Α2 Isoform Research Articles

Arsenic: A Perspective on Its Effect on Pioglitazone Bioavailability.

Urine, water, and plasma samples from a healthy population (n = 11) and a population with T2DM (n = 20) were obtained. Samples were analyzed by fluorescence spectroscopy/hydride generation (As) and HPLC (pioglitazone). Additionally, CYP 3A4 and CYP 2C8 were studied by density functional theory (DFT). The healthy and T2DM groups were exposed via drinking water to >0.010 ppm, Ka values with a factor of 4.7 higher, Cl 1.42 lower, and ABCt 1.26 times higher concerning the healthy group. In silico analysis (DFT) of CYP 3A4 and CYP 2C8 isoforms showed the substitution of the iron atom by As in the active sites of the enzymes. The results indicate that the substitution of Fe for As modifies the enzymatic function of CYP 3A4 and CYP 2C8 isoforms, altering the metabolic process of CYP 2D6 and CYP 3A4 in patients with T2DM. Consequently, the variation in metabolism alters the bioavailability of pioglitazone and the expected final effect.

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

Lipids and Secretory Vesicle Exocytosis.

In recent years, the number of studies implicating lipids in the regulation of synaptic vesicle exocytosis has risen considerably. It has become increasingly clear that lipids such as phosphoinositides, lysophospholipids, cholesterol, arachidonic acid and myristic acid play critical regulatory roles in the processes leading up to exocytosis. Lipids may affect membrane fusion reactions by altering the physical properties of the membrane, recruiting key regulatory proteins, concentrating proteins into exocytic "hotspots" or by modulating protein functions allosterically. Discrete changes in phosphoinositides concentration are involved in multiple trafficking events including exocytosis and endocytosis. Lipid-modifying enzymes such as the DDHD2 isoform of phospholipase A1 were recently shown to contribute to memory acquisition via dynamic modifications of the brain lipid landscape. Considering the increasing reports on neurodegenerative disorders associated with aberrant intracellular trafficking, an improved understanding of the control of lipid pathways is physiologically and clinically significant and will afford unique insights into mechanisms and therapeutic methods for neurodegenerative diseases. Consequently, this chapter will discuss the different classes of lipids, phospholipase enzymes, the evidence linking them to synaptic neurotransmitter release and how they act to regulate key steps in the multi-step process leading to neuronal communication and memory acquisition.

Determination of ATP1A1 Gene Polymorphism in the Turkish Holstein Cattle

Heat stress is an important factor negatively affecting the productive characteristics, immune response and reproductive performance of livestock. Sustainable livestock systems that can tolerate the impact of increasing environmental temperature are very important to ensure global food security. Oxidative stress triggered by heat stress influences plasma Na and K levels in cattle. The ATP1A1 gene encodes the α1 isoform that forms the transmembrane subunit of the NA,K ATPase enzyme. The α subunit plays a major role in maintaining sodium-potassium homeostasis in all animal cells. The aim of the study was to determine ATP1A1 gene polymorphisms in Turkish Holstein cattle. The target regions (intron 17 and exon 18) were amplified and sequenced in 50 Turkish Holstein cattle. Multiple alignments revealed three SNP. rs109703332 A>G and rs110455455 C>T were detected in intron 17 and a synonymous SNP rs110256520 C>A in exon 18. It was observed that the three SNPs were in strong linkage disequilibrium (LD) with each other and therefore had the same genotype and allele frequencies. The three SNPs were found to be highly linked in one haplotype block. This haplotype block consisted of 2 haplotypes (CCA and ATG). The frequency of the CCA haplotype was 0.860 and the ATG was 0.140. Individuals of Holstein cattle tolerate heat stress to different levels. This difference between individuals may be due to variations in the genes involved in the adaptation mechanism. Therefore, it is important to identify polymorphisms in genes involved in the heat stress tolerance mechanism. In conclusion, in this study, the three SNPs and the two haplotypes were determined on the ATP1A1 gene in Turkish Holsteins cattle.

