Glycerol-3-phosphate Dehydrogenase 1-like Research Articles

Abstract 425: GPD1L participates in glycerol-3-phosphate biogenesis and promotes tumor initiation and invasion in hepatocellular carcinoma

Abstract Background: Metabolic reprogramming in hepatocellular carcinoma (HCC) has received rising interest. However, current studies often focus on fatty acid de novo synthesis or oxidation, but less on glycerolipids metabolism. Interestingly, in HCC, we observed frequent dysregulation in enzymes responsible for glycerol-3-phosphate shuttle, which potentially links glycolysis and glycerolipids metabolism. Hence, we aimed to identify the functional significance of glycerol-3-phosphate dehydrogenase 1 like (GPD1L), an enzyme for the shuttle, and its regulation of expression in HCC. Methods: We utilized transcriptomic analysis of clinical HCC samples to validate the frequent overexpression of GPD1L in tumor cells and examined its association with clinicopathological features. In vitro and in vivo assays were performed to explore the functional significance of GPD1L in HCC initiation and progression. Furthermore, with targeted metabolomics analysis, we investigated the role of GPD1L in glycerol-3-phosphate shuttle in HCC. We also performed promoter study to understand the transcriptional regulation on GPD1L expression. Results: Among GPD family members, GPD1L was consistently upregulated at mRNA level in clinical HCC samples in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) (N=50) and our in-house RNA-sequencing cohorts (N=41), and in a separate independent in-house cohort as examined by RT-qPCR (N=80). GPD1L overexpression was associated with increased tumor invasiveness (p=0.008) and poorer overall survival (p=0.0017) in HCC patients. Functionally, knockdown of GPD1L suppressed HCC migration (fold change ≤ 0.43) and invasion (fold change ≤ 0.40). GPD1L knockdown also inhibited tumor initiation abilities, as shown by reduced colony (fold change ≤ 0.37) and sphere formation (fold change ≤ 0.41) in vitro, and lower stem cell frequency (p=1.96e-9) in vivo. Furthermore, GPD1L knockdown enhanced sensitivity of HCC cells to chemotherapeutic drugs, sorafenib and doxorubicin. Metabolically, GPD1L facilitated synthesis of glycerolipids precursor, glycerol-3-phosphate (G3P) from glycolysis intermediate dihydroxyacetone phosphate (DHAP) in HCC. Lastly, with chromatin-immunoprecipitation and Luciferase reporter assays, we demonstrated ELF1 bound to GPD1L promoter directly to enhance the gene transcription, while knockdown of ELF1 suppressed GPD1L mRNA and protein expression. Conclusion: Our study demonstrated the overexpression of the glycolysis-related gene, GPD1L, in HCC. GPD1L promoted tumor initiation, invasion and chemoresistance, and in turn might lead to worse clinical outcome. Metabolically, it was responsible for DHAP-to-G3P conversion in HCC and might serve as a linkage between glycolysis and glycerolipid metabolism. Our results also identified the novel role of ELF1 as the transcriptional activator of GPD1L in HCC. Citation Format: Tiffany Ching-Yun Yu, Luqing Zhao, Lu Tian, Charles Shing Kam, Joyce Man-Fong Lee, Daniel Wai-Hung Ho, Lo-Kong Chan, Yu-Man Tsui, Karen Man-Fong Sze, Irene Oi-Lin Ng. GPD1L participates in glycerol-3-phosphate biogenesis and promotes tumor initiation and invasion in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 425.

GPD1L inhibits renal cell carcinoma progression by regulating PINK1/Parkin-mediated mitophagy.

Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT-qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.

P-637 pseudo-targeted metabolomics of follicular fluid reveals ovarian GPD1L-mediated glycerophospholipid metabolism dysfunction in patients with biochemical premature ovarian insufficiency

