S-nitrosoglutathione reductase (GSNOR) deficiency accelerates cardiomyocyte differentiation of induced pluripotent stem cells

Introduction: Induced pluripotent stem cells (iPSCs) provide a model of cardiomyocyte (CM) maturation. Nitric oxide signaling promotes CM differentiation and maturation, although the mechanisms remain controversial. Aim: The study tested the hypothesis that in the absence of S-nitrosoglutathione reductase (GSNOR), a denitrosylase regulating protein S-nitrosylation, the resultant increased S-nitrosylation accelerates the differentiation and maturation of iPSC-derived cardiomyocytes (CMs). Methods and Results: iPSCs derived from mice lacking GSNOR (iPSC) matured faster than wildtype iPSCs (iPSC) and demonstrated transient increases in expression of murine Snail Family Transcriptional Repressor 1 gene (Snail), murine Snail Family Transcriptional Repressor 2 gene (Slug) and murine Twist Family BHLH Transcription Factor 1 gene (Twist), transcription factors that promote epithelial-to-mesenchymal transition (EMT) and that are regulated by Glycogen Synthase Kinase 3 Beta (GSK3β). Murine Glycogen Synthase Kinase 3 Beta (Gsk3β) gene exhibited much greater S-nitrosylation, but lower expression in iPSC. S-nitrosoglutathione (GSNO)-treated iPSC and human (h)iPSCs also demonstrated reduced expression of GSK3β. Nkx2.5 expression, a CM marker, was increased in iPSC upon directed differentiation toward CMs on Day 4, whereas murine Brachyury (t), Isl1, and GATA Binding Protein (Gata4) mRNA were decreased, compared to iPSC, suggesting that Page 2 of Salerno et al. J Cardiovasc Aging 2021;1:13 https://dx.doi.org/10.20517/jca.2021.19 19 GSNOR deficiency promotes CM differentiation beginning immediately following cell adherence to the culture dish-transitioning from mesoderm to cardiac progenitor. Conclusion: Together these findings suggest that increased S-nitrosylation of Gsk3β promotes CM differentiation and maturation from iPSCs. Manipulating the post-translational modification of GSK3β may provide an important translational target and offers new insight into understanding of CM differentiation from pluripotent stem cells. One sentence summary: Deficiency of GSNOR or addition of GSNO accelerates early differentiation and maturation of iPSC-cardiomyocytes.


INTRODUCTION
The maturation of cardiomyocytes (CM) derived from induced pluripotent (iPS) cells is a highly complex yet incompletely understood process. Endogenous and exogenous nitric oxide (NO) affects stem cell biology and promotes CM maturation, although the mechanism(s) remains controversial [1,2] . S-nitrosylation (S-NO) of cysteine thiols is a major signaling pathway through which NO exerts its broad regulatory actions [3,4] . Levels of S-NO in mice are physiologically regulated via the activity of S-nitrosoglutathione (GSNO) reductase [GSNOR (alcohol dehydrogenase 5)], an enzyme that promotes protein denitrosylation. Mice lacking GSNOR (GSNOR -/mice) show increased levels of S-NO proteins [5][6][7] . GSNOR -/mice have enhanced cardiomyocyte proliferation and recover better than wildtype mice post-myocardial infarction [8,9] . Here, we hypothesized that the absence of GSNOR, and the resultant increased S-nitrosylation, accelerates the differentiation and maturation of cardiomyocytes. We analyzed early cardiomyogenic differentiation of iPS cells derived from GSNOR -/and wildtype [WT (control)] mice to test this hypothesis.
Colony structural integrity is crucial for maintaining pluripotency during the expansion of induced pluripotent stem cells (iPSCs) and requires that cells do not undergo epithelial-to-mesenchymal transition (EMT). EMT is a highly ordered process that generates pools of primitive stem/progenitor cells at precise locations within the embryo [10] . EMT is initiated by the downregulation of intercellular adhesion proteins, such as E-cadherin, the prototypical epithelial adhesion protein, and by the upregulation of EMT-inducing transcription factors, such as Snail (encoded by SNAI1 and SNAI2) [11] . Glycogen Synthase Kinase 3 Beta (GSK3β), a serine/threonine kinase, plays a central role in controlling Snail Family Transcriptional Repressor 1 (Snail) expression and the Wnt/β-catenin pathway, an important initiator of EMT [12] . Modulation of the GSK and Wnt pathways (with small-molecule inhibitors) affects the CM maturation of iPSCs [13,14] . S-NO of GSK3β reduces its activity [15] , and inhibition of GSK3β activity promotes cardiac differentiation and proliferation in murine and human PSCs [16] .
Here, for the first time, we show that GSNOR loss of function promotes EMT, S-NO of GSK3β, and CM proliferation and differentiation and accelerates the maturation of iPSC-CMs. These findings provide new insights into the modulation of iPSC-cardiomyogenesis and potential cardiac repair targets and cell-based therapy.