Phase I Metabolism of Pterostilbene, a Dietary Resveratrol Derivative: Metabolite Identification, Species Differences, Isozyme Contribution, and Further Bioactivation

Pterostilbene (PTE), a dietary derivative of resveratrol, displayed pleiotropic health-promoting activities. This study aimed to explore the metabolic profiles and species differences of the phase I metabolism of PTE and to investigate subsequent detoxification after PTE bioactivation. PTE was found to be biotransformed to two pharmacologically active metabolites, pinostilbene and 3'-hydroxypterostilbene, in vivo and in vitro with substantial species differences. Human CYP1A2 was proved to be mainly responsible for the demethylation and 3'-hydroxylation of PTE, with its contribution to a demethylation of 94.5% and to a 3'-hydroxylation of 97.9%. An in vitro glutathione trapping experiment revealed the presence of an ortho-quinone intermediate formed by further oxidation of 3'-hydroxypterostilbene. Human glutathione S-transferase isoforms A2, T1, and A1 inactivated the ortho-quinone intermediate by catalyzing glutathione conjugation, implicating a potential protective pathway against PTE bioactivation-derived toxicity. Overall, this study provided a comprehensive view of PTE phase I metabolism and facilitated its further development as a promising nutraceutical.

Efficacy of the New Inotropic Agent Istaroxime in Acute Heart Failure.

Current therapeutic strategies for acute heart failure (AHF) are based on traditional inotropic agents that are often associated with untoward effects; therefore, finding new effective approaches with a safer profile is dramatically needed. Istaroxime is a novel compound, chemically unrelated to cardiac glycosides, that is currently being studied for the treatment of AHF. Its effects are essentially related to its inotropic and lusitropic positive properties exerted through a dual mechanism of action: activation of the sarcoplasmic reticulum Ca2+ ATPase isoform 2a (SERCA2a) and inhibition of the Na+/K+-ATPase (NKA) activity. The advantages of istaroxime over the available inotropic agents include its lower arrhythmogenic action combined with its capability of increasing systolic blood pressure without augmenting heart rate. However, it has a limited half-life (1 hour) and is associated with adverse effects including pain at the injection site and gastrointestinal issues. Herein, we describe the main mechanism of action of istaroxime and we present a systematic overview of both clinical and preclinical trials testing this drug, underlining the latest insights regarding its adoption in clinical practice for AHF.

Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome

ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na+, and increased affinity for K+. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of β subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some β subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating invitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

Effects of multi-walled carbon nanotubes in soil on earthworm growth and reproduction, enzymatic activities, and metabolomics

Increased production and environmental release of multi-walled carbon nanotubes (MWCNTs) increase soil exposure and potential risk to earthworms. However, MWCNT toxicity to earthworms remains unclear, with some studies identifying negative effects and others negligible effects. In this study, to determine whether exposure to MWCNTs negatively affects earthworms and to elucidate possible mechanisms of toxicity, earthworms were exposed to sublethal soil concentrations of MWCNTs (10, 50, and 100 mg/kg) for 28 days. Earthworm growth and reproduction, activities of cytochrome P450 (CYP) isoforms (1A2, 2C9, and 3A4) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione-s-transferase (GST)), and metabolomics were determined. Effects of MWCNTs on earthworms depended on exposure concentration. Exposure to MWCNTs did not significantly affect growth and reproduction of individual earthworms. Exposure to 50 mg/kg MWCNTs significantly increased activities of CYP2C9, CYP3A4, SOD, CAT, and GST but clearly reduced levels of L-aspartate, L-asparagine, and glutamine. With exposure to 100 mg/kg MWCNTs, toxic effects on earthworms were observed, with significant inhibition in activities of CYP isoenzymes and SOD, significant reductions in L-aspartate, L-asparagine, glutamine, and tryptophan, and simultaneous accumulations of citrate, isocitrate, fumarate, 2-oxoglutarate, pyruvate, D-galactose, carbamoyl phosphate, formyl anthranilate, hypoxanthine, and xanthine. Results suggest that toxicity of MWCNTs to earthworms is associated with reduced detoxification capacity, excessive oxidative stress, and disturbance of multiple metabolic pathways, including amino acids metabolism, the tricarboxylic acid cycle, pyruvate metabolism, D-galactose metabolism, and purine metabolism. The study provides new insights to better understand and predict the toxicity of MWCNTs in soil.

