scholarly journals Culture and adenoviral infection of sinoatrial node myocytes from adult mice

2015 ◽  
Vol 309 (3) ◽  
pp. H490-H498 ◽  
Author(s):  
Joshua R. St. Clair ◽  
Emily J. Sharpe ◽  
Catherine Proenza
Keyword(s):  
Action Potential ◽  
Protein Expression ◽  
Sinoatrial Node ◽  
New Method ◽  
Mouse Heart ◽  
Isolated Cells ◽  
Adenoviral Infection ◽  
Adult Mice ◽  
Spontaneous Action

Pacemaker myocytes in the sinoatrial node of the heart initiate each heartbeat by firing spontaneous action potentials. However, the molecular processes that underlie pacemaking are incompletely understood, in part because of our limited ability to manipulate protein expression within the native cellular context of sinoatrial node myocytes (SAMs). Here we describe a new method for the culture of fully differentiated SAMs from adult mice, and we demonstrate that robust expression of introduced proteins can be achieved within 24–48 h in vitro via adenoviral gene transfer. Comparison of morphological and electrophysiological characteristics of 48 h-cultured versus acutely isolated SAMs revealed only minor changes in vitro. Specifically, we found that cells tended to flatten in culture but retained an overall normal morphology, with no significant changes in cellular dimensions or membrane capacitance. Cultured cells beat spontaneously and, in patch-clamp recordings, the spontaneous action potential firing rate did not differ between cultured and acutely isolated cells, despite modest changes in a subset of action potential waveform parameters. The biophysical properties of two membrane currents that are critical for pacemaker activity in SAMs, the “funny current” ( If) and voltage-gated Ca2+ currents ( ICa), were also indistinguishable between cultured and acutely isolated cells. This new method for culture and adenoviral infection of fully-differentiated SAMs from the adult mouse heart expands the range of experimental techniques that can be applied to study the molecular physiology of cardiac pacemaking because it will enable studies in which protein expression levels can be modified or genetically encoded reporter molecules expressed within SAMs.

scholarly journals Aloe-Emodin Relieves High-Fat Diet Induced QT Prolongation via MiR-1 Inhibition and IK1 Up-Regulation in Rats

2017 ◽  
Vol 43 (5) ◽  
pp. 1961-1973 ◽  
Author(s):  
Yan Bai ◽  
Zhenli Su ◽  
Hanqi Sun ◽  
Wei Zhao ◽  
Xue Chen ◽  
...  
Keyword(s):  
Action Potential ◽  
Protein Expression ◽  
High Fat Diet ◽  
Qt Prolongation ◽  
Electrical Remodeling ◽  
High Fat ◽  
Treatment Groups ◽  
Aloe Emodin

Background/Aims: High-fat diet (HFD) causes cardiac electrical remodeling and increases the risk of ventricular arrhythmias. Aloe-emodin (AE) is an anthraquinone component isolated from rhubarb and has a similar chemical structure with emodin. The protective effect of emodin against cardiac diseases has been reported in the literature. However, the cardioprotective property of AE is still unknown. The present study investigated the effect of AE on HFD-induced QT prolongation in rats. Methods: Adult male Wistar rats were randomly divided into three groups: control, HFD, and AE-treatment groups. Normal diet was given to rats in the control group, high-fat diet was given to rats in HFD and AE-treatment groups for a total of 10 weeks. First, HFD rats and AE-treatment rats were fed with high-fat diet for 4 weeks to establish the HFD model. Serum total cholesterol and triglyceride levels were measured to validate the HFD model. Afterward, AE-treatment rats were intragastrically administered with 100 mg/kg AE each day for 6 weeks. Electrocardiogram monitoring and whole-cell patch-clamp technique were applied to examine cardiac electrical activity, action potential and inward rectifier K+ current (IK1), respectively. Neonatal rat ventricular myocytes (NRVMs) were subjected to cholesterol and/or AE. Protein expression of Kir2.1 was detected by Western blot and miR-1 level was examined by real-time PCR in vivo and in vitro, respectively. Results: In vivo, AE significantly shortened the QT interval, action potential duration at 90% repolarization (APD90) and resting membrane potential (RMP), which were markedly elongated by HFD. AE increased IK1 current and Kir2.1 protein expression which were reduced in HFD rats. Furthermore, AE significantly inhibited pro-arrhythmic miR-1 in the hearts of HFD rats. In vitro, AE decreased miR-1 expression levels resulting in an increase of Kir2.1 protein levels in cholesterol-enriched NRVMs. Conclusions: AE prevents HFD-induced QT prolongation by repressing miR-1 and upregulating its target Kir2.1. These findings suggest a novel pharmacological role of AE in HFD-induced cardiac electrical remodeling.

scholarly journals α1-adrenergic stimulation increases ventricular action potential duration in the intact mouse heart

