Statement on analysis and interpretation of clinical human gastrointestinal microbiome testing using next-generation sequencing in South Africa.

Advances in DNA sequencing technologies and computational tools over the past few years have led to vast improvements in the metagenomic analysis of the human microbiota. While this has also significantly improved our understanding of the role of the host-microbiome interaction in health and disease, the current clinical expectation is that testing, particularly of the gastrointestinal biome, can be used to diagnose, manage and treat patients. The authors outline the available technologies and highlight current limitations of these techniques to address this clinical demand. Through understanding the limitations of and need for more research and data collection, one can improve the appropriate utilisation and interpretation, as well as the current rational clinical application of these techniques.

Chronically elevated bilirubin protects from cardiac reperfusion injury in the male Gunn rat.

AIMS: Bilirubin is associated with reduced risk of cardiovascular disease, as evidenced in conditions of mild hyperbilirubinaemia (Gilbert's Syndrome). Little is known regarding myocardial stress resistance in hyperbilirubinaemic conditions or whether life-long exposure modifies cardiac function, which might contribute to protection from cardiovascular disease. METHODS: Hyperbilirubinaemic rats and littermate controls underwent echocardiography at 3, 6 and 12 months of age, with hearts subsequently assessed for resistance to 30 min of ischaemia. Heart tissue was then collected for assessment of bilirubin content. RESULTS: No difference in baseline cardiac function was evident until 6 months onwards, where Gunn rats demonstrated aortic dilatation and reduced peak ejection velocities. Additionally, duration of ventricular ejection increased progressively, indicating a negative inotropic effect of bilirubin in vivo. Ex vivo analysis of baseline function revealed reduced left ventricular pressure development (LVDP) and contractility in hyperbilirubinaemic rats. Furthermore, stress resistance was improved in Gunn hearts: post-ischaemic recoveries of LVDP (76 ± 22% vs. 29 ± 17% Control, P < 0.01) and coronary flow (96 ± 9% vs. 86 ± 16% Control, P < 0.01) were improved in Gunn hearts, accompanied by reduced infarct area (21 ± 5% vs. 47 ± 15% Control, P < 0.01), and ventricular malondialdehyde and protein carbonyl content. Expression of myocardial nitric oxide-regulating genes including Nos1 and Noa1 were not significantly different. CONCLUSIONS: These data reveal life-long hyperbilirubinaemia induces age-dependent hypocontractility in male Gunn rats, and improved stress resistance. In addition, bilirubin exerts sex-independent effects on vascular structure, myocardial function and ischaemic tolerance, the latter likely mediated via bilirubin's antioxidant properties.

Comparing laboratory costs of smear/culture and Xpert® MTB/RIF-based tuberculosis diagnostic algorithms.

SETTING: Cape Town, South Africa, where Xpert® MTB/RIF was introduced as a screening test for all presumptive tuberculosis (TB) cases. OBJECTIVE: To compare laboratory costs of smear/culture- and Xpert-based tuberculosis (TB) diagnostic algorithms in routine operational conditions. METHODS: Economic costing was undertaken from a laboratory perspective, using an ingredients-based costing approach. Cost allocation was based on reviews of standard operating procedures and laboratory records, timing of test procedures, measurement of laboratory areas and manager interviews. We analysed laboratory test data to assess overall costs and cost per pulmonary TB and multidrug-resistant TB (MDR-TB) case diagnosed. Costs were expressed as 2013 Consumer Price Index-adjusted values. RESULTS: Total TB diagnostic costs increased by 43%, from US$440 967 in the smear/culture-based algorithm (April-June 2011) to US$632 262 in the Xpert-based algorithm (April-June 2013). The cost per TB case diagnosed increased by 157%, from US$48.77 (n = 1601) to US$125.32 (n = 1281). The total cost per MDR-TB case diagnosed was similar, at US$190.14 and US$183.86, with 95 and 107 cases diagnosed in the respective algorithms. CONCLUSION: The introduction of the Xpert-based algorithm resulted in substantial cost increases. This was not matched by the expected increase in TB diagnostic efficacy, calling into question the sustainability of this expensive new technology.

