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BACKGROUND: Unlike other breast cancer subtypes that may be treated with a variety of hormonal or targeted therapies, there is a need to identify new, effective targets for triple-negative breast cancer (TNBC). It has recently been recognized that membrane potential is depolarized in breast cancer cells. The primary objective of the study is to explore whether hyperpolarization induced by opening potassium channels may provide a new strategy for treatment of TNBC. METHODS: Breast cancer datasets in cBioPortal for cancer genomics was used to search for ion channel gene expression. Immunoblots and immunohistochemistry were used for protein expression in culture cells and in the patient tissues. Electrophysiological patch clamp techniques were used to study properties of BK channels in culture cells. Flow cytometry and fluorescence microscope were used for cell viability and cell cycle studies. Ultrasound imaging was used to study xenograft in female NSG mice. RESULTS: In large datasets of breast cancer patients, we identified a gene, KCNMA1 (encoding for a voltage- and calcium-dependent large-conductance potassium channel, called BK channel), overexpressed in triple-negative breast cancer patients. Although overexpressed, 99% of channels are closed in TNBC cells. Opening BK channels hyperpolarized membrane potential, which induced cell cycle arrest in G2 phase and apoptosis via caspase-3 activation. In a TNBC cell induced xenograft model, treatment with a BK channel opener significantly slowed tumor growth without cardiac toxicity. CONCLUSIONS: Our results support the idea that hyperpolarization induced by opening BK channel in TNBC cells can become a new strategy for development of a targeted therapy in TNBC.
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Mama/patología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Oxadiazoles/farmacología , Tetrazoles/farmacología , Tiourea/análogos & derivados , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Conjuntos de Datos como Asunto , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Microscopía Intravital , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/agonistas , Potenciales de la Membrana/efectos de los fármacos , Ratones , Oxadiazoles/uso terapéutico , Técnicas de Placa-Clamp , Tetrazoles/uso terapéutico , Tiourea/farmacología , Tiourea/uso terapéutico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
How obesity or sex may affect the gene expression profiles of human cardiac hypertrophy is unknown. We hypothesized that body-mass index (BMI) and sex can affect gene expression profiles of cardiac hypertrophy. Human heart tissues were grouped according to sex (male, female), BMI (lean<25 kg/m2, obese>30 kg/m2), or left ventricular hypertrophy (LVH) and non-LVH nonfailed controls (NF). We identified 24 differentially expressed (DE) genes comparing female with male samples. In obese subgroup, there were 236 DE genes comparing LVH with NF; in lean subgroup, there were seven DE genes comparing LVH with NF. In female subgroup, we identified 1,320 significant genes comparing LVH with NF; in male subgroup, there were 1,383 significant genes comparing LVH with NF. There were seven significant genes comparing obese LVH with lean NF; comparing male obese LVH with male lean NF samples we found 106 significant genes; comparing female obese LVH with male lean NF, we found no significant genes. Using absolute value of log2 fold-change > 2 or extremely small P value (10-20) as a criterion, we identified nine significant genes (HBA1, HBB, HIST1H2AC, GSTT1, MYL7, NPPA, NPPB, PDK4, PLA2G2A) in LVH, also found in published data set for ischemic and dilated cardiomyopathy in heart failure. We identified a potential gene expression signature that distinguishes between patients with high BMI or between men and women with cardiac hypertrophy. Expression of established biomarkers atrial natriuretic peptide A (NPPA) and B (NPPB) were already significantly increased in hypertrophy compared with controls.
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Índice de Masa Corporal , Cardiomegalia/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Caracteres Sexuales , Adulto , Anciano , Cardiomiopatía Dilatada/genética , Femenino , Ontología de Genes , Redes Reguladoras de Genes , Humanos , Hipertrofia Ventricular Izquierda/genética , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/genética , Adulto JovenRESUMEN
BACKGROUND: Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. METHODS: We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. RESULTS: Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. CONCLUSIONS: We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.
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Adenocarcinoma/metabolismo , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Terapia por Estimulación Eléctrica , Neoplasias de la Mama Triple Negativas/metabolismo , Adenocarcinoma/terapia , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer. METHODS: In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive). RESULTS: Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10-20 µM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 µM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil. CONCLUSIONS: Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.