MATERIAL AND METHODS
This study was reviewed and approved by the University of Miami Institutional Animal Care and Use Committee and complies with all federal and state guidelines concerning animals in research and teaching as defined by "The Guide for the Care and Use of Laboratory Animals" [17] .

Animals
The generation of GSNOR -/mice and their backcrossing for > 10 generations into a C57Bl6/J mouse background (WT) to eliminate/minimize genetic heterogeneity has been reported [8] . Age-matched WT (C57Bl6/J) mice obtained from Jackson Laboratories were used as controls. Mice received food and water ad libitum and were on a 12-h light/dark cycle. At the endpoint, mice were euthanized by either cervical dislocation or isoflurane inhalation, followed by exsanguination.

GSNOR activity in whole heart lysates and iPSCs
The GSNOR activity assay was performed as previously described [8] . Briefly, heart tissues from WT and GSNOR -/male mice were minced/homogenized in a cell lysis buffer (Cell Signaling). The protein concentration of these samples was determined using a standard Bradford assay. Heart homogenate (200 μg/mL) were incubated with Tris-HCI (2 mmol/L, pH 8.0), Ethylenediaminetetraacetic acid (EDTA) (0.5 mmol/L), and nicotinamide adenine dinucleotide (NADH) (200 μmol/L). The reaction was started by adding S-nitrosoglutathione (GSNO) (400 μmol/L), and activity was measured as GSNO-dependent NADH consumption at an absorbance of 340 nm for 10 min.

Gene-expression analysis
Total RNA was extracted from iPSC GSNOR-/-, iPSC WT and human induced pluripotent stem cells (hiPSCs) at selected time points before and during their differentiation into cardiomyocytes, using the RNeasy mini plus kit, according to the manufacturers' instructions (Qiagen). cDNA synthesis was performed using the high-capacity cDNA reverse-transcription kit, according to the manufacturer's instructions (Applied Biosystems). Quantitative PCR was performed using Taqman Universal Master mix in an iQ5 real-time PCR (qRT-PCR) detection system (Bio Rad). All samples were run in triplicates and normalized to a GAPDH/18s endogenous control. Relative fold-change was calculated using the ΔΔCt method. In addition, Taqman Gene-expression assays were performed for the following murine genes: t, alcohol Dehydrogenase 5 (Class III) (Adh5), Snail1, Snail2, E-cadherin, POU Class 5 Homeobox gene (Oct4), Sox2, Kruppel Like  Table 1 for full details of primer/probe sequences).

MTT assay
MTT assays were carried out on Day 4 to 5 of the differentiation process. Cell viability was measured by using Vybrant® MTT Cell Proliferation Assay Kit (Invitrogen by ThermoFisher Scientific) according to the manufacturer's protocol (Quick Protocol Option). Cells were plated in 24-well plates at the density of 2.5 × 10 4 cells/well. Briefly, the cells were labeled with MTT and incubated for 4 h at 37 ℃. Then, DMSO was added to each well and incubated at 37 ℃ for 10 min to dissolve the formazan. Absorbance at a wavelength of 540 nm was measured in a Spectromax M5 spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale, CA, USA) using software: SoftMaxPro 6.5.1.

Bromodeoxyuridine proliferation assay
Bromodeoxyuridine (BrdU) is incorporated into the newly synthesized DNA strands of actively proliferating cells. According to the manufacturer's protocol, we determined cell proliferation using a BrdU Cell Proliferation enzyme-linked immunosorbent assay kit (Abcam, ab126556, USA). Briefly, 10, 20, or 30 embryoid bodies (EBs) were seeded in a 96-well plate and received BrdU (20 μL). After 3 (Day 5) or 24 (Day 4-5) h of incubation (37 ℃, 5% CO 2 ), cells were fixed, permeabilized and the DNA denatured. Next, Anti-BrdU monoclonal antibodies were added and incubated for 1 hour. Absorbance was then obtained using a Spectromax M5 spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 450 nm.

Beating cells
Video files were captured using a Zeiss Axio Vert.A1 inverted microscope under phase contrast. The resulting video file was then processed into digitized representations of moving pixels, after which the sum of moving pixels was plotted vs. time. Finally, 10-s segments of these videos were employed for the visual plots.

Migration assay
Cell migration was detected using the Transwell system (Corning, Inc., Corning, NY, USA). Cells (1.5 × 10 5 ) were trypsinized and seeded into the upper chamber. After incubation for 24 h at 37 ℃, the cells that had migrated into the lower compartment were fixed with methanol, stained with a 5% crystal violet solution, and counted. Each experiment was conducted with at least four replicates, and migrated cells were counted in a minimum of 3 microscope fields.

