Chris J. Pemberton

Characterisation of proghrelin peptides in mammalian tissue and plasma

Ghrelin is a 28 amino acid stomach peptide, derived from proghrelin(1-94), that stimulates GH release, appetite and adipose deposition. Recently, a peptide derived from proghrelin(53-75) -- also known as obestatin -- has been reported to be a physiological antagonist of ghrelin in the rat. Using four specific RIAs, we provide the first characterisation of proghrelin(1-94) peptides in human plasma, their modulation by metabolic manipulation and their distribution in mammalian tissues. ghrelin(1-28) immunoreactivity (IR) in human plasma and rat plasma/stomach consisted of major des-octanoyl and minor octanoylated forms, as determined by HPLC/RIA. Human plasma ghrelin(1-28) IR was significantly suppressed by food intake, oral glucose and 1 mg s.c. glucagon administration. ghrelin(1-28) IR and proghrelin(29-94) IR peptide distributions in the rat indicated that the stomach and gastrointestinal tract contain the highest amounts of the peptides. Human and rat plasma and rat stomach extracts contained a major IR peak of proghrelin(29-94)-like peptide as determined by HPLC/RIA, whereas no obestatin IR was observed. Human plasma proghrelin(29-94)-like IR positively correlated with ghrelin(1-28) IR, was significantly suppressed by food intake and oral glucose and shared with ghrelin(1-28) IR a negative correlation with body mass index. We found no evidence for the existence of obestatin as a unique, endogenous peptide. Rather, our data suggest that circulating and stored peptides derived from the carboxyl terminal of proghrelin (C-ghrelin) are consistent in length with proghrelin(29-94) and respond to metabolic manipulation, at least in man, in similar fashion to ghrelin(1-28).

Cardiac chymase converts rat proAngiotensin-12 (PA12) to angiotensin II: effects of PA12 upon cardiac haemodynamics

AIMS The aim of this study was to observe the direct physiological and biochemical cardiac effects in response to a newly identified putative component of the renin-angiotensin system, proangiotensin-12 (PA12); and investigate whether PA12 can serve as a substrate for Angiotensin II (AngII) generation. METHODS AND RESULTS The direct cardiac actions of PA12 and its role as a substrate for chymase-dependent AngII generation were investigated in Sprague-Dawley rats using an isolated heart model of cardiac ischaemia-reperfusion injury. PA12 potently constricted coronary arteries with no significant effect on left-ventricular contractility. PA12 impaired recovery from global ischaemia, maintaining coronary constriction and markedly increasing release of creatine kinase and troponin I (TnI), indicating greater myocardial injury. Analysis of perfusate collected after transcardiac passage revealed a marked increase in AngII production from hearts infused with PA12. Cardiac AngII production was not blocked by angiotensin-converting enzyme inhibitors, whereas inhibition of chymase with chymostatin significantly reduced AngII production and attenuated PA12-induced vasoconstriction and myocardial damage following ischaemia. Furthermore, Angiotensin II type 1 receptor (AT(1)R) blockade abolished PA12 activity. In vitro, PA12 was efficiently and precisely converted to AngII as assessed on reverse phase-high performance liquid chromatography coupled to tandem mass spectrometry. This conversion was blocked by chymostatin. CONCLUSION PA12 may act as a circulating substrate for cardiac chymase-mediated AngII production, in contrast to ACE-mediated AngII production from AngI.

Rapid Rule-out of Acute Myocardial Infarction With a Single High-Sensitivity Cardiac Troponin T Measurement Below the Limit of Detection: A Collaborative Meta-analysis

