Action Potential Parameters Research Articles

Effects of chronic exposure to usual and high doses of Bisphenol A on cardiac arrhythmias, electrophysiology, and autonomic modulation

Abstract Background Bisphenol A (BPA) is a synthetic compound found in plastic bottles, sports equipment, or water pipes. The continuous and inevitable human exposure to BPA has been related to increased cardiovascular risk. In experimental studies, acute exposure to high BPA doses was associated with ventricular arrhythmias. However, the effects of chronic exposure to usual BPA doses (i.e., relevant for usual human exposure) on cardiac arrhythmogenicity have not been properly assessed. Purpose We aimed to assess the effects of chronic exposure to high and usual BPA doses on cardiac arrhythmias, electrophysiology, and autonomic modulation in rats. Methods Adult female Wistar rats were randomized into 3 groups: Control, BPA, and BPA-HD (n=7 each). BPA and BPA-HD rats received 50 μg/kg/day and 25 mg/kg/day of BPA, respectively, for 9 weeks. Rats were implanted with radiotelemetry ECG devices and 24-h ECG recordings were performed prior to and after application of transesophageal atrial burst pacing (4,000 stimuli/min for 20 s; 15 cycles). Heart rate variability (HRV) parameters and atrial and ventricular arrhythmias (i.e., atrial premature beats [APB], atrial fibrillation [AF], ventricular premature beats [VPB], ventricular tachycardia/fibrillation [VT/VF]) were assessed based on the 24-h ECG recordings. The left atrium was then collected and atrial depolarization velocity and action potential duration to 90% (APD90) and 50% (APD50) of complete repolarization were measured. Results Mean heart rate (p=0.48), the number of VPB (p=0.90), AF inducibility (p=0.68), and atrial action potential parameters (all p>0.05) were all similar between the 3 groups. None of the animals presented VT/VF. There was no significant difference in the number of atrial or ventricular arrhythmic events nor in the HRV parameters between BPA and Control (all p>0.05). However, BPA-HD presented a significantly higher number of APB both prior to (p=0.02) and after (p=0.04) atrial pacing, and a significantly higher number of post-pacing spontaneous AF episodes (p<0.01) compared to Control. The parasympathetic indexes SDNN, RMSSD, pNN5, and the NHF of the HRV spectrum were all significantly higher (all p<0.05), whereas the LF/HF ratio, an index of sympathetic and parasympathetic interactions, was significantly lower (p=0.01) in the BPA-HD compared with the Control rats. Conclusions In rats, chronic exposure to BPA doses relevant for usual human exposure was not associated in any significant change in cardiac arrhythmogenicity, electrophysiology, or autonomic modulation. However, high-dose BPA exposure, such as that occurring in workers in the plastics industry, led to significant increase in atrial arrhythmogenicity, including AF, that did not seem to be related to atrial electrophysiology changes. Instead, given the strong atrial proarrhythmic effects of vagal hyperactivity, this effect could be linked to the significant increase in vagal modulation induced by high-dose BPA exposure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS – UEFISCDI

Aromatase and nonaromatase neurons in the zebra finch secondary auditory forebrain are indistinct in their song-driven gene induction and intrinsic electrophysiological properties.

Estrogens support major brain functions including cognition, reproduction, neuroprotection and sensory processing. Neuroestrogens are synthesized within some brain areas by the enzyme aromatase and can rapidly modulate local circuit functions, yet the cellular physiology and sensory-response profiles of aromatase neurons are essentially unknown. In songbirds, social and acoustic stimuli drive neuroestrogen elevations in the auditory forebrain caudomedial nidopallium (NCM). In both males and females, neuroestrogens rapidly enhance NCM auditory processing and auditory learning. Estrogen-producing neurons in NCM may therefore exhibit distinguishing profiles for sensory-activation and intrinsic electrophysiology. Here, we explored these questions using both immunocyctochemistry and electrophysiological recordings. Immunoreactivity for aromatase and the immediate early gene EGR1, a marker of activity and plasticity, were quantified in NCM of song-exposed animals versus silence-exposed controls. Using whole-cell patch clamp recordings from NCM slices, we also documented the intrinsic excitability profiles of aromatase-positive and aromatase-negative neurons. We observed that a subset of aromatase neurons were significantly activated during song playback, in both males and females, and in both hemispheres. A comparable population of non-aromatase-expressing neurons were also similarly driven by song stimulation. Membrane properties (i.e., resting membrane potential, rheobase, input resistance and multiple action potential parameters) were similarly indistinguishable between NCM aromatase and non-aromatase neurons. Together, these findings demonstrate that aromatase and non-aromatase neurons in NCM are indistinct in terms of their intrinsic electrophysiology and responses to song. Nevertheless, such similarities in response properties may belie more subtle differences in underlying conductances and/or computational roles that may be crucial to their function.

