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Computational analysis of Ca2+ dynamics in isolated cardiac mitochondria predicts two distinct modes of Ca2+ uptake

Journal of Physiology - 01 May 2014

Cardiac mitochondria can act as a significant Ca2+ sink and shape cytosolic Ca2+ signals affecting various cellular processes, such as energy metabolism and excitation–contraction coupling. However, different mitochondrial Ca2+ uptake mechanisms are still not well understood. In this study, we analysed recently published Ca2+ uptake experiments performed on isolated guinea pig cardiac mitochondria using a computer model of mitochondrial bioenergetics and cation handling. The model analyses of the data suggest that the majority of mitochondrial Ca2+ uptake, at physiological levels of cytosolic Ca2+ and Mg2+, occurs through a fast Ca2+ uptake pathway, which is neither the Ca2+ uniporter nor the rapid mode of Ca2+ uptake. This fast Ca2+ uptake component was explained by including a biophysical model of the ryanodine receptor (RyR) in the computer model. However, the Mg2+-dependent enhancement of the RyR adaptation was not evident in this RyR-type channel, in contrast to that of cardiac sarcoplasmic reticulum RyR. The extended computer model is corroborated by simulating an independent experimental dataset, featuring mitochondrial Ca2+ uptake, egress and sequestration. The model analyses of the two datasets validate the existence of two classes of Ca2+ buffers that comprise the mitochondrial Ca2+ sequestration system. The modelling study further indicates that the Ca2+ buffers respond differentially depending on the source of Ca2+ uptake. In particular, it suggests that the Class 1 Ca2+ buffering capacity is auto-regulated by the rate at which Ca2+ is taken up by mitochondria.

Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D-CFTR

Journal of Physiology - 01 May 2014

Cystic fibrosis (CF) is caused by dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). One strategy to restore function to CF mutants is to suppress defects in CFTR processing and function using revertant mutations. Here, we investigate the effects of the revertant mutations G550E and 4RK (the simultaneous disruption of four arginine-framed tripeptides (AFTs): R29K, R516K, R555K and R766K) on the CF mutant G551D, which impairs severely channel gating without altering protein processing and which affects a residue in the same α-helix as G550 and R555. Both G550E and 4RK augmented strongly CFTR-mediated iodide efflux from BHK cells expressing G551D-CFTR. To learn how revertant mutations influence G551D-CFTR function, we studied protein processing and single-channel behaviour. Neither G550E nor 4RK altered the expression and maturation of G551D-CFTR protein. By contrast, both revertants had marked effects on G551D-CFTR channel gating, increasing strongly opening frequency, while 4RK also diminished noticeably the duration of channel openings. Because G551D-CFTR channel gating is ATP independent, we investigated whether revertant mutations restore ATP dependence to G551D-CFTR. Like wild-type CFTR, the activity of 4RK-G551D-CFTR varied with ATP concentration, suggesting that 4RK confers some ATP dependence on the G551D-CFTR channel. Thus, the revertant mutations G550E and 4RK alter the gating pattern and ATP dependence of G551D-CFTR without restoring single-channel activity to wild-type levels. Based on their impact on the CF mutants F508del and G551D, we conclude that G550E and 4RK have direct effects on CFTR structure, but that their action on CFTR processing and channel function is CF mutation specific.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte

Journal of Physiology - 01 May 2014

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure–function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca2+ were measured. We observed the modulation of twitch force, but not of intracellular Ca2+, by both extracellular [Ca2+] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation–relaxation and force redevelopment kinetics by varied Ca2+ activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure–function relationships.

Genetic ablation of ryanodine receptor 2 phosphorylation at Ser-2808 aggravates Ca2+-dependent cardiomyopathy by exacerbating diastolic Ca2+ release

