Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Effect of racing on cardiac troponin I concentration and associations with cardiac rhythm disturbances in Standardbred racehorses.

INTRODUCTION/OBJECTIVES: Accumulating evidence indicates intense exercise can be associated with myocardial damage. Investigating the impact of maximal effort on myocardium and exploring possible association of injury with rhythm disturbance requires a high-sensitivity cardiac troponin assay. The objectives of this study were: (1) to determine the effect of racing on serum cardiac troponin I (cTnI) in Standardbred horses using a high-sensitivity assay; (2) to determine the 99th percentile of cTnI in healthy horses and investigate the effect of demographic variables on cTnI prevailing pre-race in Standardbred horses using a validated high-sensitivity assay and a contemporary assay, and; (3) to explore associations between exercise-associated arrhythmia and cTnI concentration. ANIMALS: Racehorses (n = 145). MATERIALS AND METHODS: ≤ 2 h pre-race, cTnI concentrations were measured in 158 race starts. Electrocardiogram (ECG) monitoring was applied during racing and race recovery and screened for complex ventricular arrhythmia. Associations between cTnI prevailing before racing concentration, age, sex, and gait were investigated. Demographic and performance variables were evaluated for associations with cTnI concentration post-race and rhythm disturbance. RESULTS: Incidence of arrhythmia was 11.6% (16 horses). A significant increase in median (interquartile range) cTnI concentration of 1.36 (0.49-2.81) ng/L was found post-race (p < 0.0001). Serum cardiac troponin I (cTnI) concentration prevailing pre-race was positively associated with increasing age, and gait. Serum cardiac troponin I prevailing post-race was positively associated with concentration prevailing pre-race. Interaction between arrhythmia and finishing distanced revealed horses finishing distanced and experiencing arrhythmia displayed higher cTnI release than with the presence of either alone. CONCLUSIONS: Racing increased cTnI concentration. Horses finishing distanced and also exhibiting arrhythmia may be experiencing myocardial compromise.

Ontario Racehorse Death Registry, 2003-2015: Descriptive analysis and rates of mortality.

BACKGROUND: The Province of Ontario maintains a registry of racehorse deaths occurring within 60 days of a race or trial entry that provides insight into mortality rates and costs of competition. OBJECTIVES: To characterise and quantify mortality and identify breed differences. STUDY DESIGN: Retrospective annualised cohort study. METHODS: The Ontario Death Registry for 2003-2015, containing 1713 cases, was audited and information on the relationship between death and official work added. Race and trial data from industry performance databases were used to determine mortality rates according to breed, year, age, sex and circumstances of death. RESULTS: Breed differences in mortality rate and individual risk were found. Thoroughbreds (Tb) had the greatest exercise-associated mortality (EAM) rate and risk by all measures (2.27 deaths/1000 race starts, 0.95-1.0% annual individual risk), followed by Quarter horses (Qh, 1.49, 0.60-0.69%). Rate and risk were lowest for Standardbreds (Sb, 0.28, 0.23-0.24%). Nonexercise annual individual risk was highest for the Sb (0.45%, vs. Tb 0.33%, and Qh 0.32%). Pattern and type of EAM mirrored the characteristics of competitive activity in each industry, with high Tb and Qh mortality being associated with exercise and involving musculoskeletal injuries, dying suddenly and accidents. Low Sb EAM reflected the more extensive nature of training preparation and racing for this breed. MAIN LIMITATIONS: Available data provided no information on morbidity, mortality beyond the 60-day horizon or for horses not racing. Numbers for the Qh were low. CONCLUSIONS: Race-intensity exercise is clearly hazardous for horses, with hazards varying widely between breeds and showing parallels with industry cultural and management norms. Breed differences provide insights concerning strategies that could reduce mortality, while improving welfare and reducing costs of participation. For all breeds, musculoskeletal injury was the major contributing cause of mortality.

Post-exercise cardiac troponin I release and clearance in normal Standardbred racehorses.

