Abstract Background The progression of ischemic heart disease results from various forms of cardiomyopathies, which begin with cardiac remodelling. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one of the basic kinase family components responsible for oxidative phosphorylation. However, due to the lack of a suitable research model, there is no evidence that remodelling pathogenesis in humans causes death by PDK1 knockout (KO). In the current study, we established a PDK1-deficient human cardiomyocyte (CM) model under conditions imitating the human PDK1-KO model. We determined the role of PDK1 in myocardial apoptosis induced by hypoxia and its implicit mechanism. Methods A human PDK1-KO CM’s model was established by combining CRISPR/Cas-9 gene-editing and human induced pluripotent stem cells (hiPSC) directed differentiation technology. The pathological features of PDK1-KO cardiomyocytes were assessed using a phenotypic cell model under basal and hypoxic conditions. Results We found that pluripotency and differentiation efficiency of hiPSCs after PDK1 knockout remain intact. Cardiomyocytes with a PDK1 gene knockout showed hypoxia-induced myocardial apoptosis by disturbing mitochondrial metabolism, increased oxidative stress levels, and decreased cell energy and viability. In addition, lentivirus transfection significantly improved the metabolism and cell viability in PDK1-deficient cardiomyocytes. Conclusions Our study established a PDK1 knockout model under hypoxia that exhibits mitochondrial metabolism dysregulation, elevated oxidative stress, and decreased cell viability. This model is an important tool for understanding the mechanism of hypoxia-induced myocardial apoptosis, elucidating the gene-phenotype relationship of PDK1 deficiency, and providing evidence to mitigate the damage against hypoxia. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-025-04518-9. Keywords: PDK1, Cardiomyocytes, CRISPR/Cas9, Hypoxia, Metabolism, HiPSC, Myocardial injury Background Cardiovascular diseases have been leading source of morbidity and mortality worldwide [[38]1]. and Ischemic heart failure (IHD) is considered the final common symptom of diverse cardiac abnormalities. The loss of cells is widely acknowledged as a critical pathophysiological factor in the onset and progression of IHD. Numerous studies have suggested that several forms of cell death, like autophagy, apoptosis, and necrosis, are all involved [[39]2]. However, elevated reactive oxygen species (ROS) level may induce oxidative stress and play a essential role in developing detrimental remodelling processes including apoptosis and cardiac dysfunction to IHF [[40]3]. Given the importance of apoptosis and oxidative stress in maintaining normal cardiac structure and function, interventions for multiple cell death pathways may be therapeutic strategies for preventing and treating cardiac dysfunction. Previous research reported that inhibition of the apoptosis signalling pathway in animal models of myocardial injury exerts a cardioprotective effect [[41]4, [42]5]. Despite some advanced research, the specific signalling pathways have not been elucidated completely in this study. 3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a key part of the AGC serine/threonine family of kinases, and the PDK1/AGC kinase signalling pathway has a critical role in biological processes associated with growth, proliferation, apoptosis and autophagy [[43]6]. It is well known that the heart contains numerous mitochondria, and the normal mitochondrial function plays a important protective role in myocardial damage. A previous study reported the metabolic abnormalities in cardiomyocytes of PDK1 deficiency mice [[44]7]. Nevertheless, the mechanism underlying PDK1-induced IHD under hypoxia conditions is not clear. In the present study, LC-ESI-MS/MS metabolomic and Gene set enrichment analysis (GSEA) of the proteomics dataset identified apoptotic- and metabolism-related signalling pathways are direct targets of PDK1. Notably, cardiac dysfunction closely correlates with AKT/GSK3- β level. To better look upon the role of PDK1 in metabolic characteristics, time-dependent changes in the metabolism of cardiomyocytes were determined by LC-ESI-MS/MS compared to controls. The results verify the fundamental role of PDK1 in metabolic profiles. Further, the mitochondrial functions were assessed as well [[45]7]. Using LC-ESI-MS/MS based metabolomics, we demonstrated that citric acid, cyclic AMP, Glycerol-3 Phosphate and Fructose-1,6 Bisphosphate were the major differential metabolites in the PDK1-knockout cardiomyocytes. Lactate, alanine, glycine, taurine, choline, fumarate, IMP, AMP and ATP levels were lowered as compared with controls. This metabolomic disfunction has also been linked with the onset and development of ischemic heart failure [[46]8]. Our findings revealed that PDK1 deficiency caused cardiomyocyte apoptosis and oxidative damage in vitro. Metabolic disturbances can promote cardiomyocyte death by increasing reactive oxygen species production and apoptosis, leading to structural and functional modifications in the heart. To explore the mechanism of PDK1-induced cell apoptosis and oxidative damage, we demonstrated that overexpression of PDK1 effectively attenuated apoptosis and oxidative stress in PDK1-deficient cells. Current findings emphasize on the potential of targeting PDK1 metabolism as a novel therapeutic strategy for ischemic heart disease. Materials and methods Human embryonic stem cell culture and cardiac differentiation This study approved by the Ethical Committee Anzhen Hospital, Capital Medical University (#134/18). Matrigel-coated (Corning, United States) feeder-free plates were prepared with an E8 medium (Cellapy, China) to culture human embryonic stem cell H9 (hESC-H9). Fresh medium changed daily and passaged the cells using 0.5 mM EDTA without MgCl2 or CaCl2 (HyClone, United States) routinely when 80% confluence is reaches and kept at 37◦C, 5% CO2 incubator. As previously reported, Human embryonic stem cells’ differentiation into cardiomyocytes occurred by using a small molecule-based protocol, then a metabolic selection method was used to purify cardiomyocytes [[47]9]. Generation of PDK1−/− hESC-CMs PDK1 knockout cardiomyocytes