The effects of visceral obesity and androgens on bone: trenbolone protects against loss of femoral bone mineral density and structural strength in viscerally obese and testosterone-deficient male rats.

SUMMARY: In males, visceral obesity and androgen deficiency often present together and result in harmful effects on bone. Our findings show that both factors are independently associated with adverse effects on femoral bone structure and strength, and trenbolone protects rats from diet-induced visceral obesity and consequently normalises femoral bone structural strength. INTRODUCTION: In light of the rapidly increasing incidence of obesity and osteoporosis globally, and recent conjecture regarding the effects of visceral adiposity and testosterone deficiency on bone health, we investigated the effects of increased visceral adipose tissue (VAT) mass on femoral bone mineral density (BMD), structure and strength in normal weight rats with testosterone deficiency. METHODS: Male Wistar rats (n = 50) were fed either standard rat chow (CTRL, n = 10) or a high-fat/high-sugar diet (HF/HS, n = 40). Following 8 weeks of feeding, rats underwent sham surgery (CTRL, n = 10; HF/HS, n = 10) or orchiectomy (HF/HS + ORX, n = 30). Following a 4-week recovery period, mini-osmotic pumps containing either vehicle (CTRL, n = 10; HF/HS, n = 10; HF/HS + ORX, n = 10), 2.0 mg kg day(-1), testosterone (HF/HS + ORX + TEST, n = 10) or 2.0 mg kg day(-1) trenbolone (HF/HS + ORX + TREN, n = 10) were implanted for 8 weeks of treatment. Dual-energy X-ray absorptiometry and three-point bending tests were used to assess bone mass, structure and strength of femora. RESULTS: Diet-induced visceral obesity resulted in decreased bone mineral area (BMA) and content (BMC) and impaired femoral stiffness and strength. Orchiectomy further impaired BMA, BMC and BMD and reduced energy to failure in viscerally obese animals. Both TEST and TREN treatment restored BMA, BMC, BMD and energy to failure. Only TREN reduced visceral adiposity and improved femoral stiffness and strength. CONCLUSIONS: Findings support a role for both visceral adiposity and testosterone deficiency as independent risk factors for femoral osteoporosis, adverse bone geometry and impaired bone strength in male rats. Trenbolone may be a more effective candidate for androgen replacement therapy than testosterone in viscerally obese testosterone-deficient males.

Selective pinning control of the average disease transmissibility in an HIV contact network.

Medication is applied to the HIV-infected nodes of high-risk contact networks with the aim of controlling the spread of disease to a predetermined maximum level. This intervention, known as pinning control, is performed both selectively and randomly in the network. These strategies are applied to 300 independent realizations per reference level of incidence on connected undirectional networks without isolated components and varying in size from 100 to 10,000 nodes per network. It is shown that a selective on-off pinning control strategy can control the networks studied with limited steady-state error and, comparing the medians of the doses from both strategies, uses 51.3% less medication than random pinning of all infected nodes. Selective pinning could possibly be used by public health specialists to identify the maximum level of HIV incidence in a population that can be achieved in a constrained funding environment.

Comparing multidrug-resistant tuberculosis patient costs under molecular diagnostic algorithms in South Africa.