BACKGROUND: There are currently no studies detailing cardiac troponin I (cTnI) release in normal horses post-exercise using an analytically validated assay. These data are essential for selecting appropriate sampling times in equine athletes with suspected myocardial injury. OBJECTIVE: To plot the magnitude and time course of cTnI release after maximal effort, using validated cTnI assays. STUDY DESIGN: Descriptive longitudinal study. METHODS: Five clinically normal Standardbred racehorses in race training were included in the study. Horses were exercised in harness at near-race intensity. Blood samples were taken immediately pre- and post-exercise and then hourly for 24 h. Samples were analysed using the validated high-sensitivity cTnI assay and a contemporary sensitivity cTnI assay. RESULTS: Mean resting cTnI was 1.33 ± 0.6 s.d. ng/L (range, 0.82-2.33 ng/L) using assay A. All horses were below the detection limit at rest using assay B. Peak elevation occurred 2-6 h post-exercise with both assays (mean, 4.6 ± 1.7 and 4.0 ± 2 h, respectively). Mean peak increase in cTnI was 11.96 ± 9.41 ng/L (range, 1.72-23.76 ng/L) using assay A. Peak concentrations were detectable in three of the horses using assay B and were between 0.039 and 0.051 µg/L (mean: 0.043 ± 0.006 µg/L). All horses returned to baseline within 24 h. MAIN LIMITATIONS: A small (n = 5) convenience sample was used as random sampling was not logistically possible. CONCLUSIONS: All horses experienced an increase in cTnI post-exercise, with peak occurring 2-6 h post-exercise. Cardiac troponin I elevation was detected earlier using the high-sensitivity assay, which may convey a diagnostic advantage. Targeted studies are needed to determine the significance of these increases.

Developing neurites from mouse basal forebrain gonadotropin-releasing hormone neurons use Sonic hedgehog to modulate their growth.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. GnRH neuron cell bodies reside in the basal forebrain, and most extend long neurites in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. Using in vitro neurite growth assays, histological methods, and genetic deletion strategies in mice we have analysed the role of the morphogen and neurite growth and guidance molecule, Sonic hedgehog (Shh), in the growth of GnRH neurites to their target. Immunohistochemistry revealed that Shh was present in the basal forebrain, the preoptic area (POA) and mediobasal hypothalamus (MBH) at gestational day 14.5 (GD 14.5), a time when GnRH neurites grow towards the ME. Furthermore, in situ hybridization revealed that mRNA encoding the Shh receptor, Smoothened (Smo), was present in GnRH neurons from GD 15.5, when the first GnRH neurites are extending towards the MBH. In vitro neurite growth assays using hypothalamic explants from GD 15.5 fetuses in 3-D collagen gels showed that Shh was able to significantly stimulate GnRH neurite outgrowth. Finally, genetic deletion of Smo specifically from GnRH neurons in vivo, using Cre-loxP technology, resulted in a significant decrease in GnRH neurites innervating the ME. These experiments demonstrate that GnRH neurites use Shh for their neurite development, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional hypothalamic neuronal population for which Shh/Smo signaling is developmentally important.

Estimates of heritability of atrial fibrillation in the Standardbred racehorse.

BACKGROUND: The number of Standardbred racehorses admitted to the Ontario Veterinary College Teaching Hospital (Guelph, Canada) for treatment of atrial fibrillation (AF) has been on the rise since the early 1990s. A small number of sires have been contributing to a large proportion of cases, indicating there may be a genetic predisposition to the arrhythmia in this breed. OBJECTIVES: The objectives of this study were to determine the heritability of AF in Standardbred horses and whether heritability of the arrhythmia differs across gaits and/or sexes. STUDY DESIGN: Heritability study based on retrospective review of clinical records and publicly available pedigree and racing records. METHODS: Standardbred horses admitted to hospital for treatment of AF that were born between 1978 and 2007 comprised the affected case population (n = 204). Five randomly selected racing contemporaries for each case, assumed to not suffer from the arrhythmia, comprised the control population (n = 1017). Racing contemporaries were identified by examining the race records of affected horses within the 6 months prior to their admission, and randomly selecting sex- and gait-matched horses from these races. Heritability was estimated from the sampled horses as a whole (n = 1221), as well as for both sexes and gaits, using a generalised linear mixed model. RESULTS: Heritability of AF on the underlying liability scale was estimated to be (±s.e.) 0.30±0.04 in the entire data set; 0.30±0.06 in males; 0.24±0.08 in females; and 0.32±0.05 in pacers. After conversion to the observed scale, heritability estimates were 0.14, 0.15, 0.09 and 0.15, respectively. MAIN LIMITATIONS: There were insufficient data to estimate heritability of AF for trotters. CONCLUSIONS: Modest heritability estimates were found for AF in the Standardbred horse, particularly in males and pacers, which support the hypothesis that there is a genetic contribution to the arrhythmia in this breed. The Summary is available in Chinese - See Supporting Information.