Cytosolic calcium measurement
Intracellular Ca 2+ was measured using the Ca 2+ -sensitive dye Fura-2 and a dual-excitation spectrofluorometer (IonOptix LLC, Milton, MA, USA). Briefly, cells were loaded with 2.5 μM Fura-2 for 20 min at room temperature in Tyrode's buffer containing (in mM): 144 NaCl, 1 MgCl 2 , 10 HEPES, 5.6 glucose, 5 KCl, 1.2 NaH 2 PO 4 , 1.5 CaCl 2 (adjusted to a pH 7.4 with NaOH). Then the cells were washed with fresh regular Tyrode's solution for at least 10 min. Cells were excited with a xenon lamp at 340 nm and 380 nm wavelengths. The emission fluorescence (510 ± 15 nm) was collected by a photomultiplier and subsequently integrated by the system's interface. The Ca 2+ signal was recorded under spontaneous cardiomyocyte contractile activity or electric field-paced (20 V) at different frequencies from 0.5 to 4 Hz. The calibration was performed in cardiomyocytes by superfusing a free Ca 2+ and then a Ca 2+ saturating (5 mmol/L) solutions, both containing 10 μmol/L ionomycin (Sigma, St. Louis, MO) until reaching a minimal (R min ) or a maximal (R max ) ratio value, respectively. (Ca 2+ ) i was calculated as previously in Dulce et al. [3] using the following equation: K d (dissociation constant) in adult myocytes was taken as 224 nmol/L. The scaling factors S f2 and S b2 were extracted from calibration [3] .

Calcium transient kinetics and sarcoplasmic reticulum function
We measured the time to reach the (Ca 2+ ) transient peak and the time of decaying from the peak to 50% over baseline (tt50%). We also assessed the time constant (τ) of Ca 2+ decay from a regular twitch and after a pulse of 20 mM caffeine to block the sarcoplasmic reticulum (SR) re-uptake both in regular conditions (1.5 mM CaCl 2 ) and in the absence of Na + and Ca 2+ to block the Na + /Ca 2+ exchanger (NCX) activity as well. Using these parameters, we calculated the proportional contribution of sarco(endo)plasmic reticulum Ca 2+ -ATPase, human protein (SERCA), NCX, and other non-NCX mechanisms in mouse induced pluripotent stem cells (miPSCs)-derived CMs by comparing the time constants of Ca 2+ decay in the following conditions: where K represent the time constant for each depicted mechanism.

Statistics
Statistical analyses were performed using GraphPad Prism version 9.00 for Mac. Values were analyzed using Student's t-test or two-way ANOVA. All data met the assumptions of the tests. Therefore, a P < 0.05 was considered statistically significant. All values are reported as mean ± SEM (n is noted in the figures).

S-nitrosylation status is linked to early cardiomyogenesis in murine iPSCs
Nitric oxide synthase inhibitors prevent the maturation of mESCs into differentiated cardiomyocytes, a process that can be readily rescued by NO donors [20] . To investigate the effect of NO-mediated protein Snitrosylation during early cardiomyogenesis in our miPSC model, we used an NADH-dependent GSNOR activity assay to gauge GSNOR enzymatic activity in GSNOR -/and C57Bl6/J wild type (WT) minced adult heart. As expected, in the presence of GSNO, NADH oxidation produced a sharp decrease in absorbance in WT but not in GSNOR -/lysates over time [ Figure 1A]. Murine iPSC GSNOR-/and iPSC WT from these adult heart-minced fibroblasts were generated after virus infection using the OSKM factors protocol [21] . Spontaneous differentiation of iPSCs into beating cardiomyocytes subsequent to the formation of 3dimensional EBs was seen, as depicted in the schematic experimental approach [ Figure 1B]. EBs were grown in hanging drops for the first two days and neither Gsnor gene expression nor protein levels were observed in the iPSC GSNOR-/cells on Day 0 to Day 6 of the differentiation process [ Figure 1C and D]. In addition, to provide some characterization on the pluripotency of our cells, we observed an accelerated loss of pluripotency markers such as Oct4, Sox2 and Klf4 in both groups, but, in the iPSC GSNOR-/group on Day 6, Oct4 and Sox2 were significantly lower than control iPSCs. In contrast, Klf4 expression, which is also involved in proliferation and differentiation [22] , was higher in iPSC GSNOR-/on Day 2 but not at later timepoints [ Figure 1E].
Finally, the morphology of EB differentiation was examined over a period of 6 days (Day 0-6). The formation and maintenance of the EB population remained relatively uniform until Day 4, at which point, we observed continuous proliferation/differentiation and apparent EMT only in the iPSC GSNOR-/-. Thus, we demonstrated that the genetic deletion of GSNOR is directly associated with differences at the early stages of miPSC growth.

GSNOR deletion stimulates cell proliferation and migration in murine iPSCs
NO, as described above, can influence the proliferation and differentiation cascades of certain cell types [23,24] . Indeed, S-nitrosylation may be integral to NO function during a variety of cellular processes. We previously described that GSNOR -/mice exhibit the enhanced proliferative activity of adult cardiac progenitors and cardiomyocytes post-myocardial infarction [9] . Therefore, we next examined the effect of GSNOR deletion on cell proliferation and migration in miPSCs. An MTT assay was performed on Day 5 and showed that iPSC GSNOR-/have a higher proliferative (metabolic) activity compared to iPSC WT (0.096 ± 0.006 vs. 0.12 ± 0.006; P < 0.05) [ Figure 2A]. We observed enhanced BrdU incorporation in iPSC GSNOR-/-EBs (20 and 30 EBs/well) compared with WT [ Figure 2B and C], in agreement with the results of the MTT assay, which also indicated increased proliferation. We also investigated if the absence of GSNOR activity promotes cell migration in addition to proliferation. To quantify migration, cells were seeded into the upper chamber of a transwell dish and after 24 h of incubation; cells that migrated were counted and stained with crystal violet solution. We observed that cellular migration increased by 75% in iPSC GSNOR-/compared with iPSC WT [ Figure 2D], 3.3 ± 0.16 vs. 5.8 ± 0.3; P < 0.05). Representative micrographs at Day 4 are shown in Figure 2E. These results demonstrate that iPSC GSNOR-/exhibit increased cellular migration and proliferation compared to iPSC WT .

GSNOR deletion reduces Gsk3β and increase Axin 1 and Axin 2 in iPSC GSNOR-/-
As mentioned above, S-nitrosylation of GSK3β inhibits its kinase activity independent of the canonical phospho-inhibition pathway [15] . Furthermore, inhibition of GSK3 is sufficient to induce CM cell cycle reentry, DNA synthesis, and likely proliferation [16] . We hypothesized that GSNOR loss of function increases GSK3β S-nitrosylation and decreases Gsk3β expression/functionality during the initial growth and differentiation of iPSCs. To test this hypothesis, we first measured mRNA levels of Gsk3β from Day 0 to Day 6. iPSC GSNOR-/showed significantly reduced expression levels of Gsk3β on Day 4 compared to iPSC WT [ Figure 3A]. We next performed SNO-RAC assays [19] on iPSC WT and iPSC GSNOR-/-. S-nitrosylated proteins were captured by thiol-reactive resin, and Gsk3β S-nitrosylation status was assessed by immunoblotting.
iPSC GSNOR-/demonstrated greater S-nitrosylation of Gsk3β compared to iPSC WT [ Figure 3B]. The identification of endogenous S-NO in the SNO-RAC assay was validated by the omission of ascorbate, which prevents S-nitrosothiols (SNOs) from being reduced. Furthermore, we examined if increased NO levels in iPSC WT , mimic the effects of the suppressed Gsk3β levels seen in iPSC GSNOR-/-. For these experiments,  Figure 3C]. The same experiment was performed in hiPSC using two different concentrations of GSNO (100 µM and 250 µM) for 24 h on Day 2 of cell culture, a time point that appears to be equivalent to murine iPSC on Day 4 [25] ; human Glycogen Synthase Kinase 3 Beta gene (GSK3β) expression was reduced by ~50% with 100 μM GSNO (P < 0.05) compared to control [ Figure 3D]. These studies demonstrated that increased S-nitrosylation or exposure to a pharmacologic NO donor reduces GSK3β expression in iPSCs.
Axin, a scaffold protein, that forms a complex with GSK3β and β-catenin and promotes GSK3β-dependent phosphorylation of β-catenin, thereby stimulating the degradation of β-catenin. Axin 1 and Axin 2 share a number of structural and functional properties, including homologous GSK3β-binding domains. Surprisingly, Axin 1 protein and mRNA expression on Day 4 [ Figure 3D and E], and Axin 2 mRNA expression, on Day 2 were increased in iPSC GSNOR-/compared to iPSC WT [ Figure 3F]. These results suggest that aberrant S-NO/NO levels impact multiple components of the β-catenin regulatory pathway.

EMT-related transcription factors twist, slug, and snail are upregulated, and E-cadherin is downregulated in murine iPSC GSNOR-/and in hiPSCs treated with GSNO
Previously described, inhibition of GSK3β stimulates the transcription of Snail, a repressor of E-cadherin and inducer of EMT [26] . Similar cooperative activities suggest that Twist and Snail work synergistically to induce EMT [27] . Since migration, proliferation and EMT are closely related processes, we tested whether  Figure 4E and F], although the GSK3β expression was reduced with 100 µM GSNO. These data suggest that the decreased Gsk3β expression/activity observed in iPSC GSNOR-/is associated with the early initiation of EMT and resulting in enhanced cell proliferation.

GSNOR deletion reduces Brachyury (T) and promotes early cardiac differentiation
Multiple upstream signals regulate the mesoderm marker, brachyuryexpression including Phosphatidylinositol-3 kinase (PI3K)/Akt pathway [28] and GSK3β via the Wnt/β-catenin pathway [29] . Recent work suggests that brachyury is essential for specifying cell fate but dispensable for cell survival, proliferation and EMT [30] . Thus, we investigated the effects of altered S-nitrosylation on the function of brachyury in our iPSC model. Surprisingly, brachyury mRNA expression was reduced on Day 4 in iPSC GSNOR-/-[ Figure 5A]. To confirm this result, we treated iPSC WT with 25 µM GSNO for 24 h on Day 4, which produced a 45% reduction in brachyury protein levels [ Figure 5B]. Murine brachyury gene (t) mRNA levels in iPSC WT treated with three different concentrations of GSNO (25 µM, 125 µM and 250 µM) for 3 h on Day 4 of differentiation were measured. A significant reduction in t mRNA was observed with 125 µM  Figure 5C]. Using the same approach, human brachyury gene (T) mRNA levels in hiPSC after 24 h GSNO (on Day 2) confirmed the reduction of T under conditions of increased NO availability [ Figure 5D]. However, there was no significant increase in T mRNA following treatment with 250 µM GSNO in either miPSCs or hiPSCs, a result that can likely be explained by the biphasic effect (low and high concentration) of NO on differentiation [23] . Our results suggest that increasing NO bioavailability reduces brachyury expression.

GSNO [
iPSC GSNOR-/exhibited an increased percentage of beating EBs on Days 8, 10, and 11. On Day 10, 80% of iPSC GSNOR-/-EB outgrowth area displayed beating compared with 48% of iPSC WT EBs [ Figure 5E]. Representative images of organoids [ Figure 5F] and video of beating organoids was captured by standard brightfield microscopy (Supplementary Movie 1 -beating organoids of iPSC WT ; Supplementary Movie 2beating organoids of iPSC GSNOR-/-). In addition to brachyury, mRNA for stage-specific markers of cardiac mesoderm (Isl1) and cardiac progenitors (Nkx 2.5 and Gata4) were also altered at specific timepoints in iPSC GSNOR-/compared to iPSC WT . Isl1 expression was decreased and Nkx 2.5 was significantly increased on Day 4 while Gata4 expression was decreased on Day 6 [ Figure 5G]. Mesp1 expression was not different between the two iPSCs [ Figure 5G]. Together, these results demonstrate that increased NO bioavailability in iPSC GSNOR-/promotes a faster transition from mesoderm to cardiac progenitor, as measured by increasing Nkx 2.5 expression.

GSNOR deletion impacts cardiomyocyte structural maturation
As CM differentiation progresses, there are specific isoform switches of structural proteins indicative of the maturity of differentiating CMs, e.g., increased myosin heavy chain MYH6/MYH7 and troponin I ssTNI/TNNi1 to cTNI/TNNi3 expression ratios, and the emergence of genes involved in calcium handling, such as RyR2, sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) and Na + /Ca 2+ exchanger (NCX) [31] . To understand the maturation of our undifferentiated iPSCs into cardiomyocytes, and to assess the impact of Snitrosylation on this process, we investigated the expression of these CM-related genes on Days 10 and 21 of differentiation/maturation. Tnni1 expression was increased on Day 10 in iPSC GSNOR-/compared to iPSC WT .
Tnni3 was detected at the early stage (Day 0), followed by a sharp decrease and a gradual increase thereafter. For this gene, we observed a significant increase in iPSC GSNOR-/on Day 4 and Day 10 [ Figure 6A]. Immunofluorescence analyses confirmed our previous results that troponin I expression increased on Day 10 in the iPSC GSNOR-/-[ Figure 6B]. Following typical differentiation protocols, the sarcomeres of hPSC-CMs are usually composed of a mixture of α (MYH6) and β (MYH7) isoforms of the myosin heavy chain [32] . In our study, both Myh6 or Myh7 expression were significantly increased in iPSC GSNOR-/--CMs compared to iPSC WT on Days 8 and 10 [ Figure 6A]. We evaluated the expression of markers of cardiomyocyte maturation, including structural proteins, with increasing differentiation time (Day 21). Tnni1, Tnni3, Myh6, and Myh7 were significantly increased in iPSC GSNOR-/--CMs compared to iPSC WT -CMs on Day 21 [ Figure 6C]. We next evaluated the presence and relative expression of the mature calcium-handling molecular components, RyR2 and Atp2a2, in miPSC-CMs. Both genes were expressed at significantly higher levels in iPSC GSNOR-/compared to iPSC WT . We also observed that the sarcomere appeared better organized in iPSC GSNOR-/on Day 21 as revealed by α-actinin staining [ Figure 6D]. These results of structural proteins, calcium handling genes, and sarcomeric α-actinin suggest that, at this stage of cardiomyocyte differentiation of iPSC GSNOR-/is more advanced than iPSC WT .

The absence of GSNOR impacts Calcium handling
Ca 2+ signaling within early cardiac progenitors may be important to promote sufficient differentiation for subsequent contractile function [33,34] . To obtain mechanistic insight into the onset of Ca 2+ handling in our model and its impact on CM differentiation, recording of calcium transients with fluorescent indicators was performed. We compared Ca 2+ handling in iPSC WT -and iPSC GSNOR-/--derived CMs on Day 10 and 21 of differentiation. Both strains exhibited properly shaped Ca 2+ transients (spontaneous or electrically induced), Figure 7A and B, and a functional SR, since Ca 2+ was released by activating the ryanodine receptor (RyR2) with 20 mM caffeine [ Figure 7A]. However, iPSC GSNOR-/--CMs from Day 10 responded to 4 Hz pacing, whereas iPSC WT -CMs did not, although both strains responded to 4 Hz stimulation at Day 21 [ Figure 7C]. Hence, we investigated the time dependence of changes in intracellular Ca 2+ cycling in more detail. The Ca 2+ transient peaks were higher in iPSC GSNOR-/-CMs ( Figure 7C, P < 0.0001 on Day 10 and P < 0.0001 on Day 21) and the time to reach those Ca 2+ transient peaks was shorter compared with iPSC WT CMs ( Figure 7D, P < 0.0001 on Day 10 and P < 0.0016 on Day 21), suggesting a more efficient Ca 2+ -induced Ca 2+ release (CICR) mechanism in cells lacking GSNOR. Likewise, the Ca 2+ transient decay rate (shown as tt50%, Figure 7E) was, overall, significantly lower in iPSC GSNOR-/--CMs ( Figure 7E, P < 0.0001 on Day 10). Next, we calculated the relative contribution of the three main mechanisms involved in the cytosolic Ca 2+ uptake and removal (SERCA, NCX, Non-NCX) in our miPSC lines at Day 10 and 21 of differentiation [ Figure 7F and G]. The main mechanism involved in Ca 2+ decay in fully mature cells is re-uptake into the SR. Cytosolic Ca 2+ reuptake into the SR occurs through SERCA. NCX plays a major role in Ca 2+ handling in early, developing CMs; however, it plays a more modest role in removing Ca 2+ from the CM in mature cells. Consistently, SERCA activity (involved in SR Ca 2+ re-uptake) was more prominent than the participation of Na + /Ca 2+ exchanger (NCX) or other non-NCX mechanisms (both involved in removing Ca 2+ from the cell) in both types of iPSCs. The contribution of SERCA at Day 10 was significantly higher in iPSC GSNOR-/compared to iPSC WT (91.1% vs. 85.7%, respectively), depicting an advanced SR maturation in iPSC GSNOR-/-, whereas at Day 21 both iPSCs exhibited approximately 96% contribution by SERCA. Additionally, the contribution of NCX and Non-NCX was smaller in iPSC GSNOR-/compared to iPSC WT on Day 10 and 21. Therefore, Ca 2+ re-uptake into the SR was favored due to greater participation of SERCA over NCX in both groups, indicating an advanced state of SR maturation. However, intracellular Ca 2+ was more efficiently handled in iPSC GSNOR-/either as spontaneous Ca 2+ transients or in response to electric field-stimulation. Overall, these data indicate that at Day 10 and 21 of differentiation, the iPSC GSNOR-/--CMs are more mature than iPSC WT -CMs.

DISCUSSION
Our report is the first to show that GSNOR loss of function and S-nitrosylation more generally, accelerate the early stage of cardiomyogenesis as modeled by iPSCs. iPSC GSNOR-/exhibited earlier onset EMT and increased cellular migration and proliferation. Moreover, the maturity of differentiating cardiomyocytes, as represented by the expression of structural proteins, Ca 2+ transients and emergent genes involved in calcium handling, and sarcomere organization, revealed by α-actinin expression, occurred earlier in iPSC GSNOR-/-. GSNOR loss of function increased S-nitrosylation and decreased expression and function of GSK3β, with a concomitant increase of EMT-related transcription factors (Snail, Slug and Twist) but decreased E-cadherin [ Figure 8], results that were also seen in iPSC WT and hiPSC following exposure to an NO donor. Nkx 2.5 was significantly increased upon directed differentiation toward cardiomyocytes, while brachyury, Isl1, and Gata4 mRNA were decreased in iPSC GSNOR-/on Day 4. Therefore, GSNOR loss of function promoted the transition from mesoderm to cardiac progenitor and accelerated cardiomyocyte differentiation at the transcription level, as early as the start of adherent culture.
Stem cell pluripotency and differentiation are regulated by several pathways. The differentiation and maintenance of pluripotency are dependent on NO concentration. Low NO concentrations delay stem cell differentiation, inducing the expression of Nanog, Oct4, and Sox2, and the activation of survival pathways [23] , whereas high NO concentrations, as seen in iPSC GSNOR-/-, induce differentiation [35] . Consistent with this concept, we found that iPSC GSNOR-/down-regulated Oct4 and Sox2; however, Klf4 was increased.
The major regulatory pathway of protein S-NO is via the metabolism of GSNO by GSNOR [36] . GSNO exists in a steady state with protein S-NO and cardiomyocytes use GSNO as a physiologic reservoir for NO, a major participant in trans-S-nitrosation reactions [37] . We showed that Gsnor mRNA and protein were present at Day 0 in iPSC WT but, as expected, not in iPSC GSNOR-/-, suggesting that the changes we observed are directly associated with the absence of GSNOR function.
At the start of adherent culture, on Day 4, cell proliferation/differentiation and the activation of EMT were seen only in iPSC GSNOR-/-. EMT is dependent on and orchestrated by Wnt/β-catenin signaling, consistent with its role during vertebrate heart development [38] . Wnt/β-catenin signaling may regulate the self-renewal of hPSCs [39] . Inactivation of GSK3β, a central regulator of the Wnt/β-catenin pathway, enables the accumulation of β-catenin, initially in the cytosol, and subsequently in the nucleus, where β-catenin forms a complex with transcription factor (TCF) proteins to activate Wnt pathway gene targets [40,41] . Wnt/β-catenin target genes regulate the expression of pluripotency and developmental factors associated with the primitive streak and early-stage germ layers [42] . Short-term Wnt induction maintains pluripotency, whereas long-term induction, in response to GSK3β inhibition, induces stem cell differentiation into endo-and mesoderm derivatives [43] . Increased S-nitrosylation of Gsk3β in iPSC GSNOR-/and hiPSCs treated with GSNO likely maintains long-term Wnt induction, promoting expression of the EMT transcriptional activators, Snail, Slug, and Twist and reducing E-cadherin. However, the relationship between elevated NO and EMT is complicated. NO at subtoxic concentrations inhibits EMT and metastasis in metastatic cell lines by upregulating RKIP and E-cadherin protein [44] . These opposite effects may be related to an NO "threshold" that is dependent on cell type.
The regulation of Gsk3β activity is a complex process that can be mediated by multiple mechanisms including phosphorylation/dephosphorylation, S-nitrosylation, subcellular localization, protein-protein interactions, and proteolytic cleavage [45] . Subcellular localization of GSK3β can also modulate its enzymatic activity [15] . Moreover, S-nitrosylation of GSK3β inhibits its activity [15] . CM-specific deletion of GSK3 family members induces CM proliferation [46,47] , and the degree of this proliferation is amplified in the setting of cardiac stress [48] . Our results also demonstrated that a reduction of GSK3β by S-nitrosylation resulted in increased proliferation but coupled with differentiation. However, the precise molecular mechanism for this shift from proliferating to terminally differentiated CMs is unclear, and currently of great scientific interest. Elucidating these mechanisms will provide novel strategies to reverse this terminally differentiated state, allowing CMs to resume proliferation.
Axins were significantly increased on Day 4, in iPSC GSNOR-/cells. Both Axin and its homolog Axin2 act as scaffold proteins, which bind several components of the canonical Wnt signaling pathway [49] . Axin2, a bona fide downstream target of canonical Wnt, participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal [50] . Moreover, Axin2 increases canonical TCF/LEF activity and Snail-mediated EMT progression [51] . Here, we observed increased levels of Axin2, which might indicate activation of the canonical Wnt pathway, likely secondary to the inactivation of GSK3β upon its Snitrosylation. There may be at least two pools of GSK3β in cells, one associated with Axin and refractory to phosphorylation by Akt, and another that can be regulated by Akt [52] . Our findings complement a positive regulatory function for Axin1 and Axin2 in Wnt signaling [53] in developing CMs.
The differentiation of stem cells along the cardiac lineage is characterized by the early expression of a network of mesodermal-cardiac transcription factors, that activate gene expression for structural proteins such as MHC, Mlc2v, α-actinin, and troponin. Upon directed differentiation toward cardiomyocytes, pluripotent cells first pass through a progenitor stage, identified by the expression of specific transcription factors, such as brachyury in the mesoderm stage (early phase) and Nkx 2.5, Gata4, Mef2c in the cardiac progenitor cell stage (later phase) [54] . We showed that iPSCs GSNOR-⁄expression of markers of mesoderm induction (t-brachyury); cardiac mesoderm (Isl1); and cardiac progenitors (Gata4) were reduced, whereas, Nkx 2.5, a cardiac progenitor marker, was increased. In murine embryonic stem cells, exposure to the NO donor, diethylenetriamine/nitric oxide adduct can repress brachyury, Gata4, Gata6, Fibroblast growth factor (Fgf) 5, and Fgf8 [23] . Following cardiogenic differentiation, Isl1 is progressively repressed, and only a small number of Isl1 + cells remain in the postnatal heart [55] .
Similar to other post-translational modifications (i.e., phosphorylation, methylation, acetylation), S-NO regulates the expression and function of key proteins controlling mammalian cell differentiation [5] and cellcycle activity [56] . In the adult heart, S-NO modulates signaling pathways important for vasodilation, cardiomyocyte contraction, mitochondrial function [57] , and Ca 2+ handling [3,58] . In murine embryonic stem cells [2] , an isotope labeling proteomics screening approach, demonstrated that Prdx-2 nitrosylation (following GSNO exposure) favors cardiomyocyte differentiation. Although an increased percentage of beating cells was observed in iPSCs GSNOR-/on Day 8, significant changes occur as the cells continue to develop. Therefore, we followed the progress of cell differentiation until Day 21. In addition, NO improves calcium handling in mature cardiomyocytes [59] ; however, the acute and chronic effects on differentiating cardiomyocytes have not been elucidated.
There is no current understanding regarding the onset of Ca 2+ handling and its impact on differentiation and cardiogenesis. However, Ca 2+ transients, calcium handling-related gene, protein expression, and sarcomere organization were, except for Tnnt2, all significantly increased on Days 10 and 21 in iPSC GSNOR-/compared to iPSC WT . These results are supported by the observation that the NO-donor, CysNO, upregulated βMHC, producing a higher βMHC to αMHC ratio and superior calcium handling capabilities in EBs without altering EB size or viability [1] . Our findings also corroborate that RyR2 S-nitrosylation increases the activity of this channel [60] ; whereas, decreased RyR2 S-nitrosylation is associated with reduced SR Ca 2+ release [61] .
Prolonging time in culture promotes the maturation of iPSC-CMs, including more mature morphology, structure, and physiology [62] . Our iPSC-derived CMs were grown for only 21 days. Human iPSC-derived CMs chronologically aged in vitro, exhibited accelerated senescence and functional deterioration within four months in culture, resembling cardiac tissue from a 65-year-old human [63] . During senescence and mammalian aging, GSNOR expression declines, resulting in nitrosative stress and the accumulation of damaged mitochondria [64] . In contrast, centenarians maintain high GSNOR expression [64] .
This study is the first to investigate the effects of GSNOR deficiency during cardiomyocyte differentiation as modeled by iPSC. However, there are limitations to this study. First, this study is limited to the early stages of differentiation of both murine and human iPSCs. Second, the heterogeneous composition of differentiating iPSCs could mask the magnitude of gene expression changes. Further evaluation, such as a directed differentiation with a monolayer culture, will allow for dissection of different stages of cardiac differentiation and a better understanding of the role of GSNOR at different stages of cardiomyocyte differentiation.
In conclusion, the absence of GSNOR accelerates cardiomyocyte maturation from iPSCs. We observed increased S-nitrosylation of GSK3β, cellular migration, proliferation, and induction of EMT. Together, these findings have important implications and may offer insights into improving cell therapy and enhancing disease modeling in the cardiovascular system.

Conflicts of interest
Hare JM reported having a patent for cardiac cell-based therapy. He holds equity in Vestion Inc. and maintains a professional relationship with Vestion Inc. as a consultant and member of the Board of Directors and Scientific Advisory Board. Hare JM is the Chief Scientific Officer, a compensated consultant and advisory board member for Longeveron, and holds equity in Longeveron. Hare JM is also the coinventor of intellectual property licensed to Longeveron. Longeveron LLC and Vestion Inc. did not participate in funding this work. Dr. Hare's relationships are disclosed to the University of Miami, and a management plan is in place. Other authors declared that there are no conflicts of interest.