SETTING: Ten primary health care facilities in Cape Town, South Africa, 2010-2013. OBJECTIVE: A comparison of costs incurred by patients in GenoType MDRTBplus line-probe assay (LPA) and Xpert MTB/RIF-based diagnostic algorithms from symptom onset until treatment initiation for multidrug-resistant tuberculosis (MDR-TB). METHODS: Eligible patients identified from laboratory and facility records were interviewed 3-6 months after treatment initiation and a cost questionnaire completed. Direct and indirect costs, individual and household income, loss of individual income and change in household income were recorded in local currency, adjusted to 2013 costs and converted to $US. RESULTS: Median number of visits to initiation of MDR-TB treatment was reduced from 20 to 7 (P < 0.001) and median costs fell from US$68.1 to US$38.3 (P = 0.004) in the Xpert group. From symptom onset to being interviewed, the proportion of unemployed increased from 39% to 73% in the LPA group (P < 0.001) and from 53% to 89% in the Xpert group (P < 0.001). Median household income decreased by 16% in the LPA group and by 13% in the Xpert group. CONCLUSION: The introduction of an Xpert-based algorithm brought relief by reducing the costs incurred by patients, but loss of employment and income persist. Patients require support to mitigate this impact.

Two novel HLA alleles, HLA-A*30:02:01:03 and HLA-C*08:113, identified in a South African bone marrow donor.

Genomic sequencing of HLA-A*30:02:01:03, an intronic variant, and HLA-C*08:113, an exonic variant.

Myocardial structure, function and ischaemic tolerance in a rodent model of obesity with insulin resistance.

Obesity and its comorbidities (dyslipidaemia, insulin resistance and hypertension) that together constitute the metabolic syndrome are all risk factors for ischaemic heart disease. Although obesity has been reported to be an independent risk factor for congestive heart failure, whether obesity-induced heart failure develops in the absence of increased afterload (induced by hypertension) is not clear. We have previously shown that obesity with insulin resistance decreases myocardial tolerance to ischaemia-reperfusion, but the mechanism for this decreased tolerance remains unclear. We hypothesize that obesity with insulin resistance induces adverse cardiac remodelling and pump dysfunction, as well as adverse changes in myocardial prosurvival reperfusion injury salvage kinase (RISK) pathway signalling to reduce myocardial tolerance to ischaemia-reperfusion. Wistar rats were fed an obesogenic (obese group) or a standard rat chow diet (control group) for 32 weeks. Echocardiography was performed over the 32 weeks before isolated Langendorff-perfused hearts were subjected to 40 min coronary artery ligation followed by reperfusion, and functional recovery (rate-pressure product), infarct size and RISK pathway function were assessed (Western blot analysis). Obesity with insulin resistance increased myocardial lipid accumulation but had no effect on in vivo or ex vivo left ventricular structure/function. Hearts from obese rats had lower reperfusion rate-pressure products (13115 ± 562 beats min(-1) mmHg for obese rats versus 17781 ± 1109 beats min(-1) mmHg for control rats, P < 0.05) and larger infarcts (36.3 ± 5.6% of area at risk in obese rats versus 14.1 ± 2.8% of area at risk in control rats, P < 0.01) compared with control hearts. These changes were associated with reductions in RISK pathway function, with 30-50 and 40-60% reductions in Akt and glycogen synthase kinase 3 beta (GSK-3ß) expression and phosphorylation, respectively, in obese rat hearts compared with control hearts. Total endothelial nitric oxide synthase expression was reduced by 25% in obese rats. We conclude that obesity with insulin resistance had no effect on basal cardiac structure or function but decreased myocardial tolerance to ischaemia-reperfusion. This reduction in ischaemic tolerance was likely to be due to compromised RISK pathway function in obese, insulin-resistant animals.

Tuberculosis patients in primary care do not start treatment. What role do health system delays play?

SETTING: Primary health care facilities in five provinces of South Africa. OBJECTIVE: To investigate the association between the proportion of sputum results with a prolonged smear turnaround time and the proportion of smear-positive tuberculosis (TB) cases initially lost to follow-up. DESIGN: The unit of investigation was a primary health care facility and the outcome was the initial loss to follow-up rate per facility, which was calculated by comparing the sputum register with the TB treatment register. A prolonged turnaround time was defined as more than 48 h from when the sputum sample was documented in the sputum register to receipt of the result at the facility. RESULTS: The mean initial loss to follow-up rate was 25% (95%CI 22-28). Smear turnaround time overall was inversely associated with initial loss to follow-up (P = 0.008), when comparing Category 2 (33-66% turnaround time within 48 h) with Category 1 (0-32%) (OR 0.73, 95%CI 0.48-1.13, P = 0.163) and when comparing Category 3 (67-100%) with Category 1 (OR 0.62, 95%CI 0.39-0.99, P = 0.045). The population preventable fraction of initial loss to follow-up (when turnaround time was <48 h in ≥67% of smear results) was 21%. CONCLUSION: Initial loss to follow-up should be reported as part of the TB programme to ensure that patients are initiated on treatment to prevent transmission within communities.

