Identification of a Mutation in the PFKP as a Causative Factor in Prenatal Glycolysis Defects and Embryonic Myocardial Hypoplasia

Identification of a Mutation in the PFKP as a Causative Factor in Prenatal Glycolysis Defects and Embryonic Myocardial Hypoplasia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of a Mutation in the PFKP as a Causative Factor in Prenatal Glycolysis Defects and Embryonic Myocardial Hypoplasia Yihua He, Siyao Zhang, Hairui Sun, Xiaoyan Hao, Xu Zhi, ruimin Liu, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6341289/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Congenital heart disease (CHD) is the most common birth defect worldwide, which lacks effective early preventive methods due to limited knowledge of the genetic defects involved in its development. Through genetic analysis of families with congenital heart disease (CHD), we identified a genetic correlation between the R755W variant of the platelet isoform of phosphofructokinase 1 (PFKP) and myocardial hypoplasia. Furthermore, PFKP R754W/R754W and PFKP knockout mice exhibited a myocardial hypoplasia phenotype during embryonic development. Mechanistic studies further revealed that PFKP deficiency reduces embryonic cardiac glycolysis, leading to decreased cardiomyocyte proliferation due to altered downstream metabolites and metabolic pathways. Importantly, intrauterine supplementation with fructose 1,6-bisphosphate (F-1,6-BP), a direct product of PFKP catalysis, was able to rescue myocardial hypoplasia in fetal mice. Conversely, inhibiting glycolysis using 2-deoxyglucose (2-DG) reproduced the myocardial hypoplasia phenotype in both fetal mouse hearts and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). These findings establish PFKP as a critical regulator of glycolysis during embryonic cardiac development. They also provide novel insights suggesting that glycolytic defects or intrauterine hypoglycemia may represent common causes of myocardial hypoplasia. This research highlights potential applications for genetic interventions, prenatal screening, and targeted intrauterine therapeutic strategies. Health sciences/Cardiology/Cardiovascular biology/Heart development Biological sciences/Genetics/Clinical genetics/Disease genetics congenital heart disease phosphofructokinase 1 metabolism embryonic development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Congenital heart disease (CHD) is the most common birth defect worldwide, with a prevalence of 8–12‰ 1 . The factors that contribute to CHD include environmental exposure, maternal illness, genetics, and a combination of these 2 . However, the limited understanding of congenital heart disease genetic causes results in a lack of effective prevention and intervention measures. During embryonic development, the initial myocardial contraction and myocardial proliferation and expansion rely heavily on energy metabolism 3 , 4 . Glycolysis serves as the primary energy source for developing embryonic hearts and supplies essential biosynthetic precursors required for cell proliferation 4 . The continuous proliferation of embryonic cardiomyocytes is critical for cardiac embryonic development 3 , 5 . Consequently, dysregulation of key genes associated with glycolysis has been implicated as a potential pathogenic factor in CHD, as evidenced by studies in lower organisms such as zebrafish. Therefore, the dysregulation of critical genes associated with glycolysis may be a pathogenic factor of CHD. However, the relationship between glycolysis and human embryonic heart development remains largely unexplored. This knowledge gap can be attributed to the high proportion of complex CHD cases that result in intrauterine or prehospital deaths, thereby limiting our understanding of the pathogenesis and etiology of CHD during the embryonic stage. In this study, we identified a distinct clinical manifestation of CHD, myocardial hypoplasia, through next-generation sequencing of 1,650 fetal heart disease family lines from a comprehensive 100,000-case database at the nationwide Fetal Heart Disease Maternal-Fetal Medicine Center. Myocardial hypoplasia is characterized by structural defects that do not align with coronary artery distribution and was observed to cluster in two non-consanguineous families. Furthermore, whole-genome sequencing (WGS) and functional assays revealed specific CHD phenotypes and functional defects associated with a deficiency in the platelet isoform of phosphofructokinase 1 (PFKP), a key rate-limiting enzyme in glycolysis. These findings provide new insights into the clinical and genetic basis of CHD, contributing to an expanded understanding of its pathogenesis. More importantly, these findings underscore the critical role of glycolysis in cardiac embryonic development and suggest it as a promising therapeutic target for intrauterine interventions, potentially achievable through environmental modifications. Methods Study subjects Two large pedigrees exhibiting CHD as an autosomal dominant trait with variable expressivity and complete penetrance were recruited (Fig. 1 ). Clinical evaluations of the affected fetuses, children, and adult in Family 1 and Family 2 were performed using diagnostic modalities, including echocardiography, cardiovascular magnetic resonance (CMR), and electrocardiograms. DNA samples were obtained from affected and control individuals from both families and subjected to rigorous WGS analysis. Written informed consent was obtained from all study participants or their legal guardians, and the study was approved by the institutional review board of the Medical Ethics Committee of Beijing Anzhen Hospital. The authors confirm the accuracy and completeness of the data used in this study. Genetic analysis WGS was performed with the DNA of patients and controls. Then, linkage analysis and variant calling, annotation, filtering, and prioritization were performed. The pathogenicity of sequence variants was assessed according to ACMG guidelines. Detailed protocols are provided in the supplementary materials. Mice Myh6 Cre/+ , Pfkp flox/flox and Pfkp R754W/R754W mice were acquired from the Shanghai Model Organism Centre (Shanghai, China). WT, Pfkp R754W/+ , and Pfkp R754W/R754W littermates were used as controls. Offspring from homozygous Pfkp flox/flox mice were used as controls. Additionally, heterozygous floxed mice (Pfkp flox/+ ) were generated by crossbreeding Pfkp flox/flox mice with wild-type mice. Pfkp flox/+ mice were crossed with Myh6 Cre/+ mice to produce offspring for comparisons with the Pfkp flox/flox × Myh6 Cre/+ crossbred group. The genotypes of the embryonic mice were confirmed by tail DNA extraction and subsequent genotyping using polymerase chain reaction (PCR) analysis. Pregnant wild-type C57BL/6J mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). software (Beijing, China). Mice were bred and housed in a specific pathogen-free environment with a 12-h dark/light cycle at 23 ± 2°C and 40–70% humidity. Mice whose body weight fell below two standard deviations (< 2SD) from the average weight of their littermates were not used for breeding. The detailed protocols for the construction of the mouse model and mouse genotyping are provided in the supplementary materials. Subsequent experiments, including histology, quantitative real-time PCR, Western blot, immunofluorescence, metabolomic analysis, and PFK enzymatic activity assays, were conducted to validate the findings and investigate the underlying mechanisms. All experimental procedures were approved by the Animal Subjects Committee at Beijing Anzhen Hospital. Cell culture and cardiac differentiation H9 and hiPS cells were maintained on feeder-free Matrigel (Corning) and fed E8 medium (CellaPy) daily. Cells were routinely passaged every 3 days at 70–80% confluency using 0.5 mM EDTA in phosphate-buffered saline (PBS) without MgCl 2 or CaCl 2 (HyClone, USA). The cells were cultured at 37°C with 5% CO 2 . Human pluripotent stem cells (hPSCs) were differentiated into hPSC-CMs using a small-molecule-based method as previously described 6 . The hPSC-CMs were purified using the lactate metabolic selection method 7 . Peripheral blood mononuclear cells (PBMCs) were extracted from both patients (III-4) and unaffected family members (III-7). Subsequently, the PBMCs were reprogrammed into hiPS cells, and the PFKP R755W mutation was corrected through homologous recombination. This correction yielded hiPS (PFKP Correct/+), possessing an identical genetic background to the patient but devoid of the PFKP R755W mutation. Subsequent experiments, including quantitative real-time PCR (qRT-PCR), Western blot (WB), flow cytometry, immunofluorescence, Seahorse ECAR measurement, metabolomic analysis, and PFK enzymatic activity assays, were conducted to validate the findings and investigate the underlying mechanisms. Detailed protocols are provided in the supplementary materials. Statistical analysis Descriptive statistics for continuous variables are shown as the means ± standard deviation, while categorical variables are displayed as subject counts and percentages. Student’s t-test and ANOVA (followed by Tukey’s post hoc test) were used for the analysis of continuous variables after performing the Shapiro-Wilk normality test to obtain normal distribution of the data. Categorical variables were evaluated using Fisher’s exact test. If the data did not have a normal distribution or N was too small to determine normality, statistical significance was confirmed using non-parametric tests (Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparison test or uncorrected Dunn's multiple comparison test). For non-normal data, a non-parametric Mann-Whitney U test was used. Statistical analyses were performed using the Prism 8.0 (GraphPad Software). Statistical significance was set at P < 0.05. Results 1. Identification of PFKP R755W pathogenic variants in congenital myocardial hypoplasia families We conducted in-depth examinations of families of consecutive fetuses with CHD and significant myocardial hypoplasia (Fig. 1 A). In Family 1, early pregnancy loss occurred at 3 weeks of gestation of the first fetus, with exact aetiology unknown (IV-1). At 24 weeks of gestation of the second fetus, fetal echocardiography revealed myocardial hypoplasia (Fig. 1 A, IV-2). The fetus (IV-2) had myocardial thinning in the ventricular septum and congenital ventricular aneurysms in the middle to apical segments of the left ventricular free wall (Fig. 1B1, IV-2). This fetus also exhibited secondary cardiac dysfunction, as indicated by a Cardiovascular Profile Score 8 (CVPS) of 7 points, suggesting a high probability of postnatal mortality due to the identified cardiac anomalies in the current pregnancy. Consequently, the parents opted for induced labour after receiving adequate counselling. The third fetus (Fig. 1B2, IV-3) showed a cardiac phenotype similar to that of the previous one, resulting in a CVPS score of 6 points. Based on these findings, the family decided to terminate the pregnancy for a second time and proceed with a postmortem examination, which confirmed the prenatal diagnosis (Fig. 1B3). Upon reviewing the family's medical history, it became evident that this condition affected members of three consecutive generations. Notably, the father (Fig. 1 A, III-2) exhibited a similar clinical phenotype, with 40% ejection fraction and heart failure (Table S1). The most affected family members exhibited concurrent structural cardiac abnormalities, such as thinning of the apical ventricular septum and blunt left ventricular apex (Fig. 1 C), and atrial or ventricular septal defects (ASD or VSD) (Fig. S1A) along with the right bundle branch block (Table S1, Fig. S1B). The consistent phenotype observed within this familial lineage aligned with an autosomal-dominant mode of inheritance (Fig. 1 A). WGS was performed to investigate the genetic aetiology. Initial investigations into genes associated with CHD failed to yield positive results; therefore, we proceeded with a genome-wide linkage analysis. This analysis identified a co-segregating region of 5.8 Mb on chromosome 10 (p15.3-p15.1), with a logarithm of odds (LOD) score of 2.4, exceeding the threshold of 2.3, indicating a potential genetic link between this region and the cardiac phenotype in Family 1. In this region, we identified the only rare heterozygous PFKP (NM_002627.4: c.2263C > T; NP_002618.1: p.R755W) missense variant, which co-segregated with the cardiac phenotype of Family 1. (Table S1, Fig. S1C-D). To further determine the correlation between PFKP and CHD, we screened for the PFKP variants from a local fetal heart disease exome sequence database comprising 1650 fetal heart disease pedigrees and found the other CHD family with the same PFKP variants (c.2263C > T [p.R755W]) with Family1. We observed clinical manifestations in family 1 similar to those in family 2(Table S1, Fig. 1 D). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), we considered PFKP a gene of uncertain significance, and PFKP R755W in the two families was considered a variant of the gene of uncertain significance (Table S2). Next, we functionally analysed the PFKP R755W variant. The PFKP R755 residue is highly conserved in vertebrates (Fig. 1 E), and structural analysis of PFKP proteins indicated that R755W disrupts protein conformation and function (Fig. S1E). To understand the impact of PFKP R755W in cardiac development, we generated induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) from the healthy family member (WT), a patient (PFKP R755W/+ ), and R755W corrected cells (PFKP Correct/+ ; generated using genome editing) (Fig. S2A-C). Compared to PFKP Correct/+ CMs, PFKP R755W/+ CMs showed a 23.5% reduction in PFK1 activity with no significant changes in PFKP protein levels (Fig. 1 F, Fig. S2E). 2. Pfkp mice mimic embryonic myocardial hypoplasia To investigate the role of PFKP in embryonic heart development, we analyzed the expression pattern of PFKP during mammalian heart development. Gene expression data from the Expression Atlas ( https://www.ebi.ac.uk/gxa/home ) showed that PFKP was predominantly expressed in the human embryonic heart over the other two PFK1 isoforms (PFKM and PFKL) (Fig. S3A, B). Moreover, we verified high expression levels of PFKP in both human s (Fig. S3C-E) and mouse embryonic hearts (Fig. S3F-H). Notably, PFKP expression peaked in early stages, and immature cardiomyocytes (10-day and 15-day hiPS-CMs) 9 , when these cells were highly proliferative (Fig. S3H-J). These findings indicate that PFKP plays a critical role in embryonic cardiac development. Next, we generated a mouse model carrying a PFKP R755W equivalent variant (mouse Pfkp R754W) (Fig. S4A). The hearts of Pfkp R754W/R754W mice exhibited a 34.2% reduction in PFK1 enzyme activity at embryonic day 12.5 (E12.5) compared with that in the controls (Fig. 2 B), with no significant difference in PFKP protein expression (Fig. 2 A), which effectively mimicked the reduced enzyme activity phenotype observed in patient-derived hiPS-CMs. To better mimic the myocardial phenotype caused by reduced human PFK1 enzyme activity, we selected Pfkp R754W/R754W mice for subsequent experiments. Next, we performed histological analysis of the hearts of Pfkp-mutant and WT embryos to examine heart development from E13.5 to E17.5. The heart sizes of homozygous (Pfkp R754W/R754W ) mutation mice were smaller at E17.5 compared with those of the controls (Fig.S4B). However, examination of the LV and RV compact layer thickness revealed that Pfkp R754W/R754W mice had significant thinning of the LV compact layer at E15.5 and E17.5 and the RV compact layer at E17.5 (Fig. 2 E-G, Fig.S4C). These findings indicate that PFKP mutation leads to reduced PFK1 enzyme activity and abnormal embryonic myocardial morphology, including thinning of the compact layer of the ventricular walls and reductions in heart size. To further validate the relationship between defective PFKP function and cardiac embryonic development, we created a myocardium-specific knockout (cKO) mouse model using Myh6-driven Cre to eliminate PFKP, resulting in the loss of PFKP expression in E12.5 mice hearts (Fig. 2 C). In PFKP-knockout mice, E12.5 heart PFK1 activity was decreased by 35.96% in heterozygotes and 47.07% in homozygotes (Fig. 2 D). Histological assessment demonstrated thinning of the LV and RV compact layers in the hearts of heterozygous (Pfkp cKO/+ ) and homozygous (Pfkp cKO/cKO ) knockout mice at E15.5 and E17.5 (Fig.S5A-D). The heart sizes of heterozygous (Pfkp cKO/+ ) and homozygous (Pfkp cKO/cKO ) knockout mice were smaller at E17.5 compared with those of the controls (Fig. S5E). Compared with that of Pfkp cKO/cKO mice, Pfkp cKO/+ and Pfkp R754W/R754W mice exhibited a milder cardiac phenotype, demonstrating a dose-dependent effect of PFK1 enzyme activity. Subsequently, the family (Fig. 1 A; III-1 and III-2) pursued assisted reproductive technologies (Fig. 2 H). Normal PFKP gene of the fetus were confirmed via an umbilical cord blood test, which aligned with the embryo test results. At 19 and 28 weeks of gestation, the fetal heart function cardiac structures were within the normal range for healthy reference populations. We followed up with the baby until three months after birth, and the baby's cardiac structure and function (EF: 65%) were normal (Fig. 2 I). These findings provide valid anthropological evidence that PFKP defects cause congenital myocardial hypoplasia. 3. Pfkp deficiency impairs embryonic myocardial proliferation in vivo and in vitro PFKP defects reduce tumor cell proliferation, primarily due to impaired PFK1 enzymatic activity 10 , 11 . Myocardial proliferation is a key mechanism in embryonic heart development. To investigate the mechanisms underlying PFKP-induced myocardial hypoplasia, we examined the proliferative function of patient-derived PFKP R755W/+ hiPSC-CMs. Immunofluorescence and flow cytometry results showed that PFKP R755W/+ hiPSC-CMs exhibited a decreased proliferation markers Ki67 ratio in early-stage cardiomyocytes 9 (10-day and 15-day) (Fig. 3 A-C, Fig.S6A). A similar reduction in myocardial proliferation was observed in Pfkp R754W/R754W mice at different embryonic times (Fig. S6B). To better understand the link between PFKP deficiency and embryonic myocardial proliferation, we established PFKP knockout hESC-CMs (Fig.S7B-D). Consistent with the reduced embryonic myocardial proliferation observed in PFKP knockout mice (Fig.S7A), flow cytometry analysis revealed a decrease in proliferation markers in the PFKP knockout hESC-CMs(Fig.S7F, G). Lentivirus-mediated PFKP overexpression in PFKP-KO hESC-CMs effectively restored cardiomyocyte proliferation and PFK1 enzyme activity. These findings show that PFKP deficiency leads to reduced embryonic cardiomyocyte proliferation(Fig.S7B-G). 4. Pfkp mutation leads to dysregulated glycolytic function and reduced downstream metabolites PFKP primarily functions as a key rate-limiting enzyme in glycolysis. To test whether PFKP defects result in reduced glycolytic function, we examined the glycolytic function in PFKP R755W/+ hiPSC-CMs using Seahorse ECAR experiments. The results showed that PFKP R755W/+ hiPSC-CMs exhibited significantly reduced glycolytic function, including basal glycolysis, glycolytic capacity, and glycolytic reserve (Fig. 4 A). These findings suggest that PFKP mutation impairs cardiomyocyte proliferation and glycolysis, which may contribute to abnormal myocardial development. To comprehensively assess the metabolic changes in embryonic hearts following PFKP mutation, we performed metabolomic profiling of the hearts of control and Pfkp R754W/R754W mice at E15.5 using MALDI-MSI to assess the spatial distribution of metabolites. Principal component analysis (PCA) revealed significant metabolic differences between the control and Pfkp R754W/R754W hearts (Fig. 4 B). Metabolite set enrichment analysis (MSEA) revealed that Pfkp mutation disrupted key energy metabolic pathways, including carbohydrate and amino acid metabolism in whole embryonic hearts (Fig. 4 C, and Fig. S8A). After birth, the left ventricle bears a higher pumping pressure to sustain the systemic circulation. Therefore, we first focused on metabolic changes in the left ventricular wall of Pfkp R754W/R754W mice. The results showed a significant decrease in carbohydrate metabolite levels, with a corresponding upregulation in fatty acid and hormone metabolite levels in the left ventricle wall of Pfkp R754W/R754W mice (Fig. 4 C and 4 D). Notably, the levels of succinic acid, α-KG, and PPP metabolic intermediates were significantly reduced, and these intermediates can provide precursor substances for myocardial proliferation (Fig. S8C). Additionally, fatty acid-related pathways were upregulated in Pfkp R754W/R754W mice, which may compensate for the reduced energy due to decreased glycolysis through metabolic reprogramming (Fig. 4 E, Fig. S8C). However, this shift towards fatty acid metabolism reprogrammed the myocardial epigenome and inhibited myocardial proliferation 12 . Furthermore, we analysed differential metabolites in the LV and RV of normal embryonic mice. In the control mice, compared with the LV compact layers, 26 metabolites were significantly upregulated and 27 downregulated in the RV compact layers (Fig. S8B). However, in Pfkp R754W/R754W mice, there were no metabolic differences between the left and right ventricular walls, suggesting that Pfkp mutation may disrupt distinct metabolic profiles typically observed in the left and right ventricular walls (Fig. S8B). Pfkp knockout produced similar effects on the right and left ventricular walls (Fig.S8D, E). These results suggested that Pfkp deficiency results in the downregulation of the metabolic pathways that supply essential precursors to the embryonic myocardium. We performed Seahorse ECAR experiments and targeted mass spectrometry using PFKP knockout hESC-CMs to confirm these findings. Consistent with the mouse embryonic heart results, the levels of glycolytic metabolites and byproducts of the PPP and nucleotide biosynthesis pathways were significantly reduced (Fig. S9). These data suggested that Pfkp deficiency disrupts glycolysis and its associated pathways. PFKP mutation significantly downregulate energy metabolic pathways. We hypothesised that PFKP mutation impairs myocardial proliferation by reducing ATP levels, which in turn alters AMPK and mTOR activity, which are key regulators of energy metabolism. To verify whether PFKP deficiency activates the AMPK pathway by reducing ATP levels, we analysed AMPK levels in the hearts of E15.5 embryos from Pfkp R754W/R754W mice. The results showed that PFKP mutation significantly upregulated AMPK activation (Fig. 4 F). Additionally, AMPK activation inhibits mTOR activity, ultimately reducing protein synthesis and mRNA translation 13 , which impairs myocardial proliferation during embryonic development (Fig. 4 F, G). These results suggested that PFKP mutation reduced glycolysis, activated AMPK, and inhibited the mTOR pathway, thereby diminishing myocardial proliferation. 