FGF4 and Ascorbic acid enhance the maturation of induced cardiomyocytes by activating JAK2-STAT3 signaling

doi:10.21203/rs.3.rs-3787987/v1

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FGF4 and Ascorbic acid enhance the maturation of induced cardiomyocytes by activating JAK2-STAT3 signaling

https://doi.org/10.21203/rs.3.rs-3787987/v1

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published 01 Oct, 2024

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Direct cardiac reprogramming represents a novel therapeutic strategy to convert non-cardiac cells such as fibroblasts into cardiomyocytes (CMs). This process involves essential transcription factors - Mef2c, Gata4, and Tbx5 (MGT), MESP1 and MYOCD (MGTMM). However, the small molecules responsible for inducing immature induced CM (iCMs) and the signaling mechanisms driving their maturation remain elusive. Our study explored the effects of various small molecules on iCM induction and discovered that the combination of FGF4 and ascorbic acid (FA) enhances CM markers, exhibits organized sarcomere and T-tubule structures, and improves cardiac function. Transcriptome analysis emphasized the significance of ECM-integrins-focal adhesions and the upregulation of JAK2-STAT3 and TGFB signaling pathways in FA-treated iCMs. Notably, JAK2-STAT3 knockdown affected TGFB signaling, ECMs, and downregulated mature CM markers in FA-treated iCMs. Our findings underscore the critical role of the JAK2-STAT3 signaling pathway in directly reprogrammed CMs by activating TGFB signaling and ECM synthesis.

Biological sciences/Developmental biology/Transdifferentiation

Biological sciences/Developmental biology/Reprogramming

Biological sciences/Biotechnology/Gene delivery/Genetic vectors

Cardiovascular diseases (CVDs) are a significant and widespread health challenge contributing to elevated global mortality rates. CVDs encompass a range of conditions affecting heart and blood vessels, including coronary artery disease, heart failure, hypertension, and stroke. A major type of CVD is myocardial infarction, resulting in cardiomyocyte (CM) loss and increased cardiac fibrosis. Patients with CVDs typically exhibit CM death and dysfunction via activation of fibroblasts/myofibroblasts due to myocardial dilation and decreased cardiac function (1–3). Repair or regeneration of CMs is required for CVD recovery. However, human CMs have limited regenerative capacity. Although efforts to address recovery of fibrotic CMs have advanced cardiovascular disease research and treatment, it remains a formidable global health issue that requires continuous research, prevention, and intervention strategies (3, 4).

In severe cases, traditional therapies for CVDs rely on drugs, lifestyle changes, and heart transplantation. However, scarcity of donor hearts and limitations of current treatments have spurred research into alternative strategies (5). Therefore, only a few therapies are available for CVD regeneration or repair. Efforts to discover innovative technologies for heart regeneration, including pharmacological advancements and cell therapies, such as stem cell transplantation, are ongoing (6, 7). However, these efforts have shown limited effects, mainly because of the limited supply of cells for cardiac regeneration or repair in CVDs and challenges associated with cellular mutations. Direct cardiac reprogramming is a promising therapeutic approach; it offers a method to regenerate and repair cardiomyocytes by converting mature cells, such as fibroblasts, into induced CMs (iCMs) using transcription factors, microRNAs, and small molecules (8–12). The fundamental principle of direct cardiac reprogramming involves converting somatic cells, typically fibroblasts or other non-cardiac cells, into induced cardiomyocytes (iCMs) without an intermediate pluripotent state (13). This direct conversion is achieved through regulated manipulation of cellular factors and signaling pathways, and direct reprogramming presents advantage of reducing tissue damage while generating new cardiomyocytes.

Direct cardiac reprogramming involves overexpression of transcription factors that are upregulated in cardiomyocytes. The screening results for these transcription factors indicated that GATA4, MEF2C, and TBX5 (GMT) represented optimal conditions in mouse fibroblasts (14). Wang et al. reported that combination of MEF2C, GATA4, and TBX5 (MGT) induces higher proportions of iCMs and beating cells (15). In human fibroblasts, due to inefficiency of inducing iCMs by GMT, a combination of GMT with MESP1 and MYOCD (GMTMM) has been used, resulting in significant changes in cell morphology toward a rod-like or polygonal shape and enhanced production of cardiac-specific proteins (16). However, overexpression of these transcription factors results in iCMs with a low trans-differentiation effect and immature characteristics (17, 18).

Various small molecules, such as growth factors, hormones, chemical cues, and soluble factors, have been reported to induce differentiation of human pluripotent stem cells into mature CMs (19). Inhibition of Notch signaling using DAPT increased the number of sarcomere + and ca²⁺ flux + cells and beating iCMs (20). SB431542, a transforming growth factor-beta (TGFB) inhibitor, treatment also increased cardiac gene expression, organized sarcomere, and decreased proliferation during direct cardiac reprogramming (21). Fibroblast growth factor 10 (FGF10) and vascular endothelial growth factor (VEGF) enhance quality of cardiac reprogramming by increasing population of spontaneously beating iCMs (22). However, studies on small molecules that induce mature iCMs have not been reported. Therefore, it is necessary to investigate the small molecules related to efficient conversion and maturation of iCMs. Furthermore, genes and biological signaling pathways related to maturation of iCMs have recently been identified (23).

