Sarcolemmal cardiac K(ATP) channels as a target for the cardioprotective effects of the fluorine-containing pinacidil analogue, flocalin

Abstract

Background and purpose: A class of drugs known as K(ATP) -channel openers induce cardioprotection. This study examined the effects of the novel K(ATP) -channel opener, the fluorine-containing pinacidil derivative, flocalin, on cardiac-specific K(ATP) -channels, excitability of native cardiac myocytes and on the ischaemic heart.

Experimental approach: The action of flocalin was investigated on: (i) membrane currents through cardiac-specific K(ATP) -channels (I(KATP) ) formed by K(IR) 6.2/SUR2A heterologously expressed in HEK-293 cells (HEK-293(₆.₂/₂A) ); (ii) excitability and intracellular Ca²(+) ([Ca²(+) ](i) ) transients of cultured rat neonatal cardiac myocytes; and (iii) functional and ultrastructural characteristics of isolated guinea-pig hearts subjected to ischaemia-reperfusion.

Key results: Flocalin concentration-dependently activated a glibenclamide-sensitive I(KATP) in HEK-293(₆.₂/₂A) cells with an EC₅₀= 8.1 ± 0.4 µM. In cardiac myocytes, flocalin (5 µM) hyperpolarized resting potential by 3-5 mV, markedly shortened action potential duration, reduced the amplitude of [Ca²(+) ](i) transients by 2-3-fold and suppressed contraction. The magnitude and extent of reversibility of these effects depended on the type of cardiac myocytes. In isolated hearts, perfusion with 5 µmol·L⁻¹ flocalin, before inducing ischaemia, facilitated restoration of contraction during reperfusion, decreased the number of extrasystoles, prevented the appearance of coronary vasoconstriction and reduced damage to the cardiac tissue at the ultrastructural level (state of myofibrils, membrane integrity, mitochondrial cristae structure).

Conclusion and implications: Flocalin induced potent cardioprotection by activating cardiac-type K(ATP) -channels with all the benefits of the presence of fluorine group in the drug structure: higher lipophilicity, decreased toxicity, resistance to oxidation and thermal degradation, decreased metabolism in the organism and prolonged therapeutic action.

Figure 1

Chemical structures of pinacidil and flocalin. Flocalin (B) is characterized by the presence of benzene ring with difluoromethoxy group instead of a pyridyl ring for pinacidil (A). Me – methyl (CH₃) group.

Figure 2

Flocalin activates heterologously expressed cardiac-type K_ATP channels. (A) Representative recordings of the baseline (middle panel) and 20 µmol·L⁻¹ flocalin-activated (lower panel) membrane currents in HEK-293_6.2/2A stably transfected with K_IR6.2 /SUR2A in response to the voltage-step protocol shown on top. (B) Representative recordings of the baseline current and currents in the presence of 5, 10 and 20 µmol·L⁻¹ flocalin, as well as of 20 µmol·L⁻¹ flocalin plus 10 µmol·L⁻¹ glibenclamide (Floc + Glib) in response to the voltage-ramp protocol shown on top; dashed lines in A and B denote zero current and zero voltage levels. (C) I-V relationships of 20 µmol·L⁻¹ flocalin-activated I_KATP measured from square pulses (symbols, mean ± SEM, n = 5) and ramp (smooth line) voltage-clamp protocols. (D) Dose–response relationship of I_KATP activation by flocalin (symbols – experimental data points, mean ± SEM, n = 5–7) and the fit to the Hill equation; the values of IC50 and co-operativity (Hill) coefficient, p, are shown on the plot.

