Work-loop contractions reveal that the afterload-dependent time course of cardiac Ca2+ transients is modulated by preload

Abstract

Preload and afterload dictate the dynamics of the cyclical work-loop contraction that the heart undergoes in vivo. Cellular Ca²⁺ dynamics drive contraction, but the effects of afterload alone on the Ca²⁺ transient are inconclusive. To our knowledge, no study has investigated whether the putative afterload dependence of the Ca²⁺ transient is preload dependent. This study is designed to provide the first insight into the Ca²⁺ handling of cardiac trabeculae undergoing work-loop contractions, with the aim to examine whether the conflicting afterload dependency of the Ca²⁺ transient can be accounted for by considering preload under isometric and physiological work-loop contractions. Thus, we subjected ex vivo rat right-ventricular trabeculae, loaded with the fluorescent dye Fura-2, to work-loop contractions over a wide range of afterloads at two preloads while measuring stress, length changes, and Ca²⁺ transients. Work-loop control was implemented with a real-time Windkessel model to mimic the contraction patterns of the heart in vivo. We extracted a range of metrics from the measured steady-state twitch stress and Ca²⁺ transients, including the amplitudes, time courses, rates of rise, and integrals. Results show that parameters of stress were afterload and preload dependent. In contrast, the parameters associated with Ca²⁺ transients displayed a mixed dependence on afterload and preload. Most notably, its time course was afterload dependent, an effect augmented at the greater preload. This study reveals that the afterload dependence of cardiac Ca²⁺ transients is modulated by preload, which brings the study of Ca²⁺ transients during isometric contractions into question when aiming to understand physiological Ca²⁺ handling.NEW & NOTEWORTHY This study is the first examination of Ca²⁺ handling in trabeculae undergoing work-loop contractions. These data reveal that reducing preload diminishes the influence of afterload on the decay phase of the cardiac Ca²⁺ transient. This is significant as it reconciles inconsistencies in the literature regarding the influence of external loads on cardiac Ca²⁺ handling. Furthermore, these findings highlight discrepancies between Ca²⁺ handling during isometric and work-loop contractions in cardiac trabeculae operating at their optimal length.

Keywords: Ca2+ handling; Ca2+ transient; afterload; preload; work-loop.

Graphical abstract

Figure 1.

Twitch stress and Ca²⁺ transient parameters. Each panel shows a transient computed by averaging 10 steady-state stress twitches or Ca²⁺ transients. The passive component of stress twitches has been accounted for and only the active stress is shown. A: twitch duration measurements for an averaged isometric stress profile. t_durx refers to the time that stress remained greater than X % of the peak active stress and was calculated for 10% (t_dur10), 25% (t_dur25), 50% (t_dur50), and 75% (t_dur75) of the stress amplitude. The shaded area represents the stress-time integral (STI). B: twitch duration measurements for an averaged work-loop contraction. For this work-loop twitch, the afterload was 0.31 (decimal fraction of the peak isometric stress) as achieved by setting R_p to 50 kPa·s/mL. Four measures of twitch duration (as in A) were assessed, but only t_dur10 and t_dur75 have been included on the panel to mitigate visual clutter. The shaded area represents the integral of the twitch. C: twitch duration measurements for an averaged Ca²⁺ transient. t_durx here refers to the time that the fluorescence signal remained greater than X % of the peak signal and was calculated for 10% (t_dur10), 25% (t_dur25), 50% (t_dur50), and 75% (t_dur75) of the Ca²⁺ transient amplitude. The F₃₄₀/F₃₈₀-time integral (CTI) is represented by the shaded area. D: decay time measurements for an averaged Ca²⁺ transient. t_X equates to the time to X % decay from the peak fluorescence and was calculated for 25% (t₂₅), 50% (t₅₀), 75% (t₇₅), and 90% decay (t₉₀). The maximum rate of rise for stress and Ca²⁺ transients was also computed but have not been included on any panels.