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Leptin has been proposed to modulate cardiac electrical properties via ß-adrenergic receptor activation. The presence of leptin receptors and adipocytes in myocardium raised a question as to whether leptin can directly modulate cardiac electrical properties such as heart rate and QT interval via its receptor. In this work, the role of local direct actions of leptin on heart rate and ventricular repolarization was investigated. We identified the protein expression of leptin receptors at cell surface of sinus node, atrial, and ventricular myocytes isolated from rat heart. Leptin at low doses (0.1-30 µg/kg) decreased resting heart rate; at high doses (150-300 µg/kg), leptin induced a biphasic effect (decrease and then increase) on heart rate. In the presence of high-dose propranolol (30 mg/kg), high-dose leptin only reduced heart rate and sometimes caused sinus pauses and ventricular tachycardia. The leptin-induced inhibition of resting heart rate was fully reversed by leptin antagonist. Leptin also increased heart rate-corrected QT interval (QTc), and leptin antagonist did not. In isolated ventricular myocytes, leptin (0.03-0.3 µg/ml) reversibly increased the action potential duration. These results supported our hypothesis that in addition to indirect pathway via sympathetic tone, leptin can directly decrease heart rate and increase QT interval via its receptor independent of ß-adrenergic receptor stimulation. During inhibition of ß-adrenergic receptor activity, high concentration of leptin in myocardium can cause deep bradycardia, prolonged QT interval, and ventricular arrhythmias.
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Potenciales de Acción/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Leptina/farmacología , Miocardio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Receptores de Leptina/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Antagonistas Adrenérgicos beta/farmacología , Animales , Fenómenos Electrofisiológicos/efectos de los fármacos , Corazón/efectos de los fármacos , Atrios Cardíacos/citología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Leptina/metabolismo , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Propranolol/farmacología , Ratas , Ratas Sprague-Dawley , Ratas Zucker , Nodo Sinoatrial/metabolismo , Taquicardia VentricularRESUMEN
Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) using tyrosine kinase inhibitors for cancer therapy. We report here that a widely used selective inhibitor of Src tyrosine kinases, PP2, can inhibit and prevent isoproterenol stimulation of cardiac pacemaker activity. In dissected rat sinus node, PP2 inhibited and prevented isoproterenol stimulation of spontaneous beating rate. In isolated sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (If) by negatively shifting the activation curve and decelerating activation kinetics, associated with decreased cell surface expression and reduced tyrosine phosphorylation of hyperpolarization-activated cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. In human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed isoproterenol stimulation of HCN4 and inhibited HCN4-573x, a cAMP-insensitive human HCN4 mutant. Isoprotenrenol had little effects on HCN4-573x. These results demonstrated that inhibition of presumably tyrosine Src kinase activity in heart by PP2 decreased and prevented the potential ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the key channel protein that controls the heart rate.
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Arritmias Cardíacas , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Isoproterenol/farmacología , Nodo Sinoatrial , Familia-src Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/prevención & control , Cardiotónicos/farmacología , Fármacos Cardiovasculares/farmacología , Fosforilación , Ratas , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismoRESUMEN
One of the main strategies for cancer therapy is to use tyrosine kinase inhibitors for inhibiting tumor proliferation. Increasing evidence has demonstrated the potential risks of cardiac arrhythmias (such as prolonged QT interval) of these drugs. We report here that a widely used selective inhibitor of Src tyrosine kinases, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), can inhibit and prevent ß-adrenergic stimulation of cardiac pacemaker activity. First, in dissected rat sinus node, PP2 inhibited and prevented the isoproterenol-induced increase of spontaneous beating rate. Second, in isolated rat sinus node myocytes, PP2 suppressed the hyperpolarization-activated "funny" current (traditionally called cardiac pacemaker current, I(f)) by negatively shifting the activation curve and decelerating activation kinetics. Third, in isolated rat sinus node myocytes, PP2 decreased the Src kinase activity, the cell surface expression, and tyrosine phosphorylation of hyperpolarization-activated, cyclic nucleotide-modulated channel 4 (HCN4) channel proteins. Finally, in human embryonic kidney 293 cells overexpressing recombinant human HCN4 channels, PP2 reversed the enhancement of HCN4 channels by isoproterenol and inhibited 573x, a cyclic adenosine momophosphate-insensitive human HCN4 mutant. These results demonstrated that inhibition of Src kinase activity in heart by PP2 decreased and prevented ß-adrenergic stimulation of cardiac pacemaker activity. These effects are mediated, at least partially, by a cAMP-independent attenuation of channel activity and cell surface expression of HCN4, the main channel protein that controls the heart rate.
