Introduction: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants such as D... more Introduction: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants such as Delta and Omicron. Early detection of virus and effective therapeutic treatment independent of variants help control virus spread. Hypothesis: We hypothesized that in the genome of SARS-CoV-2 two highly conserved viroporins Orf3a and E channels directly related to virus replication may be explored as a target for detection and inhibition of viral replication independent of variants using FDA-approved ion channel modulators. Methods: A combination of a fluorescence potassium ion assay with three channel modulators (4-aminopyridine, emodin-Orf3a channel blocker, gliclazide-E channel blocker) in a 96-well plate format was developed to detect SARS-CoV-2 Orf3a/E channel activity. We subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant) t...
Introduction: SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the ... more Introduction: 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. Hypothesis: SARS-CoV-2 is present in the blood of COVID-19 infected patients. Methods: We targeted two viropotins, 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, 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 foun...
If, the "cardiac pacemaker current, " is a nonselective cation channel activated on hyp... more If, the "cardiac pacemaker current, " is a nonselective cation channel activated on hyperpolarization in primary and secondary pacemaker regions of the mammalian heart. The cardiac pacing rate can be modulated by shifting the activation of If to more positive (faster pacing) or more negative (slower pacing) voltages. We report for the first time the presence of this pacemaker current in ventricular myocytes. The potential importance of this observation to the mechanism of differentiation of nonpacing regions in the heart is discussed. (Circulation Research 1993;72:232-236) KEY WORDS * ventricular myocyte * pacemaker current * pacing current T he rhythm of the mammalian heart is myogenic in origin. ' One current that contributes to pacing in both the primary (SA node) and secondary pacemakers (Purkinje fibers) is a nonselec-tive cation current activated on hyperpolarization and called If.2-4 If provides a time-dependent inward current that drives the membrane toward th...
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer about this process is available in the located, click Request Permissions in the middle column of the Web page under Services. Further information Editorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on February 27,
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that ... more The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na + and K + . There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current if. Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phos- phorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node if conduc- tance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages ne...
L'invention concerne un procede permettant d'induire un courant dans le coeur d'un pa... more L'invention concerne un procede permettant d'induire un courant dans le coeur d'un patient qui consiste a mettre en contact une cellule de son coeur avec un compose en dose suffisante pour induire un courant dans ladite cellule. L'invention concerne egalement un procede de traitement d'un etat cardiaque chez un patient qui consiste a mettre en contact une cellule du coeur de ce dernier avec un compose en dose suffisante pour accroitre l'expression du courant de la cellule, ce qui permet de traiter l'etat cardiaque du patient.
L'invention concerne une chambre et un systeme concus pour etre utilises dans le dosage des e... more L'invention concerne une chambre et un systeme concus pour etre utilises dans le dosage des effets de medicaments sur la frequence cardiaque. Cette chambre est constituee d'une serie de puits presentant tous un diametre interieur de 3 mm sur 3 mm. Des myocytes cardiaques desagreges provenant d'animaux nouveaux-nes sont plaques sur le fond de chaque puits. On les fait ensuite croitre dans des conditions de culture classiques. La chambre selon l'invention contient de 24 a 96 puits de la sorte. Lorsque des medicaments doivent etre doses, les cellules de chaque puits sont chargees au moyen d'un colorant sensible au calcium et la vitesse de battement dans chaque puits est controlee par une photodiode. Un medicament est ajoute a chaque puits selon des concentrations classees. On equilibre ensuite le medicament et on observe ses effets sur la vitesse de battement. La construction selon l'invention permet d'utiliser le dosage biologique a base de cellules dans l&...
It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward cu... more It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time-dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [...
Missense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4... more Missense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia.
I(f), the "cardiac pacemaker current," is a nonselective cation channel activated on hy... more I(f), the "cardiac pacemaker current," is a nonselective cation channel activated on hyperpolarization in primary and secondary pacemaker regions of the mammalian heart. The cardiac pacing rate can be modulated by shifting the activation of I(f) to more positive (faster pacing) or more negative (slower pacing) voltages. We report for the first time the presence of this pacemaker current in ventricular myocytes. The potential importance of this observation to the mechanism of differentiation of nonpacing regions in the heart is discussed.
Background —Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing o... more Background —Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. Methods and Results —We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12±0.18 to −0.34±0.21 mV (±SEM, P <0.05), and T-wave vector, which shifted from −37±12° to −143±4° ( P <0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P <0.05). Calcium-insensitive transient outward current ( I to ) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes...