Role of Preexisting Proarrhythmic Atrial Remodeling in Post-Coronary Artery Bypass Grafting Atrial Fibrillation

Abstract Introduction Due to its deleterious effects, early identification of patients at risk of postoperative AF (POAF) is of critical importance. Preexisting proarrhythmic atrial remodeling could contribute to this increased risk. Therefore, we aimed to evaluate the presence of preexisting proarrhythmic atrial remodeling and its impact on POAF occurrence in patients undergoing coronary artery bypass grafting (CABG). Methods Data regarding atrial structural (atrial size and histology), electrical (P-wave and atrial action potential parameters, mRNA expression of several AF-related genes), and autonomic (heart rate variability parameters) proarrhythmic remodeling were compared between patients with (AF; n=11) and without (no-AF; n=19) POAF. Impact of POAF on postoperative outcomes was also evaluated. Results No significant difference was observed in atrial electrical parameters between the two groups (all p>0.05). However, compared with no-AF, AF patients had more important subepicardial adipose infiltration (p=0.02) and higher markers of parasympathetic and sympathetic modulation (both p=0.03). Patients with POAF had longer hospital stay and more often presented postoperative renal dysfunction (both p=0.04). Conclusion These findings suggest that preexisting atrial structural (i.e., increased atrial subepicardial adiposity) and autonomic (i.e., sympatho-vagal coactivation) alterations could favor the occurrence of POAF. At its turn, POAF was associated with altered postoperative outcomes in CABG patients.

An in silico hiPSC-Derived Cardiomyocyte Model Built With Genetic Algorithm.

The formulation of in silico biophysical models generally requires optimization strategies for reproducing experimentally observed phenomena. In electrophysiological modeling, robust nonlinear regressive methods are often crucial for guaranteeing high fidelity models. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), though nascent, have proven to be useful in cardiac safety pharmacology, regenerative medicine, and in the implementation of patient-specific test benches for investigating inherited cardiac disorders. This study demonstrates the potency of heuristic techniques at formulating biophysical models, with emphasis on a hiPSC-CM model using a novel genetic algorithm (GA) recipe we proposed. The proposed GA protocol was used to develop a hiPSC-CM biophysical computer model by fitting mathematical formulations to experimental data for five ionic currents recorded in hiPSC-CMs. The maximum conductances of the remaining ionic channels were scaled based on recommendations from literature to accurately reproduce the experimentally observed hiPSC-CM action potential (AP) metrics. Near-optimal parameter fitting was achieved for the GA-fitted ionic currents. The resulting model recapitulated experimental AP parameters such as AP durations (APD50, APD75, and APD90), maximum diastolic potential, and frequency of automaticity. The outcome of this work has implications for validating the biophysics of hiPSC-CMs in their use as viable substitutes for human cardiomyocytes, particularly in cardiac safety pharmacology and in the study of inherited cardiac disorders. This study presents a novel GA protocol useful for formulating robust numerical biophysical models. The proposed protocol is used to develop a hiPSC-CM model with implications for cardiac safety pharmacology.

Ticagrelor alleviates high-carbohydrate intake induced altered electrical activity of ventricular cardiomyocytes by regulating sarcoplasmic reticulum-mitochondria miscommunication.

Metabolic syndrome (MetS) is associated with additional cardiovascular risk in mammalians while there are relationships between hyperglycemia-associated cardiovascular dysfunction and increased platelet P2Y12 receptor activation. Although P2Y12 receptor antagonist ticagrelor (Tica) plays roles in reduction of cardiovascular events, its beneficial mechanism remains poorly understood. Therefore, we aimed to clarify whether Tica can exert a direct protective effect in ventricular cardiomyocytes from high-carbohydrate diet-induced MetS rats, at least, through affectingsarcoplasmic reticulum (SR)-mitochondria (Mit) miscommunication. Tica treatment of MetS rats (150mg/kg/day for 15 days) significantly reversed the altered parameters of action potentials by reversing sarcolemmal ionic currents carried by voltage-dependent Na+ and K+ channels, and Na+/Ca2+-exchanger in the cells, expressed P2Y12 receptors. The increased basal-cytosolic Ca2+ level and depressed SR Ca2+ load were also reversed in Tica-treated cells, at most, though recoveries in the phosphorylation levels of ryanodine receptors and phospholamban. Moreover, there were marked recoveries in Mit structure and function (including increases in both autophagosomes and fragmentations) together with recoveries in Mit proteins and the factors associated with Ca2+ transfer between SR-Mit. There were further significant recoveries in markers of both ER stress and oxidative stress. Taken into consideration the Tica-induced prevention of ER stress and mitochondrial dysfunction, our data provided an important document on the pleiotropic effects of Tica in the electrical activity of the cardiomyocytes from MetS rats. This protective effect seems through recoveries in SR-Mit miscommunication besides modulation of different sarcolemmal ion-channel activities, independent of P2Y12 receptor antagonism.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Ecole doctorale Background Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. Purpose To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation, we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). Methods We performed current and voltage-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 µM). Results Nife significantly reduced the spontaneous firing under ISO perfusion in all rhythmic SANCs (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm) and 60% of dysrhythmic SANCs (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) whereas it completely stopped it in the remaining 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Interestingly, these cells exhibited a significantly slower rate and a slower slope of the diastolic depolarization under ISO perfusion compared to the remaining 25% dormant SANCs in which Nife only reduced the ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: -30.1 ± 2.5, dysrhythmic: -43.3 ± 2.3, rhythmic: -41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Conclusion Our results seem to point at a difference of expression in ionic channels (Cav1.3, HCN4) within isolated SANCs. Preliminary results on If density support this hypothesis with a lesser density in dormant SANCs compared to dysrhythmic SANCs. These results also tend to indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs.

Effects of Focal Ionizing Radiation of the Squid Stellate Ganglion on Synaptic and Axonal Transmission in the Giant-Fiber Pathway.

Ionizing radiation is clinically used to treat neurological problems and reduce pathological levels of neural activity in the brain, but its cellular-level mechanisms are not well understood. Although spontaneous and stimulated synaptic activity has been produced in rodents by clinically and environmentally relevant doses of radiation, the effects on basic excitability properties of neurons have seldom been reported. This study examined the effects of focused ionizing radiation on synaptic transmission and action potential generation in the squid giant-fiber system, which includes the giant synapse between a secondary interneuron and the tertiary giant motor axons. Radiation of 140-300 Gy was delivered to a stellate ganglion of a living squid over several minutes, with the contralateral stellate ganglion serving as an internal control. No qualitative changes in the efficacy of synaptic transmission were noted in conjunction with stimulation of the input to the giant synapse, although in one irradiated ganglion, the refractory period increased from about 5 ms to more than 45 seconds. Small but significant changes in the action potential recorded from the giant motor axon in response to electrical stimulation were associated with an increased maximum rate of fall and a shortened action potential duration. Other action-potential parameters, including resting potential, overshoot, the maximum rate of the rise, and the refractory period were not significantly changed. Attempts to account for the observed changes in the action potential were carried through a Hodgkin-Huxley model of the action potential. This approach suggests that an increase in the maximum voltage-gated potassium conductance of about 50% mimics the action potential shortening and increased rate of fall that was experimentally observed. We propose that such an effect could result from phosphorylation of squid potassium channels.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca2+ concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a ACADVL gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.

Interrelated In Vitro Mechanisms of Sibutramine-Induced Cardiotoxicity.

Consumption of illicit pharmaceutical products containing sibutramine has been reported to cause cardiovascular toxicity problems. This study aimed to demonstrate the toxicity profile of sibutramine, and thereby provide important implications for the development of more effective strategies in both clinical approaches and drug design studies. Action potentials (APs) were determined from freshly isolated ventricular cardiomyocytes with whole-cell configuration of current clamp as online. The maximum amplitude of APs (MAPs), the resting membrane potential (RMP), and AP duration from the repolarization phases were calculated from original records. The voltage-dependent K+-channel currents (IK) were recorded in the presence of external Cd2+ and both inward and outward parts of the current were calculated, while their expression levels were determined with qPCR. The levels of intracellular free Ca2+ and H+ (pHi) as well as reactive oxygen species (ROS) were measured using either a ratiometric micro-spectrofluorometer or confocal microscope. The mechanical activity of isolated hearts was observed with Langendorff-perfusion system. Acute sibutramine applications (10-8-10-5M) induced significant alterations in both MAPs and RMP as well as the repolarization phases of APs and IK in a concentration-dependent manner. Sibutramine (10μM) induced Ca2+-release from the sarcoplasmic reticulum under either electrical or caffeine stimulation, whereas it depressed left ventricular developed pressure with a marked decrease in the end-diastolic pressure. pHi inhibition by sibutramine supports the observed negative alterations in contractility. Changes in mRNA levels of different IK subunits are consistent with the acute inhibition of the repolarizing IK, affecting AP parameters, and provoke the cardiotoxicity.

Electrophysiological properties of maxillary trigeminal Aβ-afferent neurons of rats.

Aβ-afferents in maxillary or V2 trigeminal ganglion (TG) neurons are somatosensory neurons that may be involved in both non-nociceptive and nociceptive functions in orofacial regions. However, electrophysiological properties of these V2 trigeminal Aβ-afferent neurons have not been well characterized so far. Here, we used rat ex vivo trigeminal nerve preparations and applied patch-clamp recordings to large-sized V2 TG neurons to characterize their electrophysiological properties. All the cells recorded had afferent conduction velocities in the range of Aβ-afferent conduction speeds. However, these V2 trigeminal Aβ-afferent neurons displayed different action potential (AP) properties. APs showed fast kinetics in some cells but slow kinetics with shoulders in repolarization phases in other cells. Based on the derivatives of voltages in AP repolarization with time (dV/dt), we classified V2 trigeminal Aβ-afferent neurons into four types: type I, type II, type IIIa and type IIIb. Type I V2 trigeminal Aβ-afferent neurons had the largest dV/dt of repolarization, the fastest AP conduction velocities, the shortest AP and afterhyperpolarization (AHP) durations, and the highest AP success rates. In contrast, type IIIb V2 trigeminal Aβ-afferent neurons had the smallest dV/dt of AP repolarization, the slowest AP conduction velocities, the longest AP and AHP durations, and the lowest AP success rates. The type IIIb cells also had significantly lower voltage-activated K+ currents. For type II and type IIIa V2 trigeminal Aβ-afferent neurons, AP parameters were in the range between those of type I and type IIIb V2 trigeminal Aβ-afferent neurons. Our electrophysiological classification of V2 trigeminal Aβ-afferent neurons may be useful in future to study their non-nociceptive and nociceptive functions in orofacial regions.

