Increased Ca2+ Sequestration by the Sarco-/Endoplasmic Reticulum in Cardiac Purkinje Cells After Myocardial Infarction

Figures

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  Figure 1. High-resolution confocal microscopy and analysis of spontaneous Ca2+ transients in dog Purkinje cells. (A) Wavelet transients were captured by fluorescence laser scanning confocal microscopy (LSCM) at a scanning rate of 350 Hz along 25–50 µm distances. As reported previously, wavelets propagated exclusively at the periphery of Pcells in the “SubSL compartment” [15,30]. For clarity, a scan example (blue line) of the peripheral region is represented on a representative 2D (spinning disk) confocal snapshot of a propagating wavelet in (A.1). A total of 250 successive scans of the same region (3 ms/scan) were arranged into the scan-line image shown in (A.4). The time course of the transient shown in (A.2) was measured at half the distance of the wave’s overall propagation along the a-b segment represented in (A.4). A linear regression through maximal scan values verified the linearity of the propagation (see segment c-d in (A.4)) and consistency of the transient amplitude (A.3) during the propagation. The data points represented in (A.4) are the maximal values measured in the scans. Transients with amplitude fluctuation > 5% during the propagation were rejected. The parameters listed…

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  Figure 2. Ca2+ spark activity of individual Ca2+ release sites in Pcells of canine hearts with acute ischemic MI. (A) Pcells were isolated from the left ventricle of normal dog hearts and dog hearts with 48-h MI. Permanent LAD coronary occlusion induced typical transmural MI within less than two days, with no evident sign yet of scar tissue (see yellow dashed line in (A.1)). At this stage, we postulated that the MI zone comprised myocardial cells undergoing typical acute ischemia. Pcells were isolated from endocardial free-running Purkinje strands (see dashed white circle in (A.1) and yellow arrows in (A.2)). (A.3) illustrates the capture of spark activity by a 25 µm line scan (5 ms/scan) positioned randomly in the cell; for clarity, a scan line is represented on the 2D confocal (spinning disk) image of a representative Fluo-4-loaded Pcell. (B) Consecutive scans were plotted as shown in (B.1); the spark firing rate (B.2) and density (B.3) of active Ca2+ release sites were measured as indicated in (B.1). To limit photobleaching, scanning was interrupted and cell illumination was occulted for 5 ms every 1.5 s (300 scans). The firing rate (B.2) and number of sites (B.3) were typically…

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  Figure 3. Comparisons of normal vs. MI Pcells of peripheral (Wlets) and central (CWWs) Ca2+ removal. (A) Typical Wlet transients were captured by 25 µm line scans positioned in the peripheral regions of Pcells as indicated (blue line) in the representative 2D confocal snapshot in panel (A.1); the peripheral regions of the cell are underlined to emphasise the regionalisation of Wlet occurrence. The time courses of 10 normal Wlets (from 6 cells) and 16 MI Wlets (from 6 cells) are displayed in panels (A.2) and (A.3), respectively. The mean characteristics of the waves are reported in Table 1. After normalisation to the maximal Ca2+ concentration, the decay phase of each transient was isolated and represented in the insets of (A.2) and (A.3). The following one-phase exponential decay model was used to fit the data of each transient: [Ca2+] = (Y0 − P)*exp[-(1/τ) * t] + P, where Y0 is the [Ca2+] value at t = 0, P is the [Ca2+] value at infinite t, and τ is the time constant of the decay expressed in ms and reflects the rate of intracellular Ca2+ decline. The averaged τ values (mean ± SD) are reported on the graphs for each group. (B) Individual CWWs were sampled by LSCM as indicated in the…

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  Figure 4. Computational evidence of SR-Ca2+ uptake’s contribution to the alterations in peripheral Ca2+ dynamics in Pcells after MI. (A) Ten and 17 representative Wlets were combined, respectively, to produce the averaged Ca2+ transients of normal (control) (A.1) and MI (A.2) Pcells. The Purkinje-specific model of Ca2+ mobilisation described in the Supplementary Materials and references [24,32] was used to replicate the normal and MI-averaged transients; predicted (dotted line) and experimental (plain line) curves are superimposed in (A.1) and (A.2). The accuracy of the predictions was estimated by linear regressions as indicated in the corresponding insets, where arrows 1 and 2 indicate the rising and decay phases of the transients, respectively. (B) The maximal Ca2+ uptake rate (Umax) was incrementally increased (B.1) in the numerical expression of the normal Ca2+ transient shown in (A.1); the simultaneous effects of Umax increments on the Ca2+ release pulse function and overall Ca2+ transient are represented in (B.2) and (B.3), respectively. As indicated in (B.3), a complete switch from normal transient (blue trace) to MI transient (red trace) was achieved with a ~35% increase in Umax…