5. F-1,6-BP rescues PFKP mutation phenotypes F-1,6-BP is an endogenous intermediate that is generated by the PFK1 enzyme. Given that PFKP deficiency impairs glycolytic function, leading to abnormal embryonic myocardial proliferation and development, we hypothesised that F-1,6-BP supplementation would restore the glycolytic flux and alleviate myocardial hypoplasia. To test this, we treated WT, PFKP Correct/+ , PFKP R755W/+ hiPS-CMs with F-1,6-BP (5mM) on the 8th day of differentiation. Cardiomyocyte proliferation was assessed on day 15 of differentiation (Fig. 5 A). We observed that F-1,6-BP successfully rescued the PFKP mutation-induced reduced cardiomyocyte proliferation (Fig. 5 B-D). We injected each mouse fetus with F-1,6-BP (75 ng/mouse fetus) via intra-amniotic injection at E12.5 and harvested the hearts at E17.5 for comprehensive phenotypic assessment. Administration of F-1,6-BP in the early embryonic period effectively rescued the thinning of the LV and RV compact layers (Fig. 5 E, 5 G) and restored myocardial proliferation in Pfkp R754W/R754W mice (Fig. 5 F, 5 H). Overall, these findings suggest that F-1,6-BP can rescue abnormal cardiac development and decrease proliferation caused by PFKP mutation, highlighting its potential therapeutic application in CHD. 6. Impaired non-genetic glycolytic function causes abnormal embryonic myocardial proliferation To explore the effect of non-genetic glycolytic dysfunction on immature cardiomyocyte proliferation, we treated hESC-CMs in the early cardiac development stages in vitro with low glucose concentrations and the glycolytic inhibitor 2DG (Fig. 6 A). We found that lowering glucose concentrations to 7.5 mM and the addition of 3 mM 2DG decreased hiPS-CM proliferation on day 13 of differentiation (Fig. 6 B, 6 C). We further inhibited glycolytic activity in embryonic mice via intraperitoneal injection of a high concentration of 2DG (500 mg/kg per day) into pregnant mice on day E12.5 (Fig. 6 D). Compared to WT + Saline mice, WT + 2DG mice had a significantly increased ratio of embryonic abortions (Fig. 6 E). At E17.5, 2DG treatment decreased the left ventricular myocardium thickness (Fig. 6 F) and myocardial proliferation (Fig. 6 G). These results suggested that reduced glycolysis without genetic causes leads to embryonic myocardial hypoplasia and impaired cardiomyocyte proliferation, offering potential strategies for preventing CHD. Discussion CHD is the most common birth defect worldwide 1 . The lack of knowledge about the genes involved in its development has led to a shortage of effective preventive methods. Identifying the pathogenic mechanisms of CHD could aid in establishing effective prevention and intervention strategies. The dysregulation of cardiomyocyte proliferation plays a critical role in the pathogenesis of most CHD cases 14 – 16 . Our study presents, for the first time, a case with multiple manifestations of myocardial hypoplasia in the ventricular myocardium, accompanied by structural cardiac defects, specifically VSD, ASD, and arrhythmia. Cardiomyocyte proliferation is essential for embryonic heart development; however, this process halts after birth. Teratogens that disrupt cardiomyocyte proliferation during embryonic development can lead to heart malformations. After excluding congenital coronary artery lesions, we hypothesised that reduced cardiomyocyte proliferation was the likely mechanism underlying the clinical phenotype of a thinning embryonic myocardium. During embryonic development, myocardial proliferation is highly dependent on energy metabolism 3 . Unlike the energy supply mechanisms in the adult heart, glycolysis is the primary energy supply for the development of the embryonic heart 4 , 17 . Although some critical genes associated with glycolysis have been considered potential pathogenic factors for CHD, there is currently no clinical evidence directly linking the genetic variants of glycolytic genes to CHD. Studying the clinical phenotypes of the human embryonic heart and the underlying genetic mechanisms presents significant challenges. These challenges arise from the difficulty in obtaining clinical phenotypes, variations in the expression of genes related to embryonic development before and after birth, genetic compensatory mechanisms, and other factors that complicate this study. Combining the results of the linkage and WGS analyses, we identified PFKP R755W as the likely causative mutation for the cardiac phenotype observed in the family. Furthermore, offspring produced after clinically blocking the genetic transmission of PFKP R755W using assisted reproductive techniques showed no cardiac pathogenic phenotypes. This provides strong anthropological evidence for the pathogenicity of PFKP R755W. We observed that some adult patients with pathogenic PFKP variants did not show a significant reduction in cardiac ejection fraction. However, the prognosis of fetuses with PFKP mutations is poor, likely due to the polygenic nature of CHD and its interaction with the intrauterine environment. After confirming that functional defects in key glycolytic genes were associated with reduced embryonic myocardial proliferation, we identified the energy metabolic pathways and substrates that regulate this process through mechanistic studies, offering evidence for potential maternal interventions during embryonic development. Understanding the energy metabolism pathways and substrate changes underlying teratogenicity in CHD is expected to facilitate the prevention of birth defects in the future. We inhibited glycolysis by specifically knocking out PFKP in the heart during mouse embryonic development, which resulted in abnormalities in other metabolic pathways (Fig. 4 ). Downregulation of the levels of metabolites such as α-KG and succinate in PFKP-deficient mice can promote myocardial regeneration after myocardial injury 18 , 24 . The decrease in glycolysis likely triggers metabolic reprogramming to maintain energy production via alternative pathways, such as fatty acid metabolism; however, this comes at the cost of reduced nucleotide availability and impaired cell proliferation 12 . Recent research has also highlighted the co-regulation of metabolic pathways 12 , 18 – 21 , including glycolysis, as a contributing factor to myocardial proliferation and regeneration following cardiac injury. The absence of reduced glycolysis in the embryo leads to abnormalities in energy metabolic pathways such as AMPK activation and mTOR inhibition. These may be key mechanisms by which reduced glycolysis impairs cardiac proliferation. Impaired nongenetic glycolysis mimicked myocardial hypoplasia phenotypes in the hearts of fetal mice and hPSC-CMs (Fig. 6 ). This is consistent with our recent findings in an 8-million-case cohort, which indicated that hypoglycaemia increases the risk of birth defects in offspring (unpublished). Supplementation with the downstream glycolysis substrate F-1,6-BP reduced cell proliferation and myocardial hypoplasia (Fig. 5 ). Our research provides strong evidence for early intrauterine intervention in fetuses with genetic defects as well as for glycaemia management in individuals with abnormal intrauterine glycolysis. Some studies have highlighted the role of high PFKP expression in cardiac hypertrophy 22 ; however, its involvement in chronic heart failure remains unclear. Exploring the regulatory mechanisms of glycolysis in chronic heart failure may reveal potential therapeutic targets. F-1,6-BP has been used to treat chronic heart failure 23 , 24 , but not in pregnant women, requiring further safety evaluation. By identifying phenotypes, confirming the underlying genetic pathogenicity, and exploring underlying mechanisms, we have broadened the phenotypic and genetic pathogenicity spectrum of CHD and, more importantly, revealed possible shared mechanisms that influence cardiac development. These findings also suggest potential options for intrauterine interventions in the future. Declarations Disclosure of Interest None. Funding This study was supported by the National Natural Science Foundation of China (No. 82200334 to Xiaoyan Hao; No U21A20523 and 82170301 to Yihua He; No. 82100322 to Tong Yi). Acknowledgments We thank Professor Dong Zhao (Beijing Anzhen Hospital, Capital Medical University), Professors Yimin Hua and Yifei Li (West China Second University Hospital, Sichuan University, China) for their assistance. References Global, regional, and national burden of congenital heart disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. 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Wu T, Liang Z, Zhang Z, Liu C, Zhang L, Gu Y, Peterson KL, Evans SM, Fu XD and Chen J. PRDM16 Is a Compact Myocardium-Enriched Transcription Factor Required to Maintain Compact Myocardial Cardiomyocyte Identity in Left Ventricle. Circulation . 2022;145:586-602. Gaspar JA, Doss MX, Hengstler JG, Cadenas C, Hescheler J and Sachinidis A. Unique metabolic features of stem cells, cardiomyocytes, and their progenitors. Circ Res . 2014;114:1346-60. Shi Y, Tian M, Zhao X, Tang L, Wang F, Wu H, Liao Q, Ren H, Fu W, Zheng S, Jose PA, Li L and Zeng C. α-Ketoglutarate promotes cardiomyocyte proliferation and heart regeneration after myocardial infarction. Nat Cardiovasc Res . 2024;3:1083-1097. Bae J, Salamon RJ, Brandt EB, Paltzer WG, Zhang Z, Britt EC, Hacker TA, Fan J and Mahmoud AI. Malonate Promotes Adult Cardiomyocyte Proliferation and Heart Regeneration. Circulation . 2021;143:1973-1986. Gao F, Liang T, Lu YW, Pu L, Fu X, Dong X, Hong T, Zhang F, Liu N, Zhou Y, Wang H, Liang P, Guo Y, Yu H, Zhu W, Hu X, Chen H, Zhou B, Pu WT, Mably JD, Wang J, Wang DZ and Chen J. Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration. Circulation . 2023;148:1887-1906. Magadum A, Singh N, Kurian AA, Munir I, Mehmood T, Brown K, Sharkar MTK, Chepurko E, Sassi Y, Oh JG, Lee P, Santos CXC, Gaziel-Sovran A, Zhang G, Cai CL, Kho C, Mayr M, Shah AM, Hajjar RJ and Zangi L. Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration. Circulation . 2020;141:1249-1265. Vigil-Garcia M, Demkes CJ, Eding JEC, Versteeg D, de Ruiter H, Perini I, Kooijman L, Gladka MM, Asselbergs FW, Vink A, Harakalova M, Bossu A, van Veen TAB, Boogerd CJ and van Rooij E. Gene expression profiling of hypertrophic cardiomyocytes identifies new players in pathological remodelling. Cardiovasc Res . 2021;117:1532-1545. Munger MA, Botti RE, Grinblatt MA and Kasmer RJ. Effect of intravenous fructose-1,6-diphosphate on myocardial contractility in patients with left ventricular dysfunction. Pharmacotherapy . 1994;14:522-8. Alva N, Alva R and Carbonell T. Fructose 1,6-Bisphosphate: A Summary of Its Cytoprotective Mechanism. Curr Med Chem . 