Background High-sensitivity assays for cardiac troponin T (hs-cTnT) are sometimes used to rapidly rule out acute myocardial infarction (AMI). Purpose To estimate the ability of a single hs-cTnT concentration below the limit of detection (<0.005 µg/L) and a nonischemic electrocardiogram (ECG) to rule out AMI in adults presenting to the emergency department (ED) with chest pain. Data Sources EMBASE and MEDLINE without language restrictions (1 January 2008 to 14 December 2016). Study Selection Cohort studies involving adults presenting to the ED with possible acute coronary syndrome in whom an ECG and hs-cTnT measurements were obtained and AMI outcomes adjudicated during initial hospitalization. Data Extraction Investigators of studies provided data on the number of low-risk patients (no new ischemia on ECG and hs-cTnT measurements <0.005 µg/L) and the number who had AMI during hospitalization (primary outcome) or a major adverse cardiac event (MACE) or death within 30 days (secondary outcomes), by risk classification (low or not low risk). Two independent epidemiologists rated risk of bias of studies. Data Synthesis Of 9241 patients in 11 cohort studies, 2825 (30.6%) were classified as low risk. Fourteen (0.5%) low-risk patients had AMI. Sensitivity of the risk classification for AMI ranged from 87.5% to 100% in individual studies. Pooled estimated sensitivity was 98.7% (95% CI, 96.6% to 99.5%). Sensitivity for 30-day MACEs ranged from 87.9% to 100%; pooled sensitivity was 98.0% (CI, 94.7% to 99.3%). No low-risk patients died. Limitation Few studies, variation in timing and methods of reference standard troponin tests, and heterogeneity of risk and prevalence of AMI across studies. Conclusion A single hs-cTnT concentration below the limit of detection in combination with a nonischemic ECG may successfully rule out AMI in patients presenting to EDs with possible emergency acute coronary syndrome. Primary Funding Source Emergency Care Foundation.

B-Type Natriuretic Peptide Signal Peptide Circulates in Human Blood Evaluation as a Potential Biomarker of Cardiac Ischemia

Background— The diagnosis of cardiac necrosis such as myocardial infarction can be difficult and relies on the use of circulating protein markers like troponin. However, there is a clear need to identify circulating, specific biomarkers that can detect cardiac ischemia without necrosis. Methods and Results— Using specific immunoassay and tandem mass spectrometry, we show that a fragment derived from the signal peptide of B-type natriuretic peptide (BNPsp) not only is detectable in cytosolic extracts of explant human heart tissue but also is secreted from the heart into the circulation of healthy individuals. Furthermore, plasma levels of BNPsp in patients with documented acute ST-elevation myocardial infarction (n=25) rise to peak values (≈3 times higher than the 99th percentile of the normal range) significantly earlier than the currently used biomarkers myoglobin, creatine kinase-MB, and troponin. Preliminary receiver-operating characteristic curve analysis comparing BNPsp concentrations in ST-elevation myocardial infarction patients and other patient groups was positive (area under the curve=0.97; P<0.001), suggesting that further, more rigorous studies in heterogeneous chest pain patient cohorts are warranted. Conclusion— Our results demonstrate for the first time that BNPsp exists as a distinct entity in the human circulation and could serve as a new class of circulating biomarker with the potential to accelerate the clinical diagnosis of cardiac ischemia and myocardial infarction. Clinical Trial Registration— URL: http://www.anzctr.org.au. Unique identifier: ACTRN12609000040268.

High-sensitivity troponin T for early rule-out of myocardial infarction in recent onset chest pain.

Objective To investigate whether a high-sensitivity troponin assay, shown to improve early detection of acute myocardial infarction (AMI), permits accelerated rule-in/rule-out of AMI. Methods Patients who presented to the emergency department within 4 h of the onset of chest pain suggestive of acute coronary syndrome were prospectively recruited from November 2007 to April 2010. Blood samples were taken at 0, 1, 2 and 12–24 h after presentation and were analysed for clinically applied troponin I and for high-sensitivity troponin T (hsTnT). The dynamic change in hsTnT levels between time points was measured. The primary outcome was admission diagnosis of AMI. Results Of the 385 patients recruited, 82 (21.3%) had AMI. The sensitivity of hsTnT by 2 h was 95.1% (88.7–98.1%), specificity 75.6% (73.8–76.5%), positive predictive value 53.8% (50.2–55.5%) and negative predictive value 98.3% (96.0–99.3%). The sensitivity was not statistically different between peak values at 2 h and 24 h. Adding ECG results reduced the false negative rate to 1.2%. The additional application of ≥20% delta criterion over the 2 h period for 0–2 h samples increased specificity to 92.4% (90.2–94.3%) but reduced sensitivity to 56.1% (48.0–63.2%). Conclusion hsTnT taken at 0 and 2 h after presentation, together with ECG results, could identify patients suitable for early stress testing with a false negative rate for AMI of 1.2%. Further trials of such an approach are warranted. The specificity of hsTnT for diagnosing AMI could be improved by the use of a delta of ≥20%, but at the cost of major reductions in sensitivity.

