5-Hydroxymethylcytosine Signatures in Circulating Cell-Free DNA as Early Warning Biomarkers for COVID-19 Progression and Myocardial Injury

Background: The symptoms of coronavirus disease 2019 (COVID-19) range from moderate to critical conditions, leading to death in some patients, and the early warning indicators of the COVID-19 progression and the occurrence of its serious complications such as myocardial injury are limited.Methods: We carried out a multi-center, prospective cohort study in three hospitals in Wuhan. Genome-wide 5-hydroxymethylcytosine (5hmC) profiles in plasma cell-free DNA (cfDNA) was used to identify risk factors for COVID-19 pneumonia and develop a machine learning model using samples from 53 healthy volunteers, 66 patients with moderate COVID-19, 99 patients with severe COVID-19, and 38 patients with critical COVID-19.Results: Our warning model demonstrated that an area under the curve (AUC) for 5hmC warning moderate patients developed into severe status was 0.81 (95% CI 0.77–0.85) and for severe patients developed into critical status was 0.92 (95% CI 0.89–0.96). We further built a warning model on patients with and without myocardial injury with the AUC of 0.89 (95% CI 0.84–0.95).Conclusion: This is the first study showing the utility of 5hmC as an accurate early warning marker for disease progression and myocardial injury in patients with COVID-19. Our results show that phosphodiesterase 4D and ten-eleven translocation 2 may be important markers in the progression of COVID-19 disease.

circ_0023461 Silencing Protects Cardiomyocytes from Hypoxia-Induced Dysfunction through Targeting miR-370-3p/PDE4D Signaling

Background. Acute myocardial infarction (AMI) is a common cardiovascular disease with high disability and mortality. Circular RNAs (circRNAs) are implicated in the pathomechanism of multiple human diseases, including AMI. This study intended to explore the function and working mechanism of a novel circRNA circ_0023461 in hypoxia-induced cardiomyocytes. Methods. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were implemented to detect RNA and protein expression. Cell counting kit-8 (CCK8) assay and 5-ethynyl-2 ′ -deoxyuridine (Edu) assay were conducted to analyze cell viability and proliferation ability. Cell migration and apoptosis were assessed by Transwell assay and flow cytometry. Cell oxidative stress was analyzed using the commercial kits. Enzyme-linked immunosorbent assay (ELISA) was conducted to analyze cell inflammation. Cell glycolytic metabolism was evaluated using the commercial kits. Dual-luciferase reporter assay and RNA pull-down assay were conducted to verify the intermolecular interactions. Results. circ_0023461 expression was upregulated in AMI patients and hypoxia-induced AC16 cells. Hypoxia restrained the viability, proliferation, migration, and glycolysis and induced the apoptosis, oxidative stress, and inflammation of AC16 cells, and these effects were attenuated by the silence of circ_0023461. MicroRNA-370-3p (miR-370-3p) was verified as a target of circ_0023461, and circ_0023461 silencing-mediated protective effects in hypoxia-induced cardiomyocytes were partly alleviated by the knockdown of miR-370-3p. miR-370-3p interacted with the 3 ′ untranslated region (3 ′ UTR) of phosphodiesterase 4D (PDE4D), and PDE4D overexpression partly reversed miR-370-3p overexpression-induced protective effects in hypoxia-induced cardiomyocytes. circ_0023461 can upregulate PDE4D expression by acting as a molecular sponge for miR-370-3p in AC16 cells. Conclusion. circ_0023461 knockdown attenuated hypoxia-induced dysfunction in AC16 cells partly by targeting the miR-370-3p/PDE4D axis.

Abstract MP241: Roflumilast-mediated Phosphodiesterase 4d Inhibition Reverses Diabetes-associated Cardiac Dysfunction And Remodeling: Effects Beyond Glucose Lowering

Introduction: Patients with type 2 diabetes (T2DM) have a substantial risk of developing cardiovascular disease. Our recent studies reveal that hyperinsulinemia attenuates cardiac contractility by inducing expression of phosphodiesterase 4D (PDE4D) that increases cAMP degradation. Furthermore, there is growing evidence that PDE4 dysregulation is of pathophysiological importance in metabolic disorders. Hypothesis: We propose that inhibition of PDE4D might ameliorate diabetes-associated cardiac dysfunction, in addition to lowering glucose. Methods: Male C57BL/6J mice fed with high-fat diet (HFD) were treated with PDE4 inhibitor roflumilast (currently used to treat chronic obstructive pulmonary disease, COPD). Myocardial structure, contractile function and remodeling were evaluated. For mechanistic studies, specific silencing of cardiac PDE4D and overexpression of miR-1 were administrated in mice undergoing high-fat feeding. These studies were complemented by in vitro analysis in primary cultured rat cardiomyocytes and cardiac fibroblasts. Results: Mice on HFD developed greater body weight compared to normal chow diet-fed mice, manifesting hyperglycemia, hyperinsulinemia, cardiac contractile dysfunction and remodeling, and cardiac PDE4D5 upregulation by 24 weeks. The expression of PDE4D5 was also elevated in human hearts with diabetes. In comparison with vehicle-treated HFD controls, PDE4 inhibitor roflumilast (1mg/kg/day) can prevent and even reverse hyperglycemia and cardiac dysfunction, accompanied by the decrease of cardiac PDE4D expression. In addition, cardiac miR-1 level was reduced in HFD mice, which was restored by PDE4 inhibitor roflumilast treatment. Either cardiac specific PDE4D5 knockdown or miR-1 overexpression significantly reversed cardiac dysfunction in HFD-mice, despite persistence of hyperglycemia and glucose intolerance. Gain- and loss-of-function studies of PDE4D in cardiomyocytes implicated that suppression of PDE4D protected cardiac hypertrophy via SERCA2a-mediated miR-1 restoration. Moreover, inhibition of PDE4D prevented insulin-activated TGF-β1 signaling which promotes miR-1 reduction in cardiac fibroblasts and subsequent fibrosis. Conclusions: These studies elucidate a novel mechanism by which PDE4D contributes to HFD-induced heart failure through reducing miR-1 expression in both cardiomyocytes and cardiac fibroblasts and suggest a therapeutic potential of PDE4 inhibitor roflumilast in preventing or treating cardiac dysfunction associated with diabetes.

Phosphodiesterase 4D Depletion/Inhibition Exerts Anti-Oncogenic Properties in Hepatocellular Carcinoma

Isoform D of type 4 phosphodiesterase (PDE4D) has recently been associated with several human cancer types with the exception of human hepatocellular carcinoma (HCC). Here we explored the role of PDE4D in HCC. We found that PDE4D gene/protein were over-expressed in different samples of human HCCs compared to normal livers. Accordingly, HCC cells showed higher PDE4D activity than non-tumorigenic cells, accompanied by over-expression of the PDE4D isoform. Silencing of PDE4D gene and pharmacological inhibition of protein activity by the specific inhibitor Gebr-7b reduced cell proliferation and increased apoptosis in HCC cells, with a decreased fraction of cells in S phase and a differential modulation of key regulators of cell cycle and apoptosis. PDE4D silencing/inhibition also affected the gene expression of several cancer-related genes, such as the pro-oncogenic insulin growth factor (IGF2), which is down-regulated. Finally, gene expression data, available in the CancerLivER data base, confirm that PDE4D over-expression in human HCCs correlated with an increased expression of IGF2, suggesting a new possible molecular network that requires further investigations. In conclusion, intracellular depletion/inhibition of PDE4D prevents the growth of HCC cells, displaying anti-oncogenic effects. PDE4D may thus represent a new biomarker for diagnosis and a potential adjuvant target for HCC therapy.