2016;23:4396-4417. Additional Declarations There is NO Competing Interest. Supplementary Files supplementaryappendix.docx Α StructuredGraphicalAbstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6341289","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437972240,"identity":"614f9aaa-31eb-4cef-a4a3-74bf4c61e226","order_by":0,"name":"Yihua He","email":"data:image/png;base64,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","orcid":"","institution":"Capital Medical University; Beijing Anzhen Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yihua","middleName":"","lastName":"He","suffix":""},{"id":437972241,"identity":"8428efdf-a1ab-4ed2-bf2b-e936610a3acc","order_by":1,"name":"Siyao Zhang","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Siyao","middleName":"","lastName":"Zhang","suffix":""},{"id":437972242,"identity":"d46d5cdf-1a77-4946-87e6-9abb2049767b","order_by":2,"name":"Hairui Sun","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hairui","middleName":"","lastName":"Sun","suffix":""},{"id":437972243,"identity":"fd6bc923-93a2-4222-b4b4-4a44490d812d","order_by":3,"name":"Xiaoyan Hao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Hao","suffix":""},{"id":437972244,"identity":"152214ff-0cbc-43ac-9ba1-a129d85a0714","order_by":4,"name":"Xu Zhi","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Zhi","suffix":""},{"id":437972245,"identity":"96386622-eaf3-4193-b27e-92da7ce3fac5","order_by":5,"name":"ruimin Liu","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"ruimin","middleName":"","lastName":"Liu","suffix":""},{"id":437972246,"identity":"6c3e7c1a-0eac-49f7-b25d-2b9aed4ff64b","order_by":6,"name":"pengyun Gong","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"pengyun","middleName":"","lastName":"Gong","suffix":""},{"id":437972247,"identity":"f35f4e9e-c702-4b25-b9c4-e30a94020569","order_by":7,"name":"Jianbin Wang","email":"","orcid":"https://orcid.org/0000-0001-6725-7925","institution":"Tsinghua University","correspondingAuthor":false,"prefix":"","firstName":"Jianbin","middleName":"","lastName":"Wang","suffix":""},{"id":437972248,"identity":"9df6edab-9708-4c65-b42a-beb7cbbd2d0b","order_by":8,"name":"ming Hao","email":"","orcid":"","institution":"Tsinghua University, Beijing","correspondingAuthor":false,"prefix":"","firstName":"ming","middleName":"","lastName":"Hao","suffix":""},{"id":437972249,"identity":"13981170-76da-4f58-b5fb-813f2d3ae296","order_by":9,"name":"Yi Tong","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Tong","suffix":""},{"id":437972250,"identity":"f5163684-13d0-45ec-97bd-3a385558241c","order_by":10,"name":"Taoyan Liu","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Taoyan","middleName":"","lastName":"Liu","suffix":""},{"id":437972251,"identity":"f6cc2768-5556-4284-96e8-5cab3da28f86","order_by":11,"name":"Ye Zhang","email":"","orcid":"","institution":"Maternal-Fetal Medicine Center in Fetal Heart Disease, Capital Medical University; Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhang","suffix":""},{"id":437972252,"identity":"147f8dab-8de0-41c8-ac31-8da0efe4bbb6","order_by":12,"name":"Xiaoyan Gu","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"Gu","suffix":""},{"id":437972253,"identity":"91019b2e-4894-46ab-aa77-0edbdfae49d1","order_by":13,"name":"Jiancheng Han","email":"","orcid":"","institution":"Maternal-Fetal Medicine Center in Fetal Heart Disease, Capital Medical University; Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiancheng","middleName":"","lastName":"Han","suffix":""},{"id":437972254,"identity":"c9b49870-9ad0-479c-bf49-906052051a7a","order_by":14,"name":"Xiaowei Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Li","suffix":""},{"id":437972255,"identity":"7437792d-3cca-4114-a88a-fff33c5bfa07","order_by":15,"name":"Jiaqi Fan","email":"","orcid":"","institution":"Maternal-Fetal Medicine Center in Fetal Heart Disease, Capital Medical University; Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"Fan","suffix":""},{"id":437972256,"identity":"f51f28c8-3274-4f1b-b2f5-942eae84009b","order_by":16,"name":"Liying Yan","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liying","middleName":"","lastName":"Yan","suffix":""},{"id":437972257,"identity":"18be2eb1-260d-455e-98d2-88693b4179d9","order_by":17,"name":"Hankui Liu","email":"","orcid":"https://orcid.org/0000-0001-7826-0388","institution":"BGI group","correspondingAuthor":false,"prefix":"","firstName":"Hankui","middleName":"","lastName":"Liu","suffix":""},{"id":437972258,"identity":"90d8d6d2-852d-42e8-8e4f-e23e325abcf3","order_by":18,"name":"Feng Lan","email":"","orcid":"","institution":"Beijing Anzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Lan","suffix":""},{"id":437972259,"identity":"8859c72c-65b7-4ee1-8b3b-42f2a82a6f7c","order_by":19,"name":"Hong-Jia Zhang","email":"","orcid":"","institution":"Department of Cardiac Surgery, Beijing An-zhen Hospital, Capital Medical University, No. 2 An-Zhen Road, Chaoyang District, Beijing 100029, China","correspondingAuthor":false,"prefix":"","firstName":"Hong-Jia","middleName":"","lastName":"Zhang","suffix":""},{"id":437972260,"identity":"db52ebe6-9ab4-4f03-971b-74be61892776","order_by":20,"name":"Jie Qiao","email":"","orcid":"","institution":"Peking University Third Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Qiao","suffix":""}],"badges":[],"createdAt":"2025-03-31 03:35:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6341289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6341289/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80035370,"identity":"d9939b9a-fa92-4db2-a5a9-d2ae8f43a373","added_by":"auto","created_at":"2025-04-07 08:22:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2067050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the PFKP R755W variant in patients with myocardial hypoplasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. The pedigree of Family 1 who had myocardial hypoplasia. Family members for whom whole-genome sequences (WGS) were obtained are indicated as Wt/Mut or Wt/Wt. Squares represent male family members, circles represent female family members, and triangles represent the unborn fetus. Solid grey shapes indicate affected persons. Question marks indicate individuals whose clinical status was unknown. Slashes indicate deceased individuals. Arrows indicate probands. Wt/Mut indicates the PFKP R755W heterozygous variation; Wt/Wt indicates wild-type R755.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eThe echocardiograms of the affected fetuses (IV-2 and IV-3) and the pathological specimen (IV-3). B1 shows patient IV-2, who had thin myocardium of ventricular septum, middle and apical segments of the left ventricular free wall, and CA (the arrow points to the bulging ventricular septum). B2 shows the echocardiograph of patient IV-3, displaying thin myocardium in the left and right ventricular walls and ventricular septum, a bulging ventricular septum, and a middle segment of the right ventricular free wall (the arrow indicates the bulging of the ventricular septum and the right ventricular myocardium). B3 shows a section of the postmortem heart of patient IV-3, in which the septal myocardia of the left and right ventricular walls are thinner than normal. The ventricular septal myocardium and right ventricular wall are more prominent (arrowhead).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eCardiovascular magnetic resonance (CMR) images obtained from the affected adults (III-2 and II-2). C1 shows a blunt left ventricular apex (indicated by an arrowhead) and right ventricular enlargement in patient III-2. C2 shows thinning of the interventricular septum with bulges toward the right ventricle side (indicated by an arrowhead) in patient III-2. C3 shows the thinning of the right ventricular myocardium (indicated by the left arrowhead) and the ventricular septum, bulging toward the left ventricular side (indicated by the right arrowhead), and a blunt left ventricular (indicated by the up arrowhead).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eThe pedigree of Myocardial Hypoplasia Family 2. Family members with the Wt/Mut or Wt/Wt underwent whole-genome sequencing. Squares represent males, and circles represent females. Affected individuals are indicated by solid grey outlines, slashed symbols denote deceased members, and arrows indicate the probands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eAlignment of the PFKP protein sequences from different vertebrates, which demonstrates that the conservation of amino acids (highlighted in red) was affected by the missense alterations observed in the patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003ePFK1 enzyme activity of WT, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e hiPS-CMs. (n = 6 per group)\u003c/p\u003e\n\u003cp\u003eMean ± S.D, statistical significance was determined by one-way ANOVA. **means P\u0026lt;0.01. CA, congenital ventricular aneurysm; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; WGS, whole-genome sequencing.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/08f35013734e73e9cadbef61.png"},{"id":80035398,"identity":"8c4a3a73-01d7-414f-8fe9-8c3620e07e90","added_by":"auto","created_at":"2025-04-07 08:22:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2509601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMyocardial hypoplasia phenotype demonstrated in a Pfkp deficiency mice model and the healthy infant from Family 1 via assisted reproduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eProtein expression in the hearts of WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W \u003c/sup\u003emice at E12.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003ePFK1 enzyme activity in the hearts of WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W \u003c/sup\u003emice\u003csup\u003e \u003c/sup\u003eat E12.5 (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eProtein expression in Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e, Pfkp\u003csup\u003ecKO/+\u003c/sup\u003e, and Pfkp\u003csup\u003ecKO/cKO\u003c/sup\u003e mice at E12.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e PFK1 activity in the hearts of Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e, Pfkp\u003csup\u003ecKO/+\u003c/sup\u003e, and Pfkp\u003csup\u003ecKO/cKO \u003c/sup\u003emice at E12.5 (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eRepresentative images of HE-stained heart sections from WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E15.5. Scale bars = 600 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eRepresentative images of HE-stained heart sections from WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E17.5. Scale bars = 600 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;G\u003c/strong\u003e. Quantification of left and right ventricular wall thickness in WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice after different durations of embryonic development (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; H. \u003c/strong\u003eSanger sequencing results of the amplification products of the biopsy sample from the embryo. Results indicated the embryo was free of \u003cem\u003ePFKP\u003c/em\u003e c.2263C\u0026gt;T.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e. Echocardiograms of the\u003cstrong\u003e \u003c/strong\u003ehealthy offspring obtained through assisted reproduction in Family 1 at the prenatal stage, 19 weeks, 28 weeks, and the neonatal stage, which suggests a normal heart structure.\u003c/p\u003e\n\u003cp\u003eOne-way ANOVA determined statistical significance; data present the mean ± S.D, n.s., not significant (P\u0026gt;0.05); \"*\" denoted significance between the Pfkp\u003csup\u003eflox/flox \u003c/sup\u003eand Pfkp\u003csup\u003ecKO/+\u003c/sup\u003egroups; \"#\" denoted significance between Pfkp\u003csup\u003eflox/flox \u003c/sup\u003eand Pfkp\u003csup\u003ecKO/cKO\u003c/sup\u003e groups in panel D, G. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/1d5ad5a46e6b779928c1fc1f.png"},{"id":80036209,"identity":"08971b74-de20-451b-9b6b-d65dfcf7ebcd","added_by":"auto","created_at":"2025-04-07 08:30:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1835855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePfkp deficiency impairs embryonic myocardial proliferation in mice and hiPS-CMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative images of Ki67 flow cytometry in WT, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e hiPS-CMs on day 15 of differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Quantification of proliferation via Ki67 flow cytometry of WT, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e hiPS-CMs on Day 10,15,30 of differentiation (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eRepresentative images and quantification of Ki67 immunofluorescence of WT, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e hiPS-CMs on day 15 of differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eKi67 heart immunofluorescence in WT and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E13.5, E15.5, and E17.5; cardiomyocyte proliferation was significantly lower in the Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e group than in the normal control (n=5 per group). Scale bar:30μm.