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Background: Tissues, such as skeletal muscle, have been targeted for the delivery of plasmid DNA (pDNA) encoding vaccines and therapeutics. The application of electric pulses (electroporation or electrotransfer) increases cell membrane permeability to enhance plasmid delivery and expression. However, the molecular effects of DNA electrotransfer on the muscle tissue are poorly characterized. Materials and Methods: Four hours after intramuscular plasmid electrotransfer, we evaluated gene expression changes by RNA sequencing. Differentially expressed genes were analyzed by gene ontology (GO) pathway enrichment analysis. Results: GO analysis highlighted many enriched molecular functions. The terms regulated by pulse application were related to muscle stress, the cytoskeleton and inflammation. The terms regulated by pDNA injection were related to a DNA-directed response and its control. Several terms regulated by pDNA electrotransfer were similar to those regulated by pulse application. However, the terms related to pDNA injection differed, focusing on entry of the plasmid into the cells and intracellular trafficking. Conclusion: Each muscle stimulus resulted in specific regulated molecular functions. Identifying the unique intrinsic molecular changes driven by intramuscular DNA electrotransfer will aid in the design of preventative and therapeutic gene therapies.
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The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3 nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72 h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.
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Skeletal muscle is a promising tissue for therapeutic gene delivery because it is highly vascularized, accessible, and capable of synthesizing protein for therapies or vaccines. The application of electric pulses (electroporation) enhances plasmid DNA delivery and expression by increasing membrane permeability. Four hours after plasmid electroporation, we evaluated acute gene and protein expression changes in mouse skeletal muscle to identify regulated genes and genetic pathways. RNA sequencing followed by functional annotation was used to evaluate differentially expressed mRNAs. Our data highlighted immune signaling pathways that may influence the effectiveness of DNA electroporation. Cytokine and chemokine protein levels in muscle lysates revealed the upregulation of a subset of inflammatory proteins and confirmed the RNA sequencing analysis. Several regulated DNA-specific pattern recognition receptor mRNAs were also detected. Identifying unique molecular changes in the muscle will facilitate a better understanding of the underlying molecular mechanisms and the development of safety biomarkers and novel strategies to improve skeletal muscle targeted gene therapy.
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Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD+ salvage pathway and is dysregulated in diabetes. Nampt activation results in rescued NADH/NAD+ ratios and provides pharmacological changes necessary for diabetic cardioprotection. Computer docking shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) allows for enhanced Nampt dimerization and association. To test the pharmacological application, we used male leptin receptor-deficient (db/db) mice and treated them with Nampt activator P7C3. The effects of 4-week P7C3 treatment on cardiac function were evaluated along with molecular signaling changes for phosphorylated protein kinase B (p-AKT), phosphorylated endothelial nitric oxide synthase (p-eNOS), and sirtuin 1 (SIRT1). The cardiac function evaluated by ECG and echocardiography were significantly improved after 4 weeks of P7C3 treatment. Biochemically, higher NADH/NAD+ ratios in diabetic hearts were rescued by P7C3 treatment. Moreover, activities of Nampt and SIRT1 were significantly increased in P7C3-treated diabetic hearts. P7C3 treatment significantly decreased the blood glucose in diabetic mice with 4-week treatment as noted by glucose tolerance test and fasting blood glucose measurements compared with vehicle-treated mice. P7C3 activated Nampt enzymatic activity both in vitro and in the 4-week diabetic mouse hearts, demonstrating the specificity of the small molecule. P7C3 treatment significantly enhanced the expression of cardioprotective signaling of p-AKT, p-eNOS, and Beclin 1 in diabetic hearts. Nampt activator P7C3 allows for decreased infarct size with decreased Troponin I and lactose dehydrogenase (LDH) release, which is beneficial to the heart. Overall, the present study shows that P7C3 activates Nampt and SIRT1 activity and decreases NADH/NAD+ ratio, resulting in improved biochemical signaling providing cardioprotection. SIGNIFICANCE STATEMENT: This study shows that 1-(3,6-Dibromo-carbazol-9-yl)-3-phenylamino-propan-2-ol (P7C3) is effective in treating diabetes and cardiovascular diseases. The novel small molecule is antiarrhythmic and improves the ejection fraction in diabetic hearts. The study successfully demonstrated that P7C3 decreases the infarct size in hearts during myocardial infarction and ischemia-reperfusion injury. Biochemical and cellular signaling show increased NAD+ levels, along with Nampt activity involved in upregulating protective signaling in the diabetic heart. P7C3 has high therapeutic potential for rescuing heart disease.