Urotensin II and urotensin II-related peptide (URP) in cardiac ischemia-reperfusion injury

Circulating urotensin II (UII) concentrations and the tissue expression of its cognate receptor (UT) are elevated in patients with cardiovascular disease (CVD). The functional significance of elevated plasma UII levels in CVD is unclear. Urotensin-related peptide (URP) is a paralog of UII in that it contains the six amino acid ring structures found in UII. Although both peptides are implicated as bioactive factors capable of modulating cardiovascular status, the role of both UII and URP in ischemic injury is unknown. Accordingly, we provide here the first report describing the direct cardiac effects of UII and URP in ischemia-reperfusion injury. Isolated perfused rat hearts were subjected to no-flow global ischemia for 45 min after 30min preconditioning with either 1nM rUII or 10nM URP. Both rUII- and URP-induced significant vasodilation of coronary arteries before (both P<0.05) and after ischemia (both P<0.05). Rat UII alone lowered contractility prior to ischemia (P=0.053). Specific assay of perfusate revealed rUII and URP both significantly inhibited reperfusion myocardial creatine kinase (CK) release (P=0.012 and 0.036, respectively) and atrial natriuretic peptide (ANP) secretion (P=0.025). Antagonism of the UT receptor with 1muM palosuran caused a significant increase in perfusion pressure (PP) prior to and post-ischemia. Furthermore, palosuran significantly inhibited reductions in both PP and myocardial damage marker release induced by both rUII and URP. In conclusion, our data suggests rUII and URP reduce cardiac ischemia-reperfusion injury by increasing flow through the coronary circulation, reducing contractility and therefore myocardial energy demand, and inhibiting reperfusion myocardial damage. Thus, UII and URP present as novel peptides with potential cardioprotective actions.

Amino-terminal proBNP in ovine plasma: evidence for enhanced secretion in response to cardiac overload

We have recently identified a novel amino-terminal fragment of pro-brain natriuretic peptide (NT-proBNP) in the circulation of humans, the concentration of which increases progressively as the left ventricle fails. To clarify the origins of NT-proBNP in experimental animals, we have developed an RIA for NT-proBNP based on residues 52-71 of ovine proBNP-(1-103) and used it to study cardiac processing, secretion, and metabolism of BNP in sheep with cardiac overload induced by coronary artery ligation (CAL) or rapid left ventricular pacing (rLVP). The concentration of NT-proBNP in left atrial plasma extracts drawn from normal control sheep was threefold that of mature BNP. Size-exclusion and reverse-phase HPLC analyses of plasma extracts coupled to RIA revealed a single peak of immunoreactive (ir) NT-proBNP [∼8,000 relative molecular weight ( M r)], quite distinct from a single peak of ir-mature BNP (∼3,000 M r). In contrast, ovine cardiac tissue contained only a single immunoreactive peak of high-molecular-weight BNP (∼11,000 M r), consistent in size with proBNP-(1-103). Sampling from the cardiac coronary sinus in normal control sheep ( n = 5) and sheep with CAL ( n = 5) revealed that the molar ratio of NT-proBNP to mature BNP was similar. There was a significant gradient of both mature and NT-proBNP across the heart in normal sheep, whereas after CAL the gradient was significant for mature BNP only. In both forms of cardiac overload (CAL and rLVP), left atrial plasma levels of NT-proBNP were significantly increased above normal levels, in contrast with mature BNP levels, which were raised only in the rLVP group of animals. Blockade of natriuretic peptide metabolism in sheep with heart failure (induced by rLVP) raised mature BNP levels threefold but did not affect levels of NT-proBNP. In conclusion, these studies show that NT-proBNP is formed from proBNP stores during secretion and, compared with mature BNP, accumulates in plasma because metabolism of NT-proBNP appears to differ from that of mature BNP. Although its function, if any, remains unclear, plasma NT-proBNP may prove to be a sensitive marker of cardiac overload and/or decompensation.