Body mass index: A modifying factor of median nerve conduction

Background: Nerve conduction studies are important electrophysiological tests used to assess functional status of peripheral nerves. Entrapment of median nerve in carpal tunnel is a possibility in high body mass index (BMI). Aim and Objectives: The objective of this study was to determine BMI as a modifying factor of median nerve conduction. Materials and Methods: Forty-seven healthy male subjects were divided in three study groups, that is, normal, pre-obese, and obese according to BMI. Nerve conduction study was tested to compare different parameters of compound motor action potential (CMAP) and sensory nerve action potential (SNAP). Parameters used for SNAP were peak latency (PL), conduction velocity (CV), and amplitude (Amp). Parameters used for CMAP were distal motor latency (DML), CV, and amplitude (Amp). Results: In the right median nerve, CMAP between various groups represented significant prolongation of DML (P = 0.000) and slowing CV (P = 0.000) with increasing BMI. In the left median nerve, CMAP between various groups represented significant prolongation of DML (P = 0.000) and slowing CV (P = 0.000) with increasing BMI. In the right median nerve, SNAP between various groups represented significant prolongation of PL (P = 0.000) and slowing CV (P = 0.000) with increasing BMI. In the left median nerve, SNAP between various groups represented significant prolongation of PL (P = 0.000) and slowing CV (P = 0.000) with increasing BMI. Conclusion: Our study suggested BMI as the substantial factor to affect median nerve conduction at carpal tunnel. Thus, high BMI plays crucial role to develop carpal tunnel syndrome (CTS).

A novel mutation in transmembrane protein 168 causes fatal ventricular arrhythmogenesis in Brugada syndrome

Abstract Brugada syndrome (BrS) is diagnosed by a typical electrocardiography (ECG) with ST-segment elevation in precordial leads and tends to induce sudden cardiac death (SCD) due to ventricular tachycardia/fibrillation. About 20% of SCDs in non-structural cardiac diseases are considered to be caused by BrS. In patients with BrS, loss of function mutations in the Na+ channel is often observed, but the causative gene mutation is not detected for about 70% of BrS patients. Here, we investigated a family with clinically diagnosed BrS, in which no known gene mutations related to BrS had not been found, by whole exome sequencing. Novel heterozygous variant (c. 1616G>A, p. R539Q) in transmembrane protein 168 (TMEM168) was identified only in symptomatic family members. Similar to endogenous TMEM168, both wild-type and mutant TMEM168 localized at the nuclear membrane. Na+ current density in whole-cell patch-clamp recordings was significantly reduced in HL-1 cardiomyocytes transfected with TMEM168 R539Q mutant, compared with those with wild-type TMEM168. Next, heterozygous Tmem168 1616G>A knock-in mice were generated by the CRISPR/Cas9 genome editing technology. Although the knock-in mice had no abnormalities in ECG at the physiological state, the treatment with ajmaline caused various arrhythmias including ventricular tachycardia/fibrillation in the knock-in mice, but not in wild-type mice. Na+ current density and the parameters of action potentials were remarkably impaired in the cardiomyocytes of the knock-in mice. Optical mapping analysis in the whole heart showed the reduced left ventricular conduction velocity in the knock-in mice. The expression of Nav1.5, an α-subunit of the cardiac Na+ channel, was significantly decreased in the mutant TMEM168-transfected HL-1 cells and the knock-in hearts. We found that the decrease was caused by the enhanced ubiquitination of Nav1.5, which was mediated by increased binding of Nedd4–2 E3 ubiquitin ligase to Nav1.5 in the knock-in hearts. Co-immunoprecipitation experiments demonstrated that overactivity of Nedd4–2 is a result of Tmem168 mutant-mediated sequestration of a chaperon protein αB-crystallin, a Nav1.5-binding molecule that interferes with the interaction of Nedd4–2 with Nav1.5. These findings reveal the molecular mechanism of TMEM168 R539Q mutation-induced fatal ventricular arrhythmias in BrS. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): JSPS Grants-in-aid for Scientific Research

Vibratory testing with the 64 Hz Rydel-Seiffer tuning fork and its relation to the sural nerve action potential.