were derived from genetic editing of hESCs from PDK1−/− hESCs. PDK1 single guide RNA (CATTTATGTTTCTGCGGCAA) was dconstructed using online ‘CRISPR RGEN TOOLs’ ([48]http://rgenome.net). Next, cell transfection was done after the ligation of sgRNA into plasmid vector epi-CRISPR. Human stem cell Nucleofection (Lonza) was used to electroporate 1.5 × 106 dissociated H9 stem cells with an electroporation mixture containing 0.5 µg intact epiCRISPR plasmid with 100 µL electroporation solution. On the first day of electroporation, cells were cultivated in a PSCeasy medium containing Rho-kinase inhibitor Y-27,632 to improve cell survival. To identify the PDK1-/- hESCs lines positive clones were chosen for gene sequencing. Cell culture and hypoxia At 37◦C air atmosphere incubator, H9C2 cells were cultivated with 10% FBS in a humidified, 5% CO2 and supplemented with DMEM. Hypoxic conditions were given to cells as previously described [[49]10]. Concisely, H9C2 cells were placed in a hypoxia chamber (MIC-101; Billups-Rothenberg, Del Mar, CA, United States), bubbled with 5% CO2 and 95% N2 at 10 L/min for 15 min, then equilibrated for 10 min. This protocol repeatedly done until the oxygen concentration reaches up to 0.2%. As reported, H9C2 cells were kept under this condition and used for the following experiments. Immunofluorescence staining Prepare the cell slides at room temperature and fix them with 4% formaldehyde for 15 min. Next, wash the slides 3 times using PBS, 0.5% Triton X-100 (Sigma) used for 15 min permeabilization of cells followed by 3% bovine serum albumin (Sigma) to block them for half an hour. After an overnight incubation period at 4 °C with the primary antibody, rinse the cells with PBS. After that, cells were incubated with a secondary antibody at room temperature for 1 h. Wash the cells with PBS 3 times, then counterstained the cells with DAPI-containing anti-fade solution (Invitrogen) for 15 min in the dark at room temperature. Laser Scanning Confocal Microscope (Lycra) was used to take the fluorescence pictures and analyze them with ImageJ software. Table [50]S1 presented both primary and secondary antibodies. Flow cytometry According to the two-step protocol, single cells were prepared using Cardio Easy CM dissociation buffer I and II (Cellapy) and then fixed with cold fixation buffer (BD Biosciences) at room temperature for 15 min. As described previously, cell permeabilization and blocking were carried in immunofluorescence staining. Next, primary and secondary antibodies were used to stain the cells for 30 min each. Then, the cells were rinsed 3 times using PBS. After washing, resuspend the cells with PBS and measure the stained cells with a Flow Cytometer (EPICS XL, Beckman). FlowJo and CytExpert software were used to carry out the analysis and Quantification. RNA isolation and quantitative real-time PCR (qRT-PCR) TRIzolTM Reagent (Life Technologies) extracted the total RNA following the manufacturer’s instructions from about 1 × 106 cells then dissolved with RNase-free Water (Qiagen). DNase I (Life Technologies) and RNeasy Mini Kit (QIAGEN) were used to purify RNA. RNA concentration was measured by using the NanoDrop-1000 spectrophotometer (Thermo Scientific). PrimeScriptTM reverse transcription system (Takara) synthesized the cDNA according to the manufacturer’s instructions. Next, run the qRT-PCR using 2 × SYBR Master Mix (Takara) on the iCycler iQ5 (Bio-Rad). △△CT Mode was used to perform the comparative mRNA expression analyses. Table [51]S1 lists every primer sequence used for qPCR. LC-ESI-MS/MS based metabolomic analysis The samples were melted on ice, and then 500 µL of 80% methanol/water was added and mixed (precooled at -20 °C) and under 2500 r/min vortexed for 2 min. The samples were kept for 5 min in liquid nitrogen, removed and place on ice for 5 min; after that, vortexed them for 2 min. Repeat this for 3 times. Next, the samples were centrifuged for 10 min at 12,000 r/min and 4 °C. Transfer the supernatant of 300 µL into a fresh centrifuge tube and place it for 30 min in a -20 °C refrigerator. Then, at 12,000 r/min centrifuged the supernatant for 10 min at 4 °C. Transfer 200 µL of supernatant after centrifugation for further LC-MS analysis through the Protein Precipitation Plate. The LC-ESI-MS/MS experiments were performed on a QTRAP^® 6500 + LC-MS/MS System with a triple quadrupole-linear ion trap mass spectrometer. Parameters for LC-ESI-MS/MS acquisition were set as follows: temperature, 550 ℃; ion spray voltage (IS) 5500 V(Positive),-4500 V(Negative); curtain gas (CUR) was set at 35 psi, respectively. The KEGG compound database ([52]http://www.kegg.jp/kegg/compound/) was used to annotate identified metabolites, and the KEGG Pathway database ([53]http://www.kegg.jp/kegg/pathway.html) was used to map annotated metabolites. TUNEL staining and assessment of oxidative stress According to the manufacturer’s guidelines, a one-step TUNEL assay kit was used to detect the cell apoptosis. Briefly, H9C2 cells were fixed for 30 min with 4% paraformaldehyde at room temperature and then permeabilize them by adding 0.15% Triton-X on ice for 5 min, followed by TUNEL for one h at 37◦C. DAPI was used to counterstained the nuclei. Confocal fluorescent microscope was used to capture the images. The apoptotic cells are defined with green fluorescence. RNA-sequencing (RNA-seq) assay Purified cardiomyocytes of D30 were used for mRNA library establishment and transcriptome sequencing. After total RNA extraction, purification was done by using RNase H and DNase I. Purified RNA was then fbreakdown into pieces, subsequently first and second-strand cDNA synthesis. Afterwards, magnetic beads were used to purify the complementary DNA and PCR for amplification. The single-strand circle DNA (ssCir DNA) was formed after denaturation and circularization of PCR products, becoming the final library after further amplification. BGISEQ500 platform analyzed the qualified library with a 50-base reads (SE50) at a single-end. Clean reads were obtained after removing reads with low quality, contamination or large number of unknown base. Bowtie2 and HISAT2 aligned the clean reads for reference genome sequencing. Genes expressed more significantly and differentially expressed genes (DEGs) were