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Diabetes Mellitus Experimental , Infarto do Miocárdio , Animais , Glicemia , Carbazóis , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Masculino , Camundongos , Infarto do Miocárdio/tratamento farmacológico , NAD/metabolismo , Nicotinamida Fosforribosiltransferase , Proteínas Proto-Oncogênicas c-akt , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD salvage pathway is decreased in metabolic diseases, and its precise role in skeletal muscle function is not known. We tested the hypothesis, Nampt activation by P7C3 (3,6-dibromo-α-[(phenylamino)methyl]-9H-carbazol-9-ethanol) ameliorates diabetes and muscle function. METHODS: We assessed the functional, morphometric, biochemical, and molecular effects of P7C3 treatment in skeletal muscle of type 2 diabetic (db/db) mice. Nampt+/- mice were utilized to test the specificity of P7C3. RESULTS: Insulin resistance increased 1.6-fold in diabetic mice compared with wild-type mice and after 4 weeks treatment with P7C3 rescued diabetes (P < 0.05). In the db-P7C3 mice fasting blood glucose levels decreased to 0.96-fold compared with C57Bl/6J wild-type naïve control mice. The insulin and glucose tolerance tests blood glucose levels were decreased to 0.6-fold and 0.54-folds, respectively, at 120 min along with an increase in insulin secretion (1.76-fold) and pancreatic ß-cells (3.92-fold) in db-P7C3 mice. The fore-limb and hind-limb grip strengths were increased to 1.13-fold and 1.17-fold, respectively, together with a 14.2-fold increase in voluntary running wheel distance in db-P7C3 mice. P7C3 treatment resulted in a 1.4-fold and 7.1-fold increase in medium-sized and larger-sized myofibres cross-sectional area, with a concomitant 0.5-fold decrease in smaller-sized myofibres of tibialis anterior (TA) muscle. The transmission electron microscopy images also displayed a 1.67-fold increase in myofibre diameter of extensor digitorum longus muscle along with 2.9-fold decrease in mitochondrial area in db-P7C3 mice compared with db-Veh mice. The number of SDH positive myofibres were increased to 1.74-fold in db-P7C3 TA muscles. The gastrocnemius and TA muscles displayed a decrease in slow oxidative myosin heavy chain type1 (MyHC1) myofibres expression (0.46-fold) and immunostaining (6.4-fold), respectively. qPCR analysis displayed a 2.9-fold and 1.3-fold increase in Pdk4 and Cpt1, and 0.55-fold and 0.59-fold decrease in Fgf21 and 16S in db-P7C3 mice. There was also a 3.3-fold and 1.9-fold increase in Fabp1 and CD36 in db-Veh mice. RNA-seq differential gene expression volcano plot displayed 1415 genes to be up-regulated and 1726 genes down-regulated (P < 0.05) in db-P7C3 mice. There was 1.02-fold increase in serum HDL, and 0.9-fold decrease in low-density lipoprotein/very low-density lipoprotein ratio in db-P7C3 mice. Lipid profiling of gastrocnemius muscle displayed a decrease in inflammatory lipid mediators n-6; AA (0.83-fold), and n-3; DHA (0.69-fold) and EPA (0.81-fold), and a 0.66-fold decrease in endocannabinoid 2-AG and 2.0-fold increase in AEA in db-P7C3 mice. CONCLUSIONS: Overall, we demonstrate that P7C3 activates Nampt, improves type 2 diabetes and skeletal muscle function in db/db mice.
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Carbazóis , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Animais , Carbazóis/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Lipídeos , Camundongos , Músculo Esquelético , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismoRESUMO
Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine-induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.
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Cardiovascular diseases including cardiac arrhythmias lead to fatal events in patients with coronary artery disease; however, clinical associations from echocardiography, electrocardiography (ECG), and biomarkers remain unknown. We sought to identify the factors that may be related to elevated QRS intervals in patients with risk for coronary artery disease. In this study, we performed analysis of clinical data from 503 patients divided into two groups, i.e., patients with either <50% coronary artery stenosis or >50% coronary artery stenosis. We further examined patients with elevated ECG parameters such as QRS > 100 ms and QTc > 440 ms. Patients with >50% coronary artery stenosis exhibited significant increases in age, triglycerides, and troponin levels. Further, ECG parameters demonstrated increased QRS and QTc durations, while echocardiographic parameters highlighted a decrease in ejection fraction (EF) and fractional shortening (FS). Patients with QTc > 440 ms exhibited increased brain natriuretic peptide and creatinine levels with a decrease in estimated glomerular filtration rate clearance rates. Patients with QRS > 100 ms had greater left ventricular (LV) mass and LV internal diameter in systole and diastole. Multimodal logistic regression showed significant relation between QTc, age, and creatinine. These findings suggest that patients with significant coronary stenosis may have lower EF and FS with prolonged QRS intervals, demonstrating greater risk for arrhythmic events.