Abstract Study question Does the metabolic profile of follicular fluid from patients with biochemical premature ovarian insufficiency (bPOI) differ from that of healthy women? Summary answer The metabolic signature of follicular fluid reveals a significant decrease of phosphatidic acid (PA), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) in bPOI patients. What is known already Metabolites in follicular fluid have attracted extensive attention as they are important components of the microenvironment during follicular development and oocyte maturation. Studies on follicular fluid metabolomics may enhance the understanding of the pathogenesis of ovarian aging, particularly premature ovarian insufficiency. Although a variety of metabolites have been suggested to be related to cell senescence, their specific roles in regulating follicular atresia remain unclear. Study design, size, duration An observational study of follicular fluid samples collected from 120 women undergoing in vitro fertilization and embryo transfer was conducted. All enrolled women were classified into two groups: one with 60 bPOI patients and the other with 60 healthy controls. Each group was further subdivided into two subgroups (young and aged group, 30 women for each section). Participants/materials, setting, methods The follicular fluids of mature follicles (diameter over 17 millimeters) were isolated and collected for pseudo-targeted metabolic analysis. The intensities of captured metabolites were utilized for further bioinformatic analysis. The correlation between the significant differential metabolites screened and their clinical outcomes were analyzed. In vitro experiments were used to demonstrate the cellular function of mainly regulated signaling pathway. Main results and the role of chance A total of 608 metabolites were identified in 120 follicular fluid samples. There were significant differences in 143 metabolites between the bPOI and healthy controls. As revealed by metabolic pathway analysis, glycerophospholipid metabolism was the most enriched pathway in bPOI follicular fluids. The intensities of several glycerophospholipid-associated metabolites PA, PC, lysoPC and lysoPE were positively correlated with the ovarian reserve indicator anti-Müllerian hormone (AMH), female age, as well as the expression level of glycerol-3-phosphatedehydrogenase 1-like (GPD1L), a key enzyme of this pathway, in granulosa cells. Furthermore, knockdown of GPD1L induced mitochondria dysfunction and granulosa cell apoptosis via GPD1L/PKCε/ATF2/NEAT1 pathway, as well as led to follicular atresia and impaired the quality of oocytes. Limitations, reasons for caution The validity of differential intensified metabolites we detected as biomarkers for clinical diagnosis needs to be confirmed in a large-scale study. And the multiple effects of glycerophospholipid metabolic pathway posed on follicular development need to be study in-depth. Wider implications of the findings Our results suggested that a highly coordinated follicular metabolism exhibits a strong relationship with the quality of follicles. An understanding of the mechanisms underlying ovarian aging will provide a theoretical basis, as well as new ideas for POI diagnosis, treatment and intervention. Trial registration number Not applicable.

Atrial myocyte-derived exosomal microRNA contributes to atrial fibrosis in atrial fibrillation

BackgroundAtrial fibrosis plays a critical role in the development of atrial fibrillation (AF). Exosomes are a promising cell-free therapeutic approach for the treatment of AF. The purposes of this study were to explore the mechanisms by which exosomes derived from atrial myocytes regulate atrial remodeling and to determine whether their manipulation facilitates the therapeutic modulation of potential fibrotic abnormalities during AF.MethodsWe isolated exosomes from atrial myocytes and patient serum, and microRNA (miRNA) sequencing was used to analyze exosomal miRNAs in exosomes derived from atrial myocytes and patient serum. mRNA sequencing and bioinformatics analyses corroborated the key genes that were direct targets of miR-210-3p.ResultsThe miRNA sequencing analysis identified that miR-210-3p expression was significantly increased in exosomes from tachypacing atrial myocytes and serum from patients with AF. In vitro, the miR-210-3p inhibitor reversed tachypacing-induced proliferation and collagen synthesis in atrial fibroblasts. Accordingly, miR-210-3p knock out (KO) reduced the incidence of AF and ameliorated atrial fibrosis induced by Ang II. The mRNA sequencing analysis and dual-luciferase reporter assay showed that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is a potential target gene of miR-210-3p. The functional analysis suggested that GPD1L regulated atrial fibrosis via the PI3K/AKT signaling pathway. In addition, silencing GPD1L in atrial fibroblasts induced cell proliferation, and these effects were reversed by a PI3K inhibitor (LY294002).ConclusionsAtrial myocyte-derived exosomal miR-210-3p promoted cell proliferation and collagen synthesis by inhibiting GPD1L in atrial fibroblasts. Preventing pathological crosstalk between atrial myocytes and fibroblasts may be a novel target to ameliorate atrial fibrosis in patients with AF.Graphical

Calycosin Inhibits the Malignant Behaviors of Lung Adenocarcinoma Cells by Regulating the circ_0001946/miR-21/GPD1L/HIF-1α Signaling Axis.