American Journal of Physiology-Cell Physiology, 2006
The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac ( If)/neurona... more The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac ( If)/neuronal ( Ih) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and Ih channels in neurons. This raises the possibility of Ca2+ permeation in If, the Ih counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca2+ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by If in rat ventricular myocytes. We observed Ca2+ influx when HCN/ If channels were activated. Ca2+ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an If channel blocker, inhibited If and Ca2+ influx at the same time. Quantitative analysis revealed that Ca2+ flux contributed to ∼0.5% of current produce...
Introduction: MAP2K3 is a stress-induced kinase and its role cardiac hypertrophy has been controv... more Introduction: MAP2K3 is a stress-induced kinase and its role cardiac hypertrophy has been controversial: while in vitro studies have displayed a positive correlation in activity with hypertrophy, in vivo studies have shown a negative association. Its direct downstream target, p38α MAPK, has been shown to be in activated in hypertrophic and failed hearts, but this has not been explored with regard to gender or BMI. Hypothesis: Early activation of p38 MAPK in hypertrophy is influenced by gender and BMI. Methods: Human heart samples were grouped into non-failed (NF), left ventricular hypertrophy (LVH), and NF without LVH (control). BMI < 25 was considered “lean”, 25 < BMI < 30 was considered overweight, and BMI > 30 was considered “obese”. RNA-Seq was used to generate cardiac gene expression profiles. Immunoblots were used to examine protein expression of MAP2K3, p38, and activated p38 (pp38; phospho-T180 and Y182). Results: We found that MAP2K3 mRNA levels were increased i...
Introduction: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants such as D... more Introduction: SARS-CoV-2 has undergone mutations, yielding clinically relevant variants such as Delta and Omicron. Early detection of virus and effective therapeutic treatment independent of variants help control virus spread. Hypothesis: We hypothesized that in the genome of SARS-CoV-2 two highly conserved viroporins Orf3a and E channels directly related to virus replication may be explored as a target for detection and inhibition of viral replication independent of variants using FDA-approved ion channel modulators. Methods: A combination of a fluorescence potassium ion assay with three channel modulators (4-aminopyridine, emodin-Orf3a channel blocker, gliclazide-E channel blocker) in a 96-well plate format was developed to detect SARS-CoV-2 Orf3a/E channel activity. We subtracted the fluorescence signals in the absence and presence of emodin/gliclazide to detect Orf3a and E channel activity. Two FDA-approved drugs, amantadine (an antiviral) and amitriptyline (an antidepressant) t...
Introduction: SARS-CoV-2 has been found in the heart of COVID-19 patients. It is unclear how the ... more Introduction: 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. Hypothesis: SARS-CoV-2 is present in the blood of COVID-19 infected patients. Methods: We targeted two viropotins, 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, 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 foun...
If, the "cardiac pacemaker current, " is a nonselective cation channel activated on hyp... more If, the "cardiac pacemaker current, " is a nonselective cation channel activated on hyperpolarization in primary and secondary pacemaker regions of the mammalian heart. The cardiac pacing rate can be modulated by shifting the activation of If to more positive (faster pacing) or more negative (slower pacing) voltages. We report for the first time the presence of this pacemaker current in ventricular myocytes. The potential importance of this observation to the mechanism of differentiation of nonpacing regions in the heart is discussed. (Circulation Research 1993;72:232-236) KEY WORDS * ventricular myocyte * pacemaker current * pacing current T he rhythm of the mammalian heart is myogenic in origin. ' One current that contributes to pacing in both the primary (SA node) and secondary pacemakers (Purkinje fibers) is a nonselec-tive cation current activated on hyperpolarization and called If.2-4 If provides a time-dependent inward current that drives the membrane toward th...
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer about this process is available in the located, click Request Permissions in the middle column of the Web page under Services. Further information Editorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Researchin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on February 27,
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles origi... more Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at:
The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that ... more The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na + and K + . There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current if. Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phos- phorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node if conduc- tance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages ne...
L'invention concerne un procede permettant d'induire un courant dans le coeur d'un pa... more L'invention concerne un procede permettant d'induire un courant dans le coeur d'un patient qui consiste a mettre en contact une cellule de son coeur avec un compose en dose suffisante pour induire un courant dans ladite cellule. L'invention concerne egalement un procede de traitement d'un etat cardiaque chez un patient qui consiste a mettre en contact une cellule du coeur de ce dernier avec un compose en dose suffisante pour accroitre l'expression du courant de la cellule, ce qui permet de traiter l'etat cardiaque du patient.