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Agonistas Adrenérgicos beta/farmacología , Pirimidinas/farmacología , Nodo Sinoatrial/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Animales , Células Cultivadas , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/biosíntesis , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Canales de Potasio/biosíntesis , Ratas , Ratas Sprague-Dawley , Nodo Sinoatrial/enzimología , Familia-src Quinasas/metabolismoRESUMEN
The majority of diabetic patients who are overweight or obese die of heart disease. We suspect that the obesity-induced insulin resistance may lead to abnormal cardiac electrophysiology. We tested this hypothesis by studying an obese insulin-resistant rat model, the obese Zucker rat (OZR). Compared with the age-matched control, lean Zucker rat (LZR), OZR of 16-17 wk old exhibited an increase in QTc interval, action potential duration, and cell capacitance. Furthermore, the L-type calcium current (I(CaL)) in OZR exhibited defective inactivation and lost the complete inactivation back to the closed state, leading to increased Ca(2+) influx. The current density of I(CaL) was reduced in OZR, whereas the threshold activation and the current-voltage relationship of I(CaL) were not significantly altered. L-type Ba(2+) current (I(BaL)) in OZR also exhibited defective inactivation, and steady-state inactivation was not significantly altered. However, the current-voltage relationship and activation threshold of I(BaL) in OZR exhibited a depolarized shift compared with LZR. The total and membrane protein expression levels of Cav1.2 [pore-forming subunit of L-type calcium channels (LTCC)], but not the insulin receptors, were decreased in OZR. The insulin receptor was found to be associated with the Cav1.2, which was weakened in OZR. The total protein expression of calmodulin was reduced, but that of Cavß2 subunit was not altered in OZR. Together, these results suggested that the 16- to 17-wk-old OZR has 1) developed cardiac hypertrophy, 2) exhibited altered electrophysiology manifested by the prolonged QTc interval, 3) increased duration of action potential in isolated ventricular myocytes, 4) defective inactivation of I(CaL) and I(BaL), 5) weakened the association of LTCC with the insulin receptor, and 6) decreased protein expression of Cav1.2 and calmodulin. These results also provided mechanistic insights into a remodeled cardiac electrophysiology under the condition of insulin resistance, enhancing our understanding of long QT associated with obese type 2 diabetic patients.
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Calcio/metabolismo , Resistencia a la Insulina/fisiología , Síndrome de QT Prolongado/metabolismo , Miocardio/metabolismo , Obesidad/metabolismo , Potenciales de Acción/fisiología , Animales , Canales de Calcio Tipo L/metabolismo , Calmodulina/metabolismo , Modelos Animales de Enfermedad , Electrocardiografía , Hipertrofia , Síndrome de QT Prolongado/fisiopatología , Contracción Miocárdica/fisiología , Miocardio/patología , Obesidad/fisiopatología , Ratas , Ratas Zucker , Delgadez/metabolismo , Delgadez/fisiopatologíaRESUMEN
BACKGROUND: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants. HYPOTHESIS: We hypothesized that in SARS-CoV-2, two highly conserved Orf3a and E channels directly related to the virus replication were a target for the detection and inhibition of the viral replication, independent of the variant, using FDA-approved ion channel modulators. METHODS: A combination of a fluorescence potassium ion assay with channel modulators was developed to detect SARS-CoV-2 Orf3a/E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant), which are ion channel blockers, were tested as to whether they inhibited Orf3a/E channel activity in isolated virus variants and in nasal swab samples from COVID-19 patients. The variants were confirmed by PCR sequencing. RESULTS: In isolated SARS-CoV-2 Alpha, Beta, and Delta variants, the channel activity of Orf3a/E was detected and inhibited by emodin and gliclazide (IC50 = 0.42 mM). In the Delta swab samples, amitriptyline and amantadine inhibited the channel activity of viral proteins, with IC50 values of 0.73 mM and 1.11 mM, respectively. In the Omicron swab samples, amitriptyline inhibited the channel activity, with an IC50 of 0.76 mM. CONCLUSIONS: We developed an efficient method to screen FDA-approved ion channel modulators that could be repurposed to detect and inhibit SARS-CoV-2 viral replication, independent of variants.
