The pesticides paraquat (PQ) and maneb (MB) have been described as environmental risk factors for Parkinson's disease (PD), with mechanisms associated with mitochondrial dysfunction and reactive oxygen species generation. A combined exposure of PQ and MB in murine models and neuroblastoma cells has been utilized to further advance understanding of the PD phenotype. MB acts as a redox modulator through alkylation of protein thiols and has been previously characterized to inhibit complex III of the electron transport chain and uncouple the mitochondrial proton gradient. The purpose of this study was to analyze ATP-linked respiration and glycolysis in human neuroblastoma cells utilizing the Seahorse extracellular flux platform. Employing an acute, subtoxic exposure of MB, this investigation revealed a MB-mediated decrease in mitochondrial oxygen consumption at baseline and maximal respiration, with inhibition of ATP synthesis and coupling efficiency. Additionally, MB-treated cells showed an increase in nonmitochondrial respiration and proton leak. Further investigation into mitochondrial fuel flex revealed an elimination of fuel flexibility across all 3 major substrates, with a decrease in pyruvate capacity as well as glutamine dependency. Analyses of glycolytic function showed a substantial decrease in glycolytic acidification caused by lactic acid export. This inhibition of glycolytic parameters was also observed after titrating the MB dose as low as 6 μM, and appears to be dependent on the dithiocarbamate functional group, with manganese possibly potentiating the effect. Further studies into cellular ATP and NAD levels revealed a drastic decrease in cells treated with MB. In summary, MB significantly impacted both aerobic and anaerobic energy production; therefore, further characterization of MB's effect on cellular energetics may provide insight into the specificity of PD to dopaminergic neurons.
Down syndrome (DS) is a chromosomal disorder caused by trisomy of chromosome 21 (Ts21). Unbalanced karyotypes can lead to dysfunction of the proteostasis network (PN) and disrupted proteostasis is mechanistically associated with multiple DS comorbidities. Autophagy is a critical component of the PN that has not previously been investigated in DS. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts and other DS cell models. Following induction of autophagy by serum starvation, DS fibroblasts displayed impaired autophagic flux indicated by autophagolysosome accumulation and elevated p62, NBR1, and LC3-II abundance, compared to age- and sex-matched, euploid (CTL) fibroblasts. While lysosomal physiology was unaffected in both groups after serum starvation, we observed decreased basal abundance of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) family members syntaxin 17 (STX17) and Vesicle Associated Membrane Protein 8 (VAMP8) indicating that decreased autophagic flux in DS is due at least in part to a possible impairment of autophagosome-lysosome fusion. This conclusion was further supported by the observation that over-expression of either STX17 or VAMP8 in DS fibroblasts restored autophagic degradation and reversed p62 accumulation. Collectively, our results indicate that impaired autophagic clearance is a characteristic of DS cells that can be reversed by enhancement of SNARE protein expression and provides further evidence that PN disruption represents a candidate mechanism for multiple aspects of pathogenesis in DS and a possible future target for therapeutic intervention.
Cardiac myocytes isolated from adult heart tissue have a rod‐like shape with highly organized intracellular structures. Cardiomyocytes derived from human pluripotent stem cells (iPSC‐CMs), on the other hand, exhibit disorganized structure and contractile mechanics, reflecting their pronounced immaturity. These characteristics hamper research using iPSC‐CMs. The protocol described here enhances iPSC‐CM maturity and function by controlling the cellular shape and environment of the cultured cells. Microstructured silicone membranes function as a cell culture substrate that promotes cellular alignment. iPSC‐CMs cultured on micropatterned membranes display an in‐vivo‐like rod‐shaped morphology. This physiological cellular morphology along with the soft biocompatible silicone substrate, which has similar stiffness to the native cardiac matrix, promotes maturation of contractile function, calcium handling, and electrophysiology. Incorporating this technique for enhanced iPSC‐CM maturation will help bridge the gap between animal models and clinical care, and ultimately improve personalized medicine for cardiovascular diseases. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Cardiomyocyte differentiation of iPSCs Basic Protocol 2: Purification of differentiated iPSC‐CMs using MACS negative selection Basic Protocol 3: Micropatterning on PDMS
KCNH2 encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (I Kr ) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a hERG1 polypeptide (hERG1 NP ) that is targeted to the nuclei of immature cardiac cells, including human stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat cardiomyocytes. The nuclear hERG1 NP immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1 NP signal maps to the hERG1 distal C-terminal domain. KCNH2 deletion using CRISPR simultaneously abolished I Kr and the hERG1 NP signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or β1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the putative hERG1 NP peptide in the nuclei of HEK cells significantly reduced hERG1a current density, compared to cells expressing the NLS-deficient hERG1 NP or GFP. These data identify a developmentally regulated polypeptide encoded by KCNH2 , hERG1 NP , whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.
Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21 (Tri21). This unbalanced karyotype has the ability to produce proteotoxic stress and dysfunction of the proteostasis network (PN), which are mechanistically associated with several comorbidities found in the DS phenotype. Autophagy is the cellular process responsible for bulk protein degradation and its impairment could negatively impact protein quality control. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts. Both euploid (CTL) and DS fibroblasts induced autophagy successfully through serum starvation (SS), as evidenced by increased LC3-II abundance in CTL and DS. However, DS cells displayed evidence of autophagolysosome (AL) accumulation and impaired clearance of autophagosome cargo, e.g. accumulation of p62 and NBR1. Similar observations were also present in DS cells from multiple differentiation stages, implicating impeded autophagic degradation as a possible early pathologic mechanism in DS. Lysosomal pH and cathepsin B proteolytic activity were found to not differ in CTL and DS fibroblasts after SS, indicating that lysosomal dysfunction did not appear to contribute to unsuccessful autophagic clearance. Based on these results, we hypothesized that possible interference of the endosomal system with autophagy results in autophagosome fusion with endosomal vesicles and negatively impacts degradation. Consistent with this hypothesis, we observed increased abundance of the recycling endosome marker, Rab11, after SS in DS fibroblasts. Further, colocalization of autophagosome markers with resident proteins of early endosomes, late endosomes and recycling endosomes (Rab11) further support our hypothesis. In summary, our work is consistent with impairment of autophagic flux and general PN dysfunction as candidate mechanisms for pathology in DS.
hERG1 conducts cardiac IKr and is critical for repolarization of the human heart. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden cardiac death. At least two subunits combine to form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG 1a/1b subunit abundance modulates IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue have suggested that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven hiPSC-CM maturation model to investigate how changes in relative hERG 1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immature hiPSC-CMs display a two-fold greater inhibition by extracellular acidosis (pH 6.3) compared to matured hiPSC-CMs. qRT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated, and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared to immature hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by an increased in IKr density. Silencing the impact of hERG1b on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a PAS domain reduced the magnitude of IKr proton inhibition in immature hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and that hERG1 subunit abundance determines IKr sensitivity to protons in hiPSC-CMs.
region which comprises a cyclic nucleotide binding domain (CNBD). Altered cAMP responses caused either by point mutations in the protein or by nonphysiological levels of the cyclic nucleotide in the cell lead to pathological conditions both in the heart and in the nervous system. The recently obtained cryo-EM structures of human HCN1 in the cAMP-bound and unbound forms confirm previous NMR studies on cAMP-induced conformational changes and reveals the presence of two additional helices at the C-terminus of the CNBD which fold in the presence of cAMP. Inspection of cAMP-bound structure reveals the presence of several putative electrostatic and hydrophobic interactions between both the D and E helices and the CNBD's C-helix, which enable a hairpin-like conformation that holds the C-helix in place. We have previously shown, by isothermal titration calorimetry (ITC) and patch-clamp experiments, that deletion of helices D and E decreases the affinity for cAMP in HCN1, HCN2 and HCN4, the three HCN isoforms that respond to cAMP. Here, we introduce single point mutations in HCN4, designed to disrupt individual residue interactions with the aim to investigate the molecular mechanism that allows such fine regulation of cAMP response. All patch-clamp experiments were performed in whole cell configuration using a new all-in-one integrated amplifier, the ePatch.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.