Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation

Figures

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  Figure 1.. IANBD-cTnCT53C exchange into skinned cardiac porcine myofibrils. IANBD-cTnCT53C is properly incorporated into myofibrils without loss of function. (A) Structure of cTnC (PDB accession no. 4Y99). Orange ball represents the IANBD probe covalently attached to residue 53. (B) 18% SDS-PAGE gel showing: molecular weight markers (MW, lane 1), dilutions of Tn containing IANBD-cTnCT53C used for exchanges (Tn standard, lanes 2–4), non-exchanged myofibrils (N.E., lane 5), and four separate exchanges of Tn containing IANBD-cTnCT53C into myofibrils (1–4, lanes 6–10). The top image (Coomassie stain) shows the gel stained with Coomassie Blue to visualize total protein. The bottom image (fluorescence) is the region of the gel containing cTnC imaged for fluorescence signal. The level of IANBD-cTnCT53C incorporation was calculated using ratios of the labeled protein IANBD-cTnCT53C (bottom gel: fluorescent cTnC) to total cTnC (top gel: cTnC). Specifically, troponin (Tn) with IANBD-TnCT53C, prior to exchange, was used to determine the fluorescence/Coomassie ratio for cTnC in the input labeled Tn (Tn standard lanes). Ratios of fluorescence/Coomassie intensities of cTnC for 4 (1–4) exchanges were…

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  Figure 2.. Effects of Ca2+ and ATP on fluorescence intensity and lifetime in IANBD-cTnCT53C exchanged myofibrils. (A) In rigor conditions, IANBD-cTnC intensity in myofibrils is reduced by high Ca2+ (red) compared with low Ca2+ (grey; *P = 5.9 × 10−5). n = 23 repeats in wells from N = 1 experimental preparation. (B) In rigor conditions (grey and red), IANBD-cTnC lifetime is reduced by Ca2+ (*P = 4.6 × 10−46). Effects of ATP, releasing the rigor bound myosin from actin, at low Ca2+ were significant (black vs. grey; #P = 7.2 × 10−27). In the presence of ATP, the effects of Ca2+ were clear. The difference between low Ca2+ +ATP (black, weak-binding myosin [W]) and high Ca2+ +ADP@, i.e., 0.1 mM ATP hydrolyzed in pCa 4.5 after 20 min (green, strong-binding myosin [S]) was very significant (*P = 2 × 10−32). (C–F) In the mock screens (∼24 wells for each of two conditions in C–F) the average for each condition is indicated with a dashed line and 3× SD above and below the average is shown with a solid line. n = 23 repeats in wells from N = 1 experimental preparation. Shown are Ca2+ effects on (C) intensity (Z′ = −3.45) and (D) lifetime (Z′ = 0.70), (E) ATP effects on lifetime at low Ca2+ (Z′ = 0.16)…

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  Figure S1.. Effects of Ca2+ and ATP on fluorescence intensity and lifetime in IANBD-cTnCT53C exchanged myofibrils. (A) Fluorescence waveforms of IANBD-cTnCT53C exchanged myofibrils illustrate differences in intensities (peaks) due to Ca2+ and ATP. (B) Normalized fluorescence waveforms of IANBD-cTnCT53C exchanged myofibrils allow for visual comparison of lifetimes (τ = time to decay to 1/e) due to Ca2+ and ATP. The red box highlights the waveform’s region of τ, which is zoomed-in on in C. (C) Zoom-in of red box in B shows separation of lifetime decays. Data is representative of Fig. 2.

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  Figure S2.. Effects of Ca2+ and ATP on fluorescence intensity and lifetime in IANBD-cTnCT53C exchanged myofibrils. (A and B) Zoomed-in A and B from Fig. 2 (see legend) are plotted here on y-axis starting at (A) 0 mV (N = 1, n = 23) and (B) ns (N = 1, n = 23).

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  Figure 3.. Effects of Ca2+ concentration on lifetime in IANBD-cTnCT53C exchanged myofibrils. (A) Decreases in lifetime (ns) with increasing [Ca2+] (i.e., sigmoidal dose-response plot of [Ca2+] on lifetime) in solid triangles. (B) Relative change in lifetime with increasing [Ca2+] (i.e., relative lifetime-pCa plot) with pCa 9.0 at 0 and pCa 4.5 normalized to 1 in open triangles. Error bars are SE. For B, fit to a Hill equation, pCa50 was 6.07 ± 0.08 and the Hill coefficient η was 0.8 ± 0.1. N = 4, n = 16.

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  Figure 4.. Effects of W7 and Pimo on fluorescence lifetime in IANBD-cTnCT53C exchanged myofibrils in a mock screen illustrates Z′ quality of high-throughput assays. (A) IANBD-cTnC lifetime is increased with W7 at low (left blue) and high Ca2+ (right hatched blue). Pimo causes a decrease in lifetime at low (left green) and high (right hatched green) Ca2+, ***P < 0.0001, *P < 0.01. Data is mean ± SD; W7 and Pimo data are averages of N = 2 experimental preparations, whereas DMSO (control) is the average of N = 7 experiments (i.e., DMSO data was combined from Mava and OM experiments); n = 48–140. (B) At low Ca2+, W7 increased lifetime as compared to DMSO control (blue vs. grey: Z′ = 0.71) and effects of Pimo decreased lifetime as compared to DMSO (green vs. grey: Z′ = 0.22). In the mock screens (n = 23 wells for each of two conditions) the average for each condition is indicated with a dashed line and 3× SD above and below the average is shown with a solid line.

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  Figure 5.. Effects of Mava and OM on fluorescence lifetime in IANBD-cTnCT53C exchanged myofibrils in a mock screen illustrates Z′ quality of high-throughput assays. (A) IANBD-cTnC lifetime is reduced with Mava in the presence of ATP at low (left) and high (right) Ca2+. OM causes small but significant increases in lifetime at low and high Ca2+, ***P < 0.0001, *P < 0.002. Data is mean ± SD; n = 23 repeats in wells from one experimental preparation. (B) In the presence of ATP at low Ca2+, Mava reduces lifetime as compared to DMSO control and 3× SD of the Mava data is well separated from 3× SD of the DMSO data (blue vs. grey: Z′ = 0.19). For OM, 3× SD of the OM data overlap with 3× SD of the DMSO data (green vs. grey: Z′ = −2.71). In the mock screen (n = 23 wells for each of two conditions) the average for each condition is indicated with a dashed line and 3× SD above and below the average is shown with a solid line. Note that dashed green (mean + OM) line is coincident with the upper solid grey line (+3× SD +DMSO).

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  Figure S3.. Effects of Mava and OM on fluorescence lifetime in IANBD-cTnCT53C exchanged myofibrils in a mock screen illustrates Z′ quality of high-throughput assays. (A) In the presence of ATP at high Ca2+, Mava reduces lifetime as compared to DMSO control (blue vs. grey: Z′ = 0.04). For OM, 3× SD of the OM data overlap with 3× SD of the DMSO data (green vs. grey: Z′ = −5.6). (B) When lifetime changes are corrected for well-to-well variability, and percentage change in lifetime following the addition of ATP, Mava effects are further separated from DMSO control (blue vs. grey: Z′ = 0.27). In the mock screens (n = 23–24 wells for each of two conditions in A and B), the average for each condition is indicated with a dashed line and 3× SD above and below the average is shown with a solid line. @With high Ca2+ in control and +OM at 20 min, ATP has been hydrolyzed to ADP.