\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA in B and C; student’s t-test in D determined statistical significance; data present the mean ± S.D, n.s., not significant (P\u0026gt;0.05); *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/93dafd149ed6f392a88d90b0.png"},{"id":80036207,"identity":"176e69ef-ae0e-4381-8997-c566bb3bfa49","added_by":"auto","created_at":"2025-04-07 08:30:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1625453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePfkp mutation leads to dysregulated glycolytic function and reduced downstream metabolite levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eExtracellular acidification rate (ECAR) by Seahorse for glycolysis activity of WT, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+ \u003c/sup\u003ehiPS-CMs (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003ePrincipal component analysis of the left ventricular wall of the hearts of WT and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E15.5 (n = 3 per group). The two principal components (PCs) that explain the largest portion of the data variation are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eMSI differential metabolism representative diagram of the hearts of WT and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E15.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eVolcano plot of differential metabolite levels of WT and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice hearts in the left ventricular wall.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eMetabolite Set enrichment analysis (MSEA) of the LC-MS data of differentially expressed metabolites in the left ventricular wall.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003eRepresentative images and statistical analysis of AMPK, pAMPK, p70S6K, p-p70S6K protein expression levels in the hearts of WT and Pfkp\u003csup\u003eR754W/R754W \u003c/sup\u003emice at E15.5(n=3 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eSchematic diagram of reduced embryonic cardiomyocyte proliferation caused by impaired glycolysis and energy metabolism pathways under PFKP deficiency.\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined by one-way ANOVA in A; student’s t-test in F determined statistical significance; data present the mean ± S.D, n.s., not significant (P\u0026gt;0.05); *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/31627f3cfe1001bd72dadb5a.png"},{"id":80036211,"identity":"c491ade9-ceeb-4ef2-bda6-9801c071d7e4","added_by":"auto","created_at":"2025-04-07 08:30:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3800832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eF-1,6-BP supplementation reverses PFKP mutation-related myocardial hypoplasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp; \u003c/strong\u003eSchematic diagram of F-1-6-BP application in the \u003cem\u003ein vitro\u003c/em\u003e hiPS-CMs model.\u003cstrong\u003e \u003c/strong\u003ehiPS-CMs were administered 5 mM F-1,6-BP on the 8th day of differentiation, and proliferation was detected on the 15th day of differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eRepresentative immunofluorescence proliferation marker Ki67 images of hiPS-CMs in the WT, WT+ F-1,6-BP, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eR755W/+\u003c/sup\u003e+ F-1,6-BP,PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e+ F-1,6-BP groups. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u0026nbsp; \u003c/strong\u003eQuantitative analysis of Ki67 immunofluorescence staining in Panel B. PFKP mutation led to decreased cardiomyocyte proliferation, which was rescued by supplementation with F-1,6-BP (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u0026nbsp; \u003c/strong\u003eKi67 coupled fluorescence flow cytometry and Ki67-positive cell percentages of hESC-CMs in the WT, WT+ F-1,6-BP, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e, and PFKP\u003csup\u003eR755W/+\u003c/sup\u003e+ F-1,6-BP,PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e, and PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e+ F-1,6-BP groups. PFKP mutation led to decreased cardiomyocyte proliferation, which was rescued by supplementation with 5 mM F-1,6-BP (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eRepresentative images of HE-stained embryonic hearts at E17.5 from WT + Saline, Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e + F-1,6-BP, Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e+ Saline, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e + F-1,6-BP groups. Pregnant mice were injected intra-amniotically with 75 ng F-1,6-BP dissolved in 50 μl saline per fetus at E12.5. As controls, 50 μl of saline was injected intra-amniotically per fetus (WT+ Saline, Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e+ Saline). Scale bar = 600 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u0026nbsp;\u0026nbsp; \u003c/strong\u003eRepresentative images of immunofluorescence analysis of the proliferation marker Ki67 in WT + Saline, Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e + F-1,6-BP, Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e+ Saline, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e + F-1,6-BP cells at E17.5. Scale bar = 30 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eQuantitative analysis of left ventricular (LV) and right ventricular (RV) myocardial compact thickness in the four groups revealed that F-1-6-BP supplementation rescued thinning of the left and right ventricular wall myocardium in mouse embryos caused by Pfkp mutation (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003eQuantitative analysis of Ki67 immunofluorescence staining revealed that F-1,6-BP supplementation reversed the decrease in myocardial proliferation caused by Pfkp mutation during the embryonic stage (n = 6 per group). Data shown in C, D, G, and H represent the mean ± S.D. Statistical significance was determined using one-way ANOVA (n.s., not significant P\u0026gt;0.05); *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/96a55fd2d778d5d3ebb77314.png"},{"id":80035391,"identity":"891d24e2-e007-4452-9b89-0b436cda981a","added_by":"auto","created_at":"2025-04-07 08:22:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2326488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiminished non-genetic glycolytic function reduced cell proliferation in embryonicmice and hESC-CMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic of the mechanism through which the glucose concentration was reduced and 2- deoxy-D-glucose (2DG) was added during hESC-CM differentiation. Glucose deprivation or addition of 3 mM 2DG in hESC-CMs was conducted on the 13th day of differentiation, and proliferation was assessed on the 15th day of differentiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Statistical diagram of Ki67 fluorescence flow cytometry positive percentage of hESC-CM proliferation at different concentrations (1mM,5mM,7.5mM,11mM). The lower the glucose concentration, the lower was the proliferation of hESC-CMs (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC.\u003c/strong\u003e Statistical analysis of the percentage of Ki67-positive proliferating hESC-CMs in the WT + PBS and WT+2DG groups. 2DG significantly reduced hESC-CM proliferation. Scale bar = 50 μm (n = 5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003e \u0026nbsp;Schematic diagram of 2DG injection into pregnant mice. Each pregnant mouse received an intraperitoneal injection of 2DG (500 mg/kg per day) at E12.5, and fetal heart samples were collected at E17.5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Embryonic resorption rate (Abortion%) of E17.5 embryos per pregnant mouse after 2DG administration. The resorption rate was calculated as Abortion% = (number of resorbed embryos / total number of embryos) × 100(n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Representative heart HE staining and statistical analysis of the WT+ Saline and WT+ 2DG groups at E17.5. Scale bar = 600μm (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Representative images and graph of Ki67 immunofluorescence staining in WT+ Saline and WT+ 2DG groups at E17.5. Scale bar = 30 μm (n = 6 per group).\u003c/p\u003e\n\u003cp\u003eStatistical significance of the results shown in B was determined using one-way ANOVA. Statistical significance of the results shown in C, E, F, and G was determined using a student’s t-test. Data represent the mean ± S.D, n.s indicates no significance (P\u0026gt;0.05); *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/49dbb14e7bcce2a59de1c564.png"},{"id":83289111,"identity":"c9ce3a8c-91f9-4e9b-82b0-811597b2eeb6","added_by":"auto","created_at":"2025-05-22 12:30:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14726216,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/deb0b2a8-9ade-4f42-bd76-6f4c3ee9cf34.pdf"},{"id":80035399,"identity":"09312126-900c-4e3d-b23d-0986de084ccb","added_by":"auto","created_at":"2025-04-07 08:22:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5538256,"visible":true,"origin":"","legend":"\u0026#x0391;","description":"","filename":"supplementaryappendix.docx","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/90daa2edc1d8b9ede30ea525.docx"},{"id":80036206,"identity":"3a644ad5-be84-4178-99b7-4f93ba71a591","added_by":"auto","created_at":"2025-04-07 08:30:29","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":372379,"visible":true,"origin":"","legend":"","description":"","filename":"StructuredGraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6341289/v1/208062cf22a3fb7afbbce620.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Identification of a Mutation in the PFKP as a Causative Factor in Prenatal Glycolysis Defects and Embryonic Myocardial Hypoplasia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCongenital heart disease (CHD) is the most common birth defect worldwide, with a prevalence of 8\u0026ndash;12\u0026permil;\u003csup\u003e1\u003c/sup\u003e. The factors that contribute to CHD include environmental exposure, maternal illness, genetics, and a combination of these\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, the limited understanding of congenital heart disease genetic causes results in a lack of effective prevention and intervention measures. During embryonic development, the initial myocardial contraction and myocardial proliferation and expansion rely heavily on energy metabolism\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Glycolysis serves as the primary energy source for developing embryonic hearts and supplies essential biosynthetic precursors required for cell proliferation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The continuous proliferation of embryonic cardiomyocytes is critical for cardiac embryonic development\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Consequently, dysregulation of key genes associated with glycolysis has been implicated as a potential pathogenic factor in CHD, as evidenced by studies in lower organisms such as zebrafish. Therefore, the dysregulation of critical genes associated with glycolysis may be a pathogenic factor of CHD. However, the relationship between glycolysis and human embryonic heart development remains largely unexplored. This knowledge gap can be attributed to the high proportion of complex CHD cases that result in intrauterine or prehospital deaths, thereby limiting our understanding of the pathogenesis and etiology of CHD during the embryonic stage.\u003c/p\u003e \u003cp\u003eIn this study, we identified a distinct clinical manifestation of CHD, myocardial hypoplasia, through next-generation sequencing of 1,650 fetal heart disease family lines from a comprehensive 100,000-case database at the nationwide Fetal Heart Disease Maternal-Fetal Medicine Center. Myocardial hypoplasia is characterized by structural defects that do not align with coronary artery distribution and was observed to cluster in two non-consanguineous families. Furthermore, whole-genome sequencing (WGS) and functional assays revealed specific CHD phenotypes and functional defects associated with a deficiency in the platelet isoform of phosphofructokinase 1 (PFKP), a key rate-limiting enzyme in glycolysis. These findings provide new insights into the clinical and genetic basis of CHD, contributing to an expanded understanding of its pathogenesis. More importantly, these findings underscore the critical role of glycolysis in cardiac embryonic development and suggest it as a promising therapeutic target for intrauterine interventions, potentially achievable through environmental modifications.