Evidence of cardiac injury and arrhythmias in dogs with acute kidney injury

OBJECTIVES Cardiac involvement in the course of acute kidney injury is described in humans as cardiorenal syndrome type 3 but has received only limited attention in dogs. This study was designed to evaluate cardiac injury and dysfunction in acute kidney injury in dogs and its association with outcome. METHODS This prospective cohort study enrolled 24 client-owned dogs with acute kidney injury. Cardiac disorders were evaluated with thoracic radiographs, echocardiography, 24-hour Holter monitoring and cardiac troponin I concentrations within 2 days of admission and 7 to 10 days later. RESULTS Most dogs were diagnosed with leptospirosis (n=18, 75%) and presented with moderate-to-severe acute kidney injury, International Renal Interest Society grades III to V. Dogs with ê100 ventricular premature complexes per 24 hour in the first examination (n=8) had significantly higher initial cTnI concentrations (P=0·007) compared to dogs with fewer than 100. In receiver operating characteristic curve analysis, the number of ventricular premature complexes was predictive of outcome (AUC 0·83, P<0·001). CLINICAL SIGNIFICANCE Acute kidney injury seems to be associated with cardiac injury and arrhythmias in dogs. The data do not indicate a cardiac cause of poor outcome in dogs with increased number of ventricular premature complexes but the association may reflect the severity of disease.

Improved estimation of postmortem interval with Cardiac Troponin T and improved analytical methods

Accurate knowledge of the time since death, or postmortem interval (PMI), has enormous legal, criminological, and psychological impact. In this study, an investigation was made to determine whether the relationship between the degradation of the human cardiac structure protein Cardiac Troponin T and PMI could be used as an indicator of time since death, thus providing a rapid, high resolution, sensitive, and automated methodology for the determination of PMI. ^ The use of Cardiac Troponin T (cTnT), a protein found in heart tissue, as a selective marker for cardiac muscle damage has shown great promise in the determination of PMI. An optimized conventional immunoassay method was developed to quantify intact and fragmented cTnT. A small sample of cardiac tissue, which is less affected than other tissues by external factors, was taken, homogenized, extracted with magnetic microparticles, separated by SDS-PAGE, and visualized with Western blot by probing with monoclonal antibody against cTnT. This step was followed by labeling and available scanners. This conventional immunoassay provides a proper detection and quantitation of cTnT protein in cardiac tissue as a complex matrix; however, this method does not provide the analyst with immediate results. Therefore, a competitive separation method using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was developed to study the interaction between human cTnT protein and monoclonal anti-TroponinT antibody. ^ Analysis of the results revealed a linear relationship between the percent of degraded cTnT and the log of the PMI, indicating that intact cTnT could be detected in human heart tissue up to 10 days postmortem at room temperature and beyond two weeks at 4C. The data presented demonstrates that this technique can provide an extended time range during which PMI can be more accurately estimated as compared to currently used methods. The data demonstrates that this technique represents a major advance in time of death determination through a fast and reliable, semi-quantitative measurement of a biochemical marker from an organ protected from outside factors. ^

Troponin I and cardiovascular risk prediction in the general population: the BiomarCaRE consortium

AIMS: Our aims were to evaluate the distribution of troponin I concentrations in population cohorts across Europe, to characterize the association with cardiovascular outcomes, to determine the predictive value beyond the variables used in the ESC SCORE, to test a potentially clinically relevant cut-off value, and to evaluate the improved eligibility for statin therapy based on elevated troponin I concentrations retrospectively.

METHODS AND RESULTS: Based on the Biomarkers for Cardiovascular Risk Assessment in Europe (BiomarCaRE) project, we analysed individual level data from 10 prospective population-based studies including 74 738 participants. We investigated the value of adding troponin I levels to conventional risk factors for prediction of cardiovascular disease by calculating measures of discrimination (C-index) and net reclassification improvement (NRI). We further tested the clinical implication of statin therapy based on troponin concentration in 12 956 individuals free of cardiovascular disease in the JUPITER study. Troponin I remained an independent predictor with a hazard ratio of 1.37 for cardiovascular mortality, 1.23 for cardiovascular disease, and 1.24 for total mortality. The addition of troponin I information to a prognostic model for cardiovascular death constructed of ESC SCORE variables increased the C-index discrimination measure by 0.007 and yielded an NRI of 0.048, whereas the addition to prognostic models for cardiovascular disease and total mortality led to lesser C-index discrimination and NRI increment. In individuals above 6 ng/L of troponin I, a concentration near the upper quintile in BiomarCaRE (5.9 ng/L) and JUPITER (5.8 ng/L), rosuvastatin therapy resulted in higher absolute risk reduction compared with individuals <6 ng/L of troponin I, whereas the relative risk reduction was similar.

