Functionally Isolated Sarcoplasmic Reticulum in Cardiomyocytes: Experimental and Mathematical Models

Abstract

The interaction among the various Ca²⁺ transporters complicates the assessment of isolated systems in an intact cell. This article proposes the functionally isolated SR model (FISRM), a hybrid (experimental and mathematical) approach to study Ca²⁺ cycling between the cytosol and the sarcoplasmic reticulum (SR), the main source of Ca²⁺ for contraction in mammalian cardiomyocytes. In FISRM, the main transmembrane Ca²⁺ transport pathways are eliminated by using a Na⁺, Ca²⁺-free extracellular medium, and SR Ca²⁺ release is elicited by a train of brief caffeine pulses. Two compounds that exert opposite effects on the SR Ca²⁺ uptake were characterized by this approach in isolated rat ventricular cardiomyocytes. The experimental FISRM was simulated with a simple mathematical model of Ca²⁺ fluxes across the SR membrane, based on a previous model adapted to the present conditions. To a fair extent, the theoretical model could reproduce the experimental results, and confirm the main assumption of the experimental model: that the only relevant Ca²⁺ fluxes occur across the SR membrane. Thus, the FISRM seems to be a valuable framework to investigate the SR Ca²⁺ transport in intact cardiomyocytes under physiological and pathophysiological conditions, and to test therapeutic approaches targeting SR proteins.

Keywords: 2,5-di-tert-butylhydroquinone; Ca2+ transport; caffeine; cardiomyocytes; mathematical modeling; sarcoplasmic reticulum; β-adrenergic stimulation.

Figure 1

Scheme describing transition among ryanodine receptor states, where ${\mathrm{k}}_{\mathrm{x}}^{+}$ and ${\mathrm{k}}_{\mathrm{x}}^{-}$ are transition rates, and m and n are the cooperativity parameters of the Ca²⁺ binding determined experimentally [28]. The transitions C1 ↔ O1 ↔ O2 (curved blue arrows) are Ca²⁺ dependent. It is considered that most channels are in the C1 state at diastolic [Ca²⁺]i.

Figure 2

Typical Ca²⁺ transients evoked by brief (60 ms) caffeine pulses (arrows) in the experimental FISRM.

Figure 3

Ca²⁺ transients recorded for estimation of the SR Ca²⁺ content. TyN: perfusion with modified Tyrode’s solution, during which twitch transients were electrically evoked at 0.5 Hz; Ty00: perfusion with Ca²⁺, Na⁺-free Tyrode’s solution; Caff00: perfusion with Ty00 containing 10 mM caffeine sustained for at least 10 s, not preceded by (A) or following brief Caf00 pulses (downward arrows) (B).

Figure 4

Simultaneous variations in membrane potential (V_m, panel (A) and cytosolic Ca²⁺ concentration ([Ca²⁺]i, panel (B) simulated with the theoretical FISRM. Electrical stimulation (grey bar in (A) induced Ca²⁺ transients triggered by action potentials, one of which is shown on an expanded scale as the inset. After switching perfusion to Ty00 solution (downward arrow in (A)), electric diastole ensued, but Ca²⁺ transients could be evoked by application of caffeine pulses (upward arrows in (B)).

Figure 5

Ca²⁺ transients and twitch contractions recorded simultaneously at 0.5 Hz from a rat ventricular cardiomyocyte before (control) and after addition of isoproterenol (ISO, 10 nM). (A): Systolic cell shortening (ΔL, expressed as percent of resting cell length, RCL) and cytosolic Ca²⁺ concentration ([Ca²⁺]i). (B): the same traces after normalization to the respective peak value.

Figure 6

(A): Ca²⁺ transients evoked by caffeine pulses in the experimental FISRM before (control) and during exposure to 10 nM isoproterenol (ISO). (B): Single caffeine transients in an expanded timescale.

Figure 7

(A): Ca²⁺ transients generated with the mathematical FISRM, evoked by electrical stimulation and by caffeine pulses (downward arrows) in the absence and presence of isoproterenol (ISO). (B): Single caffeine-induced transients in an expanded timescale.

Figure 8

Ca²⁺ transients and twitch contractions recorded simultaneously from a rat ventricular cardiomyocyte before (control) and after addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBQ, 5 μM). (A): Systolic cell shortening (ΔL, expressed as percent of resting cell length, RCL) and cytosolic Ca²⁺ concentration ([Ca²⁺]i). (B): the same traces after normalization to the respective peak value.

Figure 9

(A): Ca²⁺ transients evoked by brief caffeine pulses in the experimental FISRM before (control) and during exposure to 5 μM 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBQ). (B): Single caffeine-induced transients in an expanded timescale.

Figure 10

(A) Ca²⁺ transients generated with the mathematical FISRM before (control) and after exposure to 5 μM 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBQ), presented as described in Figure 7. (B) Single caffeine-induced transients in an expanded timescale.