This study used H9c2 cardiomyoblasts (European Collection of Cell Cultures) derived from BDIX embryonic rat cardiac tissue. Cells were cultured in Dulbecco's Modified Eagle's Medium Ham's F-12 1:1 (DMEM/F-12; Lonza Group, Ltd.) supplemented with 10% w/v fetal bovine serum (FBS) (Gibco-Thermo Fisher Scientific, Inc.) and 1% w/v penicillin-streptomycin. All cultures were maintained at 37°C in a humidified environment with 5% CO₂ and 95% O₂. H9c2 cardiomyoblasts represent a well-established in vitro model that has been previously used to investigate the molecular mechanisms of action of numerous anticancer agents, including TKIs (23–25).

H9c2 cardiomyoblasts were treated with imatinib and ponatinib to investigate their cardiotoxic effects. Stock solutions of both drugs (50 mM) were prepared by dissolving in Hybri-Max DMSO (cat. No. D2660; MilliporeSigma). To ensure consistency, the final concentration of DMSO in the culture media was ≤0.5%. H9c2 cells were treated with imatinib and ponatinib at 2.5 and 5.0 µM. The concentrations used in this study were consistent with those used in previous studies (23,24,26–28). Control cells received an equivalent volume of vehicle (0.5% v/v DMSO) only. Following treatment for 24 h at 37°C, cells were subjected to further experiments.

The viability of H9c2 cardiomyoblasts following treatment with imatinib or ponatinib was evaluated using MTT assay (MilliporeSigma). Briefly, cells were seeded in 48-well plates at a density of 4×10⁴ cells/well and allowed to adhere for 24 h at 37°C. Medium was replaced with fresh Dulbecco's Modified Eagle's Medium Ham's F-12 1:1 (DMEM/F-12) (Lonza, Basal, Switzerland) supplemented with 10% FBS) and 1% penicillin/streptomycin. Cells were then treated with imatinib or ponatinib at concentrations of 2.5 and 5.0 µM for 24 h at 37°C. The medium was removed following treatment and 0.5 mg/ml MTT solution was added to each well. Following incubation for 3 h at 37°C, the resulting formazan crystals were dissolved in DMSO. Absorbance was measured at 570 nm using an Epoch 2 optical microplate reader (Norgen Biotek Corp.). Cell viability was presented as a percentage of vehicle control. To account for non-specific signal, the mean absorbance of blank wells containing medium only was subtracted from all other wells. Background-corrected absorbance values for each treatment group were normalized to the mean absorbance of the vehicle control (0.5% DMSO) to enable cross-group comparisons. Finally, normalized values were multiplied by 100 to present viability as a percentage of the vehicle control. The experiment was repeated three times.

Cell surface area quantification was performed as previously established (29). Briefly, H9c2 cardiomyoblasts were seeded at a density of 10,000 cells/35-mm culture dish and allowed to adhere for 24 h at 37°C. Cells were then treated with imatinib or ponatinib at concentrations of 2.5 and 5.0 µM for 24 h at 37°C. Following treatment, cells were washed with 1X phosphate-buffered saline (PBS), fixed with 4% v/v formaldehyde and stained with 0.5% w/v crystal violet solution for 20 min at room temperature. Stained cells were visualized and captured at 20× magnification using an Axiovert 40 CFL inverted confocal microscope (Carl Zeiss AG). Cell surface area analysis was performed on 15–30 randomly selected cells using AxioVision Imaging software 4.8.2 (Carl Zeiss AG). The experiment was repeated three times. The surface area of the cell was calculated by normalizing the background-corrected absorbance values of each treatment group to the mean absorbance of the vehicle control group (0.5% DMSO) for direct comparison across groups. Then, the normalized values were multiplied by 100 to express the viability of each treatment group as a percentage of the vehicle control.

H9c2 cardiomyoblasts were seeded in 35-mm dishes containing DMEM-F12 supplemented with 10% w/v FBS and 1% w/v penicillin-streptomycin. After 24 h 37°C, they were treated with imatinib or ponatinib at concentrations of 2.5 and 5.0 µM for 24 h at 37°C. Culture medium was aspirated, and monolayers were rinsed with sterile PBS to remove serum. Cells were detached using 0.25% Trypsin [Gibco-Thermo Fisher Scientific (Waltham, MA, USA)] for ≤10 min at 37°C, centrifuged (300–400 g, 5 min, at room temperature), and washed with PBS/BSA to remove debris. The final pellet was resuspended in fresh PBS. Cells were stained with annexin-V and propidium iodide (PI) (BD Biosciences) in 1X annexin binding buffer for 30 min at room temperature. Flow cytometry was performed using BD LSRFortessa™ cell analyzer (BD Biosciences) BD FACSDiva software 6.1.3 (BD Biosciences) were used to measure the cell viability. Apoptotic cells were determined as follows: Viable, PI- and annexin-FITC-negative; early stage apoptosis, PI-negative and annexin-FITC-positive; late stage apoptosis PI- and annexin-FITC positive and necrotic, PI-positive and annexin-FITC-negative as previously described (30,31). Cell size was measured with forward light scatter using the flow cytometer and the percentage of cells in early and late apoptosis was calculated as the total proportion of apoptotic cells. The experiment was repeated three times.