Objective To clarify the potential function and molecular mechanism of calycosin in lung adenocarcinoma (LUAD) cells. Methods LUAD cells (A549 and H1299) were treated with calycosin at different concentrations (25 nM, 50 nM, and 100 nM) for 24 h. The colony formation, invasion, and migration of the cells were assessed by colony formation, transwell, and scratch assays, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the mRNA expression level of circ_0001946, miR-21, glycerol-3-phosphate dehydrogenase 1 like (GPD1L), and hypoxia-inducible factor-1α (HIF-1α) in clinical tissue samples and LUAD cells. RNA pull-down assay and dual-luciferase reporter assay were performed to verify the relationship among circ_0001946, miR-21, GPD1L, and HIF-1α. Western blot was performed to detect the protein expression of epithelial-mesenchymal transition (EMT) process-related genes (E-cadherin, N-cadherin, and snail) and GPD1L as well as HIF-1α. Results Calycosin inhibited colony formation, invasion, migration, and EMT progression in A549 and H1299 cells. Besides, calycosin was able to regulate the expression of circ_0001946, miR-21, GPD1L, and HIF-1α in LUAD cells. According to the findings of QRT-PCR, the expression level of circ_0001946 and GPD1L in LUAD tissues was significantly lower than that in adjacent noncancerous normal tissues, and the expression of miR-21 and HIF-1α was also significantly increased in clinical tissue samples. In addition, there was a targeted regulatory relationship among the above four expressions. Knockdown of circ_0001946 expression in A549 cells treated with calycosin enhanced the malignant behavior of A549 cells and inhibited the anticancer effect of calycosin. However, the knockdown of miR-21 promoted the anticancer effect of calycosin and inhibited the malignant behavior of A549. Conclusion Calycosin can inhibit colony formation, invasion, migration, and EMT process of LUAD cells via regulating the circ_0001946/miR-21/GPD1L/HIF-1α signaling axis and could be a promising therapeutic drug for LUAD.

Novel variant of the glycerol-3-phosphate dehydrogenase-1 Like (GPD1-L) gene in Japanese Brugada syndrome patients

Abstract Background The incidence of Brugada syndrome (BrS) varies among racial groups. Several studies reported Glycerol-3-Phosphate Dehydrogenase 1-Like (GPD1-L) gene is associated with BrS. However, most of these studies were reported from Western countries, so the evidence about GPD1-L mutation is limited especially among Asian BrS patients. This study aimed to search for rare variants in GPD1-L among Japanese BrS patients and to investigate the pathogenicity. Method We performed whole-exome sequencing for patients with Brugada type 1 ECG pattern from Japanese multicenter BrS cohort consisting of SCN5A-negative BrS probands (n=288) and controls (n=372). We conducted patch-clamp study in human embryonic kidney (HEK) 293 cells cotransfected with the wild-type sodium channel (SCN5A) and wild-type or mutant GPD1-L expression plasmid. Results We identified a rare variant in GPD1-L, p.D262N (c.784g>a) in 2 of 288 BrS probands, which was not identified in 372 controls. The minor allele frequency of the variant is 0.0014% in the Genome Aggregation Database. One proband was a 49-year-old man and the other was 34-year-old man who both developed a ventricular fibrillation. ECGs of both probands showed Brugada Type 1 pattern after administration of the pilsicainide. In functional study, coexpression of D262N GPD1-L with SCN5A in HEK293 cells significantly reduced inward sodium currents compared with wild-type GPD1-L. Additionally, inward sodium currents with D262N were similar to those with A280V GPD1-L, which was associated with BrS in previous reports (Figure). Also, several pathogenicity prediction programs, such as SIFT (score: 0.031) and PolyPhen2 (score: 0.937) predicted deleterious effects of GPD1-L D262N. Conclusion We identified a rare variant in GPD1-L at the rate of 0.7% in Japanese BrS patients without SCN5A mutations. GPD1-L, p.D262N reduces inward sodium currents and may be a novel susceptible variant for BrS in the Japanese population. Funding Acknowledgement Type of funding sources: None. Figure 1. Current–voltage curve

GPD1L is negatively associated with HIF1α expression and predicts lymph node metastasis in oral and HPV- Oropharyngeal cancer.