Journal of Physiology - 01 May 2014

Phosphorylation of the cardiac ryanodine receptor (RyR2) by protein kinase A (PKA) at Ser-2808 is suggested to mediate the physiological ‘fight or flight’ response and contribute to heart failure by rendering the sarcoplasmic reticulum (SR) leaky for Ca2+. In the present study, we examined the potential role of RyR2 phosphorylation at Ser-2808 in the progression of Ca2+-dependent cardiomyopathy (CCM) by using mice genetically modified to feature elevated SR Ca2+ leak while expressing RyR2s that cannot be phosphorylated at this site (S2808A). Surprisingly, rather than alleviating the disease phenotype, constitutive dephosphorylation of Ser-2808 aggravated CCM as manifested by shortened survival, deteriorated in vivo cardiac function, exacerbated SR Ca2+ leak and mitochondrial injury. Notably, the deteriorations of cardiac function, myocyte Ca2+ handling, and mitochondria integrity were consistently worse in mice with heterozygous ablation of Ser-2808 than in mice with complete ablation. Wild-type (WT) and CCM myocytes expressing unmutated RyR2s exhibited a high level of baseline phosphorylation at Ser-2808. Exposure of these CCM cells to protein phosphatase 1 caused a transitory increase in Ca2+ leak attributable to partial dephosphorylation of RyR2 tetramers at Ser-2808 from more fully phosphorylated state. Thus, exacerbated Ca2+ leak through partially dephosphorylated RyR2s accounts for the prevalence of the disease phenotype in the heterozygous S2808A CCM mice. These results do not support the importance of RyR2 hyperphosphorylation in Ca2+-dependent heart disease, and rather suggest roles for the opposite process, the RyR2 dephosphorylation at this residue in physiological and pathophysiological Ca2+ signalling.

Increase in cytosolic Ca2+ produced by hypoxia and other depolarizing stimuli activates a non-selective cation channel in chemoreceptor cells of rat carotid body

Journal of Physiology - 01 May 2014

The current model of O2 sensing by carotid body chemoreceptor (glomus) cells is that hypoxia inhibits the outward K+ current and causes cell depolarization, Ca2+ influx via voltage-dependent Ca2+ channels and a rise in intracellular [Ca2+] ([Ca2+]i). Here we show that hypoxia (<5% O2), in addition to inhibiting the two-pore domain K+ channels TASK-1/3 (TASK), indirectly activates an ~20 pS channel in isolated glomus cells. The 20 pS channel was permeable to K+, Na+ and Cs+ but not to Cl– or Ca2+. The 20 pS channel was not sensitive to voltage. Inhibition of TASK by external acid, depolarization of glomus cells with high external KCl (20 mm) or opening of the Ca2+ channel with FPL64176 activated the 20 pS channel when 1 mm Ca2+ was present in the external solution. Ca2+ (10 μm) applied to the cytosolic side of inside-out patches activated the 20 pS channel. The threshold [Ca2+]i for activation of the 20 pS channel in cell-attached patches was ~200 nm. The reversal potential of the 20 pS channel was estimated to be –28 mV. Our results reveal a sequential mechanism in which hypoxia (<5% O2) first inhibits the K+ conductance and then activates a Na+-permeable, non-selective cation channel via depolarization-induced rise in [Ca2+]i. Our results suggest that inhibition of K+ efflux and stimulation of Na+ influx both contribute to the depolarization of glomus cells during moderate to severe hypoxia.

Prophylactic erythropoietin exacerbates ventilation-induced lung inflammation and injury in preterm lambs

Journal of Physiology - 01 May 2014

Ventilation-induced lung injury (VILI) of preterm neonates probably contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Erythropoietin (EPO) has been suggested as a therapy for BPD. The aim of this study was to determine whether prophylactic administration of EPO reduces VILI in preterm newborn lambs. Lambs at 126 days of gestation (term is 147 days) were delivered and ventilated with a high tidal volume strategy for 15 min to cause lung injury, then received gentle ventilation until 2 h of age. Lambs were randomized to receive intravenous EPO (5000 IU kg–1: Vent+EPO; n = 6) or phosphate-buffered saline (Vent; n = 7) soon after birth: unventilated controls (UVC; n = 8) did not receive ventilation or any treatment. Physiological parameters were recorded throughout the experimental procedure. Samples of lung were collected for histological and molecular assessment of inflammation and injury. Samples of liver were collected to assess the systemic acute phase response. Vent+EPO lambs received higher F IO 2, P aO 2 and oxygenation during the first 10 min than Vent lambs. There were no differences in physiological indices beyond this time. Total lung injury score, airway wall thickness, inflammation and haemorrhage were higher in Vent+EPO lambs than in Vent lambs. Lung inflammation and early markers of lung and systemic injury were elevated in ventilated lambs relative to unventilated lambs; EPO administration further increased lung inflammation and markers of lung and systemic injury. Prophylactic EPO exacerbates VILI, which may increase the incidence and severity of long-term respiratory disease. More studies are required before EPO can be used for lung protection in preterm infants.

Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

Journal of Physiology - 01 May 2014

In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz, 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca2+]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P < 0.01, n = 9) and LIF (P < 0.01, n = 5) fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60–75% lower than in rested control fibres (P < 0.05), whereas subsarcolemmal glycogen was similar to control. Individual fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca2+]i and the reduction in intermyofibrillar (P = 0.051) and intramyofibrillar (P = 0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca2+]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool.

Short-term sustained hypoxia induces changes in the coupling of sympathetic and respiratory activities in rats

Journal of Physiology - 01 May 2014

Individuals experiencing sustained hypoxia (SH) exhibit adjustments in the respiratory and autonomic functions by neural mechanisms not yet elucidated. In the present study we evaluated the central mechanisms underpinning the SH-induced changes in the respiratory pattern and their impact on the sympathetic outflow. Using a decerebrated arterially perfused in situ preparation, we verified that juvenile rats exposed to SH (10% O2) for 24 h presented an active expiratory pattern, with increased abdominal, hypoglossal and vagal activities during late-expiration (late-E). SH also enhanced the activity of augmenting-expiratory neurones and depressed the activity of post-inspiratory neurones of the Bötzinger complex (BötC) by mechanisms not related to changes in their intrinsic electrophysiological properties. SH rats exhibited high thoracic sympathetic activity and arterial pressure levels associated with an augmented firing frequency of pre-sympathetic neurones of the rostral ventrolateral medulla (RVLM) during the late-E phase. The antagonism of ionotropic glutamatergic receptors in the BötC/RVLM abolished the late-E bursts in expiratory and sympathetic outputs of SH rats, indicating that glutamatergic inputs to the BötC/RVLM are essential for the changes in the expiratory and sympathetic coupling observed in SH rats. We also observed that the usually silent late-E neurones of the retrotrapezoid nucleus/parafacial respiratory group became active in SH rats, suggesting that this neuronal population may provide the excitatory drive essential to the emergence of active expiration and sympathetic overactivity. We conclude that short-term SH induces the activation of medullary expiratory neurones, which affects the pattern of expiratory motor activity and its coupling with sympathetic activity.

Characterization of the anisotropic mechanical behaviour of colonic tissues: experimental activity and constitutive formulation

Experimental Physiology - 01 May 2014

The aim was to investigate the biomechanical behaviour of colonic tissues by a coupled experimental and numerical approach. The wall of the colon is composed of different tissue layers. Within each layer, different fibre families are distributed according to specific spatial orientations, which lead to a strongly anisotropic configuration. Accounting for the complex histology of the tissues, mechanical tests must be planned and designed to evaluate the behaviour of the colonic wall in different directions. Uni-axial tensile tests were performed on tissue specimens from 15 fresh pig colons, accounting for six different loading directions (five specimens for each loading direction). The next step of the investigation was to define an appropriate constitutive framework and develop a procedure for identification of the constitutive parameters. A specific hyperelastic formulation was developed that accounted for the multilayered conformation of the colonic wall and the fibre-reinforced configuration of the tissues. The parameters were identified by inverse analyses of the mechanical tests. The comparison of model results with experimental data, together with the evaluation of satisfaction of material thermomechanics principles, confirmed the reliability of the analysis developed. This work forms the basis for more comprehensive activities that aim to provide computational tools for the interpretation of surgical procedures that involve the gastrointestinal tract, considering the specific biomedical devices adopted.

AltitudeOmics: effect of ascent and acclimatization to 5260 m on regional cerebral oxygen delivery

Experimental Physiology - 01 May 2014

Cerebral hypoxaemia associated with rapid ascent to high altitude can be life threatening; yet, with proper acclimatization, cerebral function can be maintained well enough for humans to thrive. We investigated adjustments in global and regional cerebral oxygen delivery (DO2) as 21 healthy volunteers rapidly ascended and acclimatized to 5260 m. Ultrasound indices of cerebral blood flow in internal carotid and vertebral arteries were measured at sea level, upon arrival at 5260 m (ALT1; atmospheric pressure 409 mmHg) and after 16 days of acclimatization (ALT16). Cerebral DO2 was calculated as the product of arterial oxygen content and flow in each respective artery and summed to estimate global cerebral blood flow. Vascular resistances were calculated as the quotient of mean arterial pressure and respective flows. Global cerebral blood flow increased by ~70% upon arrival at ALT1 (P < 0.001) and returned to sea-level values at ALT16 as a result of changes in cerebral vascular resistance. A reciprocal pattern in arterial oxygen content maintained global cerebral DO2 throughout acclimatization, although DO2 to the posterior cerebral circulation was increased by ~25% at ALT1 (P = 0.032). We conclude that cerebral DO2 is well maintained upon acute exposure and acclimatization to hypoxia, particularly in the posterior and inferior regions of the brain associated with vital homeostatic functions. This tight regulation of cerebral DO2 was achieved through integrated adjustments in local vascular resistances to alter cerebral perfusion during both acute and chronic exposure to hypoxia.