Adult zebrafish (AB strain) were maintained in recirculating systems (AQUA NEERING ZD560) at the Qatar University Biomedical Research Center under a 14 h light/10 h dark cycle with controlled temperatures (room: 26°C, water: 28°C). All procedures adhered to approved protocols (QU-IACUC, QU-IBC-2022/014) and national/international zebrafish guidelines (27). Standard practices (32) guided breeding. Fish were maintained in reconstituted saltwater tanks and fed twice daily with fresh brine shrimp. For embryo collection, male and female zebrafish were separated overnight using a mesh barrier in a mating tank. The next morning, the barrier was removed, allowing mating for 20 min. Fertilized embryos were collected in freshly prepared E3 medium (32) and staged/fixed according to established protocols (33).

To assess the cardiac effects of TKIs in a developing organism, fertilized ZFEs were collected and maintained at 28°C in N-phenylthiourea (PTU) water at a standard concentration of 0.003% (200 µM) to suppress pigmentation and aid observation and time-lapse video acquisition. Experiments used 12-well Falcon Tissue Culture Plate, flat bottom with low evaporation lid (Corning Life Sciences, Netherlands), housing 20 embryos each. For optimal detection of potential toxicity, treatment commenced occurred at 6 h post-fertilization (hpf), corresponding to the ‘high stage’ as described by Kimmel et al (33). This stage is characterized by rapid cell division and organogenesis, rendering the developing embryo highly susceptible to disruptions caused by toxic substances due to the presence of a largely undifferentiated cell population. Following PTU removal, 2 ml drug solution was added. TKI concentrations (2.5, 5.0 and 10.0 µM) were selected based on preliminary tissue culture experiments, ensuring relevance to clinically relevant ranges. The following controls were used: Control, maintained in PTU; negative control, treated with the DMSO vehicle (0.1% v/v) and the positive control (PC), treated with known cardiotoxic agent aristolochic acid I (AA; 1 µM). All drug solutions were prepared in DMSO at a final concentration of 0.1%. Treatment was conducted at 28°C for 24 h for SR, 48 h for both SR and HR, and 72 h for SR, cardiac function assessment, and cardiac gene assay.

At 24–72 hpf, ZFEs were examined every 24 h under a SteREO Discovery V8 light microscope (Carl Zeiss AG) with a Hamamatsu Orca Flash camera (Hamamatsu Photonics UK Limited) and HCImage software 2.0.4 (Hamamatsu Photonics UK Limited) to monitor developmental stage, mortality, hatching, spontaneous movement, response to touch, presence of deformities and heart rate. The phenotypical aberrations were recorded at each point and compared with the controls. Images of the embryos were captured and the number of similar phenotypes in the experimental group noted. Survival and morphological abnormalities were assessed; opaque, coagulated embryos lacking a heartbeat were considered non-viable and removed. The mortality percentage was determined by counting the number of dead ZFEs/group at 24, 48 and 72 hpf divided by the total number of injected embryos ×100. Dead embryos were removed at each time point. Potential neurological or muscular defects at 24 hpf were assessed by tail-flicking (burst/min) using Danio Scope software, EthoVision XT9.0 (Noldus Information Technology). The hatching percentage was determined by counting the number of hatched ZFEs/group at 48 and 72 hpf, divided by the total number of injected embryos ×100.

Experiments exceeding 20% mortality in the negative control group were excluded. Observed abnormalities were documented. At 72 hpf, six embryos/group were immobilized with 3% methylcellulose at room temperature for 30 sec to conduct cardiovascular analysis. Each embryo underwent 10 sec bright field video recording at 100 frames/sec (fps) of the beating heart and the body. MicroZebralab 3.6 software (Viewpoint) was used to determine blood flow velocity, arterial pulse and vessel diameter in the dorsal aorta (DA) and posterior cardinal vein, ensuring consistency across samples (34,35) (Fig. 1).