The study is to determine if Glycerol-3-phosphate dehydrogenase 1-like (GPD1L) and hypoxia-inducible factor-1α (HIF1α) can identify high-risk patients with delayed lymph node metastasis in early-stage head and neck squamous cell carcinoma (HNSCC). The mRNA and protein expressions of markers were analyzed using fresh and paraffin embedded HNSCC specimens. The statistical analyses included chi-squared test, t test, correlation analysis, and univariate and multivariate analyses. GPD1L (downregulated) and HIF1α (upregulated) mRNA expression had a negative correlation (r=-.496, p=.001) in cT1-2N0 HNSCC. The low GPD1L+high HIF1α expression group (22.6%) showed a significant decrease in disease-free survival compared with the high GPD1L+low HIF1α expression group (71.4%) in the neck dissection group. The low GPD1L+high HIF1α expression (39.4%) resulted in a significantly higher delayed metastasis rate than the high GPD1L+low HIF1α expression (5.6%) for cT1-2N0 HNSCC in the neck observation group. GPD1L and HIF1α protein expression more accurately predicted lymph node metastasis than the WINTER hypoxia gene panel (false-negative rate in predicting metastasis: 8.1% versus 26.4%). Cox regression analyses found that the combined protein expression of GPD1L and HIF1α could predict delayed metastasis (HR:0.118, 95% CI:0.027-0.525). Low GPD1L+high HIF1α expression can serve as candidate biomarkers for high-risk populations with lymph node metastases in early-stage HNSCC.

A novel mutation in GPD1‑L associated with early repolarization syndrome via modulation of cardiomyocyte fast sodium currents.

Early repolarization syndrome (ERS) is associated with genetic mutations, but the role of the glycerol‑3‑phosphate dehydrogenase 1‑like (GPD1‑L) mutation remains unclear. The aim of the present study was to investigate the role and potential underlying mechanism of GPD1‑L mutation P112L in the pathogenesis of ERS. Whole‑genome sequencing was performed on samples from a family with ERS, and the gene sequencing results were analyzed using bioinformatics. 293cells were transfected with wild‑type (WT) or mutant‑type (MT) GPD1‑L and SCN5A plasmids. Successful transfection of GPD1‑L in 293cells was verified by western blotting. Whole‑cell patch‑clamp recording, confocal microscopic observation and western blotting were used to uncover the potential mechanism of GPD1‑L P112L in ERS. The results of western blotting indicated that the expression of the GPD1‑L protein was lower in the MT group compared with that in the WT group, but the mock group did not express the GPD1‑L protein. The whole‑cell patch‑clamp recording results indicated that the activation current density of INa (at ‑30mV) was ~60% lower in the MT group compared with the WT group (P<0.01). The mutation caused the inactivation voltage to move in a negative direction by ~3mV compared with that of the WT group. However, there were no significant between‑group differences in the steady activation, steady inactivation, and steady recovery of INa. Confocal microscopy demonstrated that MT GPD1‑L was less expressed near the cell membrane and more expressed in the cytoplasm compared with WT GPD1‑L. Both WT and MT GPD1‑L were highly expressed in the cytoplasm and in small amounts in the nucleus. In conclusion, the GPD1‑L P112L mutation decreased INa activation and GPD1‑L cell expression, including in the region near the cell membrane. These results suggest that GPD1‑L P112L may be a pathogenic genetic mutation associated with ERS.

Long Noncoding RNA Nuclear Enriched Abundant Transcript 1 (NEAT1) Regulates Proliferation, Apoptosis, and Inflammation of Chondrocytes via the miR-181a/Glycerol-3-Phosphate Dehydrogenase 1-Like (GPD1L) Axis.