Effect of South African beef production systems on post-mortem muscle energy status and meat quality.

Post-slaughter muscle energy metabolism meat colour of South African production systems were compared; steers (n=182) of Nguni, Simmental Brahman crossbreds were reared on pasture until A-, AB-, or B-age, in feedlot until A-AB-age. After exsanguination carcasses were electrically stimulated (400 V for 15 s). M. longissimus dorsi muscle energy samples were taken at 1, 2, 4 and 20 h. Post-mortem samples for meat quality studies were taken at 1, 7 and 14 days post-mortem. Production systems affected muscle glycogen, glucose, glucose-6-P, lactic acid, ATP, creatine-P glycolytic potential (P<0.05), with the muscles of feedlot carcasses having a faster glycolysis rate than pasture carcasses. Energy metabolites correlated (0.40.5) water holding capacity, drip loss, and Warner Bratzler shear force. Muscle energy only affected muscle contraction of the A-age-pasture system (shortest sarcomere length of 1.66 µm vs 1.75 µm highest WBS of 6 kg vs 5 kg 7 days post-mortem).

The effect of creatine supplementation on myocardial function, mitochondrial respiration and susceptibility to ischaemia/reperfusion injury in sedentary and exercised rats.

AIM: To investigate the effects of dietary creatine supplementation alone and in combination with exercise on basal cardiac function, susceptibility to ischaemia/reperfusion injury and mitochondrial oxidative function. There has been an increase in the use of creatine supplementation among sports enthusiasts, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but not in the myocardium. METHODS: Male Wistar rats were swim-trained for 8 weeks, 5 days per week. Hearts were excised and either freeze-clamped for biochemical analysis or perfused on the isolated heart perfusion system to assess function and ischaemia/reperfusion tolerance. Mechanical function was documented in working heart and retrograde mode. The left coronary artery was ligated and infarct size determined. Mitochondrial oxidative capacity was quantified. RESULTS: Aortic output recovery of hearts from the sedentary controls (CSed) was significantly higher than those from creatine-supplemented sedentary (CrSed), creatine-supplemented exercised (CrEx) as well as control exercised (CEx) groups. Ischaemic contracture of hearts from CrEx was significantly higher than that of CSed. There were no differences in infarct size and mitochondrial oxygen consumption. CONCLUSION: This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise-trained animals, by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation.

Melatonin and the metabolic syndrome: a tool for effective therapy in obesity-associated abnormalities?

The metabolic syndrome (MetS) is a cluster of metabolic abnormalities associated with increased risk for cardiovascular diseases. Apart from its powerful antioxidant properties, the pineal gland hormone melatonin has recently attracted the interest of various investigators as a multifunctional molecule. Melatonin has been shown to have beneficial effects in cardiovascular disorders including ischaemic heart disease and hypertension. However, its role in cardiovascular risk factors including obesity and other related metabolic abnormalities is not yet established, particularly in humans. New emerging data show that melatonin may play an important role in body weight regulation and energy metabolism. This review will address the role of melatonin in the MetS focusing on its effects in obesity, insulin resistance and leptin resistance. The overall findings suggest that melatonin should be exploited as a therapeutic tool to prevent or reverse the harmful effects of obesity and its related metabolic disorders.

A survey of the prevalence of blowfly strike and the control measures used in the Rûens area of the Western Cape Province of South Africa.