The cardiac function of treated and negative control embryos was evaluated at 72 hpf. Time-lapse video capturing beating ventricles and red blood cell (RBC) movement in major vessels was analyzed as described previously (35). An in-house algorithm implemented in Viewpoint ZebraLab software version 3.4.4 (Viewpoint) tracked RBCs, enabling the measurement of aorta blood velocity, heartbeat and aorta diameter (Fig. 1). The mean of these measurements was used to estimate frictional shear stress levels in the cardiovascular system using the formula: Shear stress (τ; dynes/cm²)=[4 × blood viscosity (dynes/cm²) × average blood velocity (µm/sec)]/vessel diameter (µm). flow rate (nl/min), was calculated as follows: Average blood velocity (µm/s) × vessel diameter (µm) (34–36).

Gene expression changes were detected by reverse transcription-quantitative PCR which involved a 2 min Uracil DNA glycosylase incubation at 50°C, a 2-min polymerase activation at 95°C, a 1-sec denaturation at 95°C, and a final 30-sec annealing at 60°C. Total RNA was isolated from TKI-treated and negative control embryos using the IBI DNA/RNA/Protein Extraction kit (cat. no. IB47702; IBI Scientific) following the manufacturer's instructions. First-strand cDNA was synthesized from the extracted RNA using the SuperScript™ IV VILO™ Master Mix kit (cat. no. 11756050; Thermo Fisher Scientific, Inc.). Quantitative analysis of specific mRNA expression was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and specific primers (Table I) and probes constructed against the genes of interest. These include atrial natriuretic peptide (ANP; Applied Biosystems; Thermo Fisher Scientific, Inc.) and brain natriuretic peptide (BNP; Applied Biosystems, Thermo Fisher Scientific, Inc.). The signal was detected using the ABI 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). mRNA levels were quantified using the 2^−ΔΔCq method (37) and normalized to the internal reference gene B2M. This approach ensured accurate and consistent gene expression analysis across all samples.

Statistical analysis was performed using GraphPad Prism software, version 8 (GraphPad LLC; Dotmatics,). D'Agostino-Pearson normality test confirmed the distribution of parametric data, which were analyzed by one-way ANOVA with Sidak's or Dunnett's post hoc test and two-way mixed ANOVA with either Sidak's or Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All data points are presented as mean ± SEM. Each experiment was performed three times.

To assess the cardiotoxic potential of imatinib and ponatinib, H9c2 cardiomyoblast viability was evaluated using an MTT assay following 24 h treatment. A significant increase in cell viability (109.19±2.35%) compared with the negative control was demonstrated with 2.5 µM imatinib treatment, however, no significant changes were demonstrated at 5 µM. Ponatinib induced significant dose-dependent reductions in cell viability, with a 22% decrease at 2.5 µM (viability, 78.24±3.80%) and a 40% decrease at 5 µM (viability, 61.46±1.49%) compared with the negative control (Fig. 2A).

Flow cytometry confirmed these contrasting effects. While both 2.5 and 5.0 µM ponatinib significantly reduced the number of live cells in the sample population for each group (59.10±2.14 and 6.83±0.07%, respectively) compared with the negative control. Imatinib treatment at each concentration showed no significant change in the number of live cells compared with negative control (Fig. 2B).

To assess the underlying mechanism of cell death, the percentage of apoptotic cells was assessed using flow cytometry and annexin-V/PI staining. Ponatinib induced significant dose-dependent increases in the percentage of apoptotic cells compared with negative control at 2.5 (30.62±2.47%) and 5.0 µM (92.4±2.96%). Imatinib did not induce a significant change in the percentage of apoptotic cells at either concentration (Fig. 2C). Representative flow cytometry plots are shown in (Fig. 2D).

The effect of imatinib and ponatinib on cell morphology and cardiomyocyte hypertrophic markers, including cell surface area and size were assessed. Treatment with 2.5 or 5.0 µM ponatinib reduced cellular density and increased cellular detachment and cellular shrinkage compared with the Negative control (DMSO 0.1% v/v) (Fig. 3A). Imatinib treatment increased cell size, indicative of hypertrophy. Ponatinib, but not imatinib, impacted H9c2 cardiomyoblast morphology. At 2.5 µM, ponatinib cause a significant reduction in cell surface area (69.23±9.86%) compared with the negative control, indicating cell shrinkage. Furthermore, 5 µM ponatinib resulted in severe cell loss, making surface area measurement impossible. (Fig. 3A and B).

To confirm these results, flow cytometry was used to measure cell size. As expected, 2.5 and 5.0 µM ponatinib significantly reduced cell size by 37.59±14.35 and 1.26±0.22%, respectively, compared with negative control. Imatinib treatment did not induce any significant change in cell size (Fig. 3C). Representative flow cytometry plots for the cell size are shown in Fig. 3D.