The microtubule network enables Src kinase interaction with the Na,K-ATPase to generate Ca2+ flashes in smooth muscle cells.

Background: Several local Ca2+ events are characterized in smooth muscle cells. We have previously shown that an inhibitor of the Na,K-ATPase, ouabain induces spatially restricted intracellular Ca2+ transients near the plasma membrane, and suggested the importance of this signaling for regulation of intercellular coupling and smooth muscle cell contraction. The mechanism behind these Na,K-ATPase-dependent “Ca2+ flashes” remains to be elucidated. In addition to its conventional ion transport function, the Na,K-ATPase is proposed to contribute to intracellular pathways, including Src kinase activation. The microtubule network is important for intracellular signaling, but its role in the Na,K-ATPase-Src kinase interaction is not known. We hypothesized the microtubule network was responsible for maintaining the Na,K-ATPase-Src kinase interaction, which enables Ca2+ flashes. Methods: We characterized Ca2+ flashes in cultured smooth muscle cells, A7r5, and freshly isolated smooth muscle cells from rat mesenteric artery. Cells were loaded with Ca2+-sensitive fluorescent dyes, Calcium Green-1/AM and Fura Red/AM, for ratiometric measurements of intracellular Ca2+. The Na,K-ATPase α2 isoform was knocked down with siRNA and the microtubule network was disrupted with nocodazole. An involvement of the Src signaling was tested pharmacologically and with Western blot. Protein interactions were validated with proximity ligation assays. Results: The Ca2+ flashes were induced by micromolar concentrations of ouabain. Knockdown of the α2 isoform Na,K-ATPase abolished Ca2+ flashes, as did inhibition of tyrosine phosphorylation with genistein and PP2, and the inhibitor of the Na,K-ATPase-dependent Src activation, pNaKtide. Ouabain-induced Ca2+ flashes were associated with Src kinase activation by phosphorylation. The α2 isoform Na,K-ATPase and Src kinase colocalized in the cells. Disruption of microtubule with nocodazole inhibited Ca2+ flashes, reduced Na,K-ATPase/Src interaction and Src activation. Conclusion: We demonstrate that the Na,K-ATPase-dependent Ca2+ flashes in smooth muscle cells require an interaction between the α2 isoform Na, K-ATPase and Src kinase, which is maintained by the microtubule network.

New Aspects in the Mechanism of Action of ALDH1A1 and 1A3 Isoforms in Carcinogenesis

The ALDH gene superfamily encodes a group of evolutionarily-related proteins catalyzing the irreversible oxidation of aldehyde substrates to their corresponding carboxylic acids. Aldehyde dehydrogenases (ALDH) isoforms 1A1 and 1A3 belong to intracellular enzymes with a broad spectrum of functions linked with an advanced stage of solid tumors and the stemness potential of the neoplastic cells.

Catecholamine-dependent hyperpolarization of the junctional membrane via β2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as β-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific β2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

H-ABC tubulinopathy revealed by label-free second harmonic generation microscopy

Hypomyelination with atrophy of the basal ganglia and cerebellum is a recently described tubulinopathy caused by a mutation in the tubulin beta 4a isoform, expressed in oligodendrocytes. The taiep rat is the only spontaneous tubulin beta 4a mutant available for the study of this pathology. We aimed to identify the effects of the tubulin mutation on freshly collected, unstained samples of the central white matter of taiep rats using second harmonic generation microscopy. Cytoskeletal differences between the central white matter of taiep rats and control animals were found. Nonlinear emissions from the processes and somata of oligodendrocytes in tubulin beta 4a mutant rats were consistently detected, in the shape of elongated structures and cell-like bodies, which were never detected in the controls. This signal represents the second harmonic trademark of the disease. The tissue was also fluorescently labeled and analyzed to corroborate the origin of the nonlinear signal. Besides enabling the description of structural and molecular aspects of H-ABC, our data open the door to the diagnostic use of nonlinear optics in the study of neurodegenerative diseases, with the additional advantage of a label-free approach that preserves tissue morphology and vitality.

