Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging

Figures

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  Fig. 1.. Overview of the TCSPC imaging approach to probe the mechanisms for SERCA2a activation. A) Snapshot of the modeled 3D structure of the two-color cardiac SERCA2a construct labeled with a maroon fluorescent protein (mMaroon) at position Met1 and with an orange fluorescent protein (OFP) at position Gly509. The approximate location of the lipid bilayer is represented by the shaded box. We note that this structure is shown for representation only. B) Fluorescence decay measured by TCSPC for two-color SERCA2a in the DMSO vehicle. We found that the TCSPC imaging approach has over 4 orders of magnitude difference between signal (intensity at ∼100, arbitrary units (AU)) and noise (intensity at ∼10−4 AU).

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  Fig. 2.. Effects of small-molecule effectors on SERCA2a ATPase activity. A) Structures of chemically diverse small-molecule effectors reported in the literature as SERCA activators. Ten-point concentration-response curves were obtained for B) CDN1163, C) istaroxime, D) CP-154526, and E) Ro 41-0960 on the ATPase activity of the cardiac SERCA2a. In all cases, the activity of the compounds at each concentration was obtained from an eight-point free Ca2+ concentration-dependent SERCA2a activity assay and normalized relative to the untreated control, as described in the experimental procedures. Data are reported as average ± SEM of three biological replicates (N = 3).

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  Fig. 3.. PLN removal does not affect the catalytic turnover of SERCA2a. A) Western blot analysis was used to detect the presence of PLN along the purification steps of SERCA2a, starting from pig cardiac SR microsomes. The Western blot analysis showed that purification of SERCA2a removes PLN initially present in the pig cardiac SR microsomes. B) Ca2+ concentration-dependent ATPase activity plot of purified SERCA2a. The Vmax calculated from the fitted data is shown inside the plot. Data are reported as the average ± SEM of four biological replicates (N = 4).

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  Fig. 4.. Competitive FRET assays to investigate the effects of CDN1163 on the dissociation of the endogenous SERCA2a–PLN complex. A) Structural representation of the dissociation and subunit models for activation of SERCA2a by CDN1163 tested in this study. Concentration-response curves at increasing CDN1163 concentrations under B) low free Ca2+ and C) high free Ca2+ conditions. For comparison, we tested the effects of AMP-PCP alone or in the presence of CDN1163. Data are reported as the average ± SEM of four biological replicates (N = 4).

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  Fig. 5.. Effects of CDN1163 on the interaction between SERCA2a and PLN. FRET assays were performed at A) low Ca2+ conditions (0.01 µM) and B) high Ca2+ conditions (10 µM). In all cases, we found that compared to the DMSO vehicle, increasing concentrations of CDN1163 (numbers above the plots) do not significantly change the FRET efficiency between fluorescently labeled SERCA2a and PLN. The addition of the ATP analog AMP-PCP to the SERCA2a- and PLN-containing microsomes does not induce significant changes in the FRET signal vs. the control at both low and high Ca2+ conditions. Treatment of the microsomes with a fixed concentration of AMP-PCP and increasing concentrations of CDN1163 does not significantly change the FRET efficiency compared to the vehicle, CDN1163, and AMP-PCP treatment groups. Data is shown as mean ± SEM of four biological replicates (N = 4). We used a two-way ANOVA followed by Dunnett's post hoc test to compare treatments (CDN1163, AMP-PCP, and CDN1163+AMP-PCP) against the DMSO vehicle; ns = not significant.

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  Fig. 6.. Structural changes of SERCA2a in response to the ATP analog AMP-PCP and the inhibitor TG. Twelve-point Ca2+ concentration-response curves were obtained in the A) absence and B) presence of AMP-PCP at four concentrations of TG. In both cases, we include a DMSO vehicle as a control. The structural change in response to ligands is shown as the % of the closed population of the headpiece. The dashed lines show the lower, midpoint, and upper boundaries of the concentration-response curve of SERCA2a in the absence of AMP-PCP and TG. The green shaded area highlights the range of Ca2+ concentrations at which SERCA2a is operational in cardiac cells. Data are reported as average ±SEM of four biological replicates (N = 4).

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  Fig. 7.. Structural changes of SERCA2a in response to CDN1163. Twelve-point Ca2+ concentration-response curves were obtained in the presence of DMSO vehicle, CDN1163, AMP-PCP, and CDN1163 + AMP-PCP. The structural change in response to ligands is shown as the % of the closed population of the headpiece. Data are reported as average ±SEM of four biological replicates (N = 4).

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  Fig. 8.. Effects of CDN1163, AMP-PCP, and CDN1163/AMP-PCP on the structural populations of SERCA2a. TCSPC assays were performed at increasing Ca2+ conditions from 0.001 µM to 100 µM. Intracellular free Ca2+ concentration in cardiac cells fluctuates in the range of 1–2.65 µM and 0.1–0.16 µM during contraction and relaxation of the heart. Therefore, these values represent the upper and lower limits of the range of physiological Ca2+ concentrations at which SERCA2a operates. The full range of Ca2+ concentrations used in this study is shown above the plots. In all cases, we used AMP-PCP and CDN1163 concentrations of 0.5 and 10 µM, respectively. Data is shown as mean ± SEM of four biological replicates (N = 4). We used a two-way ANOVA followed by the Dunnett's post hoc test to compare treatments (CDN1163, AMP-PCP, and CDN1163/AMP-PCP) against the DMSO vehicle. *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001.

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  Fig. 9.. Effects of CDN1163 on SERCA2a's affinity for Ca2+ ions. A) Eight-point Ca2+ concentration-response of SERCA2a ATPase activity curves obtained at 10 concentrations of CDN1163 (0.1 to 100 µM). The activity at each concentration of CDN1163 was normalized relative to the control at [Ca2+] = 1 µM because this is the point representing the maximal activity of the pump in our assays. B) Changes in apparent Ca2+ affinity (KCa) induced by increasing concentrations of CDN1163; the apparent affinity is expressed as the %change relative to the vehicle control. Data are reported as average ± SEM of three biological replicates (N = 3). To eliminate the confounding effects of ATPase hydrolysis, we used TCSPC imaging to measure the effects of 10 µM CDN1163 on SERCA2a's KCa in the C) absence and D) presence of the ATP analog AMP-PCP. Data is shown as mean ± SEM of four biological replicates (N = 4). Significance compared to the DMSO vehicle was determined using a two-tailed Mann–Whitney U test; *P < 0.05.