Blowfly strike and the methods used to combat blowfly strike were recorded on 33 properties in the Rûens area of South Africa during 2003/2004. Data were recorded on Merino and Dohne Merino hoggets (n = 4951) with at least 3 months' wool growth. The following data were captured: presence or absence of strike, site of the strike (body or breech), presence or absence of dermatophilosis as well as subjective scores for wool quality and wool colour. Control measures recorded include: chemical treatment (preventative and spot treatment), crutching, mulesing and the use of the Lucitrap system. Blowfly strike was not significantly influenced by gender or breed. Hoggets suffering from dermatophilosis were more likely to be struck, compared with contemporaries not suffering from the skin disorder (0.057 vs 0.027; P < 0.05). Merino hoggets generally had higher scores than their Dohne Merino contemporaries for wool quality (32.6 vs 27.4; P < 0.05) and wool colour (29.0 vs 27.2; P < 0.05). There was an indication that the Lucitrap system may have reduced flystrike, but the effect was not statistically significant (P = 0.19 for overall strikes and P = 0.12 for body strike). The Mules operation benefited overall flystrike (0.013 vs 0.110; P < 0.05); mainly through an effect on breech strike (0.010 vs 0.109; P < 0.05). The proportion of fly strikes increased with wool length, and declined with an increase in farm size in wool colour score. None of the ethically acceptable control measures assessed could substantially reduce blowfly strike on their own, and an integrated pest management programme was proposed.

The effects of gender and obesity on myocardial tolerance to ischemia.

Obesity is increasing at an alarming rate globally. Several studies have shown that premenopausal women have a reduced risk of CV disease and a reduced myocardial susceptibility to ischemia/reperfusion injury. The effect of obesity on myocardial tolerance to ischemia in women has not been established. To determine how obesity affects myocardial susceptibility to ischemia/reperfusion injury in both males and females, we fed male and female Wistar rats a high caloric diet (HCD) or a control rat chow diet (CD) for 18 weeks. Rats were subsequently fasted overnight, anesthetized and blood was collected. In separate experiments, 18-week-fed (HCD and CD) rats underwent 45 min in vivo coronary artery ligation (CAL) followed by 2 hours reperfusion. Hearts were stained with TTC and infarct size determined. Both male and female HCD fed rats had increased body and visceral fat weights. Homeostasis model assessment (HOMA) index values were 13.95+/-3.04 for CD and 33.58+/-9.39 for HCD male rats (p<0.01) and 2.98+/-0.64 for CD and 2.99+/-0.72 for HCD fed female rats. Male HCD fed rats had larger infarct sizes than CD fed littermates (43.2+/-9.3 % vs. 24.4+/-7.6 %, p<0.05). Female HCD and CD diet fed rats had comparable infarct sizes (31.8+/-4.3 % vs. 23.9+/-3.3 %). We conclude that male rats on the HCD became viscerally obese, dyslipidemic and insulin-resistant, while female HCD fed rats became viscerally obese without developing dyslipidemia or insulin resistance. Obesity increased myocardial infarct size in males but not the females.

Beyond accuracy: creating a comprehensive evidence base for TB diagnostic tools.

The need for a strong and comprehensive evidence base to support decision making with regard to the implementation of new and improved diagnostic tools and approaches has been highlighted by a number of stakeholders; these include members of the New Diagnostics Working Group (NDWG) and the Subgroup for Introducing New Approaches and Tools of the Stop TB Partnership. To compile such evidence in a systematic manner, we have developed an impact assessment framework (IAF) which links evidence on inputs to outcomes. The IAF comprises five interconnected layers: effectiveness analysis, equity analysis, health systems analysis, scale-up analysis and policy analysis. It can be used by new diagnostics developers and other interested research teams to collect as much policy-relevant data as possible prior to, during and after the demonstration phase of tool development. The evidence collated may be used by international and national policy makers to support adoption, implementation and scale-up decisions. The TREAT TB (Technology, Research, Education and Technical Assistance for TB) initiative uses the IAF in its operational research and field evaluations of new tools and approaches for TB diagnosis. It has also been incorporated into the NDWG's recent publication: 'Pathways to better diagnostics for tuberculosis: a blueprint for the development of TB diagnostics'. This article describes the IAF and the process of improving it and suggests next steps in overcoming the challenges in its implementation.

