Inhibition of cAMP-dependent protein kinase under conditions occurring in the cardiac dyad during a Ca2+ transient

Abstract

The space between the t-tubule invagination and the sarcoplasmic reticulum (SR) membrane, the dyad, in ventricular myocytes has been predicted to experience very high [Ca2+] for short periods of time during a Ca2+ transient. The dyadic space accommodates many protein kinases responsible for the regulation of Ca2+ handling proteins of the cell. We show in vitro that cAMP-dependent protein kinase (PKA) is inhibited by high [Ca2+] through a shift in the ratio of CaATP/MgATP toward CaATP. We further generate a three-dimensional mathematical model of Ca2+ and ATP diffusion within dyad. We use this model to predict the extent to which PKA would be inhibited by an increased CaATP/MgATP ratio during a Ca2+ transient in the dyad in vivo. Our results suggest that under normal physiological conditions a myocyte paced at 1 Hz would experience up to 55% inhibition of PKA within the cardiac dyad, with inhibition averaging 5% throughout the transient, an effect which becomes more pronounced as the myocyte contractile frequency increases (at 7 Hz, PKA inhibition averages 28% across the dyad throughout the duration of a Ca2+ transient).

FIGURE 1

Cartoon of the computer model representing half of a sarcomere (Z-line to M-line). The position in the sarcomere is expressed in cylindrical coordinates (r,z,θ). The model is a cylinder with radius, r = 500 nm, length, z = 1000 nm. The junctional SR is modeled as an impermeable disk with radius, r1 = 200 nm, depth = 20 nm, situated 10 nm from the Z-line surface. Longitudinal SR is represented as a cylinder with radius = 90 nm and length = 970 nm. Free diffusion was allowed to occur throughout the longitudinal SR; however, SERCA uptake also occurred in this region. In r, the model is divided into 10-nm elements; in z the element size is 10 nm above and including the SR and 20.6 nm below, radially, the model is divided into 20 even segments. Calcium enters the model through the 20 radial segments at r = 1. Free [Ca²⁺], CaATP, MgATP, CaADP, and MgATP are obtained for each element at 1-ms intervals.

FIGURE 2

Effect of [Ca²⁺]_free on PKA activity. (A) PKA (400 U) was incubated at a range of [Ca²⁺]_free (3 μM–10 mM) for 2 min in 50 mM histidine (pH 7.0) containing 5 mM (•) or 25 mM (▴) MgSO₄, 6.25 mM NaF, 1 mM EGTA, and 0.1 mM PL919Y. PL919Y was phosphorylated for 1 min by PKA at 37°C after the addition of 0.1 mM ATP-γ-³²P (final concentration to 10 μM). The reaction was terminated by the addition of 100 μl 1% (v/v) H₃PO₄. Phosphorylated PL919Y was transferred to P81 paper, and [γ-³²P] incorporation was measured by scintillation counting. The background-corrected levels of incorporation are shown and represent the mean ± SE (n = 3). (B) Effect of increasing [Ca²⁺]_free on the CaATP/MgATP at total magnesium concentrations of 5 (•) and 25 mM (▴). The concentrations of CaATP and MgATP were calculated using BAD 4.42 (25). (C) Effect of CaATP/MgATP on PKA activity. Data from A were replotted using the ratio generated in B.

FIGURE 3

[Ca²⁺]_free transients generated by the mathematical model. [Ca²⁺]_free was averaged throughout the cytosolic elements of the model. (A) Transients 9 and 10 from a train of 20. (B) Transient generated on the 10th stimulation, expanded timescale from A.

FIGURE 4

[Ca²⁺] generated within the dyad. (A) Three-dimensional plots of [Ca²⁺] at the Z-line (z = 1) as a function of time. The shaded region represents the dyadic space. (B) [Ca²⁺] generated at r = 1, akin to the 10-nm space immediately surrounding the RYR and DHPR channels. (C) [Ca²⁺] averaged across the whole of the dyad.

FIGURE 5

CaATP/MgATP generated within the model. (A and B) Ratios within the dyad; broken line represents raw CaATP/MgATP ratio, bold black line represents the PKA-CaATP/PKA-MgATP-bound occupancy ratio using the same kinetics for both CaATP and MgATP binding to PKA. The gray lines show the effect of varying CaATP-binding kinetics by ±25%. (A) The ratio at r = 1, (B) across the dyad as a whole, and (C) average ratio throughout the cytosol.

FIGURE 6

Transient inhibition of PKA activity in the dyad. (A) Three-dimensional plot of PKA activity at the Z-line (z = 1) as a function of time. The shaded region represents the dyadic space. (B) PKA activity at r = 1, akin to the 10-nm space immediately surrounding the RYR and DHPR channels and (C) averaged across the whole of the dyad. (D and E) As B and C, respectively, with double the dyadic diffusion rate. (F and G) As B and C, respectively, with half the dyadic diffusion rate. Black line represents equal CaATP and MgATP kinetics; gray lines show the effect of varying CaATP-binding kinetics by ± 25%.

FIGURE 7

Transient inhibition of PKA activity in the dyad with reduced [Ca²⁺]_dyad. PKA activity (A) at r = 1, akin to the 10-nm space immediately surrounding the RYR and DHPR channels and (B) averaged across the whole of the dyad, where [Ca²⁺]_dyad peaks a 30 μM. (C and D) As A and B, respectively, with double the dyadic diffusion rate. (E and F) As A and B, respectively, with half the dyadic diffusion rate. Black line represents equal CaATP and MgATP kinetics; gray lines show the effect of varying CaATP-binding kinetics by ± 25%.