Intermittent and continuous high-intensity exercise training induce similar acute but different chronic muscle adaptations

Experimental Physiology - 01 May 2014

High-intensity interval training (HIIT) performed in an ‘all-out’ manner (e.g. repeated Wingate tests) is a time-efficient strategy to induce skeletal muscle remodelling towards a more oxidative phenotype. A fundamental question that remains unclear, however, is whether the intermittent or ‘pulsed’ nature of the stimulus is critical to the adaptive response. In study 1, we examined whether the activation of signalling cascades linked to mitochondrial biogenesis was dependent on the manner in which an acute high-intensity exercise stimulus was applied. Subjects performed either four 30 s Wingate tests interspersed with 4 min of rest (INT) or a bout of continuous exercise (CONT) that was matched for total work (67 ± 7 kJ) and which required ~4 min to complete as fast as possible. Both protocols elicited similar increases in markers of adenosine monophosphate-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase activation, as well as Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression (main effects for time, P ≤ 0.05). In study 2, we determined whether 6 weeks of the CONT protocol (3 days per week) would increase skeletal muscle mitochondrial content to a similar extent to what we have previously reported after 6 weeks of INT. Despite similar acute signalling responses to the CONT and INT protocols, training with CONT did not increase the maximal activity or protein content of a range of mitochondrial markers. However, peak oxygen uptake was higher after CONT training (from 45.7 ± 5.4 to 48.3 ± 6.5 ml kg–1 min–1; P < 0.05) and 250 kJ time trial performance was improved (from 26:32 ± 4:48 to 23:55 ± 4:16 min:s; P < 0.001) in our recreationally active participants. We conclude that the intermittent nature of the stimulus is important for maximizing skeletal muscle adaptations to low-volume, all-out HIIT. Despite the lack of skeletal muscle mitochondrial adaptations, our data show that a training programme based on a brief bout of high-intensity exercise, which lasted <10 min per session including warm-up, and performed three times per week for 6 weeks, improved peak oxygen uptake in young healthy subjects.

Introducing a rat model of prenatal androgen-induced polycystic ovary syndrome in adulthood

Experimental Physiology - 01 May 2014

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women, with a prevalence of 8–12% during the reproductive years. In the present study, using prenatal exposure to a single dose of testosterone during the critical period of fetal development, we aimed to introduce an enhanced rat model that would exhibit both endocrine and ovarian disturbances similar to PCOS, while maintaining normal reproductive system morphology in adulthood. Ten pregnant rats were injected s.c. with 5 mg free testosterone on gestational day 20, while control rats received only solvent. The development and function of the reproductive system in female offspring were examined in adulthood. Prenatally androgenized offspring had irregular oestrous cycles compared with control animals, and their anogenital and anovaginal distances were increased compared with control rats (P < 0.001). No significant differences were observed in the lengths of the vagina and clitoris or the number of nipples between the two groups. Levels of testosterone and luteinizing hormone and the luteinizing hormone/follicle-stimulating hormone ratio were increased in prenatally androgenized offspring compared with control animals (P < 0.05). The numbers of preantral and antral follicles in the ovaries of prenatally androgenized offspring were also increased compared with control rats (P = 0.07 and P < 0.01, respectively). The number of corpora lutea was decreased in prenatally androgenized offspring compared with control rats. Cystic follicles were observed in the ovaries of prenatally androgenized offspring. Prenatal exposure to a single dose of testosterone during the critical period of fetal development could facilitate the development a functional rat model of PCOS in adulthood, with minimal morphological disorders in the reproductive system.