Isoform a4 of the vacuolar ATPase a subunit promotes 4T1-12B breast cancer cell–dependent tumor growth and metastasis in vivo

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that governs the pH of various intracellular compartments and also functions at the plasma membrane in certain cell types, including cancer cells. Membrane targeting of the V-ATPase is controlled by isoforms of subunit a, and we have previously shown that isoforms a3 and a4 are important for the migration and invasion of several breast cancer cell lines in vitro. Using CRISPR-mediated genome editing to selectively disrupt each of the four a subunit isoforms, we also recently showed that a4 is critical to plasma membrane V-ATPase localization, as well as in vitro migration and invasion of 4T1-12B murine breast cancer cells. We now report that a4 is important for the growth of 4T1-12B tumors in vivo. We found that BALB/c mice bearing a4−/− 4T1-12B allografts had significantly smaller tumors than mice in the control group. In addition, we determined that a4−/− allografts showed dramatically reduced metastases to the lung and reduced luminescence intensity of metastases to bone relative to the control group. Taken together, these results suggest that the a4 isoform of the V-ATPase represents a novel potential therapeutic target to limit breast cancer growth and metastasis.

ZY12201, A Potent TGR5 Agonist: Identification of a Novel Pan CYP450 Inhibitor Tool Compound for In-Vitro Assessment.

Identification of clinical drug-drug interaction (DDI) risk is an important aspect of drug discovery and development owing to poly-pharmacy in present-day clinical therapy. Drug metabolizing enzymes (DME) plays important role in the efficacy and safety of drug candidates. Hence evaluation of a New Chemical Entity (NCE) as a victim or perpetrator is very crucial for DDI risk mitigation. ZY12201 (2-((2-(4-(1H-imidazol-1-yl) phenoxy) ethyl) thio)-5-(2-(3, 4- dimethoxy phenyl) propane-2-yl)-1-(4-fluorophenyl)-1H-imidazole) is a novel and potent Takeda-G-protein-receptor-5 (TGR-5) agonist. ZY12201 was evaluated in-vitro to investigate the DDI liabilities. The key objective was to evaluate the CYP inhibition potential of ZY12201 for an opportunity to use it as a tool compound for pan CYP inhibition activities. In-vitro drug metabolizing enzymes (DME) inhibition potential of ZY12201 was evaluated against major CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4/5), aldehyde oxidase (AO), monoamine oxidase (MAO), and flavin-containing monooxygenase (FMO in human liver cytosol/mitochondrial preparation/ microsomes using probe substrates and Liquid Chromatography with tandem mass spectrometry (LC-MS-MS) method. The study conducted on ZY12201 at 100 µM ZY12201 was found to reduce the metabolism of vanillin (AO probe substrate), tryptamine (MAO probe substrate), and benzydamine (FMO probe substrate) by 49.2%, 14.7%, and 34.9%, respectively. ZY12201 Ki values were 0.38, 0.25, 0.07, 0.01, 0.06, 0.02, 7.13, 0.03 and 0.003 μM for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5 (substrate: testosterone) and CYP3A4/5 (substrate: midazolam), respectively. Time-dependant CYP inhibition potential of ZY12201 was assessed against CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 and no apparent IC50 shift was observed. ZY12201, at 100 µM concentration showed low inhibition potential of AO, MAO, and FMO. ZY12201 was found as a potent inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 while moderately inhibits to CYP2E1. Inhibition of CYP1A2, CYP2B6, CYP2C19, and CYP2E1 by ZY12201 was competitive, while inhibition of CYP2C8, CYP2C9, CYP2D6, and CYP3A4/5 was of mixed-mode. ZY12201 is a non-time-dependent inhibitor of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5. In summary, the reported Ki values unequivocally support that ZY12201 has a high potential to inhibit all major CYP isoforms. ZY12201 can be effectively used as a tool compound for in-vitro evaluation of CYP-based metabolic contribution to total drug clearance in the lead optimization stage of Drug Discovery Research.

