Calcium dynamics in the ventricular myocytes of SERCA2 knockout mice: A modeling study

Abstract

We describe a simulation study of Ca²(+) dynamics in mice with cardiomyocyte-specific conditional excision of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) gene, using an experimental data-driven biophysically-based modeling framework. Previously, we reported a moderately impaired heart function measured in mice at 4 weeks after SERCA2 gene deletion (knockout (KO)), along with a >95% reduction in the level of SERCA2 protein. We also reported enhanced Ca²(+) flux through the L-type Ca²(+) channels and the Na(+)/Ca²(+) exchanger in ventricular myocytes isolated from these mice, compared to the control Serca2(flox/flox) mice (flox-flox (FF)). In the current study, a mathematical model-based analysis was applied to enable further quantitative investigation into changes in the Ca²(+) handling mechanisms in these KO cardiomyocytes. Model parameterization based on a wide range of experimental measurements showed a 67% reduction in SERCA activity and an over threefold increase in the activity of the Na(+)/Ca²(+) exchanger. The FF and KO models were then validated against experimentally measured [Ca²(+)](i) transients and experimentally estimated sarco(endo)plasmic reticulum (SR) function. Simulation results were in quantitative agreement with experimental measurements, confirming that sustained [Ca²(+)](i) transients could be maintained in the KO cardiomyocytes despite severely impaired SERCA function. In silico analysis shows that diastolic [Ca²(+)](i) rises sharply with progressive reductions in SERCA activity at physiologically relevant pacing frequencies. Furthermore, an analysis of the roles of the compensatory mechanisms revealed that the major combined effect of the compensatory mechanisms is to lower diastolic [Ca²(+)](i). Finally, by using a comprehensive sensitivity analysis of the role of all cellular calcium handling mechanisms, we show that the combination of upregulation of the Na(+)/Ca²(+) exchanger and increased L-type Ca²(+) current is the most effective means to maintain diastolic and systolic calcium levels after loss of SERCA function.

Figure 1

Parameterization of SERCA in the FF and KO models. (A) Average experimentally recorded [Ca²⁺]_i transients, paced at 1 Hz, in isolated ventricular myocytes from the SERCA2 FF and KO mice as indicated. Simulated decay of the [Ca²⁺]_i transients (dashed lines) using the fitted parameter values for SERCA (1 Hz), NCX, and PMCA are superimposed. (B) Ca²⁺ flux through SERCA (J_SERCA) at 1 Hz, plotted as a function of [Ca²⁺]_i, for FF (circles) and KO (crosses) mice. Fitted (J_SERCA) are superimposed. Inset: an enlarged view comparing SERCA activity in the KO and FF. (C) Average experimentally recorded [Ca²⁺]_i transients, paced at 6 Hz, in isolated ventricular myocytes from the SERCA2 FF and KO mice as indicated. Simulated decay of the [Ca²⁺]_i transients (dashed lines) using the fitted parameter values for SERCA (6 Hz), NCX, and PMCA are superimposed. (D) Ca²⁺ flux through SERCA (J_SERCA) at 6 Hz, plotted as a function of [Ca²⁺]_i, for FF (circles) and KO (crosses) mice. Fitted (J_SERCA) are superimposed.

Figure 2

Comparison between experimentally measured and simulated [Ca²⁺]_i transients. (A) Average experimentally measured [Ca²⁺]_i transients at 1 Hz in the FF (gray) and KO (black) myocytes. (B) Simulated [Ca²⁺]_i transients at 1 Hz in the FF (gray) and KO (black) models. (C) Average experimentally measured [Ca²⁺]_i transients at 6 Hz in the FF (gray) and KO (black) myocytes. (D) Simulated [Ca²⁺]_i transients at 6 Hz in the FF (gray) and KO (black) models. (E) Time courses of the integrals of the Ca²⁺ fluxes through SERCA, NCX, and PMCA in the FF model over one cardiac cycle at 0.5 Hz. (F) Time courses of the integrals of the Ca²⁺ fluxes through SERCA, NCX, and PMCA in the KO model over one cardiac cycle at 0.5 Hz. (G) Percentage contributions of SERCA, NCX, and PMCA to Ca²⁺ removal in the FF and KO models.

Figure 3

Changes in peak (circles and solid lines) and diastolic [Ca²⁺]_i (squares and dashed lines) in response to graded reductions in the maximum uptake activity of SERCA in the FF (gray) and KO (black) models.

Figure 4

In silico perturbations to the KO model. (A) Simulated [Ca²⁺]_i transients at 1 Hz in the KO, KO-I_CaL and KO-NCX and KO-I_CaL-NCX models. (B–E) Diastolic [Ca²⁺]_i level (B), magnitude of the [Ca²⁺]_i transient (ΔCai) (C), time to half relaxation of the transient (RT₅₀) (D), and SR Ca²⁺ content (E) in the KO, KO-I_CaL, and KO-NCX models.

Figure 5

(Top) Changes in peak (solid lines) and diastolic (dotted lines) [Ca²⁺]_i in response to changes in the conductances of the persistent Na⁺ current (G_Nab), the SR Ca²⁺ leak flux (V_leak), the Na⁺/K⁺ ATPase current (G_NKA) and the NCX (V_NCX). Dashed lines indicate the experimentally measured peak and diastolic [Ca²⁺]_i in the KO cardiomyocytes. (Bottom) Changes in peak (solid lines) and diastolic (dotted lines) [Ca²⁺]_i in response to changes total buffer concentration (B_max) and affinity (K_d).

Figure 6

(A) Theoretical analysis of the decay kinetics of the [Ca²⁺]_i transient, showing the RT₅₀ of the transient as a function of peak [Ca²⁺]_i for the FF and KO models. The circles correspond to the experimentally measured peak [Ca²⁺]_i and the predicted RT₅₀ values. (B) Sensitivity of rate of [Ca²⁺]_i decay to the total buffer concentration (B_max). Predicted RT₅₀ was obtained by substituting the appropriate parameter values into Eq. 1. (C) Sensitivity of rate of [Ca²⁺]_i decay to the affinity of the buffers (K_d).