ssorted based on adjusted P-value (FDR, Q-value) < 0.001 and|log2 Fold change|≥1. Phyper used the Hypergeometric test ([54]https://en.wikipedia.org/wiki/ Hypergeometric_distribution) to perform gene Ontology and KEGG pathway enrichment analysis of these DEGs. Pathways with Q-value less than 0.05 were significantly enriched. Both R language package and online Omicshare Tools to draw bubble charts, histograms, and heat maps ([55]https://www.omicshare.com/tools/). Gene Set Enrichment Analysis was executed using the GSEA software ([56]https://www.broadinstitute.org/gsea/) with the following parameters: metric = Signal2Noise, enrichment statistic = weighted, permutation = 1000, permutation type = gene-set. Gene sets were significantly enriched identified on the basis of|NES|≥1, P-value < 5% and q-value < 25%. Cell viability assay Cell viability was measured by using Cell Count Kit-8 (CCK-8), which allows sensitive colourimetric assays for the detection of the numeral viable cells by using WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2 H-tetrazolium, monosodium salt] in cell cytotoxicity assays. Briefly, cells were passaged and cultivated in 96-well flat-bottomed plates at confluence of 5 × 103 cells/well and treated as instructed. Then, the medium was changed by a fresh 100 µL 10% FBS DMEM medium having 10 µL CCK-8 and incubate it at 37◦C for 1–2 h. The microplate reader used to measure the absorbance at 450 nm. ATP detection ATP Assay Kit was used to measure the level of ATP as indicated in the manufacturer’s guidlines. Briefly, lysis buffer lysed the harvested cultured cells, followed by centrifugation at 12,000 × g for 5 min at 4◦C. Next, for protein concentration and ATP detection the supernatant was collected. The BCA method was used to measure the protein concentration. Next, take 20 ul supernatant and add 100 µL of ATP detection working solution to determine the level of ATP. A multi-detection microplate reader was used to measure the emitted light. Then, normalized the level of ATP for total protein. Intracellular reactive oxygen species assay Component A ROS BriteTM 670 was used for intracellular reactive oxygen species assay. Briefly, cells were seeded on the confocal plate at 2 × 104 cells/dish. Next, Incubated the cells with 10 µM working solution (AAT Bioquest) after treated as indicated, according to the manufacturer’s instructions, at 37◦C incubate them for 20 min. Then, wash the cells with PBS three times and observe under fluorescence microscope. Measurement of mitochondrial permeability transition pore opening Cells were harvested on a confocal dish to examine the mitochondrial permeability transition pore’s opening (mPTP) at 2 × 104 cells/dish. After treating the cells as indicated in the manufacturer’s instruction, they loaded Hanks’ balanced salt solution with 1.0 mM CoCl2 and 1.0 µM calcein-acetomethoxy ester (Calcein AM) at 37 °C for 30 min. Images were obtained using flow cytometry. Measurement of mitochondrial respiration Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience, Chicopee, MA, USA) was used to analyze the bioenergetics. Cultured the cells in 96-well XF Seahorse cell culture plate 36 h before transfection with siRNA. XF assay medium included 5.5 mM/L pyruvate, 4 mM/L glutamine and 1.25 mM/L glucose were used in the test. The following analyses were accomplished as described previously [[57]11]. OCR is described in pmols/minute, and the results were standardized to protein content. Western blot Protein extraction lysis buffer was prepared, consisting of Mammalian Protein Extraction Reagent (Thermo, #78501),5 mmol/L EDTA (Thermo, #1861275), phosphatase inhibitor cocktail (TERMO, #1862495) and protease inhibitor cocktail (TERMO, #1861278). Cell lysis buffer mixed with cell samples, and place on ice for 30 min, every ten minutes, vortex for 15 s to fully lyse the cells. Afterward, cell lysate was centrifuged at 12,700 g/4°C for 15 min, and the final supernatant was evaluated to determine the protein concentration by BCA Protein Assay Kit (Thermo, #23227). Heat-denatured proteins are separated by SDS-PAGE and are used to exploit Mini-Protean Tetra Cell (Bio-Rad) transferred to PVDF membranes. Blocking was made with Quick Block TM Blocking Buffer/Skim milk (Beyotime) for 1 h. Next, incubation with primary and secondary antibodies will be done separately. Table [58]S2 presents both primary and secondary antibodies. Statistical analysis All data is presented as means ± SD. Student t-tests between two groups and one-way ANOVA among multiple groups with Tukey post-test are statistical test methods mainly included. Statistically significant values P <.05 was defined as (*), with**P <.01, ***P <.001, ****P <.0001. Results Deletion of PDK1 in hESCs lead to Protein dehydrogenase Kinase-1-deficient hESC-CMs without affecting myocardial differentiation To generate PDK1 knockout cell lines, a sgRNA targeting the second axon of the PDK1 gene in hESCs (H9) using the effective epiCRISPR/Cas9 gene-editing technique. After puromycin sieving, resistant clones were picked up for genotyping by DNA sequencing to generate homozygous PDK-ko cell lines. Among PDK1−/− hESCs lines is used for 1 bp insertion in the second exon, thus leading to PDK1 protein deletion and a frameshift mutation. We found that the PDK1−/− hESCs had normal clone morphology, preserved differentiation ability to develop into three germ layers, and ES-cell-like expression of gene compared to H9 cells. To verify further that the PDK1-KO gene affects myocardial differentiation, we employed a small molecule-based differentiation protocol to establish the purified hESC-CMs (WT) and PDK1−/− hESC-CMs (KO) model. Western blot results confirmed the successful knocout of PDK1 at the protein level. As PDK1 mainly acts as a bridge between glycolysis and the TCA cycle in the mitochondria of cardiomyocytes shown as metabolic protein, not in stem cells, we confirm that PDK1-KO did not affect cardiomyocyte differentiation efficiency. After 15 days of differentiation, myocardial specific marker SOX2 was detected by flow cytometry in both WT and KO groups and showed 99.55% similarity with no statistical difference. Furthermore, OCT4, SSEA4, and TRA-1-60 staining verified that the proportion of Pluripotency