Na+/K+ ATPase α1 and β3 subunits are localized to the basolateral membrane of trophectoderm cells in human blastocysts.

STUDY QUESTIONIs there a relation between specific Na+/K+ ATPase isoform expression and localization in human blastocysts and the developmental behavior of the embryo?SUMMARY ANSWERNa+/K+ ATPase α1, β1 and β3 are the main isoforms expressed in human blastocysts and no association was found between the expression level of their respective mRNAs and the rate of blastocyst expansion.WHAT IS KNOWN ALREADYIn mouse embryos, Na+/K+ ATPase α1 and β1 are expressed in the basolateral membrane of trophectoderm (TE) cells and are believed to be involved in blastocoel formation (cavitation).STUDY DESIGN, SIZE, DURATIONA total of 20 surplus embryos from 11 patients who underwent IVF and embryo transfer at a university hospital between 2009 and 2018 were analyzed.PARTICIPANTS/MATERIALS, SETTING, METHODSAfter freezing and thawing Day 5 human blastocysts, their developmental behavior was observed for 24 h using time-lapse imaging, and the expression of Na+/K+ ATPase isoforms was examined using quantitative RT-PCR (RT-qPCR). The expressed isoforms were then localized in blastocysts using fluorescent immunostaining.MAIN RESULTS AND THE ROLE OF CHANCERT-qPCR results demonstrated the expression of Na+/K+ ATPase α1, β1 and β3 isoforms in human blastocysts. Isoforms α1 and β3 were localized to the basolateral membrane of TE cells, and β1 was localized between TE cells. A high level of β3 mRNA expression correlated with easier hatching (P = 0.0261).LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThe expression of mRNA and the localization of proteins of interest were verified, but we have not been able to perform functional analysis.WIDER IMPLICATIONS OF THE FINDINGSOf the various Na+/K+ ATPase isoforms, expression levels of the α1, β1 and β3 mRNAs were clearly higher than other isoforms in human blastocysts. Since α1 and β3 were localized to the basolateral membrane via fluorescent immunostaining, we believe that these subunits contribute to the dilation of the blastocoel. The β1 isoform is localized between TE cells and may be involved in tight junction formation, as previously reported in mouse embryos.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by the JSPS KAKENHI (https://www.jsps.go.jp/english/index.html), grant number 17K11215. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no conflicts of interest.

Effect of Knock‐out of the a4 Isoform of Vacuolar H+‐ATPase following CRISPR/Cas9 on Activation of Cathepsins in MDA‐MB231 Breast Cancer Cell

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases creates a low pH in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between the isoforms of the a subunit for V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, differences in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D. To further investigate the role of V‐ATPases in the activation of cathepsins, we are using CRISPR/Cas9 to knockout the genes of the four V‐ATPase “a” subunit isoforms, a1, a2, a3 and a4. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 and a2 isoforms are found in intracellular compartments. Thus far we have made the most progress with knocking out the a4 isoform. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L.

Knockout of the a4 isoform of the V‐ATPase subunit a reduces tumor growth and metastasis in vivo

The vacuolar ATPase (V‐ATPase) is an ATP‐dependent proton pump that transports protons across intracellular membranes and, in certain cell types including cancer cells, across the plasma membrane, where it promotes tumor survival and metastasis. V‐ATPase localization is regulated by different isoforms of subunit a, and it has been shown that the a3 and a4 isoforms both target V‐ATPases to the plasma membrane and are critical for migration and invasion of different cancer cell lines in vitro. By using CRISPR mediated gene editing to selectively disrupt each of the 4 isoforms (a1‐a4), we previously reported that the a4 isoform appears to be important for plasma membrane targeting of the V‐ATPase and migration and invasion of 4T1‐12B breast cancer cells in vitro. Using BALB/C mice as an animal model, we have investigated the effect of disruption of different isoforms of subunit a on tumor growth and metastasis in vivo. The results suggest that targeting plasma membrane V‐ATPases represents a novel therapeutic approach to inhibition of tumor growth and metastasis in breast cancer patients.