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Doença da Artéria Coronariana/fisiopatologia , Estenose Coronária/fisiopatologia , Eletrocardiografia , Função Ventricular , Fatores Etários , Idoso , Biomarcadores/sangue , Doença da Artéria Coronariana/diagnóstico , Estenose Coronária/diagnóstico , Creatinina/sangue , Ecocardiografia , Feminino , Taxa de Filtração Glomerular , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Estudos Retrospectivos , Risco , Volume SistólicoRESUMO
Kvß subunits belong to the aldo-keto reductase superfamily, which plays a significant role in ion channel regulation and modulates the physiological responses. However, the role of Kvß2 in cardiac pathophysiology was not studied, and therefore, in the present study, we hypothesized that Kvß2 plays a significant role in cardiovascular pathophysiology by modulating the cardiac excitability and gene responses. We utilized an isoproterenol-infused mouse model to investigate the role of Kvß2 and the cardiac function, biochemical changes, and molecular responses. The deletion of Kvß2 attenuated the QTc (corrected QT interval) prolongation at the electrocardiographic (ECG) level after a 14-day isoproterenol infusion, whereas the QTc was significantly prolonged in the littermate wildtype group. Monophasic action potentials verified the ECG changes, suggesting that cardiac changes and responses due to isoproterenol infusion are mediated similarly at both the in vivo and ex vivo levels. Moreover, the echocardiographic function showed no further decrease in the ejection fraction in the isoproterenol-stimulated Kvß2 knockout (KO) group, whereas the wildtype mice showed significantly decreased function. These experiments revealed that Kvß2 plays a significant role in cardiovascular pathophysiology. Furthermore, the present study revealed SLC41a3, a major solute carrier transporter affected with a significantly decreased expression in KO vs. wildtype hearts. The electrical function showed that the decreased expression of SLC41a3 in Kvß2 KO hearts led to decreased Mg2+ responses, whereas, in the wildtype hearts, Mg2+ caused action potential duration (APD) shortening. Based on the in vivo, ex vivo, and molecular evaluations, we identified that the deletion of Kvß2 altered the cardiac pathophysiology mediated by SLC41a3 and altered the NAD (nicotinamide adenine dinucleotide)-dependent gene responses.
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The emergence of multiple concurrent infectious diseases localized in the world creates a complex burden on global public health systems. Outbreaks of Ebola, Lassa, and Marburg viruses in overlapping regions of central and West Africa and the co-circulation of Zika, Dengue, and Chikungunya viruses in areas with A. aegypti mosquitos highlight the need for a rapidly deployable, safe, and versatile vaccine platform readily available to respond. The DNA vaccine platform stands out as such an application. Here, we present proof-of-concept studies from mice, guinea pigs, and nonhuman primates for two multivalent DNA vaccines delivered using in vivo electroporation (EP) targeting mosquito-borne (MMBV) and hemorrhagic fever (MHFV) viruses. Immunization with MMBV or MHFV vaccines via intradermal EP delivery generated robust cellular and humoral immune responses against all target viral antigens in all species. MMBV vaccine generated antigen-specific binding antibodies and IFNγ-secreting lymphocytes detected in NHPs up to six months post final immunization, suggesting induction of long-term immune memory. Serum from MHFV vaccinated NHPs demonstrated neutralizing activity in Ebola, Lassa, and Marburg pseudovirus assays indicating the potential to offer protection. Together, these data strongly support and demonstrate the versatility of DNA vaccines as a multivalent vaccine development platform for emerging infectious diseases.