markers - positive cells in WT and KO cardiomyocytes are almost identical. We have reported this work in our previous research results [[59]12]. Current findings indicate that PDK1 deletion in hESCs did not significantly affect cardiac differentiation and its characteristics. PDK1−/−hiPSC-CMs showed myocardial remodeling phenotype Pathological conditions are caused by myocardial remodeling in cardiomyocytes. which are the primary aspects that cause ischemic heart failure. We first looked at apoptosis to observe the role of PDK1 knockout on cardiomyocyte morphology. As shown in Fig. [60]1A, almost no apoptotic cells were seen in the control. In contrast, the TUNEL-positive cells have been seen in the PDK1 knockout group (Fig. [61]1A). Current results show that PDK1 play a critical role in causing cell apoptosis by enhancing oxidative stress and affecting the functions of cardiomyocytes. Previous studies have verified these findings that PDK1 play a significant role in cell apoptosis [[62]13]. Fig. 1. [63]Fig. 1 [64]Open in a new tab PDK1−/−hiPSC-CMs showed myocardial remodeling phenotype. (A) Cell apoptosis was detected by TUNEL staining. The rate of apoptotic cells significantly increased in KO-CM’s, Tunnel (green) and DAPI (blue) (Scale bar, 200 μm) and quantification. (B) Expression of Atrial Natriuretic Peptide (ANP), (C) B-type Natriuretic Peptide (BNP), (D) B-MHC detected by Real time PCR in cardiomyocytes. (E) Cell viability determined by CCK8 assay in KO-CM’s.(F) Expression of cleaved caspase-3 was assessed by real time PCR. (G) Western Blot confirmed the expression levels of apoptosis-related protein (Cleaved Caspase-3) and quantification. Full-length blots are presented in Supplementary file 5, Fig. [65]7. Data are analyzed with two-sample t test and shown as means ± SD. **P <.01, ***P <.001 Further, we measured the atrial natriuretic factor (ANP) and brain natriuretic peptide (BNP), heart failure-related markers, in knockout cardiomyocytes [[66]14]. Using qPCR, we confirmed that in PDK1-KO cardiomyocytes, the expression of ANP and BNP was significantly higher as compared to the WT cells (Fig. [67]1B, C), suggesting the development of cardiac apoptosis. We also identified a significant increase in MHC-β, a key feature of cardiac remodeling (Fig. [68]1D). Moreover, the cell viability of CMs was determined using the CCK-8 assay, and the results, as anticipated, are consistent with cell TUNEL staining analyses (Fig. [69]1E). We further investigate whether cardiomyocyte apoptosis is caspase-dependent or not, the stimulation of apoptosis and activation of pro-apoptotic proteins because of caspase activation (cysteine proteases). We confirmed that cleaved caspase-3 was significantly upregulated in PDK1-KO cardiomyocytes by q-PCR (Fig. [70]1F). The expression of this gene was increased in PDK1 knockout CMs which was also confirmed by Western blot (Fig. [71]1G) and Quantification of WB results. Therefore, as previously studied, we determined the gene that can act as a marker in cardiac apoptosis [[72]13]. These outcomes are likely to be a key feature for illuminating PDK1-deficient phenotype. Our results are coherent with the pathological features of IHD, which further proposes that PDK1 deletion is responsible for changes in cardiomyocyte phenotypes. Metabolomic analysis of cardiomyocytes Since PDK1 regulate the critical step of glycolysis, it was the question how it would be affected in PDK1-KO cells. PDK1 inhibits the PDH (Pyruvate dehydrogenase) by phosphorylate it, thereby reduce the conversion of pyruvate into acetyl-CoA [[73]15]. It might be suggested that PDK1 suppression affect the PDH expression; hence, change in the glycolytic pathway (Supplementary file 1: Fig. [74]S1G). It confirmed that PDK1 play a criticle role in metabolism. LC-ESI-MS/MS based metabolomics was applied to explore the altered metabolic pattern in PDK1 knockout cells. (Fig. [75]2A) depicted a glycolytic and TCA cycle metabolite. A typical LC-ESI-MS/MS heatmap of all metabolites was illustrated in (Fig. [76]2B). We observed a significant difference in the pool sizes of glycolysis and citric acid cycle (TCA). We measured the fractional enrichment of various glycolytic intermediates to confirm that glycolysis could be greatly affected across the cell lines. Fig. 2. [77]Fig. 2 [78]Open in a new tab KO hiPSC-CMs showed abnormal mitochondrial metabolism and elevated oxidative stress level. (A) Schematic depicting the glycolysis and the citric acid cycle. Significantly upregulated metabolites highited by *. (B) Heatmap of all metabolites in both groups. (C) Immunostaining of ROS (red) and DAPI (blue) shows increase ROS production in KO CMs (Scale bar, 200 μm (D) The OCR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the aerobic respiration level of KO hiPSC-CMs was reduced. (E) The ECAR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the level of glycolysis is reduced in KO hiPSC-CMs. (F) ATP level has been determined by enhanced ATP assay kit (beyotime). Result shows highly significant difference between the groups. (G, H) Quantitative statistical analysis of basal and maximal respiration levels in WT and KO hiPSC-CMs indicated that basal and maximal respiration levels were decreased in KO hiPSC-CMs Data are analyzed with two-sample t-test and shown as means ± SD. ***P <.001 Heatmap of significantly upregulated metabolites shown in (Supplementary file 1: Fig. [79]S1A). The significant difference exists in pyruvate and acetyl-CoA fractional enrichment in knockout cells as compared to control group (Supplementary file 1: Fig. [80]S1B, C). We measured the fractional enrichment of glucose (Supplementary file 1: Fig. [81]S1D). Reduced flux through glycolysis may accumulate the glycolytic intermediate such as fructose-1,6-bisphosphate and glyceraldehyde-3-phosphate (Supplementary file 1: Fig. [82]S1E, F). Table [83]S3 presents the fractional enrichment data for key metabolites involved in various metabolic pathways. According to recent findings, metabolic reprogramming, which includes alteration in the metabolism of fatty acids, glucose, ketone bodies, and amino acids, may play a role in the progression of heart failure [[84]16]. Current study used LC-ESI-MS/MS based metabolomics to investigate the