Gao, Shan

Background: Previously, we discovered that DENND3, a guanine nucleotide exchange factor regulating Rab GTPases, influences cardiac ion channel trafficking. Both electrophysiological dysfunction and arrhythmic events were identified in variant inserted inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) expressing DENND3-p.R534S. This was a novel missense variant identified in a patient with familial idiopathic ventricular fibrillation (IVF). Notably, augmented membrane retention of key cardiac ion channels was observed in both DENND3-p.R534S expressing TSA201 cells and variant inserted iPSC-CMs. To further study the mechanisms underlying arrhythmogenesis associated with IVF, intracellular calcium handling was characterized in these re-engineered heart cells with DENND3-p.R534S. Methods: Here, iPSC-CMs expressing DENND3-p.R534S and isogenic controls (IC) were studied. Optical action potentials were recorded using the FluoVolt voltage-sensitive dye at a constant pacing rate of 1 Hz. Multi-electrode array (MEA) recordings assessed beat period and conduction dynamics. Intracellular calcium dynamics were analyzed using Fluo-4 calcium imaging. Results: DENND3-p.R534S iPSC-CMs exhibited frequent arrhythmic activity, including irregular beating patterns (78%, n=28/36), alternans (25%), early afterdepolarizations (EADs, 8%), and delayed afterdepolarizations (DADs, 8%) versus 0% in IC lines (n=0/36, p<0.05). MEA recordings revealed significantly prolonged and irregular beat periods (2.59±0.26s vs. 1.37±0.10s, p<0.05), indicating electrical instability. Calcium imaging further showed significantly reduced calcium transient amplitude in DENND3-p.R534S iPSC-CMs compared to ICs (0.035±0.039ΔF/F 0 vs. 0.302±0.099ΔF/F 0 , p<0.05), and slowed upstroke velocity (0.170±0.212 ΔF/F 0 ●s -1 vs. 1.340±0.463ΔF/F 0 ●s -1 ) suggesting impaired intracellular calcium release. Conclusion: The DENND3-p.R534S variant not only disrupts ion channel distribution as shown previously but also markedly perturbs intracellular calcium handling, contributing to electrophysiological instability and arrhythmogenic cellular phenotypes. These findings highlight DENND3’s novel and emerging role in maintaining cardiac cellular homeostasis and rhythm stability.

Background: We identified a novel, ultra-rare missense variant (p.R534S) in DENND3 as a potential disease-susceptibility variant in a multi-generational pedigree of familial idiopathic ventricular fibrillation (IVF). DENND3 is a guanine nucleotide exchange factor and activator for small GTPase Rab proteins involved in endosome and ion channel membrane trafficking. However, the role of DENND3 in the heart and its potential involvement in cardiac arrhythmias are unknown. Objective: To functionally characterize DENND3-p.R534S and determine its impact on channel trafficking. Methods: Heterologous co-expression studies in TSA201 cells were performed with DENND3-WT or DENND3-p.R534S and ion channels: KCNQ1 (Kv7.1, IKs), KCNH2 (Kv11.1, IKr), SCN5A (Nav1.5, INa), or CACNA1C (Cav1.2, ICa) as well as Rab proteins (Rab4, Rab5, Rab6, Rab7, Rab11, Rab12). Patient-specific DENND3-p.R534S iPSC-derived cardiomyocytes (iPSC-CMs) and isogenic controls (IC) were created. Localization and expression of ion channel and Rab proteins was assessed using immunofluorescence (IF) by super resolution AiryScan microscopy. GTPase-Glo assays measured Rab protein activity. Electrophysiology studies using patch-clamp and FluoVolt were performed on iPSC-CM models. Results: IF imaging showed significant increases in membrane expression of KCNQ1 (11714±157to 21929±378, p<0.05), KCNH2 (11378±160 to 31965±544, p<0.05), , SCN5A (10339±8 to 11225±23, p<0.05), and CACNA1C (11879±83 to 13749±138, p<0.05) in DENND3-p.R534S compared to DENND3-WT expressing TSA201 cells. Rab5 distribution was altered significantly by the DENND3-p.R534S variant, with increased dispersion away from the nucleus (0.23±0.01 vs. 0.17±0.01, p<0.05) and increased proximity to the plasma membrane (0.16±0.02 vs. 0.23±0.01, p<0.05). Additionally, Rab5 activity was enhanced significantly (57% increase) by p.R534S versus WT (p<0.05). iPSC-CMs revealed similar increases in channel membrane accumulation and irregular arrhythmic activity including frequent action potential duration alternans and afterdepolarizations in 78% of p.R534S iPSC-CMs versus none in IC cells (p<0.05). Conclusion: DENND3 is a novel ion channel trafficking protein in the heart. The DENND3-p.R534S variant disrupts cardiac ion channel trafficking. These findings highlight DENND3’s role in cardiac ion channel distribution, cellular function, and potential association with arrhythmogenic risk.

Abstract Background Myeloid cells comprise up to 50% of the total tumor mass in glioblastoma (GBM) and have been implicated in promoting tumor progression and immunosuppression. Modulating the response of myeloid cells to the tumor has emerged as a promising new approach for cancer treatment. In this regard, we focus on the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), which has recently emerged as a novel immune modulator in peripheral tumors. Methods We studied the TREM2 expression profile in various patient tumor samples and conducted single-cell transcriptomic analysis in both GBM patients and the GL261 mouse glioma model. We utilized multiple mouse glioma models and employed state-of-the-art techniques such as invivo 2-photon imaging, spectrum flow cytometry, and in vitro co-culture assays to study TREM2 function in myeloid cell-mediated phagocytosis of tumor cells, antigen presentation, and response of CD4+ T cells within the tumor hemispheres. Results Our research revealed significantly elevated levels of TREM2 expression in brain tumors compared to other types of tumors in patients. TREM2 was predominantly localized in tumor-associated myeloid cells and was highly expressed in nearly all microglia, as well as various subtypes of macrophages. Surprisingly, in preclinical glioma models, TREM2 deficiency did not confer a beneficial effect; instead, it accelerated glioma progression. Through detailed investigations, we determined that TREM2 deficiency impaired the ability of tumor-myeloid cells to phagocytose tumor cells and led to reduced expression of MHCII. This deficiency further significantly decreased the presence of CD4+ T cells within the tumor hemispheres. Conclusions Our study unveiled a previously unrecognized protective role of tumor-myeloid TREM2. Specifically, we found that TREM2 enhances the phagocytosis of tumor cells and promotes an immune response by facilitating MHCII-associated CD4+ T-cell responses against gliomas.