Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

<div><p>The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed <i>trans</i>- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the <i>cis</i>- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of <i>trans</i>- and <i>cis</i>- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na⁺) and calcium (Ca²⁺) currents and potentiation of net potassium (K⁺) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.</p></div

Effect of azoTAB on voltage-dependent Ca²⁺ currents in neonatal rat ventricular cardiomyocytes.

<p>(A) L-type Ca²⁺ currents obtained in the absence (control) and presence of 100 μM <i>trans</i>- azoTAB and after ~365 nm near-UV irradiation. Inset: original current trace in response to a voltage step from -40 to 0 mV for 300 ms. Inactivation of INav was achieved by a prestep from a holding potential HP of -80 mV to -40 mV for 100 ms. Similar results were obtained in three other cells. (B) ICavpeak recorded before and after incubation with 100 μM azoTAB, as well as after near-UV irradiation, and expressed as a percentage of that of the peak currents before the treatment. Each cardiomyocyte was incubated in the presence of azoTAB at room temperature for ~3 min in a measuring chamber. The currents were inhibited by approximately 60% relative to the control. Near-UV was applied for 90 s. The data are the means ± SEM from three cardiomyocytes, *<i>p</i>< 0.05. (C) Averaged I/V relations of the L-type Ca²⁺ currents elicited by the voltage-clamp protocol illustrated in the inset (HP = -80 mV) and plotted before (filled circles) and after (open circles) the application of azoTAB. The values are expressed as the mean ± SEM, <i>n</i> = 4. The current density is plotted as a function of the voltage.</p

Regulation of Papillary Muscle Contractility by NAD and Ammonia Interplay: Contribution of Ion Channels and Exchangers

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation&ndash;contraction coupling control. Here, we used &beta;-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM &beta;-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in &beta;-NAD effects. Meantime, 5 mM NH4Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I&ndash;V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH4Cl, being added after &beta;-NAD and having no effect on RMP, APD, and I&ndash;V curve, recovered F to the control values, indicating &beta;-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca2+-activated K+ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K+, that creates positive and negative feedback, and known ammonia and K+ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of &beta;-NAD and ammonia on rat papillary muscle contractile activity

Effect of azobenzene trimethylammonium bromide (azoTAB) on ramp currents in neonatal rat ventricular myocytes.

<p>(A) A representative TTX-sensitive current that was evoked when the voltage was increased smoothly from -120 to +50 mV for 200 ms. The cell was prepulsed to -120 mV for 100 ms from a HP of -80mV. The voltage protocol is shown above the current trace. The inset shows scaled current traces for comparison before and after the addition of 10 μM TTX. Similar results were obtained in three other cells. (B) Scaled ramp-evoked currents recorded in response to the same ramp protocol (from -120 to +50 mV, 200 ms) in the control and after the addition of 100 μM <i>trans</i>- azoTAB. Currents were recorded every 15 s after the application of the photoreactive substance. Three minutes after the application, the current was inhibited by approximately 83% relative to that of the control. Similar results were obtained in three other cells. (C) Ion currents recorded before and after incubation with 100 μM <i>trans</i>-azoTAB, as well as after near-ultraviolet (near-UV) irradiation and expressed as percentage. Each cardiomyocyte was incubated in the presence of azoTAB at room temperature for at least 3 min in a measuring chamber. Near-UV was applied for 90 s. The data represent the means ± SEM from four cardiomyocytes, *<i>p<</i> 0.05.</p

Effect of azoTAB on voltage-dependent Na⁺ currents in neonatal rat ventricular myocytes.

<p>(A) Current-voltage relationships recorded from single neonatal ventricular cardiomyocytes under control conditions (filled circles) and after exposure to 100 μM <i>trans</i>-azoTAB (open circles). Inset: the shape of the current-voltage stimulation protocol. The current density was calculated as the Na⁺ peak current divided by the membrane capacitance of each cell (<i>n</i> = 4). (B) Concentration dependency for <i>trans</i>- azoTAB-induced inhibition of INav in neonatal rat ventricular cardiomyocytes. Mean ± SEM, <i>n</i> = 3–4 for each point.</p