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Tratamiento Farmacológico de COVID-19 , Canales Iónicos , SARS-CoV-2 , Humanos , Amantadina/farmacología , Amitriptilina/farmacología , Canales Iónicos/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Reposicionamiento de MedicamentosRESUMEN
BACKGROUND: SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the virus passes from the upper respiratory tract to the myocardium. We hypothesized that SARS-CoV-2 is present in the blood of COVID-19 infected patients, spreading to other organs such as heart. METHODS: We targeted two viroporins, Orf3a and E, in SARS-CoV-2. Orf3a and E form non-voltage-gated ion channels. A combined fluorescence potassium ion assay with three channel modulators (4-aminopyridine, emodin-Orf3a channel blocker, and gliclazide-E channel blocker) was developed to detect SARS-CoV-2 Orf3a/E channel activity. In blood samples, we subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity. RESULTS: In lentivirus-spiked samples, we detected significant channel activity of Orf3a/E based on increase in fluorescence induced by 4-aminopyridine, and this increase in fluorescence was inhibited by emodin and gliclazide. In 18 antigen/PCR-positive samples, our test results found 15 are positive, demonstrating 83.3% concordance. In 24 antigen/PCR-negative samples, our test results found 21 are negative, showing 87.5% concordance. CONCLUSIONS: We developed a cell-free test that can detect Orf3a/E channel activity of SARS-CoV-2 in blood samples from COVID-19-infected individuals, confirming a hypothesis that the virus spreads to the heart via blood circulation.
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BACKGROUND: Missense mutations in the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia. OBJECTIVE: To investigate possible HCN4 channel mutation in a young patient with profound sinus bradycardia. METHODS: Direct sequencing of HCN4 and whole-exome sequencing were performed on DNA samples from the indexed patient (P), the patient's son (PS), and a family unrelated healthy long-distance running volunteer (V). Resting heart rate was 31 bpm for P, 67 bpm for PS, and 50 bpm for V. Immunoblots, flow cytometry, and immunocytofluorescence confocal imaging were used to study cellular distribution of channel variants. Patch-clamp electrophysiology was used to investigate the properties of mutant HCN1 channels. RESULTS: In P no missense mutations were found in the HCN4 gene; instead, we found two heterozygous variants in the HCN1 gene: deletion of an N-terminal glycine triplet (72GGG74, "N-del") and a novel missense variant, P851A, in the C-terminal region. N-del variant was found before and shared by PS. These two variations were not found in V. Compared to wild type, N-del and P851A reduced cell surface expression and negatively shifted voltage-activation with slower activation kinetics. CONCLUSION: Decreased channel activity HCN1 mutant channel makes it unable to contribute to early depolarization of sinus node action potential, thus likely a main cause of the profound sinus bradycardia in this patient.
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Ca(2+) entry is delicately controlled by inactivation of L-type calcium channel (LTCC) composed of the pore-forming subunit alpha1C and the auxiliary subunits beta1 and alpha2delta. Calmodulin is the key protein that interacts with the COOH-terminal motifs of alpha1C, leading to the fine control of LTCC inactivation. In this study we show evidence that a hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, can act as a nonchannel regulatory protein to narrow the L-type Ca(2+) channel current-voltage curve. In the absence of LTCC auxiliary subunits, HCN2 can induce alpha1C inactivation. Without alpha2delta, HCN2-induced fast inactivation of alpha1C requires calmodulin. With alpha2delta, the alpha1C/HCN2/alpha2delta channel inactivation does not require calmodulin. In contrast, beta1-subunit plays a relatively minor role in the interaction of alpha1C with HCN2. The NH(2) terminus of HCN2 and the IQ motif of alpha1C subunit are required for alpha1C/HCN2 channel interaction. Ca(2+) channel inactivation is significantly slowed in hippocampus neurons (HNs) overexpressing HCN2 mutant lacking NH(2) terminus and accelerated in HNs overexpressing the wild-type HCN2 compared with HN controls. Collectively, these results revealed a potentially novel protection mechanism for achieving the LTCC inactivation via interaction with HCN2.