CONCLUSION: In individuals free of cardiovascular disease, the addition of troponin I to variables of established risk score improves prediction of cardiovascular death and cardiovascular disease.

Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

Avaliação dos valores séricos de troponina cardíaca I (cTnI) e capacidade antioxidante total (TAC) em felídeos com doença renal crónica e seu valor de prognóstico : estudo preliminar

Dissertação de Mestrado Integrado em Medicina Veterinária

Biomarkers of exercise-induced myocardial stress in relation to inflammatory and oxidative stress

Increased concentrations of biomarkers reflecting myocardial stress such as cardiac troponin I and T and brain natriuretic peptide (BNP) have been observed following strenuous, long-lasting endurance exercise. The pathophysiological mechanisms are still not fully elucidated and the interpretations of increased post-exercise concentrations range from (i) evidence for exercise-induced myocardial damage to (ii) non-relevant spurious troponin elevations, presumably caused by assay imprecision or heterophilic antibodies. Several lines of evidence suggest that inflammatory processes or oxidative stress could be involved in the rise of NT-proBNP and Troponin observed in critically ill patients with sepsis or burn injury. We tested the hypothesis that inflammatory or oxidative stress is also responsible for exercise-induced cardiomyocyte strain in a large cohort of triathletes following an Ironman triathlon. However, the post-race increase in cardiac troponin T and NT-proBNP was not associated with several markers of exercise-induced inflammation, oxidative stress or antioxidant vitamins. Therefore, we clearly need more studies with other inflammatory markers and different designs to elucidate the scientific background for increases in myocardial stress markers following strenuous endurance events.

Specific role of neuronal nitric-oxide synthase when tethered to the plasma membrane calcium pump in regulating the beta-adrenergic signal in the myocardium

The cardiac neuronal nitric-oxide synthase (nNOS) has been described as a modulator of cardiac contractility. We have demonstrated previously that isoform 4b of the sarcolemmal calcium pump (PMCA4b) binds to nNOS in the heart and that this complex regulates beta-adrenergic signal transmission in vivo. Here, we investigated whether the nNOS-PMCA4b complex serves as a specific signaling modulator in the heart. PMCA4b transgenic mice (PMCA4b-TG) showed a significant reduction in nNOS and total NOS activities as well as in cGMP levels in the heart compared with their wild type (WT) littermates. In contrast, PMCA4b-TG hearts showed an elevation in cAMP levels compared with the WT. Adult cardiomyocytes isolated from PMCA4b-TG mice demonstrated a 3-fold increase in Ser(16) phospholamban (PLB) phosphorylation as well as Ser(22) and Ser(23) cardiac troponin I (cTnI) phosphorylation at base line compared with the WT. In addition, the relative induction of PLB phosphorylation and cTnI phosphorylation following isoproterenol treatment was severely reduced in PMCA4b-TG myocytes, explaining the blunted physiological response to the beta-adrenergic stimulation. In keeping with the data from the transgenic animals, neonatal rat cardiomyocytes overexpressing PMCA4b showed a significant reduction in nitric oxide and cGMP levels. This was accompanied by an increase in cAMP levels, which led to an increase in both PLB and cTnI phosphorylation at base line. Elevated cAMP levels were likely due to the modulation of cardiac phosphodiesterase, which determined the balance between cGMP and cAMP following PMCA4b overexpression. In conclusion, these results showed that the nNOS-PMCA4b complex regulates contractility via cAMP and phosphorylation of both PLB and cTnI.