To confirm cardiotoxic effects of imatinib and ponatinib, ZFEs were used as an in vivo model. Embryos were treated with TKI and toxicity analyses were conducted by monitoring the survival and morphology for up to 72 hpf. For the 2.5 µM imatinib and ponatinib treatment, several phenotypes were observed, including edema and/or lordosis. Edema was the most common disfigurement in all ponatinib concentrations. Edema typically included cardiac malformations that disrupted the sinus rhythm of the heart. These malformations usually did not result in death of the animals. As these animals aged, they maintained their bent stature but could otherwise function normally, for example swimming and feeding. The decreased diameter of DA and posterior cardinal vein (PCV) was consistently observed in 2.5 µM imatinib and ponatinib treated embryos compared with the negative control embryos. The animals that displayed this malformation exhibited notable difficulties swimming at later time points. Dorsalization was observed in rare cases in the 10 µM ponatinib-treated embryos. Embryos exhibiting this phenotype seldom survived and failed to hatch because of the extreme nature of the deformities. Exposure to TKIs significantly increased malformation rates in zebrafish embryos compared to controls treated with 1% DMSO (Fig. 4; Table II). Control embryos showed a low baseline incidence of malformations, with only 5% exhibiting edema. Imatinib impact was dose-dependent; at lower concentrations (2.5 and 5 µM), it did not significantly increase malformation rates. However, at the highest concentration (10 µM), it caused a moderate rise in edema (10%) and a substantial increase in cardiac disruptions (40%). Ponatinib demonstrated a stronger teratogenic effect than Imatinib. Even at its lowest dose (2.5 µM), Ponatinib significantly increased the prevalence of edema (20%) and cardiac disruptions (25%). The malformation rates increased as the concentration of Ponatinib rose, with 65% of embryos showing edema and 60% having cardiac issues at 5 µM. At the highest dose (10 µM), nearly all embryos displayed malformations, with 85% showing edema, 90% exhibiting lordosis, and 95% experiencing cardiac disruptions.

To assess the potential teratogenic effects of imatinib and ponatinib, treated embryos were visually inspected compared with the negative controls at 24, 48 and 72 hpf. As a PC for heart failure induction, 1 µM AA was used, consistent with previous studies (38,39). Concentration-dependent decreases in survival rate and tail-flicking activity were observed in embryos treated with imatinib and ponatinib compared with the negative control (Fig. 5A and C). Ponatinib significantly affected survival rate at 5 and 10 µM concentrations. This effect began at the first day (24 h post fertilization, hpf) and persisted until 48 and 72 hpf. Both Imatinib and Ponatinib significantly affected tail flicking at all concentrations tested. However, Ponatinib exhibited a stronger inhibitory effect on both survival rate and tail flocking compared to Imatinib.

Furthermore, changes in hatching rates were observed at 48 hpf. While 2.5 and 5.0 µM ponatinib significantly increased the hatching rate compared with the control, 10 µM ponatinib significantly decreased hatching rate compared with the control. Imatinib treatment did not significantly affect hatching at any concentration. As expected, 1 µM AA significantly reduced the hatching rate compared with the negative control (0.1% v/v DMSO) (Fig. 5B).

DA blood flow analysis demonstrated the detrimental effects of both TKIs on cardiac function. Notably, 5 and 10 µM ponatinib nearly arrested blood flow (Fig. 4I), precluding further analysis at higher concentrations. At 2.5 µM, ponatinib and imatinib significantly impaired cardiac function. Compared with negative control embryos, 2.5 µM, ponatinib and imatinib caused a significant 48% decrease in DA blood flow velocity, indicating decreased flow rate. Furthermore, 2.5 µM ponatinib and imatinib induced a significant 27% narrowing of the aorta diameter, suggesting structural abnormalities. Both TKIs caused a 73% reduction in overall flow rate compared with negative controls (Fig. 6). These findings suggest the cardiotoxic potential of both ponatinib and imatinib in developing ZFEs.

Imatinib and ponatinib significantly increased mRNA expression of ANP and BNP compared with the controls (Fig. 7). ANP and BNP are established markers of cardiac failure (40).

Ponatinib treatment significantly increased ANP and BNP mRNA expression by approximately twofold compared to the negative control. In contrast, Imatinib only caused a onefold increase in both ANP and BNP mRNA expression compared to the negative control.

These changes in gene expression mirrored those observed in embryos treated with 1 µM Aristolochic acid I (AA), a positive control for ZFE cardiotoxicity. This suggests that both TKIs (Ponatinib and Imatinib) significantly affect zebrafish embryonic development. Notably, Ponatinib's effect was comparable to the positive control (Fig. 7).