Trans-cranial placement of an amplatzer device to control intractable cardiac failure in an infant with a vein of galen anomaly. A case report.

Neonates presenting with intractable cardiac failure due to vein of Galen aneurysmal malformations (VGAMs) rapidly progress to multisystem organ failure and death if left untreated. Currently the only viable treatment option is endovascular embolization. Although intracranial embolization of a neonate is a high-risk procedure, successful treatment can reverse cardiac failure and prevent neurological complications associated with VGAMs. Embolization via the arterial route is thought to have a better outcome than embolization via the venous system. However, multiple transarterial embolizations in different sessions may well be contraindicated in neonates, because repeat access via the femoral artery, carries a risk of arterial trauma which, in turn, can jeopardize lower limbs. With this case study we show that after repeat failure of arterial embolization, the transcranial placement of an Amplatzer PFO occluder (AGA Medical, Plymouth, USA) in the aneurysm can effectively reduce intrafistular pressure and venous outflow velocity. We also propose a mathematical model that can be used to calculate flow velocity through the aneurysm, which, in turn, could be used to aid clinical decision-making. Unlike some conventional techniques, the placement of an Amplatzer occluder does not pose the risk of completely obstructing venous drainage and therefore does not increase the risk of venous breakthrough hemorrhage. We propose this endovascular technique as a treatment option for high risk neonates in need of emergency embolization of VGAMs, where multiple arterial embolizations failed to control the condition sufficiently.

Impaired contractile function and mitochondrial respiratory capacity in response to oxygen deprivation in a rat model of pre-diabetes.

AIM: Obesity is a major contributor to the global burden of disease and is closely associated with the development of type 2 diabetes and cardiovascular diseases. This study tested the hypothesis that mitochondrial respiratory capacity of the pre-diabetic heart is decreased leading to impaired contractile function and tolerance to ischaemia/reperfusion. METHODS: Eight-week-old male Wistar rats were fed a high caloric diet for 16 weeks after which anthropometric, metabolic, cardiac and mitochondrial parameters were evaluated vs. age-matched lean controls. Cardiac function (working heart perfusions) and mitochondrial respiratory capacity were assessed at baseline and in response to acute oxygen deprivation. RESULTS: Rats fed the high caloric diet exhibited increased body weight and visceral fat vs. the control group. Heart weights of obese rats were also increased. Triglyceride, fasting plasma insulin and free fatty acid levels were elevated, while high-density lipoprotein cholesterol levels were reduced in the obese group. Contractile function was attenuated at baseline and further decreased after subjecting hearts to ischaemia-reperfusion. Myocardial infarct sizes were increased while ADP phosphorylation rates were diminished in obese rats. However, no differences were found for mtDNA levels and the degree of oxidative stress-induced damage. CONCLUSIONS: These data show that decreased mitochondrial bioenergetic capacity in pre-diabetic rat hearts may impair respiratory capacity and reduce basal contractile function and tolerance to acute oxygen deprivation.

Protection of the ischaemic heart: investigations into the phenomenon of ischaemic preconditioning.