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy subjects\u003c/h2\u003e \u003cp\u003eTwo large pedigrees exhibiting CHD as an autosomal dominant trait with variable expressivity and complete penetrance were recruited (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clinical evaluations of the affected fetuses, children, and adult in Family 1 and Family 2 were performed using diagnostic modalities, including echocardiography, cardiovascular magnetic resonance (CMR), and electrocardiograms. DNA samples were obtained from affected and control individuals from both families and subjected to rigorous WGS analysis. Written informed consent was obtained from all study participants or their legal guardians, and the study was approved by the institutional review board of the Medical Ethics Committee of Beijing Anzhen Hospital. The authors confirm the accuracy and completeness of the data used in this study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetic analysis\u003c/h3\u003e\n\u003cp\u003eWGS was performed with the DNA of patients and controls. Then, linkage analysis and variant calling, annotation, filtering, and prioritization were performed. The pathogenicity of sequence variants was assessed according to ACMG guidelines. Detailed protocols are provided in the supplementary materials.\u003c/p\u003e\n\u003ch3\u003eMice\u003c/h3\u003e\n\u003cp\u003eMyh6\u003csup\u003eCre/+\u003c/sup\u003e, Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice were acquired from the Shanghai Model Organism Centre (Shanghai, China). WT, Pfkp\u003csup\u003eR754W/+\u003c/sup\u003e, and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e littermates were used as controls. Offspring from homozygous Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e mice were used as controls. Additionally, heterozygous floxed mice (Pfkp\u003csup\u003eflox/+\u003c/sup\u003e) were generated by crossbreeding Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e mice with wild-type mice. Pfkp\u003csup\u003eflox/+\u003c/sup\u003e mice were crossed with Myh6\u003csup\u003eCre/+\u003c/sup\u003e mice to produce offspring for comparisons with the Pfkp\u003csup\u003eflox/flox\u003c/sup\u003e \u0026times; Myh6\u003csup\u003eCre/+\u003c/sup\u003e crossbred group. The genotypes of the embryonic mice were confirmed by tail DNA extraction and subsequent genotyping using polymerase chain reaction (PCR) analysis. Pregnant wild-type C57BL/6J mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). software (Beijing, China). Mice were bred and housed in a specific pathogen-free environment with a 12-h dark/light cycle at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 40\u0026ndash;70% humidity. Mice whose body weight fell below two standard deviations (\u0026lt;\u0026thinsp;2SD) from the average weight of their littermates were not used for breeding. The detailed protocols for the construction of the mouse model and mouse genotyping are provided in the supplementary materials. Subsequent experiments, including histology, quantitative real-time PCR, Western blot, immunofluorescence, metabolomic analysis, and PFK enzymatic activity assays, were conducted to validate the findings and investigate the underlying mechanisms. All experimental procedures were approved by the Animal Subjects Committee at Beijing Anzhen Hospital.\u003c/p\u003e\n\u003ch3\u003eCell culture and cardiac differentiation\u003c/h3\u003e\n\u003cp\u003eH9 and hiPS cells were maintained on feeder-free Matrigel (Corning) and fed E8 medium (CellaPy) daily. Cells were routinely passaged every 3 days at 70\u0026ndash;80% confluency using 0.5 mM EDTA in phosphate-buffered saline (PBS) without MgCl\u003csub\u003e2\u003c/sub\u003e or CaCl\u003csub\u003e2\u003c/sub\u003e (HyClone, USA). The cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Human pluripotent stem cells (hPSCs) were differentiated into hPSC-CMs using a small-molecule-based method as previously described\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The hPSC-CMs were purified using the lactate metabolic selection method\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were extracted from both patients (III-4) and unaffected family members (III-7). Subsequently, the PBMCs were reprogrammed into hiPS cells, and the PFKP R755W mutation was corrected through homologous recombination. This correction yielded hiPS (PFKP Correct/+), possessing an identical genetic background to the patient but devoid of the PFKP R755W mutation. Subsequent experiments, including quantitative real-time PCR (qRT-PCR), Western blot (WB), flow cytometry, immunofluorescence, Seahorse ECAR measurement, metabolomic analysis, and PFK enzymatic activity assays, were conducted to validate the findings and investigate the underlying mechanisms. Detailed protocols are provided in the supplementary materials.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eDescriptive statistics for continuous variables are shown as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, while categorical variables are displayed as subject counts and percentages. Student\u0026rsquo;s t-test and ANOVA (followed by Tukey\u0026rsquo;s post hoc test) were used for the analysis of continuous variables after performing the Shapiro-Wilk normality test to obtain normal distribution of the data. Categorical variables were evaluated using Fisher\u0026rsquo;s exact test. If the data did not have a normal distribution or N was too small to determine normality, statistical significance was confirmed using non-parametric tests (Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparison test or uncorrected Dunn's multiple comparison test). For non-normal data, a non-parametric Mann-Whitney U test was used. Statistical analyses were performed using the Prism 8.0 (GraphPad Software). Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e1. Identification of PFKP R755W pathogenic variants in congenital myocardial hypoplasia families\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWe conducted in-depth examinations of families of consecutive fetuses with CHD and significant myocardial hypoplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In Family 1, early pregnancy loss occurred at 3 weeks of gestation of the first fetus, with exact aetiology unknown (IV-1). At 24 weeks of gestation of the second fetus, fetal echocardiography revealed myocardial hypoplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, IV-2). The fetus (IV-2) had myocardial thinning in the ventricular septum and congenital ventricular aneurysms in the middle to apical segments of the left ventricular free wall (Fig.\u0026nbsp;1B1, IV-2). This fetus also exhibited secondary cardiac dysfunction, as indicated by a Cardiovascular Profile Score\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e (CVPS) of 7 points, suggesting a high probability of postnatal mortality due to the identified cardiac anomalies in the current pregnancy. Consequently, the parents opted for induced labour after receiving adequate counselling. The third fetus (Fig.\u0026nbsp;1B2, IV-3) showed a cardiac phenotype similar to that of the previous one, resulting in a CVPS score of 6 points. Based on these findings, the family decided to terminate the pregnancy for a second time and proceed with a postmortem examination, which confirmed the prenatal diagnosis (Fig.\u0026nbsp;1B3). Upon reviewing the family's medical history, it became evident that this condition affected members of three consecutive generations. Notably, the father (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, III-2) exhibited a similar clinical phenotype, with 40% ejection fraction and heart failure (Table S1). The most affected family members exhibited concurrent structural cardiac abnormalities, such as thinning of the apical ventricular septum and blunt left ventricular apex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and atrial or ventricular septal defects (ASD or VSD) (Fig. S1A) along with the right bundle branch block (Table S1, Fig. S1B). The consistent phenotype observed within this familial lineage aligned with an autosomal-dominant mode of inheritance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eWGS was performed to investigate the genetic aetiology. Initial investigations into genes associated with CHD failed to yield positive results; therefore, we proceeded with a genome-wide linkage analysis. This analysis identified a co-segregating region of 5.8 Mb on chromosome 10 (p15.3-p15.1), with a logarithm of odds (LOD) score of 2.4, exceeding the threshold of 2.3, indicating a potential genetic link between this region and the cardiac phenotype in Family 1. In this region, we identified the only rare heterozygous PFKP (NM_002627.4: c.2263C\u0026thinsp;\u0026gt;\u0026thinsp;T; NP_002618.1: p.R755W) missense variant, which co-segregated with the cardiac phenotype of Family 1. (Table S1, Fig. S1C-D).\u003c/p\u003e \u003cp\u003eTo further determine the correlation between PFKP and CHD, we screened for the PFKP variants from a local fetal heart disease exome sequence database comprising 1650 fetal heart disease pedigrees and found the other CHD family with the same PFKP variants (c.2263C\u0026thinsp;\u0026gt;\u0026thinsp;T [p.R755W]) with Family1. We observed clinical manifestations in family 1 similar to those in family 2(Table S1, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), we considered PFKP a gene of uncertain significance, and PFKP R755W in the two families was considered a variant of the gene of uncertain significance (Table S2).\u003c/p\u003e \u003cp\u003eNext, we functionally analysed the PFKP R755W variant. The PFKP R755 residue is highly conserved in vertebrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), and structural analysis of PFKP proteins indicated that R755W disrupts protein conformation and function (Fig. S1E). To understand the impact of PFKP R755W in cardiac development, we generated induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) from the healthy family member (WT), a patient (PFKP\u003csup\u003eR755W/+\u003c/sup\u003e), and R755W corrected cells (PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e; generated using genome editing) (Fig. S2A-C). Compared to PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e CMs, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e CMs showed a 23.5% reduction in PFK1 activity with no significant changes in PFKP protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Fig. S2E).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Pfkp mice mimic embryonic myocardial hypoplasia\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of PFKP in embryonic heart development, we analyzed the expression pattern of PFKP during mammalian heart development. Gene expression data from the Expression Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/gxa/home\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/gxa/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) showed that PFKP was predominantly expressed in the human embryonic heart over the other two PFK1 isoforms (PFKM and PFKL) (Fig. S3A, B). Moreover, we verified high expression levels of PFKP in both human\u003cb\u003es\u003c/b\u003e (Fig. S3C-E) and mouse embryonic hearts (Fig. S3F-H). Notably, PFKP expression peaked in early stages, and immature cardiomyocytes (10-day and 15-day hiPS-CMs)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, when these cells were highly proliferative (Fig. S3H-J). These findings indicate that PFKP plays a critical role in embryonic cardiac development.