BackgroundOsteoarthritis (OA) is one of the most common chronic musculoskeletal diseases, yet to date it lacks effective therapeutic strategies. Increasing evidence suggests that long noncoding RNAs (lncRNAs) serve pivotal roles in the occurrence and development of OA. However, the possible molecular mechanism involving lncRNAs, such as nuclear enriched abundant transcript 1 (NEAT1), in OA progression is still unclear.Material/MethodsFirst, NEAT1 and miR-181a expression in OA synovium tissues and normal synovium tissues were detected. Then, the effect of NEAT1 on modulating growth ability, apoptosis, and inflammation in OA chondrocytes was investigated by a series of loss-function experiments. Next, the correlation between NEAT1, miR-181a, and glycerol-3-phosphate dehydrogenase 1-like (GPD1L) was fully investigated. Finally, the downregulation of miR-181a was employed as a recovery experiment to explore the functional mechanism of NEAT1 in OA.ResultsIn the present study, we found that NEAT1 expression was downregulated in OA tissues, while miR-181a expression was prominently upregulated. Moreover, reduced expression of NEAT1 suppressed cell growth while elevating the apoptotic rate and increasing the abundance of inflammatory cytokines released in OA chondrocytes. Furthermore, we clarified that miR-181a was a direct sponge of NEAT1, and GPD1L was able to bind to miR-181a. Additionally, we found that downregulation of miR-181a was able to attenuate the effect of NEAT1 on apoptosis, inflammatory response, and proliferation in OA chondrocytes.ConclusionsOur findings indicate that downregulation of NEAT1 aggravated progression of OA via modulating the miR-181a/GPD1L axis, providing a novel insight into the mechanism of OA pathogenesis.

CTGF increases vascular endothelial growth factor-dependent angiogenesis in human synovial fibroblasts by increasing miR-210 expression.

Connective tissue growth factor (CTGF, a.k.a. CCN2) is inflammatory mediator and abundantly expressed in osteoarthritis (OA). Angiogenesis is essential for OA progression. Here, we investigated the role of CTGF in vascular endothelial growth factor (VEGF) production and angiogenesis in OA synovial fibroblasts (OASFs). We showed that expression of CTGF and VEGF in synovial fluid were higher in OA patients than in controls. Directly applying CTGF to OASFs increased VEGF production then promoted endothelial progenitor cells tube formation and migration. CTGF induced VEGF by raising miR-210 expression via PI3K, AKT, ERK, and nuclear factor-κB (NF-κB)/ELK1 pathways. CTGF-mediating miR-210 upregulation repressed glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression and PHD activity and subsequently promoted hypoxia-inducible factor (HIF)-1α-dependent VEGF expression. Knockdown of CTGF decreased VEGF expression and abolished OASF-conditional medium-mediated angiogenesis in vitro as well as angiogenesis in chick chorioallantoic membrane and Matrigel-plug nude mice model in vivo. Taken together, our results suggest CTGF activates PI3K, AKT, ERK, and NF-κB/ELK1 pathway, leading to the upregulation of miR-210, contributing to inhibit GPD1L expression and prolyl hydroxylases 2 activity, promoting HIF-1α-dependent VEGF expression and angiogenesis in human synovial fibroblasts.

HypoxamiRs and Cancer: From Biology to Targeted Therapy

Significance: Hypoxia is a hallmark of the tumor microenvironment and represents a major source of failure in cancer therapy. Recent Advances: Recent work has generated extensive evidence that microRNAs (miRNAs) are significant components of the adaptive response to low oxygen in tumors. Induction of specific miRNAs, collectively termed hypoxamiRs, has become an accepted feature of the hypoxic response in normal and transformed cells. Critical Issues: Overexpression of miR-210, the prototypical hypoxamiR, is detected in most solid tumors, and it has been linked to adverse prognosis in many tumor types. Several miR-210 target genes, including iron-sulfur (Fe-S) cluster scaffold protein (ISCU) and glycerol-3-phosphate dehydrogenase 1-like (GPD1L), have been correlated with prognosis in an inverse fashion to miR-210, suggesting that their down- regulation by miR-210 occurs in vivo and contributes to tumor growth. Additional miRNAs are modulated by decreased oxygen tension in a more tissue-specific fashion, adding another level of complexity over the classic hypoxia-regulated gene network. Future Directions: From a biological standpoint, hypoxamiRs are emerging modifiers of cancer cell response to the adaptive challenges of the microenvironment. From a clinical perspective, assessing the status of these miRNAs may contribute to a detailed understanding of hypoxia-induced mechanisms of resistance and/or to the fine-tuning of future hypoxia-modifying therapies. Antioxid. Redox Signal. 21, 1220–1238.