Exposure of the heart to one or more short episodes of ischaemia/reperfusion protects the heart against a subsequent prolonged period of ischaemia, as evidenced by a reduction in infarct size and an improvement in functional recovery during reperfusion. Elucidation of the mechanism of this endogenous protection could lead to the development of pharmacological mimetics to be used in the clinical setting. The aim of our studies was therefore to gain more information regarding the mechanism of ischaemic preconditioning, using the isolated perfused working rat heart as model. A preconditioning protocol of 1 x 5 or 3 x 5 min of ischaemia, interspersed with 5 min of reperfusion was found to protect hearts exposed to 25 min of global ischaemia or 35-45 min of regional ischaemia. These models were used throughout our studies. In view of the release of catecholamines by ischaemic tissue, our first aim was to evaluate the role of the alphaadrenergic receptor in ischaemic preconditioning. However, using a multi-cycle ischaemic preconditioning protocol, we could not find any evidence for alpha-1 adrenergic or PKC activation in the mechanism of preconditioning. Cyclic increases in the tissue cyclic nucleotides, cAMP and cGMP were found, however, to occur during a multi-cycle preconditioning protocol, suggesting roles for the beta-adrenergic signalling pathway and nitric oxide (NO) as triggers of cardioprotection. This was substantiated by the findings that (1) administration of the beta-adrenergic agonist, isoproterenol, or the NO donors SNAP or SNP before sustained ischaemia also elicited cardioprotection similar to ischaemic preconditioning; (2) beta-adrenergic blockade or nitric oxide synthase inhibition during an ischaemic preconditioning protocol abolished protection. Effectors downstream of cAMP, such as p38MAPK and CREB, were also demonstrated to be involved in the triggering process. Our next step was to evaluate intracellular signalling during sustained ischaemia and reperfusion. Our results showed that ischaemic preconditioned-induced cardioprotection was associated with a significant reduction in tissue cAMP, attenuation of p38MAPK activation and increased tissue cGMP levels and HSP27 activation, compared to non-preconditioned hearts. The role of the stress kinase p38MAPK was further investigated by using the inhibitor SB203580. Our results suggested that injury by necrosis and apoptosis share activation of p38MAPK as a common signal transduction pathway and that pharmacological targeting of this kinase offers a tenable option to manipulate both these processes during ischaemia/reperfusion injury.

A role for the RISK pathway and K(ATP) channels in pre- and post-conditioning induced by levosimendan in the isolated guinea pig heart.

BACKGROUND AND PURPOSE: Myocardial reperfusion injury prevents optimal salvage of the ischaemic myocardium, and adjunct therapy that would significantly reduce reperfusion injury is still lacking. We investigated whether (1) the heart could be pre- and/or post-conditioned using levosimendan (levosimendan pre-conditioning (LPC) and levosimendan post-conditioning (LPostC)) and (2) the prosurvival kinases and/or the sarcolemmal or mitochondrial K(ATP) channels are involved. EXPERIMENTAL APPROACH: Isolated guinea pig hearts were treated with two 5 min cycles of levosimendan (0.1 microM) interspersed with vehicle perfusion, or two 5 min cycles of ischaemia/reperfusion, before coronary artery ligation (CAL) for 40 min at 36.5 degrees C. Hearts were treated with mitochondrial or sarcolemmal K(ATP) channel blockers before LPC or LPostC. For post-conditioning, hearts received three 30 s cycles of ischaemia/reperfusion or levosimendan/vehicle. Hearts were pretreated with levosimendan immediately before CAL (without washout). Cardiac function, infarct size and reperfusion injury salvage kinase activity was assessed. KEY RESULTS: LPC and LPostC halved the infarct size compared with controls (P<0.05). Treatment with K(ATP) channel blockers before LPC or LPostC reversed this decrease. Pretreating hearts with levosimendan increased activity of extracellular signal-regulated kinase (ERK) 42/44 on reperfusion and had the most marked infarct-lowering effect (P<0.05). CONCLUSIONS AND IMPLICATIONS: (1) Hearts could be pharmacologically pre- and post-conditioned with levosimendan; (2) levosimendan pretreatment is the most effective way to reduce infarct size, possibly by increasing ERK 42/44 activity; (3) benefits of LPC and LPostC were abolished by both K(ATP) channel blockers and (4) LPC may be useful before elective cardiac surgery, whereas LPostC may be used after acute coronary artery events.