cardiac metabolites in PDK1 deleted cardiomyocytes. Therefore, higher TCA intermediate levels indicated that both TCA cycle and glycolysis were affected in knockout group in comparison to controls [[85]17]. KO hiPSC-CMs showed abnormal mitochondrial metabolism and elevated oxidative stress levels Previous studies showed that disturbance in metabolism and diminished mitochondrial activity can be involved in the aetiology of heart failure in PDK1 deleted mice [[86]7]. We are anxious whether this effect would be more noticeable in the PDK1 knockout model. Aerobic respiration produces the reactive oxygen species (ROS) as a byproduct. Almost 10% of the ROS is produced during normal metabolism of cell, and unnecessary intracellular ROS can damage biological macromolecules, like proteins, lipids, and nucleic acids [[87]18]. ROS-induced oxidative stress damage following apoptosis of cardiomyocytes is a primary source of cardiomyopathy associated with pathological phenotypes such as heart failure [[88]19]. Therefore, we performed immunofluorescence staining to determine the level of ROS, and we observed that ROS level is considerably increased in PDK1-KO cardiomyocytes compared to WT cardiomyocytes (Fig. [89]2C). In healthy cardiomyocytes, mitochondrial oxidative phosphorylation is the main process that produces ATP. The Seahorse XF assay was used to determine the mitochondrial oxidative phosphorylation level in both WT and KO cardiomyocytes. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured to check the mitochondrial and glycolytic functions. These findings (Fig. [90]2D, E) revealed that the KO cardiomyocytes’ basal respiration, maximal respiration, and ATP levels lower significantly than those of WT group. Additionally, the glycolytic capacity of knockout cardiomyocytes was decreased considerably, indicating that the significant reduction in extracellular acidification rate of KO cardiomyocytes than that of WT cardiomyocytes. Seahorse experiment results revealed the OCR curves of WT and KO-CMs, representing that the level of aerobic respiration of KO-CMs was reduced (Fig. [91]2F-H). Our findings suggest that the oxidative phosphorylation and glycolytic capacity of mitochondria are markedly decreased in PDK1 knockout cells. PDK1-KO cardiomyocytes are more sensitive to hypoxia PDK1 plays a pivotal role in regulating cardiac viabilities. Previous studies concluded that mPDK1±/± mice died unexpectedly in an early period of life, having the cardiomyocytes of same number but smaller in size and ultimately developing heart failure, and those PDK1 deficint cardiomyocytes are considerably more susceptible to hypoxia [[92]20]. We first evaluate the number of apoptotic cells. The TUNEL assay observed the cell apoptosis in response to hypoxia. Results indicated that the apoptotic cell number considerably increased in PDK1-KO cells after 18 h of hypoxia (Fig. [93]3A). Next, changes in ROS production in hypoxia-treated cells to assess the roles of PDK1 in cardiomyocytes under hypoxia using immunofluorescence staining. As shown the ROS production increased after exposure to 18 h hypoxia compared to the control group (Fig. [94]3B). Quantitative real-time polymerase chain reaction showed that the molecular marker of apoptosis (Cleaved caspase-3) increased significantly in KO CMs (Fig. [95]3C). Fig. 3. [96]Fig. 3 [97]Open in a new tab PDK1 knockout cardiomyocytes are more sensitive to hypoxia (18 h). (A) Hypoxia significantly increased the number of apoptotic cells (Scale bar, 100 μm) and quantification. (B) Immunostaining of ROS (red) and DAPI (blue) shows increase ROS production in KO CMs (Scale bar, 100 μm). (C) Hypoxia treatment significantly increased cleaved caspase 3 activity confirmed by real-time PCR. (D) Cell viability determined by CCK8 assay in KO-CM’s. Result shows highly significant difference between the groups under 18 h hypoxia. (E) The expression level of cleaved caspase-3 protein was detected by Western Blot and quantification. Full-length blots are presented in Supplementary file 5, Fig. [98]2. (F) The OCR curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the aerobic respiration level of KO hiPSC-CMs was reduced as compared to WT group under 18 h hypoxia. (G) ECAR. curves of WT and KO hiPSC-CMs were detected by Seahorse experiments, suggesting that the level of glycolysis is reduced in KO hiPSC-CMs (18 h Hypoxia). (H) ATP level determined in both groups (18 h Hypoxia). (I) Quantitative statistical analysis of basal and maximal respiration levels in WT and KO hiPSC-CMs indicated that basal and maximal respiration levels were decreased in KO hiPSC-CMs than control group. Data are analyzed with two-sample t test and shown as means ± SD. ***P <.001. Full-length blots are presented in Supplementary file 2, Fig. [99]2 Additionally, the cell viability of H9C2 was assessed under hypoxic environment using the CCK-8 assay kit and, as anticipated, cell viability results were persistent with the results of TUNEL staining analyses (Fig. [100]3D). To verify whether the sensitivity of PDK1 knockout cardiomyocytes increased in hypoxia was due to apoptosis, we checked the expression level of Cleaved-Caspase-3 under 18 h hypoxia. We found a highly significant difference between the groups and quantification (Fig. [101]3E). Additionally, the oxygen consumption rate and extracellular acidification rate of knockout cardiomyocytes was lower significantly than that of WT cardiomyocytes, indicating a significant reduction in the glycolytic rate of KO cardiomyocytes, revealed that the aerobic respiration level of KO-CMs was decreased than that of WT (Fig. [102]3F, G). In knockout cardiomyocytes, the ATP level basal and maximal respiration were significantly lower than in WT cardiomyocytes under hypoxia conditions (Fig. [103]3H, I). Different transcriptome between WT and KO hESC-CMs To examine the biological role of PDK1 and the molecular mechanisms behind PDK1 knockdown, we have done transcriptome sequencing on D30 WT and KO cardiomyocytes. Around 3403 gene expression is varied between WT and KO CMs; 60.6% are upregulated, and 39.3% are down-regulated (Fig. [104]4A). Next, we perform KEGG pathway analysis to determine the enrichment of genes differentially expressed in different metabolic pathways. The