In this study, we screened various small molecules, including chemicals, cytokines, and growth factors, and discovered that FGF4 and ascorbic acid (FA) induce mature iCMs in direct cardiac reprogramming. FA treatment enhances expression of cardiac channels and cardiac metabolic markers. Moreover, we elucidated genes and signaling pathways regulating fibroblast-to-iCM conversion and iCM maturation using transcriptome analysis and gene knockdown studies. Our findings show that understanding direct cardiac reprogramming and its potential to transform fibroblasts into functional cardiomyocytes holds promise as a novel approach to tackling CVDs. This emerging field of research offers new avenues for developing regenerative therapies and treatments that can revolutionize treatment of cardiovascular diseases.

qRT-PCR

Total RNA was extracted from each group using the TRIzol reagent (TR-118; MRC Inc., Cincinnati, OH, USA) according to the manufacturer’s instructions. After adding chloroform (C2432, Sigma-Aldrich), the RNA-containing aqueous phase was transferred to a new tube, and isopropanol (2-propanol, 190764, Sigma-Aldrich) was used for RNA precipitation. Subsequently, RNA was rinsed with 75% ethanol and air-dried. RNA concentration and purity were measured using a Nanodrop spectrophotometer (ND-1000; Thermo Fisher Scientific). The mRNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (28025-013, Invitrogen, Carlsbad, CA, USA) at 37°C for 50 min in a 20 µL reaction. qRT-PCR was performed using the iQTM SYBR Green supermix (170–8880, Bio-Rad Laboratories, Hercules, CA, USA), and the results were recorded using the MYiQ2 Detection System (Bio-Rad Laboratories). Relative mRNA expression levels were calculated and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. The primer sequences used for qRT-PCR are listed in Table S3.

Immunofluorescence Staining

At week 4 of reprogramming, mouse or human iCMs were re-seeded onto gelatin-coated slides. The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 30 min. The slides were washed three times with PBS containing 0.1% Tween 20 (PBST) and blocked with 5% normal goat serum (NGS, Thermo Fisher Scientific) in PBST for 1 h at room temperature. The blocked slides were incubated at 4°C overnight with the following primary antibodies: anti-ACTN, anti-TNNI3, anti-TNNT2, and anti-JPH2 in 5% NGS in PBST. The slides were then washed three times with PBST and incubated for 2 h at room temperature with the following secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 647 goat anti-mouse IgG, and Alexa Fluor 555 goat anti-rabbit IgG. Nuclei were stained with 1 µg/mL of DAPI. The slides were then mounted with a fluorescent mounting medium (S3023, DAKO, Glostrup, Denmark). Immunofluorescence images were acquired using a fluorescence microscope and confocal fluorescence microscope (LSM900 and LSM800; Carl Zeiss, Oberkochen, Germany). Primary and secondary antibodies are listed in Table S4.

Flow cytometry

Single cells were isolated using T/E buffer, fixed with 4% PFA in PBS for 20 min, and washed three times with PBS containing 2% FBS. The cells were then permeabilized with 100% ice-cold methanol for 20 min on ice. The cells were incubated for 45 min at RT with the primary antibody against TNNT2 (1:200; MA5-12960, Thermo Fisher Scientific). The cells were then washed thrice with PBS containing 2% FBS, followed by incubation with the corresponding secondary antibody, goat anti-mouse IgG Alexa 647 (1:1000; A32728, Thermo Fisher Scientific), for 20 min. The cells were acquired using a BD FACSCanto™ II flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Western Blot analysis

Cells were washed with PBS and lysed with ice-cold 1 × cell lysis buffer (9803; Cell Signaling Technology, Danvers, MA, USA) containing 1 mM phenylmethylsulfonyl fluoride (P7626; Sigma-Aldrich). Protein concentrations were quantified using the Bradford assay dye reagent (500-0006, Bio-Rad Laboratories). Protein Samples (15–20 µg) mixed with 1x loading dye were boiled for 8 min. The proteins were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (10600023; Thermo Fisher Scientific). The membranes were blocked using 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. The membranes were incubated overnight at 4°C with the primary antibodies. Primary antibodies used for western blot analysis are listed in Table S4. The membranes were washed thrice with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody in TBST at room temperature for 1 h. The bands were visualized using ECL (32106, Thermo Fisher Scientific), ECL Plus reagents (32132, Thermo Fisher Scientific) and West Femto reagent (34095, Thermo Scientific), and exposed to the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories) using an enhanced chemiluminescence detection system. Signal intensity was analyzed using Image Lab Software (Bio-Rad Laboratory).

Transient Ca2 + analysis

For transient Ca2 + analysis, directly reprogrammed cells were re-seeded in a confocal dish (213350; SPL, Seoul, Korea) at a density of 1.5 x 10⁴ cells per dish. Ca2 + imaging was performed according to the manufacturer’s instructions. Briefly, cells were loaded for 30 min with 5 µM Fluo-4 AM (F14201, Invitrogen) in HBSS medium at 37°C. Nuclei were stained with 1 µg/mL of Hoechst 33342. Following the staining process, iCMs were washed with PBS and incubated in fresh medium for an additional 30 min to de-esterify the fluorescence. Ca2 + imaging was performed using a confocal fluorescence microscope (LSM800, Carl Zeiss).

MitoTracker

Active mitochondria in iCMs were labeled using a standard experimental protocol with MitoTracker (Red CM-H₂XRos, M7513, Thermo Fisher Scientific). Concisely, iCMs at week 4 were re-seeded onto a confocal dish (213350, SPL) with a density of 1.5 × 10⁴ cells per dish and then incubated with 300nM MitoTracker probe in DMSO for 30 min at 37°C. Nuclei were stained with 1 µg/mL of Hoechst 33342. After staining live cells with MitoTracker, the iCMs were washed with PBS and replaced with fresh, pre-warmed media. The cells were analyzed using a confocal fluorescence microscope (LSM800, Carl Zeiss).