FACETS ◽  
2021 ◽  
Vol 6 ◽  
pp. 823-836
Author(s):  
William Joyce ◽  
Koen T. Scholman ◽  
Bjarke Jensen ◽  
Tobias Wang ◽  
Bastiaan J. Boukens
Keyword(s):  
Action Potential ◽  
Action Potential Duration ◽  
Mouse Heart ◽  
Adrenergic Blockade ◽  
Adrenergic Stimulation ◽  
Isolated Cells ◽  
Intact Mouse ◽  
Adrenergic Antagonist ◽  
The Right

The role of α1-adrenergic receptors (α-ARs) in the regulation of myocardial function is less well-understood than that of β-ARs. Previous reports in the mouse heart have described that α1-adrenergic stimulation shortens action potential duration in isolated cells or tissues, in contrast to prolongation of the action potential reported in most other mammalian hearts. It has since become appreciated, however, that the mouse heart exhibits marked variation in inotropic response to α1-adrenergic stimulation between ventricles and even individual cardiomyocytes. We investigated the effects of α1-adrenergic stimulation on action potential duration at 80% of repolarization in the right and left ventricles of Langendorff-perfused mouse hearts using optical mapping. In hearts under β-adrenergic blockade (propranolol), phenylephrine or noradrenaline perfusion both increased action potential duration in both ventricles. The increased action potential duration was partially reversed by subsequent perfusion with the α-adrenergic antagonist phentolamine (1 μmol L−1). These data show that α1-receptor stimulation may lead to a prolonging of action potential in the mouse heart and thereby refine our understanding of how action potential duration adjusts during sympathetic stimulation.

Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping

2004 ◽  
Vol 287 (6) ◽  
pp. H2634-H2643 ◽  
Author(s):  
Anders Nygren ◽  
Alan E. Lomax ◽  
Wayne R. Giles
Keyword(s):  
Action Potential ◽  
Left Atrium ◽  
Right Atrium ◽  
Imaging System ◽  
Mouse Heart ◽  
Muscarinic Agonist ◽  
Voltage Sensitive Dye ◽  
Left And Right ◽  
The Right

An imaging system for di-4-ANEPPS (4-[β-[2-(di- n-butylamino)-6-naphthylvinyl]pyridinium]) voltage-sensitive dye recordings has been adapted for recording from an in vitro mouse heart preparation that consists of both atria in isolation. This approach has been used to study inter- and intra-atrial activation and conduction and to monitor action potential durations (APDs) in the left and right atrium. The findings from this study confirm some of our previous findings in isolated mouse atrial myocytes and demonstrate that many electrophysiological properties of mouse atria closely resemble those of larger mammals. Specifically, we made the following observations: 1) Activation in mouse atria originates in the sinoatrial node and spreads into the right atrium and, after a delay, into the left atrium. 2) APD in the left atrium is shorter than in the right atrium. 3) Sites in the posterior walls have longer APDs than sites in the atrial appendages. 4) Superfusion of this preparation with 4-aminopyridine and tetraethylammonium resulted in increases in APD, consistent with their inhibitory effects on the K+ currents known to be expressed in mouse atria. 5) The muscarinic agonist carbachol shortened APD in all areas of the preparation, except the left atrial appendage, in which carbachol had no statistically significant effect on APD. These results validate a new approach for monitoring activation, conduction, and repolarization in mouse atria and demonstrate that the physiological and pharmacological properties of mouse atria are sufficiently similar to those of larger animals to warrant further studies using this preparation.

scholarly journals Pluripotent Stem Cells Can Be Isolated from Human Peripheral Nerves after in vitro BMP-2 Stimulation

2019 ◽  
Author(s):  
Ren-Yi Sun ◽  
Michael H. Heggeness ◽  
Tanghong Jia ◽  
Sunaina Shrestha ◽  
Bradley Dart ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transcription Factors ◽  
Peripheral Nerves ◽  
Nerve Tissue ◽  
Isolated Cells ◽  
Adult Mice ◽  
Stem Cell Medium ◽  
Natural Mechanism ◽  
Human Nerve

AbstractWe have recently identified a population of cells within the peripheral nerves of adult mice that can respond to BMP-2 exposure or physical injury to rapidly proliferate. More importantly, these cells exhibited embryonic differentiation potentials that could be induced into osteoblastic and endothelial cells in vitro. The current study examined human nerve specimens to compare and characterize the cells after BMP-2 stimulation. Fresh pieces of human nerve tissue were minced and treated with either BMP-2 (750ng/ml) or vehicle for 12 hours at 37°C, before digested in 0.2% collagenase and 0.05% trypsin-EDTA. Isolated cells were cultured in restrictive stem cell medium. Significantly more cells were obtained from the nerve pieces with BMP-2 treatment in comparison with the non-treated controls. Cell colonies were starting to form at day 3. Expressions of the 4 transcription factors Klf4, c-Myc, Sox2 and Oct4 were confirmed at both transcriptional and translational levels. The cells can be maintained in the stem cell culture medium for at least 6 weeks without changing morphologies. When the cells were switched to fibroblast growth medium, dispersed spindle-shaped cells were noted and became fibroblast activated protein-α (FAP) positive following immunocytochemistry staining. The data suggested that human peripheral nerve tissue also contain a population of cells that can respond to BMP-2 and express all four transcription factors KLF4, Sox2, cMyc, and Oct4. These cells are capable to differentiate into FAP-positive fibroblasts. It is proposed that these cells are possibly at the core of a previously unknown natural mechanism for healing injury.