Associations of acute stress and overnight heart rate with feed efficiency in beef heifers.

Proxies have the potential to accelerate feed efficiency (residual feed intake (RFI); kg dry matter/day) improvement, assisting with the reduction of beef cattle feed costs and environmental impact. Heart rate (HR) (beats per minute (BPM)) is associated with feed efficiency and influenced by autonomic activity and peripheral metabolism, suggesting HR could be used as a proxy for feed efficiency. Objectives were to assess associations between overnight HR, lying patterns and RFI, and between acute stress HR and RFI. Heifer calves (n=107; 408±28 days of age, 341±42.2 kg) and yearling heifers (n=36; 604±92 days of age, 539±52.2 kg) were exposed to a performance test to determine productive performance. Overnight HR (electrode based) and lying patterns (accelerometer based) were monitored on a subgroup of heifer calves (n=40; 20 lowest RFI; 20 highest RFI). In the 10-min acute stress assessment, all heifers were individually exposed to the opening and closing of an umbrella and HR before (HRBEF), in response to (HRMAX), after (HRAFT) and change (HRCHG; HRAFT-HRBEF) as a result of exposure were determined. Using polynomial regression, rate of HR decrease pre-exposure (ß 1) and rates of HR increase (ß 2) and decrease (ß 3, ß 4) post-exposure were determined. Heifer calves in the overnight assessment were classified into equal RFI groups (low RFI; high RFI) and HR means were treated as repeated measures and compared using multiple regression. In the acute stress assessment, heifers were classified within cattle category into equal RFI groups (low RFI; high RFI) and means and polynomial regression parameters were compared using multiple regression. Low-RFI heifer calves had a lower overnight HR (69.2 v. 72.6 BPM), similar HR change from lying to standing intervals (8.9 v. 9.2 BPM) and similar time lying (61.1% v. 64.5%) compared with high-RFI heifer calves. Low-RFI heifer calves had a higher absolute HRMAX (162.9 v. 145.7 BPM) and ß 2 (-0.34 v. -0.20) than high-RFI heifer calves. Low-RFI yearling heifers had similar acute stress HR means and a lower ß 1 (0.003 v. 0.006) than high-RFI yearling heifers. Overnight HR and acute stress HR are potential indicators of RFI in heifer calves. However, acute stress HR results varied in yearling heifers, suggesting previous handling experience and/or age influence stress response. Pending further development (predictive ability, repeatability), the acute stress assessment could have potential for on-farm application as a feed efficiency proxy in young heifers with minimal handling experience.

Lifelong exercise and locally produced insulin-like growth factor-1 (IGF-1) have a modest influence on reducing age-related muscle wasting in mice.

The age-related loss of skeletal muscle mass and function is termed sarcopenia and has been attributed to a decline in concentrations of insulin-like growth factor-1 (IGF-1). We hypothesized that constitutively expressed IGF-1 within skeletal muscles with or without exercise would prevent sarcopenia. Male transgenic mice that overexpress IGF-1 Ea in skeletal muscles were compared with wild-type littermates. Four-month-old mice were assigned to be sedentary, or had access to free-running wheels, until 18 or 28 months of age. In wild-type mice, the mass of the quadriceps muscles was reduced at 28 months and exercise prevented such loss, without affecting the diameter of myofibers. Conversely, increased IGF-1 alone was ineffective, whereas the combination of exercise and IGF-1 was additive in maintaining the diameter of myofibers in the quadriceps muscles. For other muscles, the combination of IGF-1 and exercise was variable and either increased or decreased the mass at 18 months of age, but was ineffective thereafter. Despite an increase in the diameter of myofibers, grip strength was not improved. In conclusion, our data show that exercise and IGF-1 have a modest effect on reducing aged-related wasting of skeletal muscle, but that there is no improvement in muscle function when assessed by grip strength.

Troponin assays in the assessment of the equine myocardium.

In 2000, troponin assays were adopted as the test of choice for detection of myocardial injury in man. This decision was made after extensive testing and followed a 60 year search for a biomarker of myocardial damage with sufficient analytical sensitivity and specificity. This has led to proliferation of assays for use in human medicine, each requiring extensive testing and validation before it could be made available on the open market for human use. The search for ever-more analytically sensitive assays and for a standard reference material continues. The adoption of troponin testing in veterinary medicine followed shortly after its development for use in man, providing a much-needed means of detecting and monitoring myocardial damage in horses. However, application of these tests in veterinary medicine has exclusively involved use of assays designed for and clinically validated in human patients. There is no mandated requirement for test validation in veterinary medicine and, while many of these assays have been shown to be capable of detecting equine troponin, the wide diversity of available tests, lack of validation, absence of protocols for their use and lack of standardisation make their application problematic. The objective of this review article is to address this issue, offering guidance where data are available and encouraging caution where there are none. Ultimately, the overall goal of this review is to examine critically the use of troponin assays in the horse and to promote the accurate and appropriate interpretation of valid results.

Ventricular arrhythmias during race recovery in Standardbred Racehorses and associations with autonomic activity.

BACKGROUND: A lack of information on normal heart rhythm at maximal effort hampers investigation of poor performance and sudden death in Standardbred racing. HYPOTHESIS/OBJECTIVE: To characterize rhythm variations during scheduled racing in clinically normal Standardbred horses. ANIMALS: Two hundred and eighty-eight Standardbred horses competing in 40 scheduled races at a Southern Ontario racetrack. METHODS: Observational study, convenience sampling: Heart rhythm was monitored by ECG from harnessing to postrace recovery and assessed visually and by examining heart rate intervals. Rhythm variations were used as response variables in multivariate analysis of race records detailing signalment, race, and race outcome. RESULTS: Monitoring involved 345 individual horse or race events. Occasional, isolated premature cycles, only, occurred during the race. Postrace, sudden cardiac slowing (punctuated deceleration [PD]) appeared in 42 events (12.2%). Only premature ventricular complexes were exhibited in 40 events postrace (11.6%), whereas 55 (15.9%) exhibited complex ventricular arrhythmias (CVA) including torsades-like polymorphic ventricular tachycardia, 34.5% of these being closely associated with PD (odds ratio = 8). Predispositions to CVA were found for horses parked at the 1/2 mile (odds ratio = 3), and trotters breaking in the stretch (odds ratio = 38). Horses spontaneously reverted to sinus rhythm and no sudden death events were encountered. CONCLUSIONS AND CLINICAL IMPORTANCE: Arrhythmias occur frequently in racing Standardbreds during cardiac deceleration, often associated with sudden, rapid increases in vagal tone. Circumstances imposing unusual demand and racing at the trot appear to predispose. Findings provide insight into possible mechanisms of sudden death.

Ocular tear film bubble formation after breath-hold diving.

Bubbles in the ocular tear film have been observed following both dry-chamber, simulated compressed air dives and in-water, recreational compressed air dives. The current paper reports on the formation of tear film bubbles in a breath-hold diver following repeated, extended breath-hold excursions to a maximum depth of -28.5 m. It is believed that this is the first time that ocular tear film bubbles have been reported in breath-hold divers.

Transvenous electrical cardioversion of equine atrial fibrillation: patient factors and clinical results in 72 treatment episodes.