highly enriched PI3K-AKT pathway is more closely linked with the biological function of the PDK1 gene and has become our research main focus (Fig. [105]4B). Furthermore, after KEGG evaluation of differential genes, we identified functional 3, 12, 9 and 25 class differences in cell processes, environmental information process, human disease, metabolism and organismal system (Supplementary file 2: Fig. [106]S2A). Metabolism is one of the most important categories of biological functions, indicating that we need to pay more attention to the proteins closely related to metabolism with PDK1 (Fig. [107]4C). Similarly, the third-ranked category, ‘metabolic processes’, once again suggests the significance of PI3K-AKT-related metabolic pathways in the biological function of PDK1 (Fig. [108]4C). Metabolic stress is one of the main causes of myocardial injury leading to heart failure [[109]20]. Previous studies have illustrated a clear difference in the metabolic pattern between Pdk1 knockdown cells and controls [[110]21]. Ischemic heart disease is the result of PDK-KO. IHD affects people globally (1–2%) [[111]22]. Sequencing analysis shows the second-highest gene ratio in ischemia and myocardial infarction (Fig. [112]4D). Gene clustering analysis of the DEGs of the two groups of myocardial cells showed that the gene expression patterns between the two groups were highly homogeneous (Supplementary file 2: Fig. [113]S2B). Fig. 4. [114]Fig. 4 [115]Open in a new tab Transcriptomes are different between pyruvate dehydrogenase kinase 1-deficient and wild-type CMs. (A) Gene expression profile was examined by RNA-seq. The volcano plot shows differentially expressed genes between two groups (n = 3, respectively). Red denotes up-regulated genes, whereas green represents down-regulated genes. (B) GO analyses include three categories, cell components, molecular function and biological process are shown here. (C) Sequencing analysis show second highest gene ratio in ischemia and myocardial infarction. (D) Significantly enriched KEGG (Kyoto Encyclopedia Genes and Genomes) pathways in differential genes. The PI3-AKT pathway enriched the most differential genes with high reliability In summary, PDK1 is involved in a variety of biological processes, especially in maintaining the functional homeostasis of mitochondrial metabolism, and inhibiting the process of cardiomyocyte remodeling. At the same time, the results of RNA-seq also suggest that the levels of apoptosis of cardiomyocytes may change after PDK1 knockout, providing important clues for us to explore the molecular mechanism of remodeling caused by PDK1 deficiency. PDK1-KO induced apoptosis via a Akt-GSK3-β signaling pathway by activating mPTP opening Previous studies stated that apoptosis induced by oxidative stress is closely related to the Akt-GSK3-β signalling pathway [[116]23]. Surprisingly, in the present study, PDK1-KO could drastically reduce the level of p-Akt and cytochrome-c determined by q-PCR (Fig. [117]5A-C). Protein expression of p-Akt, cytochrome-c and GSK3-β determined by Western blot analysis under basal conditions (Fig. [118]5D) (Supplementary file 3: Fig. [119]S3A) and Quantification (Fig. [120]5E). mPTP opening is a key factor in induced mitochondrial-mediated apoptosis [[121]24]. ELISA measured the expression of Cyt-C in the cytoplasm. The result indicated that the expression of Cyt-C increased in the cytoplasm after PDK1-KO (Fig. [122]5F). The mPTP assay kit was performed to measure the openness of mPTP channels. The result of flow cytometry exhibits that the opening of mPTP increased after PDK1-KO and was more sensitive under hypoxia conditions (Fig. [123]5G). Our findings proved that mPTP is a downstream regulator of the Akt-GSK3-β signaling pathway. These results recommended that the mitochondrial mPTP channel opening was significantly increased following PDK1 knockout cardiomyocytes, hence dysregulating the mitochondrial function, consistent with the cytochrome-c release and ultimately regulating oxidative stress-induced apoptosis. Fig. 5. [124]Fig. 5 [125]Open in a new tab Apoptosis of mitochondrial pathway was activated in PDK1 knockout CM’s. (A-C) Expression of P-AKT, GSK3- β and Cyto-C detected by qPCR and suggesting that level of P-AKT and GSK3-β upregulated in knockout cardiomyocytes and Cyto-C level decreases. (D) Expression of P-AKT and Cyto-C verified by the WB results that P-AKT and Cyto-C level downregulated in knockout CM’s. Full-length blots are presented in Supplementary file 5, Fig. [126]7. (E) The quantification data of the WB result. (F) Cytochrome-C level determined by ELISA. Cytochrome-C release was significantly decreases in knockout cardiomyocytes under basal conditions. This effect is more pronounced under hypoxia. (G) As expected, flow cytometry results showed that PDK1-KO cardiomyocytes induce the opening of mPTP. Hence, mPTP served as a downstream process of Akt-GSK3β pathway. This effect is more sensitive under hypoxia. *P <.05, **P <.01, ***P <.001 PDK1 lentivirus transfection rescue the PDK1 deficiency-induced apoptosis and oxidative stress Previous studies have shown that the unbiased gene set enrichment analysis (GSEA) demonstrated that apoptotic cell clearance and metabolism-related gene sets were enriched in the PDK1 knockout cells [[127]25]. To prove that PDK1 deficiency is the cause of the abnormal function of KO cardiomyocytes, we constructed a lentiviral expression vector expressing the entire length of PDK1 to overexpress the PDK1 gene in KO cardiomyocytes (OE) and restore the expression level of PDK1. The EF1A promoter drives the overexpression vector, carries the eGFP green marker protein, and can be screened and purified by puro (Supplementary file 3: Fig. [128]S3B). To examine the role of PDK1 in apoptosis and oxidative stress, we transfected the cardiomyocytes with a PDK1 expression vector. The expression of PDK1 was verified by Western blot (Fig. [129]6A). The results show that knockout of PDK1 expression significantly increased the levels of the cleaved caspase-3 forms while PDK1 overexpression decreased their levels. Furthermore, PDK1 knockout significantly induced apoptosis. As previously discussed, the GSK3-β, caspase-3 and cyto-c expression has been verified by WB (Fig. [130]5D, Supplementary file 3: Fig. [131]S3C). After transfection partially rescued the cells (Fig. [132]6A). Quantification of expression of p-AKT, GSK3-β, Caspase-3, Cyt-C (Fig. [133]6B). These findings showed that Akt-GSK3β signaling pathway controlled the glucose oxidative stress-induced apoptosis. Hence, these results indicate that PDK1 overexpression improves the mitochondrial metabolism. Fig. 6. [134]Fig. 6 [135]Open in a new tab Improving metabolism rescues the myocardial remodeling phenotype of PDK1-KO hiPSC‐CMs by lentivirus transfection. (A) Western blot results confirm the overexpression of PDK1 in PDK1 deficient cells. P-AKT level increased in OE cells as compared to KO. (B) Quantification. Full-length blots are presented in Supplementary file 5, Fig. [136]4. (C) Tunel staining for apoptosis. Hypoxia significantly increased the number of apoptotic cells as compared to Normoxia (Scale bar, 100 μm). Overexpression partially rescue the cells and quantification. (D) ROS levels in WT, KO, and PDK1-KO-OE groups were detected, and the results showed that ROS levels in KO hiPSC-CMs were reduced to varying degrees after overexpression under both conditions. (E) The OCR curves of WT, KO, and KO-OE hiPSC-CMs were detected by Seahorse experiments, the results showed that oxidative phosphorylation was restored in KO hiPSC-CMs after PDK1 overexpression (18 h hypoxia). (F) The ECAR curves of WT, KO, and KO-OE hiPSC-CMs were detected by Seahorse experiments, the results showed that aerobic glycolysis was restored in KO hiPSC-CMs after PDK1 overexpression (18 h hypoxia). (G) The ATP content of WT, KO and PDK1-KO-OE, was detected, and the results showed that the ATP level of KO hiPSC-CMs increased to varying degrees after treatment. (H, I) The basal respiration level of KO hiPSC-CMs was restored after treatment. The maximal respiration level of KO hiPSC-CMs was restored after treatment. (J) Cell viability after lentivirus transfection determine by CCK-8 under Normoxia and 18 h hypoxia state. (K) Cytochrome-C level determined by ELISA. Cytochrome-C release was significantly decreases in knockout cardiomyocytes and partially reverse the effect after PDK1 overexpression. *P <.05, **P <.01, ***P <.001 TUNEL assay was then used to detect apoptosis in cardiomyocytes. As shown in (Fig. [137]6C, Supplementary file 4: Fig. [138]S4A), apoptosis of myocardial cells significantly increased in PDK1 knockout cells but was reduced after lentivirus transfection. Next, the reactive oxygen species level was assessed by immunofluorescence. Our results indicated a significant increase in reactive oxygen species (ROS) production in PDK1 knockout cells relative to controls. At the same time, the level of ROS was attenuated by the overexpression of PDK1 (Fig. [139]6D, Supplementary file 4: Fig. [140]S4B). Collectively, these results propose that PDK1 regulates apoptosis and oxidative stress. Next, the seahorse assay evaluates the myocardial cell group curves of OCR and ECAR. The results illustrated that overexpression of the PDK1 in KO cardiomyocytes improved the metabolism (Fig. [141]6E, F). To determine the outcomes after lentivirus transfection to restore the metabolism in KO cardiomyocytes, first, we used an ATP detection kit to measure the ATP content of each group. The ATP content was restored to varying degrees in knockout cardiomyocytes after transfection (Fig. [142]6G). We also observed that basal and maximal respiration were reused (Fig. [143]6H, I). PDK1 overexpression, cell viability was measured by using a CCK-8 assay kit. Cell viability significantly decreased in transfected cells between the groups (Fig. [144]6J). After transfection, the cytochrome-c level was also rescued in PDK1-KO cells (Fig. [145]6K). These findings verify that improved metabolism in PDK1-deficient cardiomyocytes can restore functional activity after lentivirus transfection in PDK1 deficient cardiomyocytes. Discussion The pathological state of modification in cardiac structure, function and metabolism collectively refers to cardiac remodeling. Various pathogenic factors increase the load during stimulation and lead to myocardial damage, which is the primary cause of heart failure in many cardiovascular diseases [[146]26]. Oxidative stress, necrosis, and apoptosis have been shown to induce subcellular remodeling [[147]27], and apoptosis-induced myocardial injury under hypoxia is the major form of ischemic heart disease. To explore either PDK1-KO cardiomyocytes are more sensitive to hypoxia, we expose the knockout cardiomyocytes to 18-hour hypoxia. Our findings revealed that PDK1 deficiency caused cardiomyocyte apoptosis and oxidative damage in vitro, and metabolic disturbances can promote cardiomyocyte death by increasing reactive oxygen species production and apoptosis, leading to structural and functional changes in the heart and ultimately causing IHD. hiPSCs are human pluripotent cells with the ability to self-renewal and self-replication. They can proliferate indefinitely in vitro and differentiate into cells represent- ing all germ layers, including cardiomyocytes. hiPSCs are an important type of hiPSCs. Cardiomyocytes self-differentiated from hiPSCs by specific differentiation protocols in vitro, express metabolic dysregulation, and exhibit myocardial-specific mitochondrial metabolism disturbance, which can mimic many human cardiomyocyte phenotypes. It has become an important tool for human cardiovascular disease modeling and drug screening [[148]28]. To further study the mechanism of mitochondrial dysfunction and cardiac remodeling, a hiPSC-CM cell model was established. Lawlor et al. prove that there is no heart or heart tube presnet in E9–9.5 PDK1^–/– embryos. This study postulate strong genetic verification that PDK1 is necessary for the embryonic development in mammals, as a complete absence of PDK1 in embryos causes a number of abnormalities that ultimately lead to embryonic death. PDK1^1flox/flox MerCreMer mice died of heart failure from 5 to 21 weeks after deletion of PDK1 at 8 weeks old, demonstrating that PDK1 is essential for maintaining normal cardiac function. In mice with PDK1 cardiac deletion, there is evidence that heart failure may develop as a result of increased apoptotic cell death. Previous