Despite its widespread use, little is known regarding the ability of the semi-quantitative Rydel-Seiffer tuning fork to designate peripheral nerve function. We sought to determine in a large sample of normal and abnormal nerves the relationship between vibration sense and compound sensory nerve action potential (SNAP) parameters recorded in a corresponding innervation area. Vibratory thresholds were determined on a scale of 0 to 8 with a 64 Hz Rydel-Seiffer tuning fork placed on the lateral malleolus of 303 subjects. Sural nerve sensory neurography was employed to derive SNAP parameters, which were related to vibration sense by means of multiple linear regression. ROC curve analysis was performed to determine the classification efficacy of the tuning fork in distinguishing normal from abnormal sural nerve responses. SNAP amplitude was the most significant predictor in the whole subjects group and in the subgroup of subjects with normal SNAPs, whereas conduction velocity played a major role in subjects with abnormal SNAPs. Age was significantly associated with vibration perception, particularly in subjects with normal SNAPs. With an area under the curve of 0.730, vibration sense was a fair classifier for decreased SNAP amplitudes. The optimal vibratory cutoff was 4.2. Age is a major determinant of vibratory test results, highlighting the importance of aging of central and peripheral pathways in mediating vibration sense. Hence, neurophysiological testing cannot be omitted in the context of polyneuropathy work-up, since even at the optimal cutoff threshold, vibratory examination still displays 40% false negative test results.

Minimal contribution of IP3R2 in cardiac differentiation and derived ventricular-like myocytes from human embryonic stem cells.

Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Autonomous initiation of pacemaking by L-type Cav1.3 calcium channels in mouse sino-atrial myocytes

Cardiac pacemaking relies on the spontaneous electrical activity in the right atrium of sino-atrial myocytes (SANCs). Automaticity in SANCs results from a robust interplay of membrane ion channels activity and intracellular calcium dynamics. However, only a fraction of isolated SANCs exhibit rhythmic firing, whereas most SANCs show irregular (dysrhythmic) firing or remain dormant. To study the capability of L-type Cav1.3 calcium channels to initiate automaticity in dormant SANCs under β-adrenergic stimulation we used a knock-in mouse strain in which the sensitivity of Cav1.2 α1 subunits to dihydropyridines (DHP) was inactivated (Cav1.2DHP-/-). We performed current-clamp recordings on isolated SANCs under isoprenaline (ISO, 100 nM) and in the absence or presence of the DHP blocker Nifedipine (Nife, 3 μM). In rhythmic SANCs, Nife significantly reduced the spontaneous firing under ISO perfusion (ISO: 447 ± 12, ISO + Nife: 233 ± 25 bpm). In dysrhythmic SANCs, Nife significantly reduced the spontaneous rate under ISO in 60% of the cells (ISO: 386 ± 12, ISO + Nife: 188 ± 47 bpm) and completely stopped it in 40% (295 ± 29 bpm to 0). On 25 dormant SANCs, 50% started firing after ISO perfusion (0 to 320 ± 46 bpm). Strikingly, in 75% of them, Nife totally blocked this ISO-induced firing. Moreover, dormant SANCs showed a statistically significant increase in action potential (AP) threshold under ISO compared to dysrhythmic and rhythmic SANCs (dormant: −30.1 ± 2.5, dysrhythmic: −43.3 ± 2.3, rhythmic: −41.2 ± 2.1 mV). No significant difference was observed in the other AP parameters between dormant, dysrhythmic and rhythmic SANCs under ISO. Our results indicate that Cav1.3 channels can generate pacemaker activity autonomously, at least in a particular subpopulation of SANCs, while it is responsible of about half of the automaticity in the others.

Engineering Complex Synaptic Behaviors in a Single Device: Emulating Consolidation of Short-term Memory to Long-term Memory in Artificial Synapses via Dielectric Band Engineering.

As one of the key neuronal activities associated with memory in the human brain, memory consolidation is the process of the transition of short-term memory (STM) to long-term memory (LTM), which transforms an external stimulus to permanently stored information. Here, we report the emulation of this complex synaptic function, consolidation of STM to LTM, in a single-crystal indium phosphide (InP) field effect transistor (FET)-based artificial synapse. This behavior is achieved via the dielectric band and charge trap lifetime engineering in a dielectric gate heterostructure of aluminum oxide and titanium oxide. We analyze the behavior of these complex synaptic functions by engineering a variety of action potential parameters, and the devices exhibit good endurance, long retention time (>105 s), and high uniformity. Uniquely, this approach utilizes growth and device fabrication techniques which are scalable and back-end CMOS compatible, making this InP synaptic device a potential building block for neuromorphic computing.