RNA-seq analysis

Vehicle, MGTMM, and MGTMM + FA samples were collected at week 4 of direct reprogramming for RNA-seq analysis. Total RNA was extracted using TRIzol reagent following the manufacturer’s instructions, and RNA concentration and quality were assessed using a NanoDrop spectrophotometer and an Agilent 2100 bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands). Subsequently, libraries for RNA-seq analysis were prepared using the QuantSeq Library Prep Kit (Lexogen Inc., Vienna, Austria). High-throughput sequencing was performed using a NextSeq 500 (Illumina, Inc., San Diego, CA, USA). We compared the whole transcriptomes and identified differentially expressed genes (DEGs) that exhibited more than 2-fold change in expression. DEGs were analyzed using ExDEGA software (EBIOGEN, Inc., Seoul, Korea). For gene classification, searches were performed using DAVID (http://david.abcc.ncifcrf.gov/), Gene Ontology (http://www.ebi.ac.uk/QuickGO/), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.jp/kegg/tool/map_pathway2.html), and STRING (http://www.string-db.org/).

Statistical Analyses

All studies were performed using al least three independent experiment sets and presented as mean ± Standard deviation. Statistical analysis was performed using the Student’s t-test for two-group comparisons and ANOVA for multiple group assessments to determine the significance of the differences. Data were analyzed using GraphPad Prism version 8.0.2. (GraphPad Software, Inc., San Diego, CA, USA).

Screening for maturation factors enhances conversion of mature cardiomyocte

To obtain mature CMs from mouse embryonic fibroblasts (MEFs) through direct cardiac reprogramming, we first optimized protocol by overexpressing three cardiac transcription factors (Mef2c, Gata4, and Tbx5; MGT) and screened various combinations of growth factors (24). MEFs were infected with MGT genes encoding retrovirus and transferred into iCM medium containing puromycin to select infected cells (Fig. 1a). After two weeks, we attempted to differentiate them into mature CMs by adding the test factors in maturation medium (StemPro-34) for an additional two weeks (22) (Fig. 1a).

Direct cardiac reprogramming is regulated by epigenetic modifications such as histone H3 Lysine residue position 4 (H3K4me3) and histone H3 Lysine 27 (H3K27me3). H3K27me3 silences fibroblast-specific genes and induces expression of cardiac-specific genes, whereas H3K4me3 activates cardiac-specific genes during direct mouse cardiac reprogramming (25). To examine remodeling of chromatin architecture, we analyzed levels of H3K27me3 and H3K4me3 by immunofluorescence staining at week 1. H3K27me3 was weakly detected, and H3K4me3 was strongly expressed in MGT compared to vehicle (Fig. 1b and 1c).

To investigate combination of maturation factors that promote functional iCMs, we used various small molecules, including FGF2, FGF4, FGF10, ascorbic acid (AA), VEGF, TGFb1, and 1-Thioglycerol (1-TG). We performed qRT-PCR for total CM marker (Tnnt2), atrial CM marker (Myl7), ventricular CM marker (Myl2), and t-tubule marker (Jph2) to determine whether maturation factors induced iCM maturation (Fig. 1d). We used FFV growth factors (FGF2, FGF10, and VEGF) as positive controls (22). Combination of FGF4 + AA (FA) and TGFb + AA (TbA) significantly increased levels of Tnnt2, Myl7, and Jph2 compared to MGT. Myl2 was higher in MGT + FA only than in MGT. The positive control, MGT + FFV, showed decreased levels of Myl7 compared to MGT. To enhance reprogramming efficiency, we explored best combination, FA, with/without small molecules such as TGFb1 and 1-TG (Fig. S1). Tnnt2, Myl7, and Jph2 levels were increased in MGT + FA, MGT + FATb, and MGT + FATG compared to MGT. Interestingly, Myl2 was significantly increased only in MGT + FA compared to that in MGT. Overall, we determined that combination of FGF4 and AA enhanced efficiency of direct cardiac reprogramming using MEFs.

Next, we investigated optimal timing for reprogramming MEFs into mature iCMs with FA by changing the timing of FA treatment to weeks 1–2, 2–4, and 1–4, sequentially, after MGT transfection. Expression of Tnnt2 showed a 4-fold increase after FA treatment between 2–4 weeks compared with MGT, although FA treatment for the other periods only slightly affected direct reprogramming (Fig. S2a). qRT-PCR analysis revealed that cardiac-specific maturation markers Mly7, Myl2, Cav3 and Jph2 were significantly increased after 2–4 weeks compared to MGT. These results suggest that FA enhances maturation of iCMs between–2–4 weeks of reprogramming and plays a crucial role in reprogramming MEFs into mature iCMs.

We also investigated proportion of iCMs in vehicle, MGT, and MGT + FA by flow cytometry at week 4. Flow cytometry analysis revealed that percentage of Tnnt2 + iCMs increased to approximately 8.3% in MGT and 17.4% in MGT + FA, compared to vehicle (2.8%) (Fig. 1e). Subsequently, we assessed expression of Tnnt2, Myl7, Myl2, and Jph2 using western blotting (Fig. 1f). Levels of Tnnt2, Myl7, Myl2, and Jph2 were higher in MGT than in vehicle and were further enhanced by FA treatment. Immunofluorescence staining was performed to examine morphology and cardiac structure using a Tnnt2 and a mature CM marker, Tnni3 (Fig. 1g and 1i). Tnnt2 and Tnni3 were expressed in MGT and MGT + FA but not in vehicle. Tnnt2 + and Tnni3 + iCMs in MGT + FA were larger and showed stronger expression than those in MGT. The percentage of Tnnt2 positive cells was approximately 30% in MGT + FA and 20% in MGT (Fig. 1h).