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Canales de Calcio Tipo L/metabolismo , Canales Iónicos/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Línea Celular , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Activación del Canal Iónico/fisiología , Canales Iónicos/genética , Canales de Potasio , Subunidades de Proteína , Ratas , Transducción de SeñalRESUMEN
Therapeutic strategies such as using channel blockers and reducing culture temperature have been used to rescue some long QT-associated voltage-gated potassium Kv trafficking defective mutant channels. A hyperpolarization-activated cyclic nucleotide-gated HCN4 pacemaker channel mutant (D553N) has been recently found in a patient associated with cardiac arrhythmias including long QT. D553N showed the defective trafficking to the cell surface, leading to little ionic current expression (loss-of-function). We show in this report that enhanced tyrosine phosphorylation mediated by Src, Fyn, and Yes kinases was able to restore the surface expression of D553N for normal current expression. Src or Yes, but not Fyn, significantly increased the current density and surface expression of D553N. Fyn accelerated the activation kinetics of the rescued D553N. Co-expression of D553N with Yes exhibited the slowest activation kinetics of D553N. Src, Fyn, and Yes significantly enhanced the tyrosine phosphorylation of D553N. A combination of Src, Fyn, and Yes rescued the current expression and the gating of D553N comparable with those of wild-type HCN4. In conclusion, we demonstrate a novel mechanism using three endogenous Src kinases to rescue a trafficking defective HCN4 mutant channel (D553N) by enhancing the tyrosine phosphorylation of the mutant channel protein.
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Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Proteínas Musculares/genética , Proteínas Tirosina Quinasas/fisiología , Línea Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Proteínas Musculares/metabolismo , Proteínas Mutantes/metabolismo , Mutación Missense , Fosforilación , Canales de Potasio , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-fyn/fisiología , Proteínas Proto-Oncogénicas c-yes/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
The time- and voltage-dependent inward current generated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contributes to the tissue-specific rhythmic activities in the brain and heart. Four isoforms (HCN1-HCN4) have been identified. Previous studies showed that different HCN isoforms may form functional heteromeric channels. We report here that when HCN2 and HCN4 mRNA were injected into Xenopus oocytes with various ratios of HCN2 over HCN4 at 1:1, 10:1, and 1:10, respectively, the resultant channels showed a depolarized current activation and significantly faster activation kinetics near the midpoint of activation compared with HCN4 homomeric channels. In adult rat myocytes overexpressing HCN4, there was an associated increase in HCN2 mRNA. In neonatal rat myocytes in which HCN2 was knocked down, there was also a simultaneous decrease in HCN4 mRNA. Coimmunoprecipitation experiments showed that HCN2 and HCN4 channel proteins can associate with each other in adult rat ventricles. Finally, in adult myocytes overexpressing HCN4, the hyperpolarization-activated inward current activation, I(f), was shifted to physiological voltages from non-physiological voltages, associated with faster activation kinetics. These data suggested that different ratios of HCN2 and HCN4 transcripts overlapping in different tissues also contribute to the tissue-specific properties of I(f).
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Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Iónicos/genética , Proteínas Musculares/genética , Miocitos Cardíacos/metabolismo , Canales de Potasio/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Fenómenos Biofísicos , Línea Celular , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Fenómenos Electrofisiológicos , Femenino , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Técnicas In Vitro , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/metabolismo , Potenciales de la Membrana , Ratones , Proteínas Musculares/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
Dopamine neurons in the ventral tegmental area (VTA) are powerful regulators of depression-related behavior. Dopamine neuron activity is altered in chronic stress-based models of depression, but the underlying mechanisms remain incompletely understood. Here, we show that mice subject to chronic mild unpredictable stress (CMS) exhibit anxiety- and depressive-like behavior, which was associated with decreased VTA dopamine neuron firing in vivo and ex vivo. Dopamine neuron firing is governed by voltage-gated ion channels, in particular hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Following CMS, HCN-mediated currents were decreased in nucleus accumbens-projecting VTA dopamine neurons. Furthermore, shRNA-mediated HCN2 knockdown in the VTA was sufficient to recapitulate CMS-induced depressive- and anxiety-like behavior in stress-naïve mice, whereas VTA HCN2 overexpression largely prevented CMS-induced behavioral deficits. Together, these results reveal a critical role for HCN2 in regulating VTA dopamine neuronal activity and depressive-related behaviors.