\u003c/p\u003e \u003cp\u003eNext, we generated a mouse model carrying a PFKP R755W equivalent variant (mouse Pfkp R754W) (Fig. S4A). The hearts of Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice exhibited a 34.2% reduction in PFK1 enzyme activity at embryonic day 12.5 (E12.5) compared with that in the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), with no significant difference in PFKP protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which effectively mimicked the reduced enzyme activity phenotype observed in patient-derived hiPS-CMs. To better mimic the myocardial phenotype caused by reduced human PFK1 enzyme activity, we selected Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice for subsequent experiments. Next, we performed histological analysis of the hearts of Pfkp-mutant and WT embryos to examine heart development from E13.5 to E17.5. The heart sizes of homozygous (Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e) mutation mice were smaller at E17.5 compared with those of the controls (Fig.S4B). However, examination of the LV and RV compact layer thickness revealed that Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice had significant thinning of the LV compact layer at E15.5 and E17.5 and the RV compact layer at E17.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G, Fig.S4C). These findings indicate that PFKP mutation leads to reduced PFK1 enzyme activity and abnormal embryonic myocardial morphology, including thinning of the compact layer of the ventricular walls and reductions in heart size.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further validate the relationship between defective PFKP function and cardiac embryonic development, we created a myocardium-specific knockout (cKO) mouse model using Myh6-driven Cre to eliminate PFKP, resulting in the loss of PFKP expression in E12.5 mice hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In PFKP-knockout mice, E12.5 heart PFK1 activity was decreased by 35.96% in heterozygotes and 47.07% in homozygotes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Histological assessment demonstrated thinning of the LV and RV compact layers in the hearts of heterozygous (Pfkp\u003csup\u003ecKO/+\u003c/sup\u003e) and homozygous (Pfkp\u003csup\u003ecKO/cKO\u003c/sup\u003e) knockout mice at E15.5 and E17.5 (Fig.S5A-D). The heart sizes of heterozygous (Pfkp\u003csup\u003ecKO/+\u003c/sup\u003e) and homozygous (Pfkp\u003csup\u003ecKO/cKO\u003c/sup\u003e) knockout mice were smaller at E17.5 compared with those of the controls (Fig. S5E). Compared with that of Pfkp\u003csup\u003ecKO/cKO\u003c/sup\u003e mice, Pfkp\u003csup\u003ecKO/+\u003c/sup\u003e and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice exhibited a milder cardiac phenotype, demonstrating a dose-dependent effect of PFK1 enzyme activity.\u003c/p\u003e \u003cp\u003eSubsequently, the family (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; III-1 and III-2) pursued assisted reproductive technologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Normal \u003cem\u003ePFKP\u003c/em\u003e gene of the fetus were confirmed via an umbilical cord blood test, which aligned with the embryo test results. At 19 and 28 weeks of gestation, the fetal heart function cardiac structures were within the normal range for healthy reference populations. We followed up with the baby until three months after birth, and the baby's cardiac structure and function (EF: 65%) were normal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). These findings provide valid anthropological evidence that PFKP defects cause congenital myocardial hypoplasia.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3. Pfkp deficiency impairs embryonic myocardial proliferation in vivo and in vitro\u003c/h2\u003e \u003cp\u003ePFKP defects reduce tumor cell proliferation, primarily due to impaired PFK1 enzymatic activity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Myocardial proliferation is a key mechanism in embryonic heart development. To investigate the mechanisms underlying PFKP-induced myocardial hypoplasia, we examined the proliferative function of patient-derived PFKP\u003csup\u003eR755W/+\u003c/sup\u003e hiPSC-CMs. Immunofluorescence and flow cytometry results showed that PFKP\u003csup\u003eR755W/+\u003c/sup\u003e hiPSC-CMs exhibited a decreased proliferation markers Ki67 ratio in early-stage cardiomyocytes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (10-day and 15-day) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C, Fig.S6A). A similar reduction in myocardial proliferation was observed in Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at different embryonic times (Fig. S6B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the link between PFKP deficiency and embryonic myocardial proliferation, we established PFKP knockout hESC-CMs (Fig.S7B-D). Consistent with the reduced embryonic myocardial proliferation observed in PFKP knockout mice (Fig.S7A), flow cytometry analysis revealed a decrease in proliferation markers in the PFKP knockout hESC-CMs(Fig.S7F, G). Lentivirus-mediated PFKP overexpression in PFKP-KO hESC-CMs effectively restored cardiomyocyte proliferation and PFK1 enzyme activity. These findings show that PFKP deficiency leads to reduced embryonic cardiomyocyte proliferation(Fig.S7B-G).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4. Pfkp mutation leads to dysregulated glycolytic function and reduced downstream metabolites\u003c/h2\u003e \u003cp\u003ePFKP primarily functions as a key rate-limiting enzyme in glycolysis. To test whether PFKP defects result in reduced glycolytic function, we examined the glycolytic function in PFKP\u003csup\u003eR755W/+\u003c/sup\u003e hiPSC-CMs using Seahorse ECAR experiments. The results showed that PFKP\u003csup\u003eR755W/+\u003c/sup\u003e hiPSC-CMs exhibited significantly reduced glycolytic function, including basal glycolysis, glycolytic capacity, and glycolytic reserve (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings suggest that PFKP mutation impairs cardiomyocyte proliferation and glycolysis, which may contribute to abnormal myocardial development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo comprehensively assess the metabolic changes in embryonic hearts following PFKP mutation, we performed metabolomic profiling of the hearts of control and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice at E15.5 using MALDI-MSI to assess the spatial distribution of metabolites. Principal component analysis (PCA) revealed significant metabolic differences between the control and Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Metabolite set enrichment analysis (MSEA) revealed that Pfkp mutation disrupted key energy metabolic pathways, including carbohydrate and amino acid metabolism in whole embryonic hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, and Fig. S8A).\u003c/p\u003e \u003cp\u003eAfter birth, the left ventricle bears a higher pumping pressure to sustain the systemic circulation. Therefore, we first focused on metabolic changes in the left ventricular wall of Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice. The results showed a significant decrease in carbohydrate metabolite levels, with a corresponding upregulation in fatty acid and hormone metabolite levels in the left ventricle wall of Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Notably, the levels of succinic acid, α-KG, and PPP metabolic intermediates were significantly reduced, and these intermediates can provide precursor substances for myocardial proliferation (Fig. S8C). Additionally, fatty acid-related pathways were upregulated in Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice, which may compensate for the reduced energy due to decreased glycolysis through metabolic reprogramming (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Fig. S8C). However, this shift towards fatty acid metabolism reprogrammed the myocardial epigenome and inhibited myocardial proliferation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, we analysed differential metabolites in the LV and RV of normal embryonic mice. In the control mice, compared with the LV compact layers, 26 metabolites were significantly upregulated and 27 downregulated in the RV compact layers (Fig. S8B). However, in Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice, there were no metabolic differences between the left and right ventricular walls, suggesting that Pfkp mutation may disrupt distinct metabolic profiles typically observed in the left and right ventricular walls (Fig. S8B). Pfkp knockout produced similar effects on the right and left ventricular walls (Fig.S8D, E). These results suggested that Pfkp deficiency results in the downregulation of the metabolic pathways that supply essential precursors to the embryonic myocardium. We performed Seahorse ECAR experiments and targeted mass spectrometry using PFKP knockout hESC-CMs to confirm these findings. Consistent with the mouse embryonic heart results, the levels of glycolytic metabolites and byproducts of the PPP and nucleotide biosynthesis pathways were significantly reduced (Fig. S9). These data suggested that Pfkp deficiency disrupts glycolysis and its associated pathways.\u003c/p\u003e \u003cp\u003ePFKP mutation significantly downregulate energy metabolic pathways. We hypothesised that PFKP mutation impairs myocardial proliferation by reducing ATP levels, which in turn alters AMPK and mTOR activity, which are key regulators of energy metabolism. To verify whether PFKP deficiency activates the AMPK pathway by reducing ATP levels, we analysed AMPK levels in the hearts of E15.5 embryos from Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice. The results showed that PFKP mutation significantly upregulated AMPK activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Additionally, AMPK activation inhibits mTOR activity, ultimately reducing protein synthesis and mRNA translation\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which impairs myocardial proliferation during embryonic development (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). These results suggested that PFKP mutation reduced glycolysis, activated AMPK, and inhibited the mTOR pathway, thereby diminishing myocardial proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5. F-1,6-BP rescues PFKP mutation phenotypes\u003c/h2\u003e \u003cp\u003eF-1,6-BP is an endogenous intermediate that is generated by the PFK1 enzyme. Given that PFKP deficiency impairs glycolytic function, leading to abnormal embryonic myocardial proliferation and development, we hypothesised that F-1,6-BP supplementation would restore the glycolytic flux and alleviate myocardial hypoplasia. To test this, we treated WT, PFKP\u003csup\u003eCorrect/+\u003c/sup\u003e, PFKP\u003csup\u003eR755W/+\u003c/sup\u003e hiPS-CMs with F-1,6-BP (5mM) on the 8th day of differentiation. Cardiomyocyte proliferation was assessed on day 15 of differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We observed that F-1,6-BP successfully rescued the PFKP mutation-induced reduced cardiomyocyte proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). We injected each mouse fetus with F-1,6-BP (75 ng/mouse fetus) via intra-amniotic injection at E12.5 and harvested the hearts at E17.5 for comprehensive phenotypic assessment. Administration of F-1,6-BP in the early embryonic period effectively rescued the thinning of the LV and RV compact layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and restored myocardial proliferation in Pfkp\u003csup\u003eR754W/R754W\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Overall, these findings suggest that F-1,6-BP can rescue abnormal cardiac