To examine conversion of fibroblasts into structural-matured iCMs, we stained a cardiac sarcomere marker, Actn, and Jph2 in MGT and MGT + FA. Actn and Jph2 were co-expressed in MGT + FA, and their sarcomeres and T-tubules were regularly arranged compared to MGT (Fig. 1j). Percentage of Tnnt2/Jph2-double positive cells was approximately 1.3 times higher in MGT + FA than in MGT (data not shown). These results indicate that combination of FGF4 and AA induces formation of structurally mature iCMs from immature MEFs.

FA treated iCMs show elevated expression of ion channels genes

Cardiac functions, including rhythmic contraction, electrical activity, and ion channels, such as sodium, potassium, and Ca2 + channels, generate and regulate electrical impulses that drive heartbeat (26, 27). To evaluate effect of FA on generation of functionally mature iCMs from fibroblasts, we analyzed expression of L-type Ca2 + channel markers (ryanodine receptor 2; Ryr2), Ca2 + ATPase markers (Ca2+-ATPase; Atp2b1 and Phospholamban; Pln), plasma membrane Ca2 + pump (Atp2b1), and Na+-Ca2 + exchanger marker (Ncx1) using qRT-PCR (Fig. 2a and 2b). Expression levels of Ryr2, Atp2b1, and Pln were significantly higher in MGT + FA than in MGT (Fig. 2a). mRNA levels of Atp2b1 and Ncx1 also increased approximately 2-fold in MGT + FA compared to MGT (Fig. 2b). Furthermore, we employed Flou-4 AM staining to analyze intracellular Ca2 + flux in MGT and MGT + FA at week 4. A bright green fluorescence is observed in presence of high and low intracellular Ca2 + levels. To quantify Ca2 + level of MGT + FA compared to MGT, we determined fluorescence intensity; treatment with FA triggered maximal Ca2 + oscillation, lasting nearly 1 min, whereas rare Ca2 + oscillations were found in MGT (Fig. 2c).

In mature ventricular CMs, potassium channels play an important role in repolarization phase of action potentials (28). qRT-PCR analysis revealed that potassium channel markers Kcnj2, potassium inwardly rectifying channel; and Kcnh2, potassium voltage-gated channel were significantly upregulated in MGT + FA compared to MGT (Fig. 2d). These findings suggested that combination of FGF4 and AA enhanced cardiac function by promoting cardiac ion channel function during direct cardiac reprogramming of MEFs.

FA increases mitochondrial biogenesis, ROS production and activates mitochondrial Ca2+

In CMs, mitochondria perform major biological processes, including metabolic regulation, Ca2 + handling, and redox generation. To explore effect of FA treatment on mitochondrial biogenesis (29), we evaluated expression of mitochondrial biogenesis markers (Tfam1, Ppargc1a, Nfe2l1, and Nfe2l2) using qRT-PCR. Tfam1, Ppargc1a, Nfe2l1 and Nfe2l2 were elevated in MGT + FA compared to MGT. (Fig. 2e). Furthermore, mRNA levels of mitochondrial metabolism and ROS markers (Sirt1 and Sirt3) were significantly increased in MGT + FA than in MGT (Fig. 2g). We further examined protein levels of mitochondrial biogenesis markers by western blotting. Ppargc1a was more strongly detected in MGT + FA than in MGT but not Tfam (Fig. 2f). Ca2 + regulates mitochondrial function in energy production for cardiac activity (30, 31). Therefore, to evaluate function of cardiac contraction through Ca2 + regulation in mitochondria, we examined mitochondrial Ca2 + uniporter channel markers (Mcu, Micu1, and Micu2) using qRT-PCR. Mcu, Micu1, and Micu2 were upregulated in MGT + FA than MGT (Fig. 2h). In addition, we used Ca2 + transient markers (Fluo-4AM) and mitochondrial markers (MitoTracker) to evaluate mitochondrial structure and function in Ca2 + uptake. The Mitochondria in immature CMs are small and widely distributed in cytoplasm and are present in perinuclear region, whereas mature CMs exhibit larger mitochondria that are well-organized, primarily within intermyofibrillar or subsarcolemmal region (32, 33). Our results showed that treating iCMs with FA can increase mitochondrial content and arrange cristae. Furthermore, upon merging the two indicators (Fluo-4AM and MitoTracker), a distinct overlap appeared in yellow in MGT + FA, while there was no alignment between distribution of Fluo-4AM and MitoTracker in MGT. This result indicates that mitochondrial Ca2 + function was observed in mature cardiomyocytes (white arrow in Fig. 2i). These results demonstrate that overexpression of MGT transcription factor can induce differentiation of fibroblasts into CMs through cell conversion and that treatment with FA can induce high maturation and conversion rates in iCMs.