L'invention concerne une chambre et un systeme concus pour etre utilises dans le dosage des e... more L'invention concerne une chambre et un systeme concus pour etre utilises dans le dosage des effets de medicaments sur la frequence cardiaque. Cette chambre est constituee d'une serie de puits presentant tous un diametre interieur de 3 mm sur 3 mm. Des myocytes cardiaques desagreges provenant d'animaux nouveaux-nes sont plaques sur le fond de chaque puits. On les fait ensuite croitre dans des conditions de culture classiques. La chambre selon l'invention contient de 24 a 96 puits de la sorte. Lorsque des medicaments doivent etre doses, les cellules de chaque puits sont chargees au moyen d'un colorant sensible au calcium et la vitesse de battement dans chaque puits est controlee par une photodiode. Un medicament est ajoute a chaque puits selon des concentrations classees. On equilibre ensuite le medicament et on observe ses effets sur la vitesse de battement. La construction selon l'invention permet d'utiliser le dosage biologique a base de cellules dans l&...
It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward cu... more It is generally assumed that in cardiac Purkinje fibers the hyperpolarization activated inward current i(f) underlies the pacemaker potential. Because some findings are at odds with this interpretation, we used the whole cell patch clamp method to study the currents in the voltage range of diastolic depolarization in single canine Purkinje myocytes, a preparation where many confounding limitations can be avoided. In Tyrode solution ([K+]o = 5.4 mM), hyperpolarizing steps from Vh = -50 mV resulted in a time-dependent inwardly increasing current in the voltage range of diastolic depolarization. This time-dependent current (iKdd) appeared around -60 mV and reversed near EK. Small superimposed hyperpolarizing steps (5 mV) applied during the voltage clamp step showed that the slope conductance decreases during the development of this time-dependent current. Decreasing [K+]o from 5.4 to 2.7 mM shifted the reversal potential to a more negative value, near the corresponding EK. Increasing [...
Missense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4... more Missense mutations in the hyperpolarization‐activated cyclic nucleotide‐modulated (HCN) channel 4 (HCN4) are one of the genetic causes of cardiac sinus bradycardia.
I(f), the "cardiac pacemaker current," is a nonselective cation channel activated on hy... more I(f), the "cardiac pacemaker current," is a nonselective cation channel activated on hyperpolarization in primary and secondary pacemaker regions of the mammalian heart. The cardiac pacing rate can be modulated by shifting the activation of I(f) to more positive (faster pacing) or more negative (slower pacing) voltages. We report for the first time the presence of this pacemaker current in ventricular myocytes. The potential importance of this observation to the mechanism of differentiation of nonpacing regions in the heart is discussed.
Background —Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing o... more Background —Cardiac memory refers to an altered T-wave morphology induced by ventricular pacing or arrhythmias that persist for variable intervals after resumption of sinus rhythm. Methods and Results —We induced long-term cardiac memory (LTM) in conscious dogs by pacing the ventricles at 120 bpm for 3 weeks. ECGs were recorded daily for 1 hour, during which time pacing was discontinued. At terminal study, the heart was removed and the electrophysiology of left ventricular epicardial myocytes was investigated. Control (C) and LTM ECG did not differ, except for T-wave amplitude, which decreased from 0.12±0.18 to −0.34±0.21 mV (±SEM, P <0.05), and T-wave vector, which shifted from −37±12° to −143±4° ( P <0.05). Epicardial action potentials revealed loss of the notch and lengthening of duration at 20 days (both P <0.05). Calcium-insensitive transient outward current ( I to ) was investigated by whole-cell patch clamp. No difference in capacitance was seen in C and LTM myocytes...
American Journal of Physiology-Cell Physiology, 2006
The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac ( If)/neurona... more The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac ( If)/neuronal ( Ih) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and Ih channels in neurons. This raises the possibility of Ca2+ permeation in If, the Ih counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca2+ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by If in rat ventricular myocytes. We observed Ca2+ influx when HCN/ If channels were activated. Ca2+ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an If channel blocker, inhibited If and Ca2+ influx at the same time. Quantitative analysis revealed that Ca2+ flux contributed to ∼0.5% of current produce...
Introduction: MAP2K3 is a stress-induced kinase and its role cardiac hypertrophy has been controv... more Introduction: MAP2K3 is a stress-induced kinase and its role cardiac hypertrophy has been controversial: while in vitro studies have displayed a positive correlation in activity with hypertrophy, in vivo studies have shown a negative association. Its direct downstream target, p38α MAPK, has been shown to be in activated in hypertrophic and failed hearts, but this has not been explored with regard to gender or BMI. Hypothesis: Early activation of p38 MAPK in hypertrophy is influenced by gender and BMI. Methods: Human heart samples were grouped into non-failed (NF), left ventricular hypertrophy (LVH), and NF without LVH (control). BMI < 25 was considered “lean”, 25 < BMI < 30 was considered overweight, and BMI > 30 was considered “obese”. RNA-Seq was used to generate cardiac gene expression profiles. Immunoblots were used to examine protein expression of MAP2K3, p38, and activated p38 (pp38; phospho-T180 and Y182). Results: We found that MAP2K3 mRNA levels were increased i...
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