescence among different groups, we performed β-galactosidase staining (β-Gal) according to the manufacturer's protocol of senescence β-galactosidase staining kit (#9860, Cell Signaling Technology). Briefly, cells were fixed in 1x Fixative Solution for 15 min and washed with PBS twice. Subsequently, cells were treated with galactosidase staining solution and placed in a 37 ℃ incubator overnight. The next day, the blue color (indicative of senescence) occurs, and pictures were taken under microscope.

Data were analyzed with GraphPad Prism 9 and comparisons were performed with t-test (non-parametric tests) and a two-way ANOVA. Data were presented as mean ± SEM. p < 0.05 is considered as significantly different.

The Dox inducible cardiac reprogramming model in both MEFs and hFCFs were established and verified as described previously [19]. After initiation of cardiac reprogramming by Dox exposure, we noticed cells display signs of cell cycle arrest and apoptosis. To investigate whether direct cardiac reprogramming by GMT overexpression induced cellular senescence, we performed β-Gal staining and observed indeed an increased senescence signal, both in MEFs and hFCFs upon initiation of cardiac reprogramming (Fig. 1A, D). To further validate, we also determined the level of senescence markers GLB1 and P16 and observed that their expression level was also significantly increased (Fig. 1B, E). To determine if direct cardiac reprogramming triggers cell apoptosis, Annexin V/propidium iodide (PI) double-staining was used, and the apoptosis rate was analyzed by flow cytometry analyses. We observed that the number of Annexin V⁺/PI⁺ cells increased significantly upon exposure to Dox at different time point (Fig. 1C), indicating that initiation of cardiac reprogramming causes a significant cell death over time.

To further investigate which pathways and genes are involved in direct cardiac reprogramming, we performed RNA sequencing to identify the differentially expressed genes between GMT Dox (+) group and other control groups. Transcriptional differences within these groups were visualized in a PCA plot (Fig. 2A) and, as expected, GMT Dox (+) is distinct from both the GMT Dox (-) as empty vector controls. To investigate the transcriptional differences between these groups further, differential gene expression analysis was performed. Heatmap analyses showed that gene sets related to cellular senescence were significantly upregulated in GMT Dox (+) group in comparison to control groups. This includes genes such as RB1, RBBP4, RBBP7, CBX8 and CDKN1B (Fig. 2B). We performed GSEA to identify statistically enriched gene sets in transcriptomic data between these groups and found that in GMT Dox (+) group a significant enrichment of genes associated with cellular senescence during cardiac reprogramming was present compared to other control groups (Fig. 2C).

Given that cellular senescence hampers iPSCs reprogramming and is induced upon cardiac reprogramming, we hypothesize that inhibition of senescence may improve cardiac reprogramming efficiency. To investigate this hypothesis, senescence causal genes RB1, RBBP4, RBBP7, CBX8 and CDKN1B were suppressed in both hFCFs and MEFs with shRNA constructs as indicated in Figs. 3A and 4A, respectively. For each candidate, a pool of two independent shRNA constructs that target different regions within the gene were used and the knockdown efficiency was validated by RT-PCR (Figs. 3B and 4B), the expression of each target gene was normalized to the negative control. Individual shRNA pools were then transduced into MEFs, and the GFP signal was clearly visible when treated for 7 days with Dox (Fig. 4C). These initial results were further validated by flow cytometry by quantifying the percentage of GFP⁺ cells. Although to various degrees, RB1, RBBP4, RBBP7 and CBX8 inhibition led to a significant increase in the proportion of GFP⁺ in MEFs (Fig. 4D, E). To determine if the finding that senescence impairs successful cardiac reprogramming is applicable in hFCFs, we specifically knocked down these genes in hFCFs with shRNA and observed that RB1, RBBP7, CBX8 and CDKN1B inhibition displayed a consistent increase in α-actinin⁺ cells proportion detected by FACS (Fig. 3C, D). Based on these results, both RB1 and RBBP7 inhibition seem to contribute to a significant increase in reprogramming efficiency of both MEFs and hFCFs. Since RB1 inhibition showed a consistent and more robust cardiac-inducing effect and has been also reported to facilitate iPSCs reprogramming [20], we selected RB1 as our target gene for subsequent follow-up study.

To further confirm that RB1 is responsible for the cellular senescence, we measured the expression level of senescence marker P16 and GLB1 with RT-PCR and performed β-Gal staining as well after RB1 silencing. We observed that RB1 inhibition led to a significant reduction in senescence, as indicated by β-Gal staining in MEFs (Fig. 5A) and hFCFs (Fig. 5C) during cardiac reprogramming and associated with a significant decrease in P16 and GLB1 expression in MEFs (Fig. 5B) and hFCFs (Fig. 5D). In order to explore how the absence of the RB1 protein might render MEFs more amenable to reprogramming, as RB1 is a transcriptional suppressor (Fig. 6A), we found that RB1 expression increased significantly during cardiac reprogramming (Fig. 6B) and also determined the consequence of RB1 loss on endogenous TF gene expression. The PiggyBac system drives exogenous GMT expression while endogenous genes are separately regulated. Interestingly, we observed a significant increase in endogenous GATA4 and MEF2C expression in RB1-deficient MEFs as determined by RT-PCR (Fig. 6C), whereas TBX5 expression is not affected.