FA enhances transdifferentiation efficiency in adult mouse cardiac fibroblasts

Transfection of MGT into MEFs has been demonstrated to directly reprogram fibroblasts into iCMs without regression to a stem/progenitor state. According to other studies, efficiency of cardiac reprogramming in adult cardiac fibroblasts (MCFs) is lower than in MEFs (17, 34). Thus, we tested effect of FA on MCFs to determine whether it directly converts fibroblasts into CM-like cells. The schematic representation of experimental design is shown in Fig. 3a. To investigate chromatin architecture remodeling, we conducted immunofluorescence staining to examine levels of H3K27me3 and H3K4me3 at week 1. In MGT, we observed weak H3K27me3 expression and strong H3K4me3 expression compared to vehicle (Fig. 3b and 3c). We performed qRT-PCR to evaluate mRNA expression of cardiac-related markers, including Tnnt2, Myl7, Myl2 and Jph2. These results demonstrated that FA treatment upregulated Tnnt2, Myl7, and Jph2 compared to MGT but slightly affect Myl2 (Fig. 3d). Next, we confirmed that FA improved structural maturation of CMs by immunofluorescence staining with cardiac marker Tnnt2. MGT + FA demonstrated a higher quantity of MGT-infected cells stained with Tnnt2, unlike MGT, where such expression was not observed, as seen in vehicle (Fig. 3e). To determine whether iCMs exhibit functional characteristics typical of CMs, we analyzed Ryr2 and Kcnh2, along with genes Ppargc1a and Tfam, using qRT-PCR. mRNA expression of the genes significantly increased in MGT + FA but Kcnh2 was not significant (Fig. 3f). Finally, we used Fluo-4AM marker and mitochondrial marker MitoTracker to detect presence of mitochondrial Ca2+ (yellow; white arrow in Fig. 3h). We quantified expression of mitochondrial Ca2 + when it peaked maximally and found a significant increase in MGT + FA compared to MGT (Fig. 3g). These results indicate that combination of FA improves direct cardiac reprogramming efficiency and successfully converts fibroblasts into functional CMs, even in adult cardiac fibroblasts, known for their lower reprogramming efficiency.

FA enhances human fibroblasts to iCMs cardiac reprogramming efficiency

To investigate effects of FA on cardiac induction in human fibroblasts, we optimized a direct cardiac reprogramming protocol using transcription factors and FA. In humans, MGT, MESP1, and MYOCD (MGTMM) enhance efficiency of iCM reprogramming, promoting conversion of fibroblasts into iCMs (15, 16). We transduced Normal human dermal fibroblasts (NHDFs) with three lentiviruses encoding five-core cardiac-specific factors (MGTMM; Fig. 4a and Fig. S3a-c). We first investigated efficiency of MGT, MESP1, and MYOCD vectors by qRT-PCR in 293T cells (Fig. S3d). MEF2C, GATA4, and TBX5 were highly expressed in MGT vector-transfected 293T cells, MESP1 was significantly upregulated in MESP1 vector-transfected 293T cells, and MYOCD was upregulated in MYOCD vector-transfected 293T cells. Furthermore, we treated cells with FA at week 2 and analyzed them at week 4 (Fig. 4a). Distinct morphological changes were observed in each group, and levels of GATA4, MEF2C, TBX5, MESP1, and MYOCD were significantly increased in MGTMM and MGTMM + FA compared to vehicle (Fig. S4).

To evaluate effect of MGTMM overexpression on reprogramming, we examined expression levels of H3K4me3 using immunofluorescence staining on day 7 (Fig. 4b). H3K4me3 was more strongly expressed in MGTMM than in vehicle. Percentage of H3K4me3 area relative to total nuclei decreased in MGTMM compared to that in vehicle (Fig. 4c). Protein level of H3K4me3 was also higher in MGTMM than in vehicle (Fig. 4d).

We examined effects of FA combination on expression levels of TNNT2, MYL7 and MYL2 using qRT-PCR and western blot analyses (Fig. 4e and 4f). mRNA levels of TNNT2, MYL7, and MYL2 were significantly higher in MGTMM + FA than in MGTMM (Fig. 4e). Protein levels of TNNT2, MYL7, and MYL2 were also higher in MGTMM + FA than in MGTMM (Fig. 4f). Interestingly, nodal CM marker TBX18 revealed no significant differences between vehicle, MGTMM, and MGTMM + FA (Fig. S5a). Next, we investigated proportions of TNNT2 + iCMs in vehicle, MGTMM, and MGTMM + FA by immunofluorescence staining (Fig. 4g). Large TNNT2 + iCMs (5.6%) and long sarcomere structures (1.2 µm) were more frequently observed in MGTMM + FA compared to MGTMM (5.6% and 1.7 µm, respectively; Fig. 4h and 4i). We further assessed different cardiac markers (TNNI3, MYH6, and MYH7) and T-tubule markers (BIN1, CAV3, and JPH2) using qRT-PCR (Fig. S5b and S5c). TNNI3, MYH6, MYH7, BIN1, CAV3, and JHP2 mRNA expression was markedly increased in MGTMM + FA compared to MGTMM. A higher expression of CAV3 was also observed in MGTMM + FA than in MGTMM by western blotting (Fig. S5d). To investigate ultrastructural array between T-tubules and sarcomeres, JPH2 and TNNT2 were examined by immunofluorescence staining. Cross structures of T-tubules, Z-lines, and co-localization areas of JPH2 and TNNT2 were more frequently observed in MGTMM + FA than in MGTMM (Fig. S5e). These results demonstrate that FA treatment enhanced cardiac gene expression at reprogramming stage, indicating potential of FA to enhance efficiency of direct cardiac reprogramming of human fibroblasts.