The prognostic value of glycerol‐3‐phosphate dehydrogenase 1‐like expression in head and neck squamous cell carcinoma

In this study, we sought to determine the prognostic significance of glycerol-3-phosphate dehydrogenase 1-like (GPD1L) expression in head and neck squamous cell carcinoma (HNSCC). The mRNA levels of GPD1L were measured in 70 paired HNSCC and corresponding adjacent normal tissues using real-time PCR. GPD1L protein levels were evaluated in HNSCC from 135 patients using immunohistochemical staining. Correlations were analysed between GPD1L levels and local recurrence rate, regional recurrence rate, second primary malignancy rate), disease-free survival (DFS) and disease-specific survival (DSS). The results of real-time PCR showed that, compared with the paired normal tissues, mRNA levels of GPD1L were decreased significantly in HNSCC (P < 0.001). Patients whose tumours showed high GPD1L protein expression had a significantly better prognosis than those whose tumours showed low expression (61.3% versus 21.4%, P < 0.001 for DFS; 68% versus 39.3%, P = 0.001 for DSS). High GPD1L expression was associated with a lower local recurrence rate than low GPD1L expression (P = 0.049). Multivariate survival analysis also showed that GPD1L expression was an independent prognostic factor (P = 0.001). Our results indicate that the GPD1L expression is a strong predictor for local recurrence and survival in HNSCC.

A Hypoxia-Induced Positive Feedback Loop Promotes Hypoxia-Inducible Factor 1α Stability through miR-210 Suppression of Glycerol-3-Phosphate Dehydrogenase 1-Like

ABSTRACT Oxygen-dependent regulation of the transcription factor HIF-1α relies on a family of prolyl hydroxylases (PHDs) that hydroxylate hypoxia-inducible factor 1α (HIF-1α) protein at two prolines during normal oxygen conditions, resulting in degradation by the proteasome. During low-oxygen conditions, these prolines are no longer hydroxylated and HIF-1α degradation is blocked. Hypoxia-induced miRNA-210 (miR-210) is a direct transcriptional target of HIF-1α, but its complete role and targets during hypoxia are not well understood. Here, we identify the enzyme glycerol-3-phosphate dehydrogenase 1-like (GPD1L) as a novel regulator of HIF-1α stability and a direct target of miR-210. Expression of miR-210 results in stabilization of HIF-1α due to decreased levels of GPD1L resulting in an increase in HIF-1α target genes. Altering GPD1L levels by overexpression or knockdown results in a decrease or increase in HIF-1α stability, respectively. GPD1L-mediated decreases in HIF-1α stability can be reversed by pharmacological inhibition of the proteasome or PHD activity. When rescued from degradation by proteasome inhibition, elevated amounts of GPD1L cause hyperhydroxylation of HIF-1α, suggesting increases in PHD activity. Importantly, expression of GPD1L attenuates the hypoxic response, preventing complete HIF-1α induction. We propose a model in which hypoxia-induced miR-210 represses GPD1L, contributing to suppression of PHD activity, and increases of HIF-1α protein levels.

Reactive Oxygen Species Originating from Mitochondria Regulate the Cardiac Sodium Channel

Background: Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein, a gene associated with Brugada Syndrome and Sudden Infant Death Syndrome, cause reduced cardiac sodium channel (Nav1.5) function by modulating NAD(H). NAD(H) regulates the cardiac Na+ channel through generation of reactive oxygen species (ROS). Here, we investigated the source of ROS induced by elevated NADH.

Reactive Oxygen Species Originating From Mitochondria Regulate the Cardiac Sodium Channel

Pyridine nucleotides regulate the cardiac Na(+) current (I(Na)) through generation of reactive oxygen species (ROS). We investigated the source of ROS induced by elevated NADH. In human embryonic kidney (HEK) cells stably expressing the cardiac Na(+) channel, the decrease of I(Na) (52±9%; P<0.01) induced by cytosolic NADH application (100 μmol/L) was reversed by mitoTEMPO, rotenone, malonate, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), PK11195, and 4'-chlorodiazepam, a specific scavenger of mitochondrial superoxide and inhibitors of the mitochondrial complex I, complex II, voltage-dependent anion channels, and benzodiazepine receptor, respectively. Anti-mycin A (20 μmol/L), a complex III inhibitor known to generate ROS, decreased I(Na) (51±4%, P<0.01). This effect was blocked by NAD(+), forskolin, or rotenone. Inhibitors of complex IV, nitric oxide synthase, the NAD(P)H oxidases, xanthine oxidases, the mitochondrial permeability transition pore, and the mitochondrial ATP-sensitive K(+) channel did not change the NADH effect on I(Na). Analogous results were observed in cardiomyocytes. Rotenone, mitoTEMPO, and 4'-chlorodiazepam also blocked the mutant A280V GPD1-L (glycerol-3-phosphate dehydrogenase 1-like) effect on reducing I(Na), indicating a role for mitochondria in the Brugada syndrome caused by this mutation. Fluorescent microscopy confirmed mitochondrial ROS generation with elevated NADH and ROS inhibition by NAD(+). Altering the oxidized to reduced NAD(H) balance can activate mitochondrial ROS production, leading to reduced I(Na). This signaling cascade may help explain the link between altered metabolism, conduction block, and arrhythmic risk.

