Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes. Effects Of thapsigargin and isoproterenol - PubMed (original) (raw)

Control of maximum sarcoplasmic reticulum Ca load in intact ferret ventricular myocytes. Effects Of thapsigargin and isoproterenol

K S Ginsburg et al. J Gen Physiol. 1998 Apr.

Abstract

In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 micro M) or slowed by thapsigargin (0.2-0.4 micro M). Under control conditions, SR Ca content reached a limit of 137 micro mol.liter cytosol-1 (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents lintegraINaCaXdt) and of 119 micro mol.liter cytosol-1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (integralINaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (integralINaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.

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Figures

Figure 1

Figure 1

Experimental protocol showing voltage and solution changes applied to priorly Nai-depleted cells. The sequence of initially emptying the SR, loading, and then measuring the load was repeated on each cell for various numbers of pulses, first in control condition, and then after application of TG or ISO.

Figure 2

Figure 2

ICa was larger, while corresponding [Ca]i transients were larger and decayed faster with ISO treatment. (A and C) Control; (B and D) ISO, 1 μM. (A and B) ICa and corresponding [Ca]i transients during moderate Ca loading of the SR by a sequence of 10 pulses. Dotted line marks 0 current level. Only pulses 1, 5, and 10 shown. (C and D) Corresponding responses to caffeine after loading. Dotted line marks nonexchange (leakage) current flowing during Na-free portion of caffeine application.

Figure 3

Figure 3

Decay time constant τ of Ca current (single exponential fit) shortened as SR Ca loading increased. τ was first measured for ICa at pulse 1, for which the previously existing SR Ca load was 0. τ was then measured for Ca currents evoked by 2, 3, 6, and 11 pulses, for which just previous SR Ca load measurements were available (at 1, 2, 5, and 10 pulses, respectively). τ was also measured for ICa evoked by 20 and 40 pulses, but since load measurements were not done at 19 or 39 pulses and the SR Ca load was close to steady state, the respective SR Ca load measurements for 20 or 40 pulses were those made just afterward. τ for the various numbers of pulses were normalized to τ of the first (0 load) pulse and plotted against the corresponding SR Ca loads. A single regression line was fitted to all the data.

Figure 4

Figure 4

TG and ISO modulated the SR Ca pumping rate effectively. (top) Decay rates (λ = 1/τ = ln 2/t 1/2, where t 1/2 was obtained from straight line fits) for [Ca]i transients during loading decreased with increasing number of loading pulses and were longer with TG or shorter with ISO than in control. (bottom) Paired comparisons of λ before and after treatment, 5-pulse loading. Decay was 74 ± 5% (SEM, n = 5) faster in ISO or 39 ± 16% (SEM, n = 5) slower in TG. SR uptake is the dominant process reducing [Ca]i during loading.

Figure 5

Figure 5

TG - and ISO-dependent rate changes were not artifacts of simultaneous changes in [Ca]i. (A) Loading [Ca]i transients, control and TG conditions, from one cell. (B) Loading [Ca]i transients, control and ISO conditions, from another cell. Each transient was the last evoked in a 10-pulse sequence. (C and D) _d_[Ca]i/dt as dependent on [Ca]i corresponding with A and B, respectively. TG slowed the decay rate at all [Ca]i above rest; correspondingly, ISO sped the decay at all [Ca]i. Noise in the rate plots was reduced by fitting the decaying phase of each transient in A and B to a single exponential before differentiating.

Figure 6

Figure 6

Diastolic [Ca]i increases during loading in ventricular myocytes of both species, but less so in ferret than in rabbit. Original chart records of fluorescence ratio during execution of protocol in Fig. 1. Solid bars mark caffeine applications.

Figure 7

Figure 7

SR Ca content grows quickly with added Ca for small Ca influxes but reaches a limiting value below proportionality for large additions. Ruptured patch recording (K5-Indo) from one cell treated with ISO (A and C showing ∫INaCaXdt and Δ[Ca]T methods, respectively), and from a different cell treated with TG (B and D showing ∫INaCaXdt and Δ[Ca]T methods, respectively). Symbols show individual measurements; solid lines show least squares fits to the purely descriptive function [Ca]SR(total) = Bmax/(1 + K 1/2/∫ICadt). Bmax was practically unaffected by treatment (for values see text).

Figure 8

Figure 8

Composite data from several cells showing how SR Ca load saturated as cumulated amount of Ca added increased. (top) ∫INaCaXdt method (n = 6, TG; n = 4, ISO). (bottom) Peak fluorescence (Δ[Ca]T) method (n = 6, TG; n = 4, ISO). For clarity, data points were grouped in influx ranges of 0–50, 51–100, 101– 200, and 201–500 μmol·liter cytosol−1, but curves were fit to individual data. Parameters of fits appear in Table I. Heavy lines mark unity slope that would result if all added Ca were accounted for in the SR.

Figure 9

Figure 9

Absolute maximum SR Ca loads, regardless of stimulation history, with and without TG or ISO treatment, were similar. (top) ∫INaCaXdt method; (bottom) Peak fluorescence (Δ[Ca]T) method. *Load with TG was significantly lower (P < 0.05) in paired comparisons with control data. For illustration, maximum load with ISO or TG treatment was expressed as a fraction of the maximum load in same cell before treatment.

Figure 10

Figure 10

Model based predictions of changes in SR Ca load with TG or ISO due to leakage. Maximum SR Ca load decreases with increasing leak flux, but at the experimentally observed leak rate of 0.3 μmol·liter cytosol−1 · s−1, the effect is minor. (A) Loss of SR Ca load as leak is increased, with leak rate expressed as a fraction of forward pump rate. (B) Same, expressed using absolute leak rate, log scale. (C) Normalized and expanded version of B. The forward pump rate stays relatively constant as Vleak increases, but the reverse rate decreases, maintaining Vforward − Vreverse − Vleak = 0.

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