An upgrade on the rabbit model of anthracycline-induced cardiomyopathy: shorter protocol, reduced mortality, and higher incidence of overt dilated cardiomyopathy

Current protocols of anthracycline-induced cardiomyopathy in rabbits present with high premature mortality and nephrotoxicity, thus rendering them unsuitable for studies requiring long-term functional evaluation of myocardial function (e.g., stem cell therapy). We compared two previously described protocols to an in-house developed protocol in three groups: Group DOX2 received doxorubicin 2 mg/kg/week (8 weeks); Group DAU3 received daunorubicin 3 mg/kg/week (10 weeks); and Group DAU4 received daunorubicin 4 mg/kg/week (6 weeks). A cohort of rabbits received saline (control). Results of blood tests, cardiac troponin I, echocardiography, and histopathology were analysed. Whilst DOX2 and DAU3 rabbits showed high premature mortality (50% and 33%, resp.), DAU4 rabbits showed 7.6% premature mortality. None of DOX2 rabbits developed overt dilated cardiomyopathy; 66% of DAU3 rabbits developed overt dilated cardiomyopathy and quickly progressed to severe congestive heart failure. Interestingly, 92% of DAU4 rabbits showed overt dilated cardiomyopathy and 67% developed congestive heart failure exhibiting stable disease. DOX2 and DAU3 rabbits showed alterations of renal function, with DAU3 also exhibiting hepatic function compromise. Thus, a shortened protocol of anthracycline-induced cardiomyopathy as in DAU4 group results in high incidence of overt dilated cardiomyopathy, which insidiously progressed to congestive heart failure, associated to reduced systemic compromise and very low premature mortality.

Determinação dos marcadores cardíacos e de lipoperoxidação em equinos sob influência de exercícios de alta e baixa intensidade em esteira de alta velocidade e suplementação com vitamina E (dl-alfa-tocoferol)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Metabolismo oxidativo e cardíaco de equinos submetidos a exercício de baixa intensidade antes e após suplementação com antioxidante

The present study aimed to evaluate cardiac and lipoperoxidation markers in horses subjected to low intensity and long duration (TLD) exercise test, before and after vitamin E supplementation. For this purpose, 10 horses were used, subjecting them to the first TLD with a workload based on individual maximal oxygen uptake (VO2max). Then, horses received vitamin E (dl-alpha-tocopherol) during 59 days at a daily oral dose of 1,000IU, and thereafter they performed a second TLD with the same protocol as the first. Blood samples were collected to determine plasma malondialdehyde (MDA) as an index of lipoperoxidation, serum cardiac troponin I (cTnI) and creatine kinase MB isoenzyme (CK-MB) as cardiac markers. As a result of the exercise, there was no significant increase in MDA or cTnI, but serum CK-MB increased suggesting myocardial stress. The supplementation was able to minimize reactive oxygen species production, as evidenced by lower concentrations of MDA at all times evaluated, but it didn't cause protective effect on the myocardium. It was concluded that the low intensity and long duration exercise promoted light myocardial stress in horses and vitamin E supplementation reduced lipoperoxidation.

Efeito da suplementação com vitamina E sobre os metabolismos oxidativo e cardíaco em equinos submetidos a exercício de alta intensidade

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biomarcadores cardíacos em cães com degeneração mixomatosa da valva mitral submetidos a um programa de condicionamento físico

Pós-graduação em Medicina Veterinária - FCAV

Antioxidant treatment protects against matrix metalloproteinase activation and cardiomyocyte injury during acute pulmonary thromboembolism

Increased reactive oxygen species (ROS) promote matrix metalloproteinase (MMP) activities and may underlie cardiomyocyte injury and the degradation of cardiac troponin I (cTI) during acute pulmonary thromboembolism (APT). We examined whether pretreatment or therapy with tempol (a ROS scavenger) prevents MMP activation and cardiomyocyte injury of APT. Anesthetized sheep received tempol infusion (1.0 mg kg(-1) min(-1), i.v.) or saline starting 30 min before or 30 min after APT (autologous blood clots). Control animals received saline. Hemodynamic measurements were performed. MMPs were studied in the right ventricle (RV) by gelatin zymography, fluorimetric activity assay, and in situ zymography. The ROS levels were determined in the RV and cTI were measured in serum samples. APT increased the pulmonary arterial pressure and pulmonary vascular resistance by 146 and 164 %, respectively. Pretreