Limited effects of exogenous glucose during severe hypoxia and a lack of hypoxia-stimulated glucose uptake in isolated rainbow trout cardiac muscle

Abstract

We examined whether exogenous glucose affects contractile performance of electrically paced ventricle strips from rainbow trout under conditions known to alter cardiomyocyte performance, ion regulation and energy demands. Physiological levels of d-glucose did not influence twitch force development for aerobic preparations (1) paced at 0.5 or 1.1 Hz, (2) at 15 or 23°C, (3) receiving adrenergic stimulation or (4) during reoxygenation with or without adrenaline after severe hypoxia. Contractile responses to ryanodine, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum, were also not affected by exogenous glucose. However, glucose did attenuate the fall in twitch force during severe hypoxia. Glucose uptake was assayed in non-contracting ventricle strips using 2-[(3)H] deoxy-d-glucose (2-DG) under aerobic and hypoxic conditions, at different incubation temperatures and with different inhibitors. Based upon a lack of saturation of 2-DG uptake and incomplete inhibition of uptake by cytochalasin B and d-glucose, 2-DG uptake was mediated by a combination of facilitated transport and simple diffusion. Hypoxia stimulated lactate efflux sixfold to sevenfold with glucose present, but did not increase 2-DG uptake or reduce lactate efflux in the presence of cytochalasin B. Increasing temperature (14 to 24°C) also did not increase 2-DG uptake, but decreasing temperature (14 to 4°C) reduced 2-DG uptake by 45%. In conclusion, exogenous glucose improves mechanical performance under hypoxia but not under any of the aerobic conditions applied. The extracellular concentration of glucose and cold temperature appear to determine and limit cardiomyocyte glucose uptake, respectively, and together may help define a metabolic strategy that relies predominantly on intracellular energy stores.

Keywords: 2-deoxyglucose; adrenaline; contractility; glucose; heart; hypoxia; lactate; rainbow trout; reoxygenation.

Fig. 1.

Normalized twitch force development of cardiac muscle from rainbow trout exposed to different treatments sequentially. In each experiment (N=6) four strips from each ventricle were run in parallel. After 30 min without exogenous substrate, two strips electrically stimulated at 0.5 Hz received 5 mmol l⁻¹ glucose (Glu) and the other two 5 mmol l⁻¹ mannitol (Mann). One strip receiving Glu and one in Mann were exposed to 10 μmol l⁻¹ ryanodine (Rya). After 40 min, stimulation was turned off for 5 min and resumed to assess post-rest potentiation (post-rest). Stimulation rate was then increased from 0.5 Hz to 1.1 Hz and switched back to 0.5 Hz after 15 min. After 15 min, 10 μmol l⁻¹ adrenaline (Adr) was added to all preparations. Ten minutes later the stimulation rate was increased to 1.1 Hz for 15 min and then reduced back to 0.5 Hz for 15 min. Horizontal bars at time zero set the references for relative force development. Vertical arrows pointing down indicate the time of addition of different chemicals or the change in temperature. Horizontal arrows indicate the presence of compounds or a particular temperature during the rest of the experiment. During stimulation at 1.1 Hz, maximal force and the force at the end of exposure to 1.1 Hz are depicted as ‘Max’ and ‘End’, respectively. The time at which maximal force occurred varied between experiments and is therefore not indicated on the x-axis. Results are means ± s.e.m. (A) Twitch force development at 15°C (acclimation temperature). (B) Effects of the elevation of stimulation rate on twitch force calculated as the difference between the maximal force at 1.1 Hz and the preceding force at 0.5 Hz divided by the preceding force at 0.5 Hz at 15°C in the absence and presence of Adr. *Significant effect of ryanodine versus baseline (P<0.01, t-test). (C) Twitch force development after acute elevation of temperature to 23°C. Six new experiments were run as above except that temperature was increased from 15 to 23°C 30 min after the addition of Glu or Mann. *Significant effect of ryanodine in the absence of Adr (P<0.05, one-way ANOVA with Tukey's test). (D) Effects of increased stimulation rate on twitch force calculated as in B at 23°C, in the absence and presence of Adr. In contrast to results at 15°C, there was no effect of Rya in the presence of Adr.

Fig. 2.

