Journal of Physiology

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Species-dependent adaptation of the cardiac Na+/K+ pump kinetics to the intracellular Na+ concentration

15 December 2014

The Na+/K+ ATPase (NKA) plays a critical role in maintaining ionic homeostasis and dynamic function in cardiac myocytes, within both the in vivo cell and in silico models. Physiological conditions differ significantly between mammalian species. However, most existing formulations of NKA used to simulate cardiac function in computational models are derived from a broad range of experimental sources spanning many animal species. The resultant inability of these models to discern species-specific features is a significant obstacle to achieving a detailed quantitative and comparative understanding of physiological behaviour in different biological contexts. Here we present a framework for characterising the steady-state NKA current using a biophysical mechanistic model specifically designed to provide a mechanistic explanation of the NKA flux supported by self-consistent species-specific data. We thus compared NKA kinetics specific to guinea- pig and rat ventricular myocytes. We observe that the apparent binding affinity for sodium in the rat is significantly lower, whereas the overall pump cycle rate is doubled, in comparison to the guinea pig. This sensitivity of NKA to its regulatory substrates compensates for the differences in Na+ concentrations between the cell types. NKA is thereby maintained within its dynamic range over a wide range of pacing frequencies in these two species, despite significant disparities in sodium concentration. Hence, by replacing a conventional generic NKA model with our rat-specific NKA formula into a whole-cell simulation, we have, for the first time, been able to accurately reproduce the action potential duration and the steady-state sodium concentration as functions of pacing frequency.

Recruitment of G{beta}{gamma} controls the basal activity of G-protein coupled inwardly rectifying potassium (GIRK) channels: crucial role of distal C terminus of GIRK1

15 December 2014

The G-protein coupled inwardly rectifying potassium (GIRK, or Kir3) channels are important mediators of inhibitory neurotransmission via activation of G-protein coupled receptors (GPCRs). GIRK channels are tetramers comprising combinations of subunits (GIRK1–4), activated by direct binding of the Gβ subunit of Gi/o proteins. Heterologously expressed GIRK1/2 exhibit high, Gβ-dependent basal currents (Ibasal) and a modest activation by GPCR or coexpressed Gβ. Inversely, the GIRK2 homotetramers exhibit low Ibasal and strong activation by Gβ. The high Ibasal of GIRK1 seems to be associated with its unique distal C terminus (G1-dCT), which is not present in the other subunits. We investigated the role of G1-dCT using electrophysiological and fluorescence assays in Xenopus laevis oocytes and protein interaction assays. We show that expression of GIRK1/2 increases the plasma membrane level of coexpressed Gβ (a phenomenon we term ‘Gβ recruitment’) but not of coexpressed Gαi3. All GIRK1-containing channels, but not GIRK2 homomers, recruited Gβ to the plasma membrane. In biochemical assays, truncation of G1-dCT reduces the binding between the cytosolic parts of GIRK1 and Gβ, but not Gαi3. Nevertheless, the truncation of G1-dCT does not impair activation by Gβ. In fluorescently labelled homotetrameric GIRK1 channels and in the heterotetrameric GIRK1/2 channel, the truncation of G1-dCT abolishes Gβ recruitment and decreases Ibasal. Thus, we conclude that G1-dCT carries an essential role in Gβ recruitment by GIRK1 and, consequently, in determining its high basal activity. Our results indicate that G1-dCT is a crucial part of a Gβ anchoring site of GIRK1-containing channels, spatially and functionally distinct from the site of channel activation by Gβ.

Vitamin C and E supplementation alters protein signalling after a strength training session, but not muscle growth during 10 weeks of training

15 December 2014

This study investigated the effects of vitamin C and E supplementation on acute responses and adaptations to strength training. Thirty-two recreationally strength-trained men and women were randomly allocated to receive a vitamin C and E supplement (1000 mg day–1 and 235 mg day–1, respectively), or a placebo, for 10 weeks. During this period the participants’ training involved heavy-load resistance exercise four times per week. Muscle biopsies from m. vastus lateralis were collected, and 1 repetition maximum (1RM) and maximal isometric voluntary contraction force, body composition (dual-energy X-ray absorptiometry), and muscle cross-sectional area (magnetic resonance imaging) were measured before and after the intervention. Furthermore, the cellular responses to a single exercise session were assessed midway in the training period by measurements of muscle protein fractional synthetic rate and phosphorylation of several hypertrophic signalling proteins. Muscle biopsies were obtained from m. vastus lateralis twice before, and 100 and 150 min after, the exercise session (4 x 8RM, leg press and knee-extension). The supplementation did not affect the increase in muscle mass or the acute change in protein synthesis, but it hampered certain strength increases (biceps curl). Moreover, increased phosphorylation of p38 mitogen-activated protein kinase, Extracellular signal-regulated protein kinases 1 and 2 and p70S6 kinase after the exercise session was blunted by vitamin C and E supplementation. The total ubiquitination levels after the exercise session, however, were lower with vitamin C and E than placebo. We concluded that vitamin C and E supplementation interfered with the acute cellular response to heavy-load resistance exercise and demonstrated tentative long-term negative effects on adaptation to strength training.

Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity

15 December 2014

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca2+ channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

Low birth weight followed by postnatal over-nutrition in the guinea pig exposes a predominant player in the development of vascular dysfunction

15 December 2014

The association between intrauterine growth restriction (IUGR) and hypertension is well established, yet the interaction between IUGR and other pathogenic contributors remains ill-defined. This study examined the independent and interactive effects of fetal growth reduction resulting in low birth weight (LBW), and postnatal Western diet (WD) on vascular function. Growth reduction was induced in pregnant guinea pigs by uterine artery ablation. LBW and normal birth weight (NBW) offspring were randomly assigned to a control diet (CD) or a WD. In young adulthood, length–tension curves were generated in aortic rings and responses to methacholine (MCh) were evaluated in the carotid and aorta using wire myography. Relative to NBW/CD, aortae of NBW/WD offspring were stiffer, as determined by a leftward shift in the length–tension curve, yet the shift in the LBW/CD curve was considerably greater. Aortic stiffening was most severe in LBW/WD (slope: NBW/CD, 1.97 ± 0.04; NBW/WD, 2.16 ± 0.04; LBW/CD, 2.28 ± 0.05; LBW/WD, 2.34 ± 0.07). Maximal responses (Emax) to MCh were significantly blunted in the aorta of LBW/CD vs. NBW/CD (P < 0.05) and in LBW/WD vs. NBW/WD offspring (P < 0.05); but WD alone had no influence on MCh responses. Emax values for carotid responses to MCh were reduced in LBW/CD vs. NBW/CD (P < 0.05). Thus, aortic stiffening was influenced more by LBW than by a postnatal WD and the most severe stiffening was observed in LBW/WD offspring. In contrast, blunted endothelial responses in LBW/CD offspring were not exacerbated by WD. IUGR may have a greater independent impact on vascular function than a postnatal WD.

{beta}2-Adrenergic stimulation enhances Ca2+ release and contractile properties of skeletal muscles, and counteracts exercise-induced reductions in Na+-K+-ATPase Vmax in trained men

15 December 2014

The aim of the present study was to examine the effect of β2-adrenergic stimulation on skeletal muscle contractile properties, sarcoplasmic reticulum (SR) rates of Ca2+ release and uptake, and Na+–K+-ATPase activity before and after fatiguing exercise in trained men. The study consisted of two experiments (EXP1, n = 10 males, EXP2, n = 20 males), where β2-adrenoceptor agonist (terbutaline) or placebo was randomly administered in double-blinded crossover designs. In EXP1, maximal voluntary isometric contraction (MVC) of m. quadriceps was measured, followed by exercise to fatigue at 120% of maximal oxygen uptake (VO2, max ). A muscle biopsy was taken after MVC (non-fatigue) and at time of fatigue. In EXP2, contractile properties of m. quadriceps were measured with electrical stimulations before (non-fatigue) and after two fatiguing 45 s sprints. Non-fatigued MVCs were 6 ± 3 and 6 ± 2% higher (P < 0.05) with terbutaline than placebo in EXP1 and EXP2, respectively. Furthermore, peak twitch force was 11 ± 7% higher (P < 0.01) with terbutaline than placebo at non-fatigue. After sprints, MVC declined (P < 0.05) to the same levels with terbutaline as placebo, whereas peak twitch force was lower (P < 0.05) and half-relaxation time was prolonged (P < 0.05) with terbutaline. Rates of SR Ca2+ release and uptake at 400 nm [Ca2+] were 15 ± 5 and 14 ± 5% (P < 0.05) higher, respectively, with terbutaline than placebo at non-fatigue, but declined (P < 0.05) to similar levels at time of fatigue. Na+–K+-ATPase activity was unaffected by terbutaline compared with placebo at non-fatigue, but terbutaline counteracted exercise-induced reductions in maximum rate of activity (Vmax) at time of fatigue. In conclusion, increased contractile force induced by β2-adrenergic stimulation is associated with enhanced rate of Ca2+ release in humans. While β2-adrenergic stimulation elicits positive inotropic and lusitropic effects on non-fatigued m. quadriceps, these effects are blunted when muscles fatigue.

Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by {beta}2-adrenoceptor stimulation

15 December 2014

The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling.