FA improves iCM functions by enhancing Ca²⁺ channel and metabolism

We investigated whether FA improves cardiac function upon direct human cardiac reprogramming. To assess cardiac function, we analyzed expression levels of ion channels using qRT-PCR (Fig. 5a and 5b). Ca2 + channel markers (CACNA1C and RYR2) exhibited increased expression in MGTMM + FA compared to MGTMM (Fig. 5a). However, potassium channel markers (KCNH2 and KCNJ2) were not significantly different between two groups (Fig. 5b). Within CMs, mitochondria play a crucial role by producing over 90% of ATP required for cardiac function (35). Consequently, we investigated expression levels of mitochondrial biogenesis markers (TFAM, PPARGC1A, and SIRT1) by qRT-PCR (Fig. 5c). TFAM, PPARGC1A, and SIRT1 were upregulated in MGTMM + FA compared to MGTMM. Protein levels of TFAM and PPARGC1A were elevated in MGTMM compared to vehicle, and further elevated in MGTMM + FA (Fig. 5d). To quantify mitochondrial content in each group, we analyzed ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) by qRT-PCR. We found a significant increase in mtDNA/nDNA ratio in MGTMM + FA compared to MGTMM (Fig. 5e).

Ca2 + are crucial for CM function, and dynamics of mitochondrial Ca2 + cycling and storage during excitation-contraction coupling (ECC) are strongly linked to cardiac physiology and pathophysiology (36–38). Mitochondrial Ca2 + uptake is crucial in regulating cellular metabolism and provides the necessary energy for contraction (39–42). To analyze relationship between mitochondria and Ca2 + handling, we investigated MitoTracker and Fluo-4 AM using immunofluorescence staining (Fig. 5f and 5g). Co-localized MitoTracker and Fluo-4 AM signals were increased in MGTMM + FA compared with MGTMM.

Mature CMs are characterized by fatty acid metabolism, oxidative phosphorylation, and mitochondrial biogenesis. FA treatment increased mRNA level of CPT1B, CD36, and PPARA and protein level of CPT1B (Fig. 5h and 5i). These findings indicate that FA enhanced cardiac function by improving ion channel function, mitochondrial biogenesis, and metabolism during cardiac reprogramming.

Transcriptome analysis exhibits MGTMM + FA impact on cardiac reprogramming pathway

To understand genes and signaling pathways involved in maturation resulting from co-treatment with FGF4 and ascorbic acid, we analyzed transcriptome of vehicle, MGTMM, and MGTMM + FA. Among 25,737 genes, 358 genes in MGTMM compared to vehicle, 747 in MGTMM + FA compared to vehicle, and 757 in MGTMM + FA compared to MGTMM were upregulated (fold changes > 2; Fig. 6a). In contrast, 497 genes in MGTMM compared to vehicle, 921 in MGTMM + FA compared to vehicle, and 882 in MGTMM + FA compared to MGTMM were downregulated (fold changes > 2). Scatter plots highlighting upregulated cardiac-specific genes (TNNT2, CAV3, JPH2, CACNA1A, CACNA1C, and GJA1) are shown in Fig. 6b. Heatmap analysis further revealed upregulation of cardiac-specific genes in MGTMM + FA compared to MGTMM (Fig. 6c). To elucidate biological processes regulated by differentially expressed genes (DEG), we performed gene ontology (GO; Fig. 6d) and also analyzed Kyoto Encyclopedia of genes of genomes (KEGG) pathway databases to identify active signaling pathway involved in reprogramming processes (Fig. S6a). The most significantly regulated GO biological processes and KEGG pathway associated with these processes are shown in Table S1 and S2.

RNA-seq analysis reveals potential of JAK-STAT3 signaling in iCM maturation

To explore common signaling pathways involved in reprogramming induced by MGTMM and FA treatment, we performed KEGG pathway analysis and examined genes involved (Table S1 and S2). Results revealed that MGTMM upregulated MAPK, PI3K-AKT, VEGF, Ca2+, and p53 signaling pathway compared to vehicle. Furthermore, MGTMM + FA upregulated JAK-STAT, TGFB, and Toll-like receptor signaling pathways.

To validate these findings, we investigated ECM-receptor interaction and focal adhesions (Fig. S6b-d). COL1A1 and Laminin in ECMs, ITGAV, ITGA7, and ITGB3 in integrins were increased in MGTMM and MGTMM + FA compared to vehicle (Fig. S6b and S6c). However, FN1 was decreased in MGTMM and MGTMM + FA compared with vehicle. ROCK1, ROCK2, TLN1, and DES in focal adhesions were highly expressed in MGTMM and MGTMM + FA compared to vehicle, whereas VCL and TLN2 showed no significant differences between vehicle, MGTMM, and MGTMM + FA (Fig. S6d). These results suggest that MGTMM induces cardiac reprogramming by activating VEGF, ECM proteins, integrins, focal adhesions, and Ca2 + signaling.

To examine signaling pathways related to maturation of iCMs following FA treatment, we analyzed JAK-STAT and TGFB signaling pathways (Fig. 6f-j). TGFB1, TGFB2, and GDF5 were elevated in MGTMM + FA compared to MGTMM (Fig. 6f and 6h). Expression levels of BMP2 and BMP4 were not significantly different between groups. Therefore, we further analyzed downstream signaling of TGFB1 and 2, SMAD2 and SMAD3, and downstream signaling of GDF5, SMAD1/5 (Fig. 6g and 6i). The results revealed an increase in p-SMAD3 in MGTMM + FA compared to that in MGTMM, while p-SMAD1/5 showed no significant difference. Additionally, p-JAK2, p-STAT3, and p21 in JAK-STAT3 levels were increased in MGTMM + FA compared to MGTMM, whereas p-cJun showed no significant difference between groups (Fig. 6j). These findings indicate that FA contributes to maturation of iCMs by influencing JAK-STAT and TGFB signaling cascades.

JAK2-STAT3 signaling pathway modulates iCM maturation upon treatment of FA

Additionally, owing to the remarkable activation of JAK2-STAT3 signaling pathway in MGTMM + FA, which exhibited both structural and functional maturation compared to MGTMM, we further investigated the roles of JAK2-STAT3 signaling pathway in iCMs maturation. We treated MGTMM and MGTMM + FA cells with cryptotanshinone, a JAK2-STAT3 inhibitor, from week 2 to week 4 and performed qRT-PCR and western blot analyses (Fig. 7a). mRNA expression levels of FGFR1 and FGFR2 were significantly higher in MGTMM + FA and MGTMM + FA + inh than in MGTMM and MGTMM + inh. These results indicated that FGF signaling pathway was activated in FA-treated iCMs. To analyze effect of inhibitor, cryptotanshinone, we investigated JAK2-STAT3 signaling pathway using western blot analysis (Fig. 7b). Protein levels of p-JAK2, JAK2, p-STAT3, and STAT3 were lower in MGTMM + inh and MGTMM + FA + inh than in MGTMM and MGTMM + FA. These results demonstrated that cryptotansbinone inhibited JAK2-STAT3 signaling pathway.

JAK2-STAT3 signaling pathway promotes transcription of TGFB, collagen, and MYH7. Next, to investigate whether TGFB signaling was affected during iCM maturation, we analyzed markers of TGFB signaling pathway by qRT-PCR (Fig. 7c). TGFB1, TGFB2, TGFBR1, and TGFBR2 were significantly decreased in MGTMM + inh compared with MGTMM + FA, and there was no significant difference in MGTMM + Inh compared with MGTMM. TGFB2, TGFBR1, and TGFBR2 were decreased in MGTMM + FA + Inh compared to MGTMM + FA, but TGFB1 was not significantly different between MGTMM + FA and MGTMM + FA + Inh. We also observed enhanced phosphorylation of SMAD3 in MGTMM + FA compared to that in MGTMM and decreased phosphorylation in MGTMM + Inh and MGTMM + FA + Inh compared to that in MGTMM + FA (Fig. 7d). To examine regulation of collagen by JAK2-STAT3 inhibition, we performed western blotting for ECM markers (Laminin, COL1A1, and FN1) (Fig. 7e). Laminin and COL1A1 were decreased in MGTMM + FA + Inh compared to MGTMM + FA, whereas FN1 was not significantly different between MGTMM + FA and MGTMM + FA + Inh. These findings demonstrated that during direct cardiac reprogramming, JAK2-STAT3 signaling pathway regulates TGFB2, TGFBR1, TGFBR2, Laminin, and COL1A1.

To examine whether JAK2-STAT3 signaling pathway induces cardiac differentiation in FA-treated iCMs, we analyzed cardiac markers (MYH7, TNNT2, and MYL2) using qRT-PCR (Fig. 7f). MYH7, TNNT2, and MYL2 levels were lower in MGTMM + Inh mice than in MGTMM + FA mice. TNNT2 levels were not significantly different between MGTMM + FA and MGTMM + FA + Inh, whereas MYH7 and MYL2 were lower. Protein levels of TNNT2, MYL7, and MYL2 were also decreased in MGTMM + Inh and MGTMM + FA + Inh compared to those in MGTMM + FA (Fig. 7g). To investigate induction of cardiac maturation via JAK2-STAT3 signaling, we examined T-tubule markers (CAV3 and JPH2) and mitochondrial biogenesis markers (PPARGC1A and TFAM) using qRT-PCR (Fig. 7h and 7i). mRNA levels of CAV3, JPH2, PPARGC1A, and TFAM were significantly decreased in MGTMM + Inh and MGTMM + FA + Inh compared to MGTMM + FA; protein expression of JPH2 and PPARGC1A was also (Fig. 7j). Collectively, these results demonstrate that JAK2-STAT3 signaling pathway contributes to maturation of iCMs by regulating TGFB (TGFB2, TGFBR1, and TGFBR2) signaling pathway.

Our findings suggest that combination of FGF4 and ascorbic acid can effectively promote maturation and direct reprogramming of fibroblasts into cardiomyocytes via three (in mice) or five (in humans) cardiac transcription factors: MGT in mice and MGTMM in humans. FGF4 and ascorbic acid synergistically enhance expression of these factors, resulting in significant improvements in cardiac function. Specifically, during direct cardiac reprogramming, this combination facilitates critical processes, including maturation of cardiomyocytes, formation of T-tubule structures, regulation of Ca2 + exchange dynamics, and induction of mitochondrial biogenesis.

Moreover, integration of small molecules has demonstrated potential to enhance reprogramming in conjunction with transcription factors, thereby playing a pivotal role in promoting cardiomyocyte maturation during cardiac differentiation (11). An encouraging strategy for enhancing efficiency, quality, and speed of direct reprogramming involves simultaneous suppression of TGFB and WNT signaling pathways (43). Furthermore, targeting signaling pathways, such as ROCK, NOTCH, C-C chemokines, p38 mitogen-activated protein kinase, and PI3K/AKT pathway, has been identified as a means of amplifying reprogramming efficiency (20, 22, 43–46). A noteworthy discovery was effectiveness of FFV in overcoming obstacles encountered in later stages of cardiac reprogramming (22). Our findings highlight that combination of FA results in a significant population of iCMs and a substantial induction of mature iCMs.