studies use PDK1 germline and conditional knockout mouse models to explore the functions of PDK1. In contrast, our CRISPR-Cas9-mediated PDK1 KO model offers a simplified and precise approach to investigate the direct, cell-intrinsic effects of PDK1 loss, particularly under controlled metabolic conditions such as normoxia and hypoxia. This strategy provides mechanistic insights that complement and extend findings from in vivo models [[149]29]. LC-ESI-MS/MS metabolomic analysis revealed significant difference in glycolysis and TCA cycle intermediates, as shown in the heatmap. How PDK1 affect the cardiomyocyte’s glucose oxidation and fuel maintenance has previously been unclear, although PDK1 isoform is highly abundant in the heart [[150]30]. As anticipated, our findings of the metabolomic analysis reveal that significant difference exists in fractional enrichments of metabolites of glycolysis. Hence, it proved that PDK1 dysregulate the glucose metabolism. The data indicate that abnormalities in PDK1 knockout cardiomyocytes may be associated with various metabolic disorders and lowers the oxygen consumption rates. Here, we propose a hypothesis that deletion of PDK1 in cardiomyocytes leads to metabolism dysfunction, which can contribute to irregular heart function and heart failure [[151]11]. Mitochondria is the principal site of myocardial metabolism; therefore, maintaining mitochondrial function and integrity is essential for preserving myocardial structure and biological function [[152]31]. Mitochondria uphold redox balance maintenance and apoptotic signal production. mPTP opening is a key factor for inducing apoptosis mediated by mitochondria [[153]32]. Therefore, we hypothesized that apoptosis induced by oxidative stress is caused by mPTP opening. mPTP is a downstream regulator of GSK3-B and AKT, activated by reactive oxygen species. Cytochrome-c is released from mitochondria to cytosol more rapidly after the opening of mPTP. According to this research, oxidative stress-induced apoptosis was regulated by mPTP opening (Fig. [154]7). Fig. 7. [155]Fig. 7 [156]Open in a new tab Schematic diagram of the proposed mechanism. Mitochondrial oxidative damage via the PDK1/Akt- GSK3-β signaling pathway to exacerbate cardiomyocytes from apoptosis To explore the mechanism of PDK1-induced cell apoptosis and oxidative damage, high-throughput transcriptome sequencing analysis of PDK1 deficiency cardiomyocytes and controls was used. The volcano analysis and KEGG enriched pathway displayed that the AKT pathway was the major contributor to the abnormal metabolic pattern. In several disease models, AKT attenuates apoptosis and oxidative stress-mediated injuries [[157]33]. Additionally, low AKT levels have been strongly associated with cardiac dysfunction and the development of IHD. Consistent with Yan et al. study, oxidative increases cell apoptosis and decreases cell viability significantly, as indicated by the results of flow cytometry, TUNEL assays, and Western blot [[158]34]. In addition, PDK1 deficiency increased the levels of pro-apoptotic proteins (caspase-3), GSK3- β and oxidative stress in cardiomyocytes. Previous studies stated that mitochondrial redox homeostasis and apoptosis are affected by the Akt-GSK3-β signaling pathway [[159]35]. Thus, we investigate whether or not this pathway was involved in oxidative stress-induced apoptosis in cardiomyocytes. Our findings showed significant suppression of Akt and downregulate the phosphorylation of GSK3-β induced by oxidase stress. Further, we showed that Akt-GSK3-β acts as the upstream regulator of mPTP channels, which was constant with the previous studies on animal models with heart ischemia-reperfusion injury [[160]36]. Current study found that myocardial cells lacking PDK1 showed cardiac remodeling phenotypes in vitro, such as abnormal ROS production and oxidative respiratory dysfunction (functional abnormalities), increased mitochondrial damage, upregulated expression of apoptosis-related genes (molecular biological abnormalities), etc., providing evidence to prove that PDK1 deficiency in the myocardium is related to cardiac remodeling. Based on the pathological mechanism, we evaluated that lentivirus transfection can improve mitochondrial metabolic pathways for potential therapeutics and treatment. The metabolic dysregulation of PDK1-knockout cardiomyocytes can be restored partially, ATP production enhanced, and apoptosis and oxidative stress were reduced. Therefore, we predict that after transfection, it can better control the pathological progression of myocardial damage and achieve a good therapeutic effect. Conclusions we provide the foremost genetic evidence that PDK1 plays various roles in regulating cardiac viability. “Our CRISPR-mediated PDK1 knockout model underscores the critical role of PDK1 in Ischemic Heart Disease. These findings are consistent with previous studies using PDK1 KO and conditional lethal models, which have demonstrated significant phenotypic consequences in various disease contexts, including tumorigenesis and renal pathophysiology (e.g., Faubert et al., 2013; Lawlor et al., 2002). Such models in cancer and kidney disease have highlighted the essential function of PDK1 in cell survival and metabolism, reinforcing the translational relevance of our findings [[161]28, [162]37]. Our study suggests that PDK1-KO cardiomyocytes develop the phenotype of IHD and are more sensitive to hypoxia. Current study verified that oxidative stress aggravated mPTP opening through an inhibited Akt-GSK3-β signaling pathway, causing hypoxia-induced apoptosis. These findings highlight the potential target of PDK1 metabolism as a novel therapeutic strategy for (IHD). Study outcomes contribute to our understanding of the pathological mechanism behind ischemic heart injury and may have consequences for clinical treatment. Electronic supplementary material Below is the link to the electronic supplementary material. [163]Supplementary Material 1^ (1.5MB, mzml) [164]Supplementary Material 2^ (1.5MB, mzml) [165]Supplementary Material 3^ (1.5MB, mzml) [166]Supplementary Material 4^ (1.5MB, mzml) [167]Supplementary Material 5^ (1.5MB, mzml) [168]Supplementary Material 6^ (1.5MB, mzml) [169]Supplementary Material 7^ (4.6MB, docx) Acknowledgements