Cardiac Metabolic State and Brugada Syndrome

See related article, pages 737–745 Brugada syndrome (BrS) is an inherited cardiac disorder with variable ECG features characteristic of right bundle branch bloc (RBBB) and ST segment elevation in the right precordial leads (V1-V3) at rest, which was first described by the Brugada brothers as a clinical entity with a genetic background.1 In Southeast Asia, BrS causes a high risk of sudden unexplained nocturnal death syndrome (SUNDS) in young adults.2,3 In genetically determined forms of BrS, a number of mutations in the SCN5A gene located on chromosome 3p21-23 have been implicated in BrS and account for 20% to 30% of BrS in genotyped families.4 More recently, mutations in other genes such as those coding for glycerol-3-phosphate dehydrogenase 1-like (GPD1-L),5 the α-subunit of the Cav1.2 L-type calcium channel ( CACNA1C ), the β2 calcium channel regulatory subunit ( CACNB2 ),6,7 and the hERG potassium channel ( KCNH2 )8 have been shown to be associated with BrS clinical phenotypes. Mutations in the SCN5A gene are associated with loss-of-function caused by trafficking defects, truncated sodium channel proteins, or changes in the biophysical properties of sodium channels. Loss-of-function mutations of the Cav1.2 L-type calcium channel and its regulatory subunit, which are encoded by CACNA1C and CACNB2 , respectively (A39V- and G490R/ CACNA1C and S481L/ CACNB2 ), have been reported to be associated with a BrS phenotype combined with shorter QT intervals. Two gain-of-function mutations in the KCNH2 (hERG) of the cardiac potassium channel (G873S and N985S) have been reported in patients with BrS syndrome. Glycerol-3-phosphate dehydrogenase (GPD) catalyzes the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate. GPD uses nicotinamide adenine dinucleotide (NADH) as a cofactor for this …

Cardiac Na + Current Regulation by Pyridine Nucleotides

Mutations in glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) protein reduce cardiac Na+ current (I(Na)) and cause Brugada Syndrome (BrS). GPD1-L has >80% amino acid homology with glycerol-3-phosphate dehydrogenase, which is involved in NAD-dependent energy metabolism. Therefore, we tested whether NAD(H) could regulate human cardiac sodium channels (Na(v)1.5). HEK293 cells stably expressing Na(v)1.5 and rat neonatal cardiomyocytes were used. The influence of NADH/NAD+ on arrhythmic risk was evaluated in wild-type or SCN5A(+/-) mouse heart. A280V GPD1-L caused a 2.48+/-0.17-fold increase in intracellular NADH level (P<0.001). NADH application or cotransfection with A280V GPD1-L resulted in decreased I(Na) (0.48+/-0.09 or 0.19+/-0.04 of control group, respectively; P<0.01), which was reversed by NAD+, chelerythrine, or superoxide dismutase. NAD+ antagonism of the Na+ channel downregulation by A280V GPD1-L or NADH was prevented by a protein kinase (PK)A inhibitor, PKAI(6-22). The effects of NADH and NAD+ were mimicked by a phorbol ester and forskolin, respectively. Increasing intracellular NADH was associated with an increased risk of ventricular tachycardia in wild-type mouse hearts. Extracellular application of NAD+ to SCN5A(+/-) mouse hearts ameliorated the risk of ventricular tachycardia. Our results show that Na(v)1.5 is regulated by pyridine nucleotides, suggesting a link between metabolism and I(Na). This effect required protein kinase C activation and was mediated by oxidative stress. NAD+ could prevent this effect by activating PKA. Mutations of GPD1-L may downregulate Na(v)1.5 by altering the oxidized to reduced NAD(H) balance.