Figure 2.

Stress-length work loops and Ca²⁺ transients of a representative trabecula at two different preloads. Steady-state twitch stress (A and E), muscle length (B and F), and Ca²⁺ transient (C and G) for a set of work loops over a range of afterloads at fixed preloads set by holding the muscle at L₁₀₀ (A–D) and L₉₅ (E–H). In all panels, twitches are overlaid. The solid dark line represents the isometric twitch. The dashed line represents the work-loop twitch at the lowest afterload. D and H: steady-state stress-length work loops are revealed when stress and length traces are plotted parametrically for each preload.

Figure 3.

Preload and afterload dependence of the steady-state stress production. Data from 14 different experimental conditions on n = 7 muscle samples were analyzed to extract the following parameters: twitch durations (A–D), stress-time integral (STI; E), and the maximum rate of rise of stress development (dS/dt; F). The fixed-effect results of the linear mixed-effects model fitting are indicated for each of the two muscle lengths: L₁₀₀ is indicated with a solid black line, and L₉₅ is indicated with a dashed red line. The pale shading surrounding each fitted line indicates the 95% confidence band, calculated from the covariance matrix [as described previously (30)]. STI against relative ESS was fitted with a first-order polynomial; all other stress parameters were fitted with a second-order polynomial. These data are plotted against afterload, expressed as “relative ESS.” Each panel contains an inset that indicates how the respective metric was calculated. Data were analyzed using linear mixed-effect models implemented in SAS with muscle number treated as a random effect, and the initial muscle length (preload) and relative ESS (afterload) treated as fixed effects. *Significant afterload dependence at L₁₀₀; red ^osignificant afterload dependence at L₉₅; †significant preload dependence. ESS, end-systolic stress.

Figure 4.

Preload and afterload dependence of Ca²⁺ transient. Data from 14 different experimental conditions on n = 7 muscle samples were analyzed to extract the following parameters: F₃₄₀/F₃₈₀ time integral (CTI; A), peak (upper lines) and diastolic (lower lines) F₃₄₀/F₃₈₀ signal (B), the F₃₄₀/F₃₈₀ signal amplitude (C), and the maximum rate of rise of the F₃₄₀/F₃₈₀ signal (D). The fixed-effect results of the linear mixed-effects model fitting for each of the two muscle lengths, L₁₀₀ is indicated with a solid black line and L₉₅ is indicated with a dashed red line. The pale shading surrounding each fit indicates the 95% confidence band, calculated from the covariance matrix [as described previously (30)]. These data are plotted against afterload, expressed as relative ESS. ESS, end-systolic stress. Data were analyzed using linear mixed-effect models implemented in SAS with muscle number treated as a random effect, and the initial muscle length (preload) and relative ESS (afterload) treated as fixed effects.

Figure 5.

Preload and afterload dependence of the time course of Ca²⁺ transient. Data from 14 different experimental conditions on n = 7 muscle samples were analyzed to extract the following parameters: Ca²⁺ transient durations (A–D), time to F₃₄₀/F₃₈₀ signal Ca²⁺ (E), and the time to X % decay from the peak F₃₄₀/F₃₈₀ (F–I). The fixed-effect results of the linear mixed-effects model fitting for each of the two muscle lengths, L₁₀₀ is indicated with a solid black line and L₉₅ is indicated with a dashed red line. The pale shading surrounding each fit indicates the 95% confidence band, calculated from the covariance matrix [as described previously (30)]. These data are plotted against afterload (“relative ESS”). Each panel contains an inset that indicates how each metric was calculated. Data were analyzed using linear mixed-effect models implemented in SAS with muscle number treated as a random effect, and the initial muscle length (preload) and relative ESS (afterload) treated as fixed effects. *Significant afterload dependence at L₁₀₀, red ^osignificant afterload dependence at L₉₅, †significant preload dependence. ESS, end-systolic stress.