Figure 3

The effects of flocalin on excitability of the rat neonatal cultured cardiac myocytes. (A–C) Representative recordings of the control AP, AP in the presence of 5 µmol·L⁻¹ flocalin and AP after flocalin washout in the ventricular epicardial- (A), endocardial- (B) as well as in atrial-type (C) cardiac myocytes; the insets on panels A–C show the dynamics of the action potential duration (APD₉₀) and resting potential (V_r) changes in response to flocalin application (marked by horizontal bars). (D, E) Quantification of the changes of the AP amplitude (D) and of its maximal rate of rise (E) in the ventricular epicardial-, endocardial- as well as atrial-type cardiac myocytes in response to flocalin (5 µmol·L⁻¹) and following its washout relative to the control pre-drug values; mean ± SEM, n = 5–7.

Figure 4

The effects of flocalin on excitability of the rat neonatal cultured cardiac myocytes are antagonized by glibenclamide. (A–C) Representative recordings of the control AP, AP in the presence of 5 µmol·L⁻¹ flocalin and AP after addition of glibenclamide (5 µmol·L⁻¹) to flocalin-containing solution in the ventricular epicardial- (A), endocardial- (B) as well as in atrial-type (C) cardiac myocytes; in the panel C flocalin and flocalin plus glibenclamide curves coincide.

Figure 5

The effects of flocalin on spontaneously beating rat neonatal cultured cardiac myocytes. (A) Representative recordings of the electrical activity of the spontaneously beating endocardial-type ventricular myocyte during application of 10 µmol·L⁻¹ flocalin (marked by horizontal bar). (B) The dynamics of the action potential duration (APD₅₀) shortening in the same myocyte in response to flocalin (open symbols – pre-drug, filled symbols – flocalin); the insets show the recordings of the APs at expanded time-scale in the absence and in the presence of flocalin taken at time periods marked by short horizontal bars at A and B. (C) Representative recordings of the [Ca²⁺]_i transients from another Fluo-4-loaded myocyte during application of 10 µmol·L⁻¹ flocalin (marked by horizontal bar).

Figure 6

The effects of flocalin on the functional properties of isolated guinea-pig hearts. (A, B) Heart perfusion with flocalin (5 µmol·L⁻¹) prior to ischaemia accelerates time to resuming contractions in response to reperfusion (A) and reduces the number of extrasystoles during reperfusion (B) compared to the control, non-flocalin conditions (Ctrl); mean ± SEM, n = 26. (C, D) Heart perfusion with flocalin prior to ischaemia improves recovery of the developed (C) and systolic (D) pressure during reperfusion compared to the control, non-flocalin conditions (Ctrl); mean ± SEM, n = 26; time ‘0’ corresponds to the beginning of reperfusion; smooth lines are the best fits of experimental data points with exponential functions with time constants shown near the curves.

Figure 7

Flocalin improves the recovery of cardiac haemodynamics during reperfusion of the ischaemic myocardium. (A, B) Changes of the coronary artery perfusion pressure (CPP), left ventricle pressure (LVP) and of dP/dt contractility index during ischaemia-reperfusion of isolated guinea-pig hearts under control conditions (A) and with flocalin (5 µM) infusion prior to inducing ischaemia (B, shown by arrow). Flocalin facilitates the re-initiation of the contractions of ischaemic myocardium, prevents coronary vasoconstriction and appearance of extrasystoles (shown by arrows in A) during reperfusion.

Figure 8

Flocalin reduces post-ischaemic damage to the guinea heart at the ultrastructural level. (A) Electron microphotograph (magnification 8000) showing profound contractures of the myofilaments (MF), mitochondria (M) swelling and irregular cristae structure, high counts of colloid lanthanum particles in the cytoplasm and outer membrane of mitochondria (arrows) due to impairment of plasma membrane integrity in the cardiac myocyte of the control post-ischaemic heart; Er – erythrocyte. (B) Electron micrograph (magnification 8000) of the subcellular structures of cardiac myocytes from post-ischaemic heart pre-treated with flocalin (5 µM): myofilaments (MF) and mitochondria (M) are well preserved, colloid lanthanum particles (arrows) are localized extracellularly; Eth – endothelium of the capillary.