Na/K‐ATPase Alpha1 Isoform Signaling in Cardiac Metabolism

Understanding cardiac metabolic signaling and its relation to pathological hypertrophy is critical to develop new therapeutic intervention in heart failure (HF). Decreased Na/K‐ATPase (NKA) activity and expression have long been associated with HF in experimental models and in patients. Classically known for its crucial role in transmembrane ion transport, electrical excitability, and contractility, NKA also supports non‐enzymatic receptor functions that differ among major cardiac isoforms (α1 and α2 in rodents). Studies from our group and others have suggested a role of these receptor functions in cardiac myocyte hypertrophy. Using epithelial (LLC‐PK1)‐derived cell lines, we have previously observed a NKA α1‐specific ability to modulate metabolic reserve and capacity. Mechanistically, this property is highly dependent upon NKA α1 ability to regulate the signaling partner Src kinase and is not supported by NKA α2, which lacks the Src binding domain. In this work, our goal was to assess the impact of NKA α1 ablation in the heart and demonstrate the importance of NKA α1 signaling function for proper cardiomyocyte metabolic function. We used a Myh6‐Cre/Lox approach to delete NKA α1 in the mouse heart (cardio α1‐/‐). These animals (3‐month‐old male mice) presented a normal cardiac structure, reduced NKA α1 expression (more than 100‐fold; n = 6‐7; p < 0.01) and increased NKA α2 expression (10‐fold; n = 6‐7; p < 0.01) by Western blot. RNA sequencing followed by gene set enrichment analysis revealed that cardio α1‐/‐ hearts have downregulated pathways related to mitochondrial metabolism, such as cytochrome complex, NADH dehydrogenase complex, respiratory chain, and mitochondrial fatty acid β‐oxidation (n = 3; FDR < 0.05). We next generated human induced pluripotent stem cells (hiPSCs) with a mutant form of the NKA α1 isoform that can pump ions but have blunted Src signaling (A420P mutant) using a CRISPR/Cas9‐mediated knock‐in approach. Genotyping and sequencing confirmed the substitution of alanine to proline in the NaKtide sequence to generate the desired mutation. These cells were differentiated into cardiomyocytes, as confirmed by overexpression of the cardiac markers myosin heavy chain 6 (MYH6) and cardiac muscle troponin T (TNNT2) (more than 10‐fold; n = 3; p < 0.05). Metabolic phenotyping using the Agilent Seahorse Extracellular Flux technology suggested that the A420P mutant hiPSC‐derived cardiomyocytes (A420P iCM) have decreased basal respiration, maximal respiration, spare respiratory capacity, and ATP production compared to the controls (WT iCM) based on changes in oxygen consumption rate (OCR) using the Agilent Seahorse XF Cell Mito Stress Test Kit. In addition, A420P iCM have reduced glycolytic capacity and reserve, based on changes in extracellular acidification rate (ECAR) using the Agilent Seahorse XF Glycolysis Stress Test Kit. Taken together, these results suggest that NKA α1 isoform signaling modulates the cardiomyocyte metabolism. Further studies may reveal a link between NKA/Src signaling, metabolic flexibility, and structural cardiac remodeling in the context of HF and cardiac hypertrophy.