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Culicidae/virologia , Ebolavirus/imunologia , Vacinas Combinadas/imunologia , Vacinas de DNA/imunologia , África Ocidental , Animais , Anticorpos Antivirais/imunologia , Arenavirus do Novo Mundo/imunologia , Vírus da Dengue/imunologia , Epidemias , Feminino , Cobaias , Doença pelo Vírus Ebola/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Febre Lassa/imunologia , Marburgvirus/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Vacinação/métodos , Vacinas Virais/imunologia , Zika virus/imunologia , Infecção por Zika virus/imunologiaRESUMO
Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvß subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvß subunits regulate myocardial K+ currents and repolarization. Here, we tested the hypothesis that Kvß2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvß2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvß2-/-) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced Ito, IK,slow1, and IK,slow2 densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvß2-/- mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)+ accelerated Kv inactivation in wild type, but not Kvß2-/- myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH]i and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvß2-/-, hearts. Taken together, these results suggest that Kvß2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate Ito and IK,slow, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.
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Ativação do Canal Iônico , Miocárdio/metabolismo , Subunidades Proteicas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Testes de Função Cardíaca , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Nucleotídeos/metabolismo , Oxirredução , Piridinas/metabolismo , Canais de Potássio Shal/metabolismoRESUMO
Glucocorticoids, such as fluticasone propionate (FP), are used for the treatment of inflammation and alleviation of nasal symptoms and allergies, and as an antipruritic. However, both short- and long-term therapeutic use of glucocorticoids can lead to muscle weakness and atrophy. In the present study, we evaluated the feasibility of the nanodelivery of FP with poly(dl-lactide-co-glycolide) (PLGA) and tested in vitro function. FP-loaded PLGA nanoparticles were prepared via nanoprecipitation and morphological characteristics were studied via scanning electron microscopy. FP-loaded nanoparticles demonstrated an encapsulation efficiency of 68.6% ± 0.5% with a drug loading capacity of 4.6% ± 0.04%, were 128.8 ± 0.6 nm in diameter with a polydispersity index of 0.07 ± 0.008, and displayed a zeta potential of -19.4 ± 0.7. A sustained in vitro drug release pattern was observed for up to 7 days. The use of fluticasone nanoparticle decreased lipopolysaccharide (LPS)-induced lactate dehydrogenase release compared with LPS alone in C2C12 treated cells. FP also decreased expression of LPS-induced inflammatory genes in C2C12 treated cells as compared with LPS alone. Taken together, the present study demonstrates in vitro feasibility of PLGA-FP nanoparticle delivery to the skeletal muscle cells, which may be beneficial for treating inflammation.
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Portadores de Fármacos/química , Fluticasona/química , Fluticasona/farmacologia , Nanopartículas/química , Animais , Linhagem Celular , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , L-Lactato Desidrogenase/metabolismo , Camundongos , Tamanho da PartículaRESUMO
Glucocorticoids are utilized for their anti-inflammatory properties in the skeletal muscle and arthritis. However, the major drawback of use of glucocorticoids is that it leads to senescence and toxicity. Therefore, based on the idea that decreasing particle size allows for increased surface area and bioavailability of the drug, in the present study, we hypothesized that nanodelivery of dexamethasone will offer increased efficacy and decreased toxicity. The dexamethasone-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared using nanoprecipitation method. The morphological characteristics of the nanoparticles were studied under scanning electron microscope. The particle size of nanoparticles was 217.5 ± 19.99 nm with polydispersity index of 0.14 ± 0.07. The nanoparticles encapsulation efficiency was 34.57% ± 1.99% with in vitro drug release profile exhibiting a sustained release pattern over 10 days. We identified improved skeletal muscle myoblast performance with improved closure of the wound along with increased cell viability at 10 nmol/L nano-dexamethasone-PLGA. However, dexamethasone solution (1 µmol/L) was injurious to cells because the migration efficiency was decreased. In addition, the use of dexamethasone nanoparticles decreased lipopolysaccharide-induced lactate dehydrogenase release compared with dexamethasone solution. Taken together, the present study clearly demonstrates that delivery of PLGA-dexamethasone nanoparticles to the skeletal muscle cells is beneficial for treating inflammation and skeletal muscle function.
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Composição de Medicamentos/métodos , Glucocorticoides/farmacologia , Miosite/tratamento farmacológico , Nanopartículas/química , Cicatrização/efeitos dos fármacos , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Liberação Controlada de Fármacos , Glucocorticoides/uso terapêutico , Ácido Láctico/química , Camundongos , Microscopia Eletrônica de Transmissão , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/lesões , Mioblastos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RatosRESUMO
Hypotension, cardiac depression, and elevated microvascular permeability are known problems that complicate resuscitation of patients following traumatic injury, particularly those who are also intoxicated from alcohol consumption. A conscious rat model of combined alcohol intoxication and hemorrhagic shock has been used to study the hemodynamic mechanisms involved. Here, we describe using this model to study microvascular leakage and cardiac electrical activity.