A plasminogen activator inhibitor type 1 mutant retards diabetic nephropathy in db/db mice by protecting podocytes

Experimental Physiology - 01 May 2014

A mutant non-inhibiting plasminogen activator inhibitor type 1 (PAI-1), termed PAI-1R, which reduces endogenous PAI-1 activity, has been shown to inhibit albuminuria and reduce glomerulosclerosis in experimental diabetes. The mechanism of the reduction of albuminuria is unclear. This study sought to determine whether the administration of PAI-1R protected podocytes from injury directly, thereby reducing albuminuria in the db/db mouse, a model of type 2 diabetes. Untreated uninephrectomized db/db mice developed significant mesangial matrix expansion and albuminuria at week 22 of age, associated with segmental podocyte foot-process effacement, reduction of renal nephrin, podocin and zonula occludin-1 production and induction of renal desmin and B7-1 generation. In contrast, treatment with PAI-1R at 0.5 mg (kg body weight)–1 i.p., twice daily from week 20 to 22, reduced glomerular matrix accumulation, fibronectin and collagen production and albuminuria by 36, 62, 65 and 31%, respectively (P < 0.05), without affecting blood glucose level or body weight. Podocyte morphology and protein markers were also significantly attenuated by PAI-1R administration. Importantly, recombinant PAI-1 downregulated nephrin and zonula occludin-1 but increased desmin and B7-1 mRNA expression and protein production by podocytes in vitro, similar to the effects of transforming growth factor-β1. These observations provide evidence that PAI-1, in a manner similar to transforming growth factor-β1, directly induces podocyte injury, particularly in the setting of diabetes, where elevated PAI-1 may contribute to the progression of albuminuria. Reducing the increased PAI-1 activity by administration of PAI-1R, in fact, reduces podocyte injury, thereby reducing albuminuria. Therefore, PAI-1R provides an additional therapeutic effect in slowing the progression of diabetic nephropathy via the protection of podocytes.

Renal epithelial sodium channel is critical for blood pressure maintenance and sodium balance in the normal late pregnant rat

Experimental Physiology - 01 May 2014

Normal pregnancy is a state marked by avid sodium retention and plasma volume expansion. Insufficient plasma volume expansion results in the compromised maternal state of intrauterine growth restriction, which afflicts ~5% of all human pregnancies. We have recently shown that renal epithelial sodium channel (ENaC) activity in vivo in the late pregnant (LP) rat is increased. To determine the importance of the renal versus extrarenal ENaC in sodium retention and blood pressure regulation during pregnancy, we have chronically blocked the ENaC pharmacologically with daily subcutaneous injections of benzamil and genetically using intrarenal transfection of αENaC short hairpin RNA. Compared with untreated LP control animals, LP rats treated with benzamil retain less sodium and have reduced mean arterial blood pressure. Furthermore, LP rats treated with benzamil had lower maternal body weight gain. Intrarenal transfection of αENaC short hairpin RNA versus scrambled small RNA successfully decreased renal αENaC mRNA expression in LP rats. Intrarenal transfection of αENaC short hairpin RNA reduced maternal sodium retention, body weight gain and pup weight. Redundant physiological systems that protect blood pressure and sodium homeostasis were unable to compensate for the loss of ENaC activity in the pregnant rat. These findings demonstrate that the renal ENaC is necessary for maintaining pregnancy-mediated sodium retention, volume expansion and blood pressure regulation.

Testosterone potentiates the hypoxic ventilatory response of adult male rats subjected to neonatal stress

Experimental Physiology - 01 May 2014

Neonatal stress disrupts development of homeostatic systems. During adulthood, male rats subjected to neonatal maternal separation (NMS) are hypertensive and show a larger hypoxic ventilatory response (HVR), with greater respiratory instability during sleep. Neonatal stress also affects sex hormone secretion; hypoxia increases circulating testosterone of NMS (but not control) male rats. Given that these effects of NMS are not observed in females, we tested the hypothesis that testosterone elevation is necessary for the stress-related increase of the HVR in adult male rats. Pups subjected to NMS were placed in an incubator for 3 h per day from postnatal day 3 to 12. Control pups remained undisturbed. Rats were reared until adulthood, and the HVR was measured by plethysmography (fractional inspired O2 = 0.12, for 20 min). We used gonadectomy to evaluate the effects of reducing testosterone on the HVR. Gonadectomy had no effect on the HVR of control animals but reduced that of NMS animals below control levels. Immunohistochemistry was used to quantify androgen receptors in brainstem areas involved in the HVR. Androgen receptor expression was generally greater in NMS rats than in control rats; the most significant increase was noted in the caudal region of the nucleus tractus solitarii. We conclude that the abnormal regulation of testosterone is important in stress-related augmentation of the HVR. The greater number of androgen receptors within the brainstem may explain why NMS rats are more sensitive to testosterone withdrawal. Based on the similarities of the cardiorespiratory phenotype of NMS rats and patients suffering from sleep-disordered breathing, these results provide new insight into its pathophysiology, especially sex-based differences in its prevalence.

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