Regulation Of Cardiac Na+ Channel By NAD+/NADH

Introduction: Glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) mutations have been shown to reduce cardiac Na+ current and cause Brugada Syndrome. Our previous work suggests that mutations in GPD1-L act through NAD(H) to alter Na+ current. NADH results in downregulation of Na+ current and NAD+ can reverse the downregulation of Na+ current by mutant (MT) GPD1-L or NADH. Here, we studied potential signaling pathways between GPD1-L, NAD(H), and Na+ channel.Methods: Currents were measured using whole-cell patch clamp of HEK cells stably expressing the human cardiac sodium channel with and without chelerythrine (a PKC inhibitor, IC50 = 660 nM), PKAI6-22 (a PKA inhibitor, IC50 = 1.6 nM), apocynin (a NADPH oxidase inhibitor), or elevated intracellular Ca2+ in the pipette solution. MT GPD1-L (A280V) was co-transfected with red fluorescent protein with Fugene6 to HEK cells 40 hours prior to measuring current.Results: The 2-fold reduction in Na+ current mediated by NADH (100 μM) was inhibited by 200 μM apocynin (50±6% vs. 86±15% of the control current, n=14, p<0.001), 6 mM [Ca2+]i (107±17% of control, n=12, p<0.001), or 10 μM chelerythrine (86±14% of control, n=10, p<0.05). PKAI6-22 blocked the NAD+-mediated upregulation of Na+ currents in the presence of MT GPD1-L (n=10, p<0.001). Chelerythrine (10-50 μM) or PKAI6-22 (50 nM - 5 μM) alone did not affect the peak currents of Na+ channels.Conclusions: Our experiments suggest that NAD(H) can alter Na+ currents. Downregulation by NADH seems to involve the NADPH oxidase and PKC. PKA appears to be involved in NAD+-dependent current upregulation. This implies that redox/metabolic state can influence Na+ current.

Abstract 5346: Regulation of Cardiac Na + Current by Pyridine Nucleotides: A Possible Explanation of Glycerol-3-Phosphate Dehydrogenase-Linked Brugada Syndrome

Recently, glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) gene mutations have been shown to reduce cardiac Na + current and cause Brugada Syndrome (BrS). The glycerol-3-phosphate dehydrogenase (GPD) family of genes is involved in nicotinamide adenine dinucleotides (NAD)-dependent energy metabolism, and GDP1-L has &gt;80% amino acid homology with GPD. Therefore, we tested whether mutations in GPD1-L could be acting through NAD (H) to alter Na + current. Human embryonic kidney (HEK) cells stably expressing the human cardiac sodium channel (SCN5A) were used to assess the effects of wild-type (WT) and mutant (MT) GPD1-L on cellular NAD(H) and the effects of NAD(H) on Na + current. A mouse model of BrS was used to assess the effect of reduced NAD + on arrhythmic risk. MT GPD1-L raised cellular NADH level by 4.3 fold (p&lt;0.01) and reduced Na + current by 69.9% (p&lt;0.01). Extracellular NADH (300 μM) raised cellular NADH by 5.3 fold and decreased whole cell peak conductance by 71.4% (p&lt;0.001). Extracellular NAD + (300 μM) raised conductance by 30.3% (p&lt; 0.001). Fluorescent microscopy showed parallel changes in membrane-associated, GFP-tagged Na + channels. Intracellular application of NADH or NAD + resulted in an immediate change in Na + current of −55.5% (p &lt; 0.01) and +66.6% (p &lt; 0.01), respectively. External NAD + could prevent the reduction in Na + current caused by MT GPD1-L. Apocynin (100 μM), an NAD(P)H oxidase inhibitor, or the reducing agent, dithiothreitol (DTT), prevented the NADH-induced reduction in Na + current (p &lt; 0.01). Application of 100 μM NAD + to a mouse model of BrS reduced the programmed electrical stimulation induced ventricular tachycardia. GPD1-L mutations may cause BrS through alterations in cellular NAD(H), and NAD + might represent a novel treatment for BrS.

Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene ( GPD1-L ) Decreases Cardiac Na + Current and Causes Inherited Arrhythmias

Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause approximately 20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by approximately 50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31+/-5% (P=0.01). GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.