BACKGROUND: Transvenous electrical cardioversion (TVEC) has been developed for treatment of atrial fibrillation (AF) in horses. The relationship among patient variables, treatment response, and outcome in a typical referral population has not been evaluated. HYPOTHESIS: Patient variables such as age, sex, weight, and duration of arrhythmia affect prognosis for response to treatment and the energy level at which cardioversion occurs. ANIMALS: TVEC was applied to 72 episodes of lone AF in 63 client-owned performance horses, with the majority (54) being Standardbred racehorses. METHODS: Catheterization of the right atrium (RA) and pulmonary artery (PA) through the jugular vein was used for electrode placement before horses were placed under general anesthesia. Biphasic, truncated exponential shock waves were delivered at incremental energy levels until cardioversion was achieved or a maximum single-energy level of 300 J was reached (cumulative energy 50-1,960 J). A multivariate model was constructed to evaluate influence of patient factors on cardioversion energy. RESULTS: Cardioversion was achieved in 71 of 72 episodes (62 of 63 horses) at a mean energy of 165.43 +/- 8.75 J. Cardioversion energy was higher for females than for males, and for interaction terms, weight was negatively related to energy in females and positively related in males. Age was positively related to cardioversion energy in females. No relationship was identified between duration of arrhythmia before treatment and prognosis for response or cardioversion energy. CONCLUSIONS AND CLINICAL IMPORTANCE: TVEC is highly effective in the treatment of lone AF in horses. Although age and sex influence cardioversion energy level, duration of arrhythmia does not.

Prevalence and severity of external auditory exostoses in breath-hold divers.

OBJECTIVE: To explore the prevalence and severity of external auditory exostoses in a population of experienced breath-hold divers, and to compare these to the same parameters within surfing and self-contained underwater breathing apparatus diving populations. DESIGN: A stepwise, multiple regression analysis of cross-sectional data examining the relative contributions of sea surface temperature, latitude of exposure and years of exposure to the prevalence and severity of stenosis due to external auditory exostoses. A chi-square analysis of the prevalence and severity of external auditory exostosis stenosis in the breath-hold divers was compared with previously published data for surfers and self-contained underwater breathing apparatus divers. SUBJECTS: Seventy-six male and thirty-five female breath-hold divers attending an international 'freedive' competition completed a questionnaire describing aquatic sports habits, geography of participation and symptomatology. Those completing the questionnaire (111/154 attendees) were examined otoscopically for evidence of external auditory exostoses. Images were digitally recorded, scored and graded. RESULTS: Exostoses were evident in 87.7 per cent of the 204 ears scored and graded for severity of stenosis due to external auditory exostoses. The prevalence of exostoses was no different from that found in previous studies of surfers and self-contained underwater breathing apparatus divers (p = 0.101). However, the pattern of affliction was more similar to that found in surfers. The severity of exostoses was significantly less than that found in surfing populations (p < or = 0.001 to 0.007), but greater than that found in self-contained underwater breathing apparatus diving populations (p < or = 0.001). Sea surface temperature at the location of open-water exposure was the most significant predictor of the prevalence and severity of external auditory exostoses in breath-hold divers (p = 0.019). CONCLUSION: The prevalence and severity patterns of stenosis due to external auditory exostoses in breath-hold divers are more similar to previously published results for surfing populations than to previously published results for self-contained underwater breathing apparatus diving populations. In breath-hold divers, sea surface temperature is the strongest predictor of severity of stenosis due to external auditory exostoses.

Effects of sodium bicarbonate on voluntary face immersion breath-hold times.

The purpose of this study was to examine the effects of orally ingested sodium bicarbonate (NaHCO3) on voluntary breath-hold time with facial immersion. Eight non-elite breath-hold divers aged 20-41 (26.75 +/- 7.83 years) were recruited to undertake two bouts of three monitored, facial-immersion breath-holds, one week apart. Subjects were randomly allocated to double-blind sessions of ingesting an experimental (0.3 g x kg(-1) NaHCO3) or placebo (8 g NaCl) solution. Heart rate, pre- and post-breath-hold end-tidal gases, peripheral oxygen saturation (SpO2) at right index finger and maximum breath-hold times (Max-BHT) were recorded. Max-BHT was established during the third breath-hold in both trials. Max-BHT of 168.2 (+/- 20.7) sec and 145.7 (+/- 21.4) see were recorded for the experimental and placebo conditions, respectively (P = 0.019). Average bradycardic response did not differ between the experimental and placebo trials (P = 0.166). Whilst there was a significant difference in post-breath-hold end-tidal oxygen (P(ET) O2), one was not noted in end-tidal carbon dioxide (P(ET) CO2) measures (P = 0.048, and; P = 0.530, respectively). There was significant difference in lowest recorded SpO2 (P = 0.008) between the trials. It is suggested that ingestion of NaHCO3, prior to facially immersed breath-holds, has an ergogenic effect, prolonging Max-BHT by approximately 8.6%.

