End‐diastolic force pre‐activates cardiomyocytes and determines contractile force: role of titin and calcium

Figures

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  Figure 1. Protein analysis in isolated cardiomyocytes from WT and HET hearts A, in HET cardiomyocytes a longer titin isoform is seen. B, Phos‐tag analysis of cardiac troponin I was performed to assess its phosphorylation state. No difference in cTnI phosphorylation was seen between WT (N = 6) and HET (N = 5). C, immunoblot analysis was performed to measure PLN expression levels and phosphorylation. D and E, no difference was seen in expression levels (D) and phosphorylation at Ser16 (E) between WT (N = 6) and HET (N = 5). Data are shown as means ± SEM.

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  Figure 2. Maximal work in RBM+/− cardiomyocytes only reduced at low pacing frequencies A and B, example tracings of work loops from WT and HET cardiomyocytes at 4 different preloads paced at 1 Hz (A) or 8 Hz (B). C and D, peak work at 1 Hz (C) and 8 Hz (D) was plotted against the end‐diastolic SL at which peak work was achieved (n = 8). End‐diastolic sarcomere length (EDSL) was always higher in HET. E, example tracings of force and length changes of contractions at 1 and 8 Hz for WT and HET. F, peak work over the range of pacing frequencies measured. Lower peak work in HET compared with WT only at low pacing frequencies. * P < 0.05. G and H, kinetics of contraction during the isometric contraction phase at different preloads were plotted for WT (G) and HET (H) cardiomyocytes (for each group n = 7). The force scales in A and B are relative; it was not possible to reliably measure and track zero force after the myocytes were attached.

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  Figure 3. End‐diastolic force not SL determines length‐dependent activation During the work loop protocol cells are stretched to different preload (end‐diastolic force; EDF), while SL is recorded. A, the EDF to SL relationship shows that HET cardiomyocytes require much less force to be stretched, which holds true at different pacing frequencies (N = 6, n = 18 WT; N = 6, n = 17 HET). B, as developed force is measured during the subsequent contraction, the developed force to end‐diastolic SL relationship can be measured. This relationship is a measure of length‐dependent activation. This is severely blunted in HET cardiomyocytes (N = 6, n = 17 WT; N = 6 n = 9 HET). C, however, if EDF is plotted against subsequent developed force, only a small non‐significant decrease is seen in HET cardiomyocytes compared with WT (N = 6, n = 21 WT; N = 6, n = 10 HET).

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  Figure 4. Pre‐activation of HET cardiomyocytes occurs at higher diastolic Ca2+ A, membrane‐permeabilized cardiomyocytes were glued between a force transducer and a length motor. Using a theta‐glass perfusion system, we could rapidly switch solutions to mimic a cardiac contraction. During the protocol we use one activation solution (2 µm Ca2+) and five different diastolic solutions (ranging from 1 to 400 nm Ca2+). Cells were perfused with diastolic solution until the force trace is stable and the pipette was rapidly switched so that the cell was exposed to activation solution for 1 s, before moving back to diastolic solution. After repeating this 3 times, the diastolic Ca2+ was changed to a different concentration. B, example tracing from a WT permeabilized cardiomyocyte undergoing the pre‐activation protocol. A permeabilized cardiomyocyte was constantly perfused with 1 nm diastolic calcium solutions and then exposed to 2 s perfusion of activating Ca2+ (2 µm). The same cell was then perfused with 80 nm diastolic Ca2+ until the force signal was stable. When switching to the same activating Ca2+, almost double the force was produced. For measurements of active force production at different…