Effects of 100 min of severe hypoxia on normalized twitch force development in rainbow trout ventricle strips with 5 mmol l⁻¹ exogenous glucose (Glu) or mannitol (Mann), in the absence (A) and presence (B) of 10 μmol l⁻¹ adrenaline (Adr). After stabilization for 60 min at 0.5 Hz and 15°C, two strips of four from each heart (N=10) received Glu and two Mann. Horizontal bars at time zero set the references for relative force development. The horizontal lines above the bars indicate either the period of severe hypoxia (N₂) or the presence of different compounds. After 15 min, severe hypoxia was imposed by replacing O₂ with N₂. After 10 min of hypoxia, one of the two strips with Glu and Mann was exposed to Adr. Results are means ± s.e.m. Significant differences (P<0.05, one-way ANOVA with Tukey's test) occurred between ventricle strips receiving Glu and Mann and varied with time. (C) Normalized twitch force of oxygenated ventricle strips serving as controls (N=4).

Fig. 3.

Normalized twitch force in response to increasing adrenaline (Adr) concentrations in the presence of various compounds during reoxygenation after severe hypoxia. Horizontal bars at time zero set the references for relative force development. The horizontal lines above the bars indicate either the period of severe hypoxia (N₂) or the presence of different compounds. Four strips from each ventricle of rainbow trout (N=6) were stimulated at 0.5 Hz and subjected to 60 min of severe hypoxia (N₂) at 15°C. Five minutes before reoxygenation, preparations received exogenous substrate [i.e. 5 or 10 mmol l⁻¹ glucose (Glu)], or 5 mmol l⁻¹ mannitol (Mann). The fourth strip (not shown) received 5 mmol l⁻¹ l-glucose (N=4), 5 mmol l⁻¹ sucrose (N=1) or nothing (N=1). After 60 min of reoxygenation, Adr was added stepwise with stabilization of force at each concentration. (A) Effects of exogenous Glu (5 or 10 mmol l⁻¹) or Mann. (B) Twitch force response to Adr in the presence of Glu (5 or 10 mmol l⁻¹), or no substrate (Mann) and continuous oxygenation, but otherwise as in A (N=10). Results are means ± s.e.m. and demonstrate that exogenous Glu had no effect on contractile performance.

Fig. 4.

Post-hypoxia twitch force recovery with 5 mmol l⁻¹ glucose (Glu), or 2 mmol l⁻¹ butyrate (Buty), acetate (Ac) or octanoate (Oct). Four ventricle strips were prepared from each of six hearts and stimulated at 0.5 Hz. Horizontal bars at time zero set the references for relative force development. The period of severe hypoxia (N₂) or the presence of different substrates is indicated by horizontal lines above the bars. After stabilization for 45–60 min, O₂ was replaced by N₂. After 55 min with N₂, the four exogenous substrates were given one to each strip (N=6). Five minutes later, N₂ was replaced by O₂ and strips remained under aerobic conditions for 60 min. Results are means ± s.e.m. *Significantly different from Glu and Ac (P<0.05, one-way ANOVA with Tukey's test).

Fig. 5.

Effects of increasing concentrations of 2-deoxyglucose (2-DG) on 2-DG uptake in isolated, non-contracting ventricle strips from rainbow trout. After a 60 min recovery period, excised cardiac tissue was rinsed for 10 min to remove extracellular glucose and then assayed for 2-DG uptake at 14°C for 20 min in medium containing 2 mmol l⁻¹ sodium pyruvate and the presence or absence of cytochalasin B (CB, 25 μmol l⁻¹, final concentration). Values are means ± s.e.m. for two to five ventricle strips per point. The absence of error bars indicates that s.e.m. fell with the symbol area.

Fig. 6.

Acute effects of temperature and cytochalasin B (CB) on 2-deoxyglucose (2-DG) uptake in isolated, non-contracting ventricle strips from rainbow trout. After a 60 min recovery period at 4, 14 or 24°C, excised cardiac tissue was rinsed for 10 min at the same temperature to remove extracellular glucose and then assayed simultaneously for 2-DG uptake at 4, 14 or 24°C for 20 min in medium containing 2 mmol l⁻¹ sodium pyruvate, in the presence or absence of CB (25 μmol l⁻¹, final concentration). Results are means ± s.e.m., N=6 for each group. Dissimilar letters and numbers denote significant differences in 2-DG uptake for ventricle strips in the absence or presence of CB, respectively (P<0.05, one-way ANOVA with Tukey's test).