Cardiac electrophysiological effects of ibuprofen in dog and rabbit ventricular preparations: possible implication to enhanced proarrhythmic risk.

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug, which has recently been associated with increased cardiovascular risk, but its electrophysiological effects have not yet been properly studied in isolated cardiac preparations. We studied the effects of ibuprofen on action potential characteristics and several transmembrane ionic currents using the conventional microelectrode technique and the whole-cell configuration of the patch-clamp technique on cardiac preparations and enzymatically isolated ventricular myocytes. In dog (200 µM; n= 6) and rabbit (100 µM; n= 7) papillary muscles, ibuprofen moderately but significantly prolonged repolarization at 1Hz stimulation frequency. In dog Purkinje fibers, repolarization was abbreviated and maximal rate of depolarization was depressed in a frequency-dependent manner. Levofloxacin (40 µM) alone did not alter repolarization, but augmented the ibuprofen-evoked repolarization lengthening in rabbit preparations (n= 7). In dog myocytes, ibuprofen (250 µM) did not significantly influence IK1, but decreased the amplitude of Ito and IKr potassium currents by 28.2% (60mV) and 15.2% (20mV), respectively. Ibuprofen also depressed INaL and ICa currents by 19.9% and 16.4%, respectively. We conclude that ibuprofen seems to be free from effects on action potential parameters at lower concentrations. However, at higher concentrations it may alter repolarization reserve, contributing to the observed proarrhythmic risk in patients.

Abstract 542: Chronic Doxorubicin Cardiotoxicity Assessed in Engineered Cardiac Tissues Generated in Biowire™ II Platform

The anthracycline doxorubicin is an effective anti-tumor agent widely used in both adults and children. One major adverse effect of doxorubicin therapy is dose-dependent cardiotoxicity, ranging from asymptomatic reduction in left ventricular ejection fraction to more serious, potentially fatal symptoms including arrythmias and congestive heart failure. The exact mechanism of doxorubicin-induced cardiotoxicity remains unknown. Recently, human induced pluripotent stem cells (hiPSC) have emerged as a potential tool to model cardiac toxicity, but their fetal-like phenotype raises concerns about the translatability of in vitro data to in vivo cardiotoxicity. To overcome this limitation, Biowire™ II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-derived cardiomyocytes and human cardiac fibroblasts. Using long-term electrical stimulation, ECTs with a phenotype approaching that of adult human myocardium were obtained. The ECTs were then exposed to 1 μM doxorubicin for 8 days followed by 7 days of washout. Measurements of contractile force amplitude at 1 Hz stimulation showed a transient increase in force within 24 hours of doxorubicin exposure followed by decrease in force after 2 days. Intracellular recordings of action potential (AP) showed a decrease in maximum upstroke velocity (dV/dt), AP amplitude (APA), and resting membrane potential (RMP) after 8 days of doxorubicin treatment. In addition, action potential duration (APD) at 30% (APD30) repolarization was increased in doxorubicin-treated ECTs, whereas APD50 and APD90 were decreased. Following 7 days of washout, no difference in force or AP parameters was found between doxorubicin and vehicle-treated ECTs with the exception of APD50 and APD90 which remained abbreviated. A global untargeted analysis of the conditioned media from doxorubicin-treated ECTs identified 204 analytes and revealed an upregulation of redox homeostasis, differential fatty acid metabolism, altered glycolysis and TCA cycle metabolites, and decreased nucleoside metabolism compared to vehicle-treated ECTs. These results show that doxorubicin not only increases oxidative stress, but also irreversibly affects action potential duration which may predispose to cardiac arrhythmias.

