Effects of tertiapin-Q (TQ) on the effect of ET-1 on spontaneous APs and ET-1 activated current.

<p>A. Continuous recording of spontaneous activity in control, in the presence of TQ (300 nM) before and with application of 10 nM ET-1 in the maintained presence of TQ. Note the absence of immediate hyperpolarisation and cessation of APs evident in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033448#pone-0033448-g001" target="_blank">Figure 1</a>. Bi, ii and iii show expanded records from recording in A, at time-points indicated: i taken during control, ii near the end of TQ alone and iii is taken at ∼13 seconds of ET-1 application (at which time-point cells exposed to ET-1 alone had hyperpolarised and become quiescent). Similar results were obtained from 7 cells. C. Mean I–V relationships for the 10 nM ET-1 activated instantaneous current in absence (filled triangles, n = 14) and in presence of 300 nM TQ (open triangles, n = 7. except at −80 mV where n = 6). TQ prevented this action of ET-1. Asterisks in C denote statistical significance (p<0.05 *, p<0.001 ***).</p

Effects of ET-1 on spontaneous APs.

<p>A. Slow time-base recording of APs before, during and after rapid application of 10 nM ET-1. B. Expanded (faster time-base) portions of the recording extracted from numbered sections of panel A (indicated labels i, ii, iii). Similar results were observed in 9 experiments.</p

ET-1 effects on the hyperpolarisation-activated current I_f.

<p>A. Upper traces show currents elicited −120 mV in an I_f-expressing cell in control solution and 10 nM ET-1 by protocol shown in bottom trace. Note outward shift in holding current in presence of ET-1. Closed circles indicate control trace; open circles indicate trace in ET-1. B. Mean I–V relationships (n = 7) for I_f, plotted as time-dependent current during command pulses, in absence (control, filled circles) and presence of 10 nM ET-1 (open circles). The activating effect of ET-1 was significant only at −120, −110 and −100 mV. C. Mean I–V relationships for the instantaneous current recorded at the beginning of the test-pulse (Ci: in absence (control, filled circles) and presence (open circles) of 10 nM ET-1). Cii shows I–V relation for the ET-1 activated instantaneous current (Cii, filled circles), in cells also showing I_f (n = 7). ET-1 activates a large inwardly rectifying current. D. I–V relations for I_f in presence of 1 µM BQ-123 (n = 11) without (filled squares) and with 10 nM ET-1 (open squares, n = 11 at all potentials except at −50 mV, where n = 10). BQ-123 prevented stimulation of I_f by ET-1. E. Inhibitory effect of 1 µM BQ-123 on the ET-1 activated current in cells exhibiting showing I_f (open squares, n = 12 except at −50 mV where n = 11). Asterisks denote statistical significance (p<0.001 ***).</p

Modulation by ET-1 of instantaneous current in cells lacking I_f.

<p>Ai. Currents recorded in the absence (control) and the presence of 10 nM ET at −120 mV (upper traces) when a voltage command was applied from −40 mV for 500 ms (lower trace). Note outward shift in holding current with ET-1. Closed circles indicate control trace; open circles indicate trace in ET-1. Aii. ET-1 activated currents (elicited at −90, −100 and −120 mV) obtained by digital subtraction of control from ET-1 records (same cell as Ai). B. Mean current-voltage (I–V) relationships for current measured at the start of applied voltage commands in absence (control, filled circles) and presence (open circles) of 10 nM ET-1 (n = 7). Asterisks denote statistical significance (p<0.05 *, p<0.01 **, p<0.001 ***). C. Plot of the mean I–V relationship for ET-1 sensitive difference (ET-1 activated) calculated from the same cells shown in B. D. Plot of ET-1 sensitive current when ET-1 was applied after 1 µM BQ-123 (n = 4).</p

Effects of ET-1 on rapid delayed rectifier K⁺ current tails.