development and decrease proliferation caused by PFKP mutation, highlighting its potential therapeutic application in CHD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e6. Impaired non-genetic glycolytic function causes abnormal embryonic myocardial proliferation\u003c/h2\u003e \u003cp\u003eTo explore the effect of non-genetic glycolytic dysfunction on immature cardiomyocyte proliferation, we treated hESC-CMs in the early cardiac development stages in vitro with low glucose concentrations and the glycolytic inhibitor 2DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We found that lowering glucose concentrations to 7.5 mM and the addition of 3 mM 2DG decreased hiPS-CM proliferation on day 13 of differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further inhibited glycolytic activity in embryonic mice via intraperitoneal injection of a high concentration of 2DG (500 mg/kg per day) into pregnant mice on day E12.5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Compared to WT\u0026thinsp;+\u0026thinsp;Saline mice, WT\u0026thinsp;+\u0026thinsp;2DG mice had a significantly increased ratio of embryonic abortions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). At E17.5, 2DG treatment decreased the left ventricular myocardium thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) and myocardial proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These results suggested that reduced glycolysis without genetic causes leads to embryonic myocardial hypoplasia and impaired cardiomyocyte proliferation, offering potential strategies for preventing CHD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCHD is the most common birth defect worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The lack of knowledge about the genes involved in its development has led to a shortage of effective preventive methods. Identifying the pathogenic mechanisms of CHD could aid in establishing effective prevention and intervention strategies. The dysregulation of cardiomyocyte proliferation plays a critical role in the pathogenesis of most CHD cases\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur study presents, for the first time, a case with multiple manifestations of myocardial hypoplasia in the ventricular myocardium, accompanied by structural cardiac defects, specifically VSD, ASD, and arrhythmia. Cardiomyocyte proliferation is essential for embryonic heart development; however, this process halts after birth. Teratogens that disrupt cardiomyocyte proliferation during embryonic development can lead to heart malformations. After excluding congenital coronary artery lesions, we hypothesised that reduced cardiomyocyte proliferation was the likely mechanism underlying the clinical phenotype of a thinning embryonic myocardium.\u003c/p\u003e \u003cp\u003eDuring embryonic development, myocardial proliferation is highly dependent on energy metabolism\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Unlike the energy supply mechanisms in the adult heart, glycolysis is the primary energy supply for the development of the embryonic heart\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Although some critical genes associated with glycolysis have been considered potential pathogenic factors for CHD, there is currently no clinical evidence directly linking the genetic variants of glycolytic genes to CHD. Studying the clinical phenotypes of the human embryonic heart and the underlying genetic mechanisms presents significant challenges. These challenges arise from the difficulty in obtaining clinical phenotypes, variations in the expression of genes related to embryonic development before and after birth, genetic compensatory mechanisms, and other factors that complicate this study. Combining the results of the linkage and WGS analyses, we identified PFKP R755W as the likely causative mutation for the cardiac phenotype observed in the family. Furthermore, offspring produced after clinically blocking the genetic transmission of PFKP R755W using assisted reproductive techniques showed no cardiac pathogenic phenotypes. This provides strong anthropological evidence for the pathogenicity of PFKP R755W. We observed that some adult patients with pathogenic PFKP variants did not show a significant reduction in cardiac ejection fraction. However, the prognosis of fetuses with \u003cem\u003ePFKP\u003c/em\u003e mutations is poor, likely due to the polygenic nature of CHD and its interaction with the intrauterine environment. After confirming that functional defects in key glycolytic genes were associated with reduced embryonic myocardial proliferation, we identified the energy metabolic pathways and substrates that regulate this process through mechanistic studies, offering evidence for potential maternal interventions during embryonic development.\u003c/p\u003e \u003cp\u003eUnderstanding the energy metabolism pathways and substrate changes underlying teratogenicity in CHD is expected to facilitate the prevention of birth defects in the future. We inhibited glycolysis by specifically knocking out PFKP in the heart during mouse embryonic development, which resulted in abnormalities in other metabolic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Downregulation of the levels of metabolites such as α-KG and succinate in PFKP-deficient mice can promote myocardial regeneration after myocardial injury\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The decrease in glycolysis likely triggers metabolic reprogramming to maintain energy production via alternative pathways, such as fatty acid metabolism; however, this comes at the cost of reduced nucleotide availability and impaired cell proliferation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Recent research has also highlighted the co-regulation of metabolic pathways\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, including glycolysis, as a contributing factor to myocardial proliferation and regeneration following cardiac injury. The absence of reduced glycolysis in the embryo leads to abnormalities in energy metabolic pathways such as AMPK activation and mTOR inhibition. These may be key mechanisms by which reduced glycolysis impairs cardiac proliferation. Impaired nongenetic glycolysis mimicked myocardial hypoplasia phenotypes in the hearts of fetal mice and hPSC-CMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This is consistent with our recent findings in an 8-million-case cohort, which indicated that hypoglycaemia increases the risk of birth defects in offspring (unpublished). Supplementation with the downstream glycolysis substrate F-1,6-BP reduced cell proliferation and myocardial hypoplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our research provides strong evidence for early intrauterine intervention in fetuses with genetic defects as well as for glycaemia management in individuals with abnormal intrauterine glycolysis.\u003c/p\u003e \u003cp\u003eSome studies have highlighted the role of high PFKP expression in cardiac hypertrophy\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e; however, its involvement in chronic heart failure remains unclear. Exploring the regulatory mechanisms of glycolysis in chronic heart failure may reveal potential therapeutic targets. F-1,6-BP has been used to treat chronic heart failure\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, but not in pregnant women, requiring further safety evaluation. By identifying phenotypes, confirming the underlying genetic pathogenicity, and exploring underlying mechanisms, we have broadened the phenotypic and genetic pathogenicity spectrum of CHD and, more importantly, revealed possible shared mechanisms that influence cardiac development. These findings also suggest potential options for intrauterine interventions in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDisclosure of Interest\u003c/h2\u003e \u003cp\u003eNone.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China (No. 82200334 to Xiaoyan Hao; No U21A20523 and 82170301 to Yihua He; No. 82100322 to Tong Yi).\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank Professor Dong Zhao (Beijing Anzhen Hospital, Capital Medical University), Professors Yimin Hua and Yifei Li (West China Second University Hospital, Sichuan University, China) for their assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGlobal, regional, and national burden of congenital heart disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. \u003cem\u003eLancet Child Adolesc Health\u003c/em\u003e. 2020;4:185-200.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wang J, Zhao J, Huang G, Liu K, Pan W, Sun L, Li J, Xu W, He C, Zhang Y, Li S, Zhang H, Zhu J and He Y. 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Effect of intravenous fructose-1,6-diphosphate on myocardial contractility in patients with left ventricular dysfunction. \u003cem\u003ePharmacotherapy\u003c/em\u003e. 1994;14:522-8.\u003c/li\u003e\n\u003cli\u003eAlva N, Alva R and Carbonell T. Fructose 1,6-Bisphosphate: A Summary of Its Cytoprotective Mechanism. \u003cem\u003eCurr Med Chem\u003c/em\u003e. 2016;23:4396-4417.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"congenital heart disease, phosphofructokinase 1, metabolism, embryonic development","lastPublishedDoi":"10.21203/rs.3.rs-6341289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6341289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCongenital heart disease (CHD) is the most common birth defect worldwide, which lacks effective early preventive methods due to limited knowledge of the genetic defects involved in its development. Through genetic analysis of families with congenital heart disease (CHD), we identified a genetic correlation between the R755W variant of the platelet isoform of phosphofructokinase 1 (PFKP) and myocardial hypoplasia. Furthermore, PFKP\u003csup\u003eR754W/R754W\u003c/sup\u003e and PFKP knockout mice exhibited a myocardial hypoplasia phenotype during embryonic development. Mechanistic studies further revealed that PFKP deficiency reduces embryonic cardiac glycolysis, leading to decreased cardiomyocyte proliferation due to altered downstream metabolites and metabolic pathways. Importantly, intrauterine supplementation with fructose 1,6-bisphosphate (F-1,6-BP), a direct product of PFKP catalysis, was able to rescue myocardial hypoplasia in fetal mice. Conversely, inhibiting glycolysis using 2-deoxyglucose (2-DG) reproduced the myocardial hypoplasia phenotype in both fetal mouse hearts and human embryonic stem-cell-derived cardiomyocytes (hESC-CMs). These findings establish PFKP as a critical regulator of glycolysis during embryonic cardiac development. They also provide novel insights suggesting that glycolytic defects or intrauterine hypoglycemia may represent common causes of myocardial hypoplasia. This research highlights potential applications for genetic interventions, prenatal screening, and targeted intrauterine therapeutic strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Identification of a Mutation in the PFKP as a Causative Factor in Prenatal Glycolysis Defects and Embryonic Myocardial Hypoplasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 08:22:24","doi":"10.21203/rs.3.rs-6341289/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7254243-7f53-4465-a6a3-86ff6bb8f4ee","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46638212,"name":"Health sciences/Cardiology/Cardiovascular biology/Heart development"},{"id":46638213,"name":"Biological sciences/Genetics/Clinical genetics/Disease genetics"}],"tags":[],"updatedAt":"2025-05-22T12:21:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 08:22:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6341289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6341289","identity":"rs-6341289","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}