A simple efficient method for separating murine uterine epithelial and mesenchymal cells

1986 ◽  
Vol 251 (5) ◽  
pp. E630-E636 ◽  
Author(s):  
R. M. Bigsby ◽  
P. S. Cooke ◽  
G. R. Cunha
Keyword(s):  
Epithelial Cells ◽  
Efficient Method ◽  
Mesenchymal Cells ◽  
Uterine Epithelium ◽  
Cell Uptake ◽  
Luminal Epithelium ◽  
Isolated Cells ◽  
Neonatal Mice ◽  
Adult Mice

A simple, and very efficient, method for isolating pure uterine epithelium from neonatal, immature, or adult mice and pure uterine mesenchyme from neonatal mice is described. The technique uses mild tryptic digestion of the tissues to loosen the adherence of the epithelium to its underlying mesenchyme followed by gentle mechanical manipulation to effect removal of the luminal epithelium as an intact tube of cells. The epithelial fraction collected by this method was free of stromal cell contamination as judged by microscopic examination of the freshly isolated cells and of cell cultures made from that fraction. The mesenchymal fraction was consistently devoid of epithelium when collected from neonatal mice (5 days old or younger), but mesenchyme from uteri of mice greater than or equal to 10 days old was usually contaminated with epithelial cells due to retention of glandular epithelial crypts in the mesenchyme following removal of the luminal epithelium. Both epithelial and mesenchymal cells obtained by this method are viable, as judged by their ability to attach, spread, and synthesize DNA in vitro. Epithelial cells isolated from 20-day-old or adult animals have a full complement of estrogen receptors, as assessed by whole cell uptake of [3H]estradiol. The technique described here has clear advantages over previously described methods for obtaining pure uterine epithelium and in addition allows mesenchymal tissue free of epithelial contamination to be obtained when applied to uteri of animals less than or equal to 5 days old.

GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro

1998 ◽  
Vol 813 (1) ◽  
pp. 88-96 ◽  
Author(s):  
Nurhadi Ibrahim ◽  
Izumi Shibuya ◽  
Narutoshi Kabashima ◽  
V.Sutarmo Setiadji ◽  
Yoichi Ueta ◽  
...  
Keyword(s):  
Action Potential ◽  
Gabab Receptor ◽  
Spontaneous Action Potential ◽  
Spontaneous Action ◽  
Supraoptic Neurons

scholarly journals Activity of Cerebellar Nuclei Neurons Correlates with ZebrinII Identity of Their Purkinje Cell Afferents

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2686
Author(s):  
Gerrit C. Beekhof ◽  
Simona V. Gornati ◽  
Cathrin B. Canto ◽  
Avraham M. Libster ◽  
Martijn Schonewille ◽  
...  
Keyword(s):  
Action Potential ◽  
Bimodal Distribution ◽  
Cerebellar Nuclei ◽  
Firing Frequency ◽  
Action Potential Firing ◽  
Adult Mice ◽  
Electrophysiological Recordings ◽  
Potential Firing

Purkinje cells (PCs) in the cerebellar cortex can be divided into at least two main subpopulations: one subpopulation that prominently expresses ZebrinII (Z+), and shows a relatively low simple spike firing rate, and another that hardly expresses ZebrinII (Z–) and shows higher baseline firing rates. Likewise, the complex spike responses of PCs, which are evoked by climbing fiber inputs and thus reflect the activity of the inferior olive (IO), show the same dichotomy. However, it is not known whether the target neurons of PCs in the cerebellar nuclei (CN) maintain this bimodal distribution. Electrophysiological recordings in awake adult mice show that the rate of action potential firing of CN neurons that receive input from Z+ PCs was consistently lower than that of CN neurons innervated by Z– PCs. Similar in vivo recordings in juvenile and adolescent mice indicated that the firing frequency of CN neurons correlates to the ZebrinII identity of the PC afferents in adult, but not postnatal stages. Finally, the spontaneous action potential firing pattern of adult CN neurons recorded in vitro revealed no significant differences in intrinsic pacemaking activity between ZebrinII identities. Our findings indicate that all three main components of the olivocerebellar loop, i.e., PCs, IO neurons and CN neurons, operate at a higher rate in the Z– modules.

Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin

2006 ◽  
Vol 291 (6) ◽  
pp. H2669-H2679 ◽  
Author(s):  
J. Guo ◽  
S. Zhan ◽  
J. Somers ◽  
R. E. Westenbroek ◽  
W. A. Catterall ◽  
...  
Keyword(s):  
Sudden Death ◽  
Action Potential ◽  
Sodium Channel ◽  
Heart Block ◽  
Mouse Heart ◽  
Channel Activity ◽  
Wild Type ◽  
The Common

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (d V/d tmax) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents ( INa) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42–50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of INa. All of these treatments rescued INa to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of INa. To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died ( P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.

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