Metformin increases plasma ghrelin in Type 2 diabetes.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Metformin, unlike the other major antihyperglycaemic drugs, is not associated with weight gain. * Ghrelin is an appetite-stimulating hormone whose concentrations vary in relation to food, obesity and diabetes control. * Reports are conflicting about how metformin affects ghrelin concentrations, and this study was aimed at resolving this issue in patients with Type 2 diabetes. WHAT THIS STUDY ADDS * In this study an increase in ghrelin concentrations was seen in response to metformin treatment in patients with Type 2 diabetes. * This effect was opposite to what might be expected if the effect of metformin on weight control was mediated via suppression of ghrelin. * It is likely that the ghrelin response was secondary to improved glycaemic control. * Meal time changes in appetite and satiety did not correlate with changes in ghrelin, which suggests ghrelin may not be important in meal initiation. AIMS Metformin treatment of Type 2 diabetes is not usually associated with weight gain, and may assist with weight reduction. Plasma ghrelin concentrations are inversely associated with obesity and food intake. Metformin might therefore affect ghrelin concentrations, although previous studies have shown variable results in this regard. The primary aim of this study was to determine the effect of metformin on plasma ghrelin, appetite and satiety in patients with Type 2 diabetes. METHODS Eighteen patients with Type 2 diabetes were studied before and after 6 weeks of metformin treatment, which was titrated to 1 g b.d. On the study days patients were fed standard meals of 390 kcal at 08.00 and 12.30 h, plasma samples were collected at 15- and 30-min intervals, and appetite and satiety were measured on visual analogue scales. Changes in the area under the concentration-time curves (AUCs) of plasma ghrelin, insulin, glucose, appetite and satiety were assessed and examined for correlations with metformin AUCs. Changes in fasting adiponectin and leptin were also measured. RESULTS Treatment with metformin increased the mean AUC (07.30-16.30 h) of plasma ghrelin by 24% (P= 0.003), while decreasing those of glucose by 19% (P < 0.001) and insulin by 19% (P= 0.001). No changes were detected in hunger and satiety, or in fasting adiponectin or leptin concentrations. There were no clear correlations between metformin plasma concentrations (AUC) and changes in plasma glucose, insulin or ghrelin. CONCLUSIONS Treatment of Type 2 diabetes with metformin was associated with increased plasma ghrelin concentrations, without associated changes in hunger and satiety.

Cardiovascular effects of native and non-native urotensin II and urotensin II-related peptide on rat and salmon hearts

Urotensin II (UII) was first discovered in the urophyses of goby fish and later identified in mammals, while urotensin II-related peptide (URP) was recently isolated from rat brain. We studied the effects of UII on isolated heart preparations of Chinook salmon and Sprague-Dawley rats. Native rat UII caused potent and sustained, dose-dependent dilation of the coronary arteries in the rat, whereas non-native UII (human and trout UII) showed attenuated vasodilation. Rat URP dilated rat coronary arteries, with 10-fold less potency compared with rUII. In salmon, native trout UII caused sustained dilation of the coronary arteries, while rat UII and URP caused significant constriction. Nomega-nitro-(l)-arginine methyl (l-NAME) and indomethacin significantly attenuated the URP and rat UII-induced vasodilation in the rat heart. We conclude that UII is a coronary vasodilator, an action that is species form specific. We also provide the first evidence for cardiac actions of URP, possibly via mechanisms common with UII.

Direct cardiac actions of erythropoietin (EPO): effects on cardiac contractility, BNP secretion and ischaemia/reperfusion injury.

EPO (erythropoietin) has recently been shown to have protective actions upon the myocardium; however, the direct effects of EPO upon cardiac contractile and secretory functions are unknown and the signalling mechanisms are not well defined. In the present study, we provide the first evidence of direct cardiac contractile actions of EPO. In isolated perfused Sprague-Dawley rat hearts, a 30 min infusion of EPO significantly increased contractility in a dose-dependent fashion (maximal change 18+/-2% with 1 unit/ml EPO; P<0.005 compared with vehicle). Perfusate ET-1 (endothelin-1) increased transiently during EPO infusion, and the ET(A/)ET(B) antagonist bosentan abolished the inotropic response to EPO. BNP (B-type natriuretic peptide) secretion (28+/-8%; P<0.05) and nuclear transcription factor GATA-4 DNA-binding activity (51%; P<0.05) were both significantly increased by EPO and blocked by bosentan. In a model of global ischaemic injury, delivery of 1 unit/ml EPO during reperfusion significantly attenuated creatine kinase release (28+/-12%; P<0.05) and significantly improved contractile recovery (P<0.001), independent of ET(A) blockade. Apoptotic indices [assessed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)/cleaved caspase-3-positive cells] were significantly decreased (P<0.01) by 1 unit/ml EPO during reperfusion alone, coincident with significantly increased phosphorylation of myocardial JAK2 (Janus kinase 2) and STAT3 (signal transducer and activator of transcription 3). Thus EPO directly enhances cardiac contractility and BNP secretion and alleviates ischemia/reperfusion injury via ET-1-dependent and -independent mechanisms respectively.