Ultrarapid Delayed Rectifier K+ Channelopathies in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. About 5–15% of AF patients have a mutation in a cardiac gene, including mutations in KCNA5, encoding the Kv1.5 α-subunit of the ion channel carrying the atrial-specific ultrarapid delayed rectifier K+ current (IKur). Both loss-of-function and gain-of-function AF-related mutations in KCNA5 are known, but their effects on action potentials (APs) of human cardiomyocytes have been poorly studied. Here, we assessed the effects of wild-type and mutant IKur on APs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We found that atrial-like hiPSC-CMs, generated by a retinoic acid-based differentiation protocol, have APs with faster repolarization compared to ventricular-like hiPSC-CMs, resulting in shorter APs with a lower AP plateau. Native IKur, measured as current sensitive to 50 μM 4-aminopyridine, was 1.88 ± 0.49 (mean ± SEM, n = 17) and 0.26 ± 0.26 pA/pF (n = 17) in atrial- and ventricular-like hiPSC-CMs, respectively. In both atrial- and ventricular-like hiPSC-CMs, IKur blockade had minimal effects on AP parameters. Next, we used dynamic clamp to inject various amounts of a virtual IKur, with characteristics as in freshly isolated human atrial myocytes, into 11 atrial-like and 10 ventricular-like hiPSC-CMs, in which native IKur was blocked. Injection of IKur with 100% density shortened the APs, with its effect being strongest on the AP duration at 20% repolarization (APD20) of atrial-like hiPSC-CMs. At IKur densities < 100% (compared to 100%), simulating loss-of-function mutations, significant AP prolongation and raise of plateau were observed. At IKur densities > 100%, simulating gain-of-function mutations, APD20 was decreased in both atrial- and ventricular-like hiPSC-CMs, but only upon a strong increase in IKur. In ventricular-like hiPSC-CMs, lowering of the plateau resulted in AP shortening. We conclude that a decrease in IKur, mimicking loss-of-function mutations, has a stronger effect on the AP of hiPSC-CMs than an increase, mimicking gain-of-function mutations, whereas in ventricular-like hiPSC-CMs such increase results in AP shortening, causing their AP morphology to become more atrial-like. Effects of native IKur modulation on atrial-like hiPSC-CMs are less pronounced than effects of virtual IKur injection because IKur density of atrial-like hiPSC-CMs is substantially smaller than that of freshly isolated human atrial myocytes.

Electrophysiological study of posterior antebrachial cutaneous nerve in a sample of normal subjects

BackgroundsPosterior antebrachial cutaneous nerve sensory conduction study is clinically essential for the assessment of its integrity and for the localization of radial nerve lesions. The aim of the study was to assess posterior antebrachial cutaneous nerve sensory antidromic conduction technique and to obtain normal reference values for different sensory nerve action potential parameters of this nerve among a sample of Egyptian population.ResultsThe current study included 120 upper limbs of 60 apparently healthy subjects [35 (58.3%) women]. Their mean age was 39.20 ± 11.95 years. The posterior antebrachial cutaneous nerve was recorded in all upper limbs (100%). The values (mean ± standard deviation) for the posterior antebrachial cutaneous nerve sensory nerve action potential onset latency were 2.05 ± 0.25 ms, peak latency was 2.63 ± 0.28 ms, conduction velocity was 60.50 ± 5.38 m/s, amplitude was 11.04 ± 4.26 μV, and inter-side sensory nerve action potential amplitude ratio was 0.69 ± 0.17. There were no statistically significant differences between men and women, as well as between right and left upper limbs regarding different posterior antebrachial cutaneous sensory nerve action potential parameters. There were no statistically significant correlations between participants’ age and anthropometric measures (i.e., height, weight, and body mass index) with different posterior antebrachial cutaneous nerve sensory nerve action potential parameters. There were no statistically significant adjusted effects of age, gender, and anthropometric measures on different posterior antebrachial cutaneous nerve sensory nerve action potential parameters in multiple linear regression analysis by controlling all other physiologic factors.ConclusionsThis research provides an applicable electrophysiological technique and normal reference values for the posterior antebrachial cutaneous nerve sensory conduction study.