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Depresión/fisiopatología , Neuronas Dopaminérgicas/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Canales de Potasio/metabolismo , Estrés Fisiológico , Área Tegmental Ventral/fisiología , Potenciales de Acción , Animales , Expresión Génica , Técnicas de Silenciamiento del Gen , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Ratones , Canales de Potasio/genéticaRESUMEN
Breast cancer is a dangerous disease that results in high mortality rates for cancer patients. Many methods have been developed for the treatment and prevention of this disease. Determining the expression patterns of certain target genes in specific subtypes of breast cancer is important for developing new therapies for breast cancer. In the present study, we performed a holistic approach to screening the mRNA expression of six members of the cell division cycle-associated gene family (CDCA) with a focus on breast cancer using the Oncomine and The Cancer Cell Line Encyclopedia (CCLE) databases. Furthermore, Gene Expression-Based Outcome for Breast Cancer Online (GOBO) was also used to deeply mine the expression of each CDCA gene in clinical breast cancer tissue and breast cancer cell lines. Finally, the mRNA expression of the CDCA genes as related to breast cancer patient survival were analyzed using a Kaplan-Meier plot. CDCA3, CDCA5, and CDCA8 mRNA expression levels were significantly higher than the control sample in both clinical tumor sample and cancer cell lines. These highly expressed genes in the tumors of breast cancer patients dramatically reduced patient survival. The interaction network of CDCA3, CDCA5, and CDCA8 with their co-expressed genes also revealed that CDCA3 expression was highly correlated with cell cycle related genes such as CCNB2, CDC20, CDKN3, and CCNB1. CDCA5 expression was correlated with BUB1 and TRIP13, while CDCA8 expression was correlated with BUB1 and CCNB1. Altogether, these findings suggested CDCA3, CDCA5, and CDCA8 could have a high potency as targeted breast cancer therapies.
RESUMEN
CONTEXT: Arsenic poisoning commonly occurs through exposure to water contaminated with arsenic and causes long-term symptoms. Of all the arsenic derivatives, arsenite is the one of the most toxic compounds. However, the toxicity of arsenite during developmental stages is still unclear. OBJECTIVE: In this study, we performed a metabolomic analysis of arsenite responses in embryonic zebrafish. MATERIALS AND METHODS: Embryonic zebrafish were used as an animal model in this study. They were exposed to sodium arsenite under different concentrations (0.5, 1.0, 2.0, and 5.0â¯mg/L) in 24â¯h, 48â¯h and 72â¯h post fertilization. Changes in morphology were observed through a light microscope. Changes in metabolomics were identified using an ultraperformance liquid chromatography quadrupole time-of-flight system. RESULTS: The IC50 range was 0.75⯱â¯0.25â¯mg/L. Compared with the control group, the embryonic lethality rate decreased to 33.3% under 1.0â¯mg/L of arsenite treatment, whereas it decreased to 20.0% under 2.0â¯mg/L of arsenite treatment. Numerous body axis curvatures were also observed under treatment with 2.0 and 5.0â¯mg/L of arsenic. Pericardium and yolk sac edema were randomly discovered and found to worsen over time. Moreover, the 10 metabolites with the highest variable importance in projection score were identified as potential biomarkers for arsenic exposure. CONCLUSION: Arsenic exposure not only leads to a change in the morphology of embryonic zebrafish but also disturbs the metabolism of zebrafish in early developmental stages.
Asunto(s)
Arsenitos/toxicidad , Embrión no Mamífero/efectos de los fármacos , Metabolómica , Pez Cebra/embriología , Animales , Ácidos Araquidónicos/fisiología , Biomarcadores , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/metabolismo , Endocannabinoides/fisiología , Glicéridos/fisiología , Curva ROCRESUMEN
Diabetes is a risk factor that increases the occurrence and severity of cardiovascular events. Cardiovascular complications are the leading cause of mortality of 75% of patients with diabetes >40 years old. Sesamin, the bioactive compound extracted from Sesamum indicum, is a natural compound that has diverse beneficial effects on hypoglycemia and reducing cholesterol. The aim of this study is to investigate sesamin effects to diabetes-inducing cardiac hypertrophy. In the present study bioinformatics analysis demonstrated cardiac hypertrophy signaling may be the most important pathway for upregulating genes in sesamin-treated groups. To verify the bioinformatics prediction, sesamin was used as the main bioactive compound to attenuate the impact of diabetes induced by streptozotocin (STZ) on cardiac function in a rat model. The results revealed that oral administration of sesamin for 4 weeks (100 and 200 mg/kg body weight) marginally improved blood glucose levels, body weight and significantly ameliorated the effects on heart rate and blood pressure in rats with type 1 diabetes relative to control rats. The QT interval of sesamin was also reduced relative to the control group. The findings indicated that sesamin has potential cardioprotective effects in the STZ-induced diabetes model. This suggested that this can be used as a novel treatment for patients with diabetes with cardiac dysfunction complication.