Increased Connexin 43 expression in AMPK deficient cardiomyocytes in response to Doxorubicin treatment

ContextDoxorubicin (DOX) is an anthracycline anticancer drug that can cause dose‐dependent cardiomyopathy and chronic heart failure. DOX cardiotoxicity has been attributed to excess production of ROS and genotoxicity that leads to cell death. AMPK is a kinase composed of α, β and γ subunits. AMPK has been proposed to modulate DOX‐induced cardiotoxicity, but the downstream mediators or effectors remain unclear. Connexin 43 (Cx43) is a gap junction protein abundantly expressed in the cardiomyocyte and is essential for heart function. Studies have suggested a role for Cx43 in DOX cardiotoxicity, but it remains unknown whether AMPK interacts with Cx43 to regulate cardiac response to DOX chemotherapy.ObjectiveTo determine the role of AMPK in the regulation of the Cx43 in response to DOX‐induced cardiotoxicity.MethodsH9c2 rat cardiomyocytes were seeded on 6‐well plates and cultured in DMEM with 6.5% fetal bovine serum. To investigate the role of AMPK, the cells were transfected with siRNAs targeting different AMPK isoforms using Lipofectamine RNAiMAX and Opti‐MEM I reduced serum medium. After 48 hours of transfection, half of the wells received 0.5 µM DOX treatment, and the other half received equal amounts of saline. Protein samples were collected 16 hours later using the Cell lysis buffer. Western blot analysis was performed to confirm the knockdown of AMPK isoforms and to determine the protein expression levels of Cx43.ResultsOur findings showed that siRNA transfections effectively reduced the protein levels of AMPK isoform a1 and a2, respectively, as indicated by the Western blot analysis. DOX treatment increased the expression levels of the Cx43 in cardiomyocytes. Also, knockdown (KD) of AMPKα1 isoform resulted in a similar increase in Cx43 expression levels as did DOX, but AMPKα2 KD did not significantly alter Cx43 levels, suggesting an AMPKa1 specific effect on Cx43 expression. Interestingly, AMPKα1 KD, together with DOX treatment, did not produce a synergistic effect to increase Cx43 expression levels further, suggesting the possibility that DOX might have elevated the Cx43 expression through its effect on AMPK.ConclusionOur results confirmed the previous studies showing that treatment of the cardiomyocytes with DOX increased the expression of Cx43. However, our investigations produced a novel finding that knocking down AMPKα1 also increased the Cx43 but did not enhance the effect of DOX on Cx43 expression, suggesting a role for AMPK in the pathway that regulates the Cx43 to mediate the DOX‐induced cardiotoxicity. Our investigations also showed an isoform‐specific effect of AMPK on Cx43 expression since only α1 but not α2 KD increased Cx43 levels. Whether the increase in Cx43 is due to increased gene expression or inhibited protein degradation remains to be determined. Further investigation is also warranted to elucidate the roles of other AMPK subunits or isoforms in regulating the Connexin 43 expression in the context of doxorubicin cardiotoxicity.

A real-time PCR method to genotype mutant mouse models with altered affinity for cardiotonic steroids on the Na,K-ATPase.

The highly conserved, cardiotonic steroid binding site (also termed ouabain binding site) on the primary α subunit of Na,K-ATPase plays a receptor signaling role in a range of vital cell processes and is a therapeutic target for human disease. Mouse lines with altered affinity for cardiotonic steroids on the α1 or α2 subunit isoform of Na,K-ATPase, without any change in pump activity, were developed by the late Jerry B Lingrel and are a valuable tool for studying its physiological roles and drug actions. In one model, the normally ouabain resistant α1 isoform was rendered sensitive to ouabain binding. In a second model, the normally sensitive α2 isoform was rendered resistant to ouabain binding. Additional useful models are obtained by mating these mice. To further advance their use, we developed a rapid, real-time PCR method that detects mutant alleles using specific primers and fluorescent probes. PCR is performed in fast mode with up to 15 samples processed in 40 min. The method was validated by Sanger sequencing using mice of known genotype, and by comparing results with a previous two-step method that used PCR amplification followed by gel electrophoresis. In addition, we clarified inconsistencies in published sequences, updated numbering to current reference sequences, and confirmed the continued presence of the mutations in the colony. It is expected that a wider availability of these models and a more efficient genotyping protocol will advance studies of the Na,K-ATPase and its cardiotonic steroid receptor.