The determinants of transverse tubular volume in resting skeletal muscle

15 December 2014

The transverse tubular (t)-system of skeletal muscle couples sarcolemmal electrical excitation with contraction deep within the fibre. Exercise, pathology and the composition of the extracellular fluid (ECF) can alter t-system volume (t-volume). T-volume changes are thought to contribute to fatigue, rhabdomyolysis and disruption of excitation–contraction coupling. However, mechanisms that underlie t-volume changes are poorly understood. A multicompartment, history-independent computer model of rat skeletal muscle was developed to define the minimum conditions for t-volume stability. It was found that the t-system tends to swell due to net ionic fluxes from the ECF across the access resistance. However, a stable t-volume is possible when this is offset by a net efflux from the t-system to the cell and thence to the ECF, forming a net ion cycle ECF->t-system->sarcoplasm->ECF that ultimately depends on Na+/K+-ATPase activity. Membrane properties that maximize this circuit flux decrease t-volume, including PNa(t) > PNa(s), PK(t) < PK(s) and N(t) < N(s) [P, permeability; N, Na+/K+-ATPase density; (t), t-system membrane; (s), sarcolemma]. Hydrostatic pressures, fixed charges and/or osmoles in the t-system can influence the magnitude of t-volume changes that result from alterations in this circuit flux. Using a parameter set derived from literature values where possible, this novel theory of t-volume was tested against data from previous experiments where t-volume was measured during manipulations of ECF composition. Predicted t-volume changes correlated satisfactorily. The present work provides a robust, unifying theoretical framework for understanding the determinants of t-volume.

The effects of dexamethasone on post-asphyxial cerebral oxygenation in the preterm fetal sheep

15 December 2014

Exposure to clinical doses of the glucocorticoid dexamethasone increases brain activity and causes seizures in normoxic preterm fetal sheep without causing brain injury. In contrast, the same treatment after asphyxia increased brain injury. We hypothesised that increased injury was in part mediated by a mismatch between oxygen demand and oxygen supply. In preterm fetal sheep at 0.7 gestation we measured cerebral oxygenation using near-infrared spectroscopy, electroencephalographic (EEG) activity, and carotid blood flow (CaBF) from 24 h before until 72 h after asphyxia induced by 25 min of umbilical cord occlusion. Ewes received dexamethasone intramuscularly (12 mg 3 ml–1) or saline 15 min after the end of asphyxia. Fetuses were studied for 3 days after occlusion. During the first 6 h of recovery after asphyxia, dexamethasone treatment was associated with a significantly greater fall in CaBF (P < 0.05), increased carotid vascular resistance (P < 0.001) and a greater fall in cerebral oxygenation as measured by the difference between oxygenated and deoxygenated haemoglobin (delta haemoglobin; P < 0.05). EEG activity was similarly suppressed in both groups. From 6 to 10 h onward, dexamethasone treatment was associated with a return of CaBF to saline control levels, increased EEG power (P < 0.005), greater epileptiform transient activity (P < 0.001), increased oxidised cytochrome oxidase (P < 0.05) and an attenuated increase in [delta haemoglobin] (P < 0.05). In conclusion, dexamethasone treatment after asphyxia is associated with greater hypoperfusion in the critical latent phase, leading to impaired intracerebral oxygenation that may exacerbate neural injury after asphyxia.

Influence of high altitude on cerebral blood flow and fuel utilization during exercise and recovery

15 December 2014

We examined the hypotheses that: (1) during incremental exercise and recovery following 4–6 days at high altitude (HA) global cerebral blood flow (gCBF) increases to preserve cerebral oxygen delivery (CDO2) in excess of that required by an increasing cerebral metabolic rate of oxygen ( CM RO2); (2) the trans-cerebral exchange of oxygen vs. carbohydrates (OCI; carbohydrates = glucose + 1/2lactate) would be similar during exercise and recovery at HA and sea level (SL). Global CBF, intra-cranial arterial blood velocities, extra-cranial blood flows, and arterial–jugular venous substrate differences were measured during progressive steady-state exercise (20, 40, 60, 80, 100% maximum workload (Wmax)) and through 30 min of recovery. Measurements (n = 8) were made at SL and following partial acclimatization to 5050 m. At HA, absolute Wmax was reduced by ~50%. During submaximal exercise workloads (20–60% Wmax), despite an elevated absolute gCBF (~20%, P < 0.05) the relative increases in gCBF were not different at HA and SL. In contrast, gCBF was elevated at HA compared with SL during 80 and 100% Wmax and recovery. Notwithstanding a maintained CDO2 and elevated absolute CM RO2 at HA compared with SL, the relative increase in CM RO2 was similar during 20–80% Wmax but half that of the SL response (i.e. 17 vs. 27%; P < 0.05 vs. SL) at 100% Wmax. The OCI was reduced at HA compared with SL during 20, 40, and 60% Wmax but comparable at 80 and 100% Wmax. At HA, OCI returned almost immediately to baseline values during recovery, whereas at SL it remained below baseline. In conclusion, the elevations in gCBF during exercise and recovery at HA serve to maintain CDO2. Despite adequate CDO2 at HA the brain appears to increase non-oxidative metabolism during exercise and recovery.

Rebuttal from David C. Poole

01 December 2014