Ca2+ increases cardiac muscle viscoelasticity independent of active force development

Figures

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  Figure 1. Viscoelastic property of cardiac muscle. Example records of changes in muscle length (A), sarcomere length (B), and muscle force (C) in response to a linear muscle stretch under relaxing conditions. The response to stretch had viscoelastic and elastic components. Muscle length was normalized to a length at which the sarcomere length was 2.0 μm.

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  Figure 2. Velocity-sensitive viscoelastic property. (A) Example data of peak and steady-state force levels after stretches of relaxed muscle with velocities ranging over three orders of magnitude; note the logarithmic abscissa scale for stretch velocity in muscle lengths per second (ML/s). (B) For all muscles in relaxing conditions, the viscoelastic force (peak force minus steady-state force) increased with increasing stretch velocity (p < 0.01, n = 6, repeated-measures one-way ANOVA). Statistical comparisons are shown relative to the slowest stretch (∗p < 0.05 and ∗∗p < 0.01 with Bonferroni correction for multiple comparisons). A different symbol is used for each muscle. For relaxed muscle, the effect of stretch on viscoelastic force was not different for males (closed symbols, n = 3) versus females (open symbols, n = 3) (p > 0.05, repeated-measures two-way ANOVA). (C) Pooled data (mean ± SE, n = 6) comparing relaxed muscle viscoelastic force measured before versus after addition of cross-bridge inhibitors (ns, no significant difference, p > 0.05, repeated-measures two-way ANOVA).

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  Figure 3. Inhibitors PNB + Mava eliminated force development. (A) Example records of muscle force during a transition from low Ca2+ solution (relax) to high Ca2+ solution (pCa 4.5). Note that the force record was affected by motion artifacts during the transition between solutions (≈1.5 s). In pCa 4.5 solution, the development of force in the absence of inhibitors (black trace) was completely prevented in the presence of the inhibitors PNB plus Mava (red trace). (B) Pooled data (mean ± SE, n = 6) show that muscle force at pCa 4.5 (active) was abolished by the inhibitors PNB + Mava (∗∗∗p < 0.001, paired t-test). A different symbol is used for each muscle. Solid symbols show males, and open symbols show females. The inset with the expanded y axis scale shows no difference in force level with low Ca2+ versus high Ca2+ in the presence of inhibitors (ns, p > 0.05, n = 6, paired t-test).

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  Figure 4. Viscoelastic property was sensitive to Ca2+. (A) Example records of muscle length, sarcomere length, and force in the presence of PNB + Mava. Peak force after a large amplitude muscle stretch was markedly increased by high Ca2+ level (red trace) compared to low Ca2+ level (relax). (B) Pooled data (mean ± SE, n = 5) show that sarcomere length measured immediately after stretch was not increased as much in high Ca2+ as in low Ca2+ (relax) (∗p < 0.05, paired t-test).

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  Figure 5. Velocity- and Ca2+-sensitive viscoelastic property. (A) Pooled data (mean ± SE, n = 6) showing the relation of muscle viscoelastic force (peak-minus steady-state level) versus muscle stretch velocity for experiments performed in the presence of cross-bridge inhibitors and low Ca2+ (relax) or high Ca2+ (∗∗∗∗p < 0.0001, repeated-measures two-way ANOVA). (B) Viscoelastic force response to the fastest stretch (velocity: 2.25 ML/s) of trabeculae from males versus females in the presence of cross-bridge inhibitors and high Ca2+ (ns, p > 0.05, n = 3 per group, unpaired t-test). (C) Force level 60 s after 0.1 s stretch/hold protocol in the presence of cross-bridge inhibitors at both low Ca2+ (relax) and high Ca2+ (ns, p > 0.05, n = 6, paired t-test).

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  Figure 6. Ca2+ regulation of viscoelastic property. (A) Effect of Ca2+ level (expressed as pCa, −log[Ca2+]) on the viscoelastic force response to the fastest stretch (velocity: 2.25 ML/s) in the presence of cross-bridge inhibitors (mean ± SE, n = 6, error bars smaller than symbols). For each experiment, viscoelastic force was normalized to the force measured at pCa 4.5. Pooled data were fit to the Hill equation. (B) In two additional experiments, the developed force-pCa relation was measured in the absence of inhibitors (closed symbols). Then, in the same two muscles, the viscoelastic force-pCa relation was measured in the presence of inhibitors (open symbols). Values were expressed as an increase relative to the values in relaxing solution (means ± SE, n = 2). Muscle length was the same (1.175 Lo) for both measurements. Nonnormalized values for both experiments are shown in the graphs on the right (different symbol for each experiment).

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  Figure 7. No effect of temperature on Ca2+-activated viscoelastic property. (A) Ca2+-activated viscoelastic force was not decreased by lowering temperature from 21°C to 10°C (ns p > 0.05, n = 6, paired t-test). (B) Stress relaxation half time (t½) measured directly from records for trabeculae in the presence of inhibitors and both low Ca2+ (relax) and high Ca2+ and with temperature set first to 10°C and then 21°C (∗p < 0.05 and ∗∗p < 0.01, n = 6, paired t-tests). (C) Low temperature reduced developed force and slowed the rate constant of force redevelopment after cross-bridges were mechanically disrupted.

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  Figure 8. Effect of Ca2+ to increase viscoelastic force was lost after extraction of myofilaments. Viscoelastic force measured in the presence of inhibitors at low and high Ca2+ levels for trabeculae before (intact) and after high-salt extraction of the myofilaments (shaded boxes indicate mean and high and low values, ∗∗p < 0.01 and ns p > 0.05, n = 6, paired t-tests).