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Intoxicação Alcoólica , Permeabilidade Capilar , Microcirculação , Ressuscitação/métodos , Choque Hemorrágico , Intoxicação Alcoólica/patologia , Intoxicação Alcoólica/fisiopatologia , Intoxicação Alcoólica/terapia , Animais , Modelos Animais de Doenças , Humanos , Ratos , Choque Hemorrágico/patologia , Choque Hemorrágico/fisiopatologia , Choque Hemorrágico/terapiaRESUMO
We previously demonstrated the role of Kvß1.1 subunit of voltage-activated potassium channel in heart for its sensory roles in detecting changes in NADH/NAD and modulation of ion channel. However, the pharmacological role for the association of Kvß1 via its binding to ligands such as cortisone and its analogs remains unknown. Therefore, we investigated the significance of Kvß1.1 binding to cortisone analogs and AR inhibitor epalrestat. In addition, the aldose reductase (AR) inhibitor epalrestat was identified as a pharmacological target and modulator of cardiac activity via binding to the Kvß1 subunit. Using a combination of ex vivo cardiac electrophysiology and in silico binding, we identified that Kvß1 subunit binds and interacts with epalrestat. To identify the specificity of the action potential changes, we studied the sensitivity of the action potential prolongation by probing the electrical changes in the presence of 4-aminopyridine and evaluated the specificity of pharmacological effects in the hearts from Kvß1.1 knock out mouse. Our results show that pharmacological modulation of cardiac electrical activity by cortisone analogs and epalrestat is mediated by Kvß1.1.
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Potenciais de Ação/efeitos dos fármacos , Aldeído Redutase/antagonistas & inibidores , Cortisona/farmacologia , Inibidores Enzimáticos/farmacologia , Canal de Potássio Kv1.1/metabolismo , Miocárdio/metabolismo , Rodanina/análogos & derivados , Tiazolidinas/farmacologia , Potenciais de Ação/genética , Animais , Canal de Potássio Kv1.1/genética , Camundongos , Camundongos Knockout , Rodanina/farmacologiaRESUMO
The present study investigates the physiological role of Kvß1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvß1.1 knockout (KO) or wild-type (WT) mice and established that Kvß1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvß1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvß1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvß1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvß1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvß1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvß1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart.NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvß1.1 subunit. In the present study, we identify Kvß1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.
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Coração/efeitos dos fármacos , Canal de Potássio Kv1.1/metabolismo , Miocárdio/metabolismo , Nucleotídeos/metabolismo , Piridinas/metabolismo , Potenciais de Ação/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Animais , Células COS , Chlorocebus aethiops , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Isoproterenol/farmacologia , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Knockout , NAD/metabolismo , Técnicas de Patch-Clamp , Ratos , Canais de Potássio ShalRESUMO
NEW FINDINGS: What is the central question of this study? The goal of this study was to evaluate sex differences and the role of the potassium channel ß1 (Kvß1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvß1.1 in females led to cardiac hypertrophy characterized by increased heart size, prolonged monophasic action potentials, elevated blood pressure and increased myosin heavy chain α (MHCα) expression. In contrast, male mice showed only electrical changes. Kvß1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvß1.1 upregulated MHCα. Cardiovascular disease is the leading cause of death and debility in women in the USA, and cardiac arrhythmias are a major concern. Voltage-gated potassium (Kv) channels along with the binding partners; Kvß subunits are major regulators of the action potential (AP) shape and duration (APD). The regulation of Kv channels by the Kvß1 subunit is unknown in female hearts. In the present study, we hypothesized that the Kvß1 subunit is an important regulator of female cardiac physiology. To test this hypothesis, we ablated (knocked out; KO) the KCNAB1 isoform 1 (Kvß1.1) subunit in mice and evaluated cardiac function and electrical activity by using ECG, monophasic action potential recordings and echocardiography. Our results showed that the female Kvß1.1 KO mice developed cardiac hypertrophy, and the hearts were structurally different, with enlargement and increased area. The electrical derangements caused by Kvß1.1 KO in female mice included long QTc and QRS intervals along with increased APD (APD20-90% repolarization). The male Kvß1.1 KO mice did not develop cardiac hypertrophy, but they showed long QTc and prolonged APD. Molecular analysis showed that several genes that support cardiac hypertrophy were significantly altered in Kvß1.1 KO female hearts. In particular, myosin heavy chain α expression was significantly elevated in Kvß1.1 KO mouse heart. Using a small interfering RNA strategy, we identified that knockdown of Kvß1 increases myosin heavy chain α expression in H9C2 cells. Collectively, changes in molecular and cell signalling pathways clearly point towards a distinct electrical and structural remodelling consistent with cardiac hypertrophy in the Kvß1.1 KO female mice.