<p>A. Upper traces show currents elicited on depolarisation to +30 mV and subsequent repolarization to −40 mV by protocol shown in lower trace. Deactivating tail currents on repolarization represent the I_Kr ‘tail’. Currents are shown in control solution and in presence of 10 nM ET-1. Insert shows an expanded portion of the traces to highlight the ‘tail’ currents (the horizontal arrow in the inset denotes the zero current level). B. Mean ‘tail’ current I–V relationships for 5 cells, in absence (control, filled circles) and presence (open circles) of 10 nM ET-1. I–V curves were fitted with equation 2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033448#s2" target="_blank">Methods</a>) to derive V_0.5 values of −15.7±3.6 mV in control and −0.7±4.3 mV in ET-1 (p<0.05), with respective k values of 6.4±2.6 mV and 6.9±3.9 mV (p>0.9). The ‘tail’ current was significantly reduced in presence of ET-1 at all voltages ranging from −20 to +50 mV except +20 mV. C. Mean I–V plots for I_Kr tails in the presence of 1 µM BQ-123 without (filled squares; n = 5) and with 10 nM ET-1 (open squares, n = 5 for all, except at +40 and +50 mV where n = 4). Derived V_0.5 values were −12.0±5.1 mV and −2.9±6.4 mV for BQ-123 and BQ-123+ET-1, respectively (P>0.3), with associated k values of 4.9±4.4 and 8.4±5.8 (p>0.6). Asterisks in B denote statistical significance (p<0.05 *, p<0.01 **, p<0.001 ***).</p

NCX in myocytes from the area adjacent to MI.

<p>A. NCX function was measured as the tail current upon repolarization to −70 mV (arrow) after a step to +10 mV in CTRL and MI. B. Averaged NCX current density in CTRL (N_pigs = 9, n_cells = 30) and MI (N_pigs = 7, n_cells = 19), with [Ca²⁺]_i measured simultaneously. * denotes P<0.05.</p

Subcellular spark properties in myocytes from the area adjacent to MI.

<p>A. Spark frequency, and, B, duration in early vs. delayed release areas in MI, with the MI-dependent change in each area (CTRL N_pigs = 12, n_cells = 41 vs. MI N_pigs = 7, n_cells = 33). C. Example of long spark in 3D and fraction of cells showing long-lasting sparks (>47.6 ms) in CTRL (24/43 cells with long sparks) and MI (22/31 cells with long sparks). * denotes P<0.05.</p

Ca²⁺ removal by NCX during caffeine application.

<p>A. Example of current and [Ca²⁺]_i transient recording obtained during the last conditioning pulse from -70 to +10 mV and caffeine application (left). The decay of the current was fit by a 1- or 2-exponential according to the goodness of fit (R²>0.95) and with the 2 amplitudes being negative. The right panel is a typical example of a 2-exponential decay in a CTRL myocyte. B. Example of monophasic I_NCX decay (left) and mean data on incidence of biphasic decay (middle). The percentage of cells better fit by a biphasic exponential was significantly higher in CTRL than in MI (CTRL, N_pigs = 4, n_cells = 17; MI, N_pigs = 4, n_cells = 12, P<0.05). In addition, Tau of fast component of I_NCX decay tended to be faster in CTRL than in MI (right, CTRL, N_pigs = 4, n_cells = 8, <i>vs</i>. MI, N_pigs = 2, n_cells = 4, P = NS). * denotes P<0.05.</p

Effect of proximity of TTs to RyR on spontaneous Ca²⁺ sparks.

<p>A. Typical example of a line scan image during and after 1 Hz stimulation. After loading the SR with conditioning pulses from −70 to +10 mV at 1 Hz, stimulation was stopped and 15 seconds of diastole were recorded for Ca²⁺ sparks. Sparks were assigned to early (blue) and delayed (red) release areas corresponding to their position on the scan line. B. Spark frequency and morphology in early vs. delayed release areas in CTRL pigs (N_pigs = 12, n_cells = 41). * denotes P<0.05.</p

Modulation of Ca²⁺ sparks with TT loss during culture.

<p>A. Representative confocal images of TT staining with di-8-ANEPPS (left) and RyR staining (right) after 48 hours of culture. B. Representative line scan image of Ca²⁺ spark protocol in CULT. The type of release areas is marked in blue and red for early and delayed release areas respectively. C. Comparison of spark duration in early (left) and delayed (right) release areas in CTRL (N_pigs = 7, n_cells = 29) vs. CULT (N_pigs = 7, n_cells = 14). D. Fraction of cells showing long-lasting sparks (>47.6 ms) in CTRL (17/35 cells with long sparks) and CULT (10/15 cells with long sparks). E. NCX current density in CTRL (N_pigs = 4, n_cells = 14) and CULT (N_pigs = 4, n_cells = 7). * denotes P<0.05.</p