Pharmacological profile of the sodium current in human stem cell-derived cardiomyocytes compares to heterologous Nav1.5+β1 model

The cardiac Nav1.5 mediated sodium current (I-Na) generates the upstroke of the action potential in atrial and ventricular myocytes. Drugs that modulate this current can therefore be antiarrhythmic or proarrhythmic, which requires preclinical evaluation of their potential drug-induced inhibition or modulation of Nav1.5. Since Nav1.5 assembles with, and is modulated by, the auxiliary beta 1-subunit, this subunit can also affect the channel's pharmacological response. To investigate this, the effect of known Nav1.5 inhibitors was compared between COS-7 cells expressing Nav1.5 or Nav1.5+beta 1 using whole-cell voltage clamp experiments. For the open state class Ia blockers ajmaline and quinidine, and class Ic drug flecainide, the affinity did not differ between both models. For class Ib drugs phenytoin and lidocaine, which are inactivated state blockers, the affinity decreased more than a twofold when beta 1 was present. Thus, beta 1 did not influence the affinity for the class Ia and Ic compounds but it did so for the class Ib drugs. Human stem cell-derived cardiomyocytes (hSC-CMs) are a promising translational cell source for in vitro models that express a representative repertoire of channels and auxiliary proteins, including beta 1. Therefore, we subsequently evaluated the same drugs for their response on the I-Na in hSC-CMs. Consequently, it was expected and confirmed that the drug response of I-Na in hSC-CMs compares best to I-Na expressed by Nav1.5+beta 1

The resting membrane potential of hSC-CM in a syncytium is more hyperpolarised than that of isolated cells

Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the I-K1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of I-K1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as I-K1. By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately -20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased

Increase in dopamine release from the nucleus accumbens in response to feeding: A model to study interactions between drugs and naturally activated dopaminergic neurons in the rat brain

The aim of the present study was to investigate the interactions between the in vivo release of dopamine and certain drugs, during conditions of increased dopaminergic activity. Dopaminergic neurons in the nucleus accumbens were activated by feeding hungry rats. 48-96 h after implantation of a microdialysis probe 30 min food ingestion by hungry rats induced an immediate eating response that was accompanied with a reproducible and long-lasting increase in extracellular dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC). The effect of various drugs (infused into the nucleus accumbens via the microdialysis probe), on the extracellular levels of dopamine and DOPAC were recorded, and the effect of eating was determined. Infusion of 5 mu mol/l nomifensine and 3.4 mmol/l calcium increased dopamine release respectively 5.4 and 2-fold but did not modify the eating related increase in dopamine and DOPAC release. Infusion (1 mu mol/l) as well as intraperitoneal administration (20 mg/kg) of sulpiride induced an increase in basal dopamine release to 220 and 195% of controls, respectively. Both routes of sulpiride pretreatment enhanced the eating related increase in extracellular dopamine and DOPAC. The results of the sulpiride experiments indicate that a behaviorally induced stimulation of dopamine release is modified by autoinhibition