Tension delivery from short fibers in long muscles.

Many of the muscles of larger mammals (including humans) feature fibers that terminate without direct tendinous insertion, so fibers in these muscles must deliver their tension into the muscle belly. This raises many important issues regarding the pathway of tension delivery from the sarcomere to the tendon and the role of series-fiber and connective tissue compliance in the properties of the tension delivered. These issues have potentially great implications for understanding motor control and for strategies for reanimation of inactive muscles.

Frequency domain analysis of heart rate variability in horses at rest and during exercise.

The pattern of variation in heart rate on a beat-to-beat basis contains information concerning sympathetic (SNS) and parasympathetic (PNS) contributions to autonomic nervous system (ANS) modulation of heart rate (HR). In the present study, heart period (RR interval) time series data were collected at rest and during 3 different treadmill exercise protocols from 6 Thoroughbred horses. Frequency and spectral power were determined in 3 frequency bands: very low (VLF) 0-< or = 0.01, low (LO) >0.01-< or = 0.07 and high (HI) >0.07-< or = 0.5 cycles/beat. Indicators of sympathetic (SNSI = LO/HI) and parasympathetic (PNSI = HI/TOTAL) activity were calculated. Power in all bands fell progressively with increasing exercise intensity from rest to trot. At the gallop VLF and LO power continued to fall but HI power rose. SNSI rose from rest to walk, then fell with increasing effort and was lowest at the gallop. PNSI fell from rest to walk, then rose and was highest at the gallop. Normalised HI power exceeded combined VLF and LO power at all gaits, with the ratio HI to LO power being lowest at the walk and highest at the gallop. ANS indicators showed considerable inter-horse variation, and varied less consistently than raw power with increasing physical effort. In the horses studied, the relationship between power and HR changed at exercise intensities associated with heart rates above approximately 120-130 beats/min. At this level, humoral and other non-neural mechanisms may become more important than autonomic modulation in influencing heart rate and heart rate variability (HRV). HRV at intense effort may be influenced by respiratory-gait entrainment, energetics of locomotion and work of breathing. HRV analysis in the frequency domain would appear to be of potential value as a noninvasive means of assessing autonomic modulation of heart rate at low exercise intensities, only. The technique may be a sensitive method for assessing exercise response to experimental manipulations and disease states.

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells.

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations.

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.

Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion.

Mutations and deletions in mitochondrial DNA (mtDNA) lead to a number of human diseases characterized by neuromuscular degeneration. Accumulation of truncated mtDNA molecules (delta-mtDNA) lacking a specific 4977-bp fragment, the common deletion, leads to three related mtDNA diseases: Pearson's syndrome; Kearns-Sayre syndrome; and chronic progressive external ophthalmoplegia (CPEO). In addition, the proportion of delta-mtDNA present increases with age in a range of tissues. Consequently, there is considerable interest in the effects of the accumulation of delta-mtDNA on cell function. The 4977-bp deletion affects genes encoding 7 polypeptide components of the mitochondrial respiratory chain, and 5 of the 22 tRNAs necessary for mitochondrial protein synthesis. To determine how the accumulation of delta-mtDNA affects oxidative phosphorylation we constructed a series of cybrids by fusing a human osteosarcoma cell line depleted of mtDNA (rho0) with enucleated skin fibroblasts from a CPEO patient. The ensuing cybrids contained 0-86% delta-mtDNA and all had volumes, protein contents, plasma-membrane potentials and mitochondrial contents similar to those of the parental cell line. The bioenergetic consequences of accumulating delta-mtDNA were assessed by measuring the mitochondrial membrane potential, rate of ATP synthesis and ATP/ADP ratio. In cybrids containing less than 50-55% delta-mtDNA, these bioenergetic functions were equivalent to those of cybrids with intact mtDNA. However, once the proportion of delta-mtDNA exceeded this threshold, the mitochondrial membrane potential, rate of ATP synthesis, and cellular ATP/ADP ratio decreased. These bioenergetic deficits will contribute to the cellular pathology associated with the accumulation of delta-mtDNA in the target tissues of patients with mtDNA diseases.