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Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Coração/fisiopatologia , Hemodinâmica/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/metabolismo , Potenciais de Ação/fisiologia , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Linhagem Celular , Ecocardiografia/métodos , Feminino , Ativação do Canal Iônico/fisiologia , Masculino , Camundongos , RatosRESUMO
Ventricular arrhythmias account for high mortality in cardiopulmonary patients in intensive care units. Cardiovascular alterations and molecular-level changes in response to the commonly used oxygen treatment remains unknown. In the present study we investigated cardiac hypertrophy and cardiac complications in mice subjected to hyperoxia. Results demonstrate that there is a significant increase in average heart weight to tibia length (22%) in mice subjected to hyperoxia treatment vs. normoxia. Functional assessment was performed in mice subjected to hyperoxic treatment, and results demonstrate impaired cardiac function with decreased cardiac output and heart rate. Staining of transverse cardiac sections clearly demonstrates an increase in the cross-sectional area from hyperoxic hearts compared with control hearts. Quantitative real-time RT-PCR and Western blot analysis indicated differential mRNA and protein expression levels between hyperoxia-treated and control left ventricles for ion channels including Kv4.2 (-2 ± 0.08), Kv2.1 (2.54 ± 0.48), and Scn5a (1.4 ± 0.07); chaperone KChIP2 (-1.7 ± 0.06); transcriptional factors such as GATA4 (-1.5 ± 0.05), Irx5 (5.6 ± 1.74), NFκB1 (4.17 ± 0.43); hypertrophy markers including MHC-6 (2.17 ± 0.36) and MHC-7 (4.62 ± 0.76); gap junction protein Gja1 (4.4 ± 0.8); and microRNA processing enzyme Drosha (4.6 ± 0.58). Taken together, the data presented here clearly indicate that hyperoxia induces left ventricular remodeling and hypertrophy and alters the expression of Kv4.2 and MHC6/7 in the heart.
Assuntos
Cardiomegalia/metabolismo , Ventrículos do Coração/metabolismo , Hiperóxia/complicações , Canais de Potássio Shal/metabolismo , Animais , Débito Cardíaco , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Conexina 43/genética , Conexina 43/metabolismo , Frequência Cardíaca , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/fisiopatologia , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Canais de Potássio Shab/genética , Canais de Potássio Shab/metabolismo , Canais de Potássio Shal/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , UltrassonografiaRESUMO
Diabetes is a metabolic disorder that ultimately results in major pathophysiological complications in the cardiovascular system. Diabetics are predisposed to higher incidences of sudden cardiac deaths (SCD). Several studies have associated diabetes as a major underlying risk for heart diseases and its complications. The diabetic heart undergoes remodeling to cope up with the underlying changes, however ultimately fails. In the present study we investigated the changes associated with a key ion channel and transcriptional factors in a diabetic heart model. In the mouse db/db model, we identified key transcriptional regulators and mediators that play important roles in the regulation of ion channel expression. Voltage-gated potassium channel (Kv4.2) is modulated in diabetes and is down regulated. We hypothesized that Kv4.2 expression is altered by potassium channel interacting protein-2 (KChIP2) which is regulated upstream by NFkB and miR-301a. We utilized qRT-PCR analysis and identified the genes that are affected in diabetes in a regional specific manner in the heart. At protein level we identified and validated differential expression of Kv4.2 and KChIP2 along with NFkB in both ventricles of diabetic hearts. In addition, we identified up-regulation of miR-301a in diabetic ventricles. We utilized loss and gain of function approaches to identify and validate the role of miR-301a in regulating Kv4.2. Based on in vivo and in vitro studies we conclude that miR-301a may be a central regulator for the expression of Kv4.2 in diabetes. This miR-301 mediated regulation of Kv4.2 is independent of NFkB and Irx5 and modulates Kv4.2 by direct binding on Kv4.2 3'untranslated region (3'-UTR). Therefore targeting miR-301a may offer new potential for developing therapeutic approaches.