Potential impacts of the cardiac troponin I mobile domain on myofilament activation and relaxation

Abstract

The cardiac thin filament is regulated in a Ca²⁺-dependent manner through conformational changes of troponin and tropomyosin (Tm). It has been generally understood that under conditions of low Ca²⁺ the inhibitory peptide domain (IP) of troponin I (TnI) binds to actin and holds Tm over the myosin binding sites on actin to prevent crossbridge formation. More recently, evidence that the C-terminal mobile domain (MD) of TnI also binds actin has made for a more complex scenario. This study uses a computational model to investigate the consequences of assuming that TnI regulates Tm movement via two actin-binding domains rather than one. First, a 16-state model of the cardiac thin filament regulatory unit was created with TnI-IP as the sole regulatory domain. Expansion of this to include TnI-MD formed a 24-state model. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than would be required for IP acting alone. Indeed, setting actin affinities of the IP and MD to 25% of that assumed for the IP in the single-site model was sufficient to achieve precisely the same degree of Ca²⁺ regulation. We also tested the 24-state model's ability to represent steady-state experimental data in the case of disruption of either the IP or MD. We were able to capture qualitative changes in several properties that matched what was seen in the experimental data. Lastly, simulations were run to examine the effect of disruption of the IP or MD on twitch dynamics. Our results suggest that both domains are required to keep diastolic cross-bridge activity to a minimum and accelerate myofilament relaxation. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to complete Ca²⁺-dependent regulation of the thin filament.

Keywords: Cardiac thin filament; Cardiac troponin I; Computational modeling; Mobile domain.

Figure 1:. Model Schematic.

(A) Diagram showing the schema of thin filament structure used to formulate the model; (B) Simplified regulatory unit (RU) with the protein domains relevant to this model: N-lobe of TnC, inhibitory peptide, switch peptide, and mobile domain of TnI, actin filament, tropomyosin, and myosin; (C) Allowed states for each protein domain and their binary encoding in the model; (D) Representative loops from within the Markov chain diagram showing microscopic reversibility of the biasing constants λ, η, and μ; (E) Full Markov chain models for the 16-state and 24 -state models. Transitions with biasing constants applied highlighted in red (λ), green (η), and blue (μ). * some transitions in 24-state model omitted from diagram for clarity; (F) Example of coupled RUs in series in various states to form a thin filament. RUs 1 and 26 remain fixed in the initial state, and neighboring RUs are coupled through Tm position.

Figure 2:. Comparison of steady-state convergence.

(A) Average force plotted over time at $\left[C{a}_{cytosolic}^{2+}\right]$ ranging from 7.5 to 4.5. Data shown for repetitions ranging from 32 to 1920. (B) Overlay of the average steady-state force values for each of the 6 simulations from A. ● used for output of model, ▬ used for fit of Hill equation.

Figure 3:. Comparison of steady-state behavior of 16-state vs. 24-state models.

(A) Steady-state force-pCa plots were produced for the 16-state and 24-state model through a range of γ values to compare behavior. ● used for output of model, ▬ used for fit of Hill equation; (B) Kinetic rates of transition were compared between the single regulatory domain of the 16-state, and the two regulatory domains of the 24-state. Unbinding rates include both when the SP is unbound from TnC and when the SP is bound TnC and the biasing constants η and μ are applied to the rates; (C) Calcium transient used to produce twitch simulations; (D) Normalized force plots of 16-state and 24-state models.

Figure 4:. Steady-state comparison of 24-state model and experimental data.

(A) Steady-state force-pCa curves of the 24-state model with both regulatory domains, with the MD deleted, and with the IP deleted. ● used for output of model, ▬ used for fit of Hill equation; (B) Kinetic rate equilibrium constants for the IP and MD both when the SP is unbound from TnC and when the SP is bound TnC and the biasing constants η and μ are applied to the rates; (C) Comparison of steady-state force-pCa curve characteristics (ΔpCa50, maximum activation, ratio of minimum activation and maximum activation, and hill coefficient) for IP disruption between experimental data and the model; (D) Comparison of steady-state force-pCa curve characteristics MD disruption between experimental data and the model.

Figure 5:. 24-state model dynamic simulations.

(A) Calcium transient used to produce twitch simulations; (B) Force output of model simulation with both regulatory domains, with the MD deleted [(−) MD], and with the IP deleted [(−) IP]; (C) Normalized force plots; (D) Comparison of twitch characteristics: Diastolic force, peak force, time from stimulus to peak force, time from peak force to 50% relaxation (RT50).