Transcriptional profiling data indicate upregulation of pathways such as MAPK, PI3K-AKT, VEGF, Ca2+, and NF-κB signaling, ECM-receptor interaction, Focal adhesion, HIF1 signaling, and p53 signaling in MGTMM compared with vehicle. Furthermore, our results demonstrate that MGTMM induces transcription of cardiac genes, ECMs, integrins, focal adhesions, and Ca2 + channels during direct cardiac reprogramming of human fibroblasts. ECMs interact with integrins, initiating downstream signaling pathways such as focal adhesions and actin-cytoskeletal regeneration. Our results revealed increased ROCK1, ROCK2, TLN1, and DES levels in MGTMM compared to those in vehicle. These results indicate that MGTMM promotes transcription of cardiac genes, ECMs, and Ca2 + channels, followed by activation of focal adhesion and actin cytoskeleton markers during transdifferentiation of fibroblasts into cardiomyocytes.

Our research further underscores pivotal role of JAK2-STAT3 signaling pathway in cardiac maturation. JAK2-STAT3 pathway, activated by FGF signaling, is critical in various physiological processes, including immune responses, cell division, cell death, and tumorigenesis (47). Within heart, JAK2-STAT3 pathway is essential for maintaining cardiac homeostasis and responds to various cytokines, such as IL6, granulocyte colony-stimulating factor, leptin, and erythropoietin (48). Transcriptional profiling of our experimental data revealed a significant upregulation of genes associated with JAK2-STAT3 signaling pathway in MGTMM + FA compared to MGTMM. Based on this observation, we conclude that JAK2-STAT3 signaling pathway is crucial for maturation of cardiomyocytes activated by combination of FA during direct reprogramming.

In cardiac hypertrophy and fibrotic response, JAK2-STAT3 signaling pathway activates TGFB, COL1A1, and MYH7 transcription, contributing to cardiac remodeling and dysfunction (49). Our results show no significant difference in TGFB signaling pathways, such as TGFB1, TGFBR1, and TGFBR2, between MGTMM + FA and MGTMM + FA + Inh. However, TGFB2, TGFBR2, and TGFB2-downstream signaling (p-SMAD3) are reduced in MGTMM + FA + Inh compared with MGTMM + FA. These findings suggest that TGFB2-TGFBR2-p-SMAD3 activation occurs via JAK2-STAT3 signaling pathway during FA-mediated direct cardiac reprogramming. During heart development, TGFB plays critical roles in populating embryonic heart with cardiomyocytes (50). TGFB signaling pathway triggers expression of cardiogenic markers in fibroblasts and promotes maturation of cardiomyocytes (51). In present study, cardiac genes, T-tubule markers, and mitochondrial biogenesis markers were downregulated in MGTMM + FA + Inh compared with MGTMM + FA. These findings demonstrate that TGFB2-TGFBR2 signaling pathway promotes cardiomyocyte maturation through FA via JAK2-STAT3 signaling pathway. Thus, generating iCMs using FA could be pivotal in treating CVDs.

ACKNOWLEDGEMENTS

This research was supported by Energy Cloud R&D Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (2019R1A2C2087198).

S.M.J., M.H.S., S.C.C., and D.S.L. designed the experiments. S.M.J. and M.H.S. performed experiments. D.E.Y. and K.M.K. designed the human vectors. S.M.J., M.H.S., J.M.N., and K.S.K analyzed RNA-seq data. S.C.C., J.H.P., K.M.K., and D.S.L. contributed scientific discussion. S.M.J. and M.H.S. analyzed data and prepared the paper.

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

Supplementary information accompanies the manuscript on the Experimental & Molecular Medicine` website (http://www.nature.com/emm/)

Correspondence and requests for materials should be addressed to Do-Sun Lim

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FGF4 and Ascorbic acid enhance the maturation of induced cardiomyocytes by activating JAK2-STAT3 signaling

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

qRT-PCR

Immunofluorescence Staining

Flow cytometry

Western Blot analysis

Transient Ca2 + analysis

MitoTracker

RNA-seq analysis

Statistical Analyses

RESULTS

Screening for maturation factors enhances conversion of mature cardiomyocte

FA treated iCMs show elevated expression of ion channels genes

FA increases mitochondrial biogenesis, ROS production and activates mitochondrial Ca2+

FA enhances transdifferentiation efficiency in adult mouse cardiac fibroblasts

FA enhances human fibroblasts to iCMs cardiac reprogramming efficiency

FA improves iCM functions by enhancing Ca²⁺ channel and metabolism

Transcriptome analysis exhibits MGTMM + FA impact on cardiac reprogramming pathway

RNA-seq analysis reveals potential of JAK-STAT3 signaling in iCM maturation

JAK2-STAT3 signaling pathway modulates iCM maturation upon treatment of FA

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

FGF4 and Ascorbic acid enhance the maturation of induced cardiomyocytes by activating JAK2-STAT3 signaling

Status:

Journal Publication

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

qRT-PCR

Immunofluorescence Staining

Flow cytometry

Western Blot analysis

Transient Ca2 + analysis

MitoTracker

RNA-seq analysis

Statistical Analyses

RESULTS

Screening for maturation factors enhances conversion of mature cardiomyocte

FA treated iCMs show elevated expression of ion channels genes

FA increases mitochondrial biogenesis, ROS production and activates mitochondrial Ca2+

FA enhances transdifferentiation efficiency in adult mouse cardiac fibroblasts

FA enhances human fibroblasts to iCMs cardiac reprogramming efficiency

FA improves iCM functions by enhancing Ca2+ channel and metabolism

Transcriptome analysis exhibits MGTMM + FA impact on cardiac reprogramming pathway

RNA-seq analysis reveals potential of JAK-STAT3 signaling in iCM maturation

JAK2-STAT3 signaling pathway modulates iCM maturation upon treatment of FA

DISCUSSION

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

FA improves iCM functions by enhancing Ca²⁺ channel and metabolism