Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle
- King’s College London, United Kingdom
- King's College London, United Kingdom
Figures
Figure 1
Sarcomere length-force relationships in rat cardiac trabeculae.
(a) Passive force (○) and maximum Ca2+-activated force (●) at five sarcomere lengths (n = 6 trabeculae). Force is normalised to maximum force measured at SL 1.9 μm (T/T0). (b) pCa-force relationships at sarcomere lengths 1.9 μm (○) and 2.3 μm (●). The data were summarised from experiments for Figure 2a and b and fit to the Hill Equation (n = 10). Dotted line is the Hill curve at SL 2.3 μm normalised to its maximum force. Increases in SL resulted in increases in maximum Ca2+-activated force and Ca sensitivity (leftward shift of pCa-force curve). Error bars denote SEM.
Figure 2
Ca2+-dependence of force and troponin orientation, <P2>, in cardiac trabeculae.
(a, c) BR-cTnC-C; (b, d) BR-cTnC-E. Open symbols, SL 1.9 μm; filled symbols, SL 2.3 μm. Error bars denote SEM.
Figure 3
Effects of sarcomere length on maximum Ca2+-activated force, orientation (<P2>) of cTnC-BR probes in solutions with the highest [Ca2+] where trabeculae remained relaxed (pCa 6) and the maximum [Ca2+] (pCa 4.5), and the Hill parameter pCa50.
(a, c, e) BR-cTnC-C; (b, d, f) BR-cTnC-E. Open bars, SL 1.9 μm; grey bars, SL 2.3 μm. Mean ± SEM (n = 5). The statistical significance of differences was assessed using a two-tailed paired t-test.
Figure 4
Effects of active force inhibition by 25 μM blebbistatin on the orientation of cTnC probes and their length-dependence.
(a, c) BR-cTnC-C; (b, d) BR-cTnC-E. (a–b) Continuous lines denote Hill fits to data at SL 1.9 μm (data not shown for clarity). Circles denote <P2> in the presence of blebbistatin at SL of 1.9 μm (○) and 2.3 μm (●). (c–d) Fitted Hill parameter, pCa50, for the control at 1.9 μm SL (white) and in the presence of blebbistatin at 1.9 μm SL (gray) and 2.3 μm (black). Mean ± SEM (n = 5–7).
Figure 5
Force and orientation (<P2>) of the BSR-RLC-BC probe in relaxing (pCa 6.6) and activating (pCa 4.5) solution at sarcomere lengths of 1.9 (white) and 2.3 μm (gray).
Mean ± SEM (n = 5). Statistical significance was assessed using a two-tailed paired t-test.
Tables
Table 1
Ca2+-dependence of force and the orientation parameter <P2> and the effects of sarcomere length. Mean ± SEM. pCa50 and nH are fitted parameters of Hill equation. Δ<P2> , changes in <P2> during Ca2+-activation from pCa 6 to 4.5. Comparisons: between sarcomere lengths 1.9 and 2.3 μm (t test, two-tailed; *p<0.05).
| BR-cTnC-C | BR-cTnC-E | |||
|---|---|---|---|---|
| SL (μm) | 1.9 | 2.3 | 1.9 | 2.3 |
| Force | ||||
| (mN/mm2) | 22.7 ± 1.8 | 32.4 ± 1.5* | 23.2 ± 3.5 | 33.1 ± 6.0* |
| pCa50 | 5.37 ± 0.03 | 5.50 ± 0.03* | 5.37 ± 0.02 | 5.49 ± 0.03* |
| nH | 4.03 ± 0.20 | 4.29 ± 0.22* | 4.12 ± 0.24 | 4.33 ± 0.16* |
| <P2> | ||||
| pCa50 | 5.37 ± 0.02 | 5.48 ± 0.01* | 5.34 ± 0.02 | 5.45 ± 0.02* |
| nH | 3.28 ± 0.16 | 3.42 ± 0.10 | 3.59 ± 0.25 | 3.24 ± 0.18 |
| at pCa 6 | 0.108 ± 0.005 | 0.113 ± 0.005 | 0.283 ± 0.009 | 0.310 ± 0.010* |
| at pCa 4.5 | 0.028 ± 0.004 | 0.036 ± 0.003* | 0.182 ± 0.007 | 0.208 ± 0.008* |
| Δ<P2> | −0.081 ± 0.005 | −0.077 ± 0.005 | −0.101 ± 0.007 | −0.101 ± 0.006 |
| n = 5 | n = 5 | |||
Table 2
Effects of force inhibition by 25 μM blebbistatin on Ca2+-dependence of force and the cTnC orientation parameter <P2>. Mean ± SEM. pCa50 and nH are fitted parameters of Hill equation. Δ<P2>, changes in <P2> during Ca2+-activation from pCa 6 to 4.5. Comparisons (paired t test, two-tailed): before and after addition of blebbistatin (*p<0.05); between sarcomere lengths 1.9 and 2.3 μm in the presence of blebbistatin (#p<0.05).
| BR-cTnC-C | BR-cTnC-E | |||||
|---|---|---|---|---|---|---|
| SL (μm) | 1.9 | 1.9 | 2.3 | 1.9 | 1.9 | 2.3 |
| 25 μM Blebbistatin | − | + | + | − | + | + |
| Force | ||||||
| pCa50 | 5.39 ± 0.03 | 5.39 ± 0.04 | ||||
| nH | 3.72 ± 0.23 | 4.23 ± 0.20 | ||||
| <P2> | ||||||
| pCa50 | 5.40 ± 0.01 | 5.32 ± 0.02 * | 5.41 ± 0.03 # | 5.35 ± 0.02 | 5.28 ± 0.03 * | 5.36 ± 0.02 # |
| nH | 3.29 ± 0.10 | 2.74 ± 0.20 * | 2.49 ± 0.15 | 3.57 ± 0.22 | 2.90 ± 0.19 * | 2.60 ± 0.17 |
| at pCa 6.0 | 0.091 ± 0.004 | 0.088 ± 0.005 | 0.086 ± 0.004 | 0.276 ± 0.010 | 0.282 ± 0.010 * | 0.305 ± 0.009 # |
| at pCa 4.5 | 0.009 ± 0.002 | 0.016 ± 0.002 | 0.024 ± 0.003 | 0.168 ± 0.008 | 0.202 ± 0.007 * | 0.225 ± 0.006 # |
| Δ<P2> | 0.082 ± 0.004 | 0.069 ± 0.004 * | 0.062 ± 0.003 # | 0.098 ± 0.007 | 0.080 ± 0.004 * | 0.080 ± 0.005 |
| n = 5 | n = 7 | |||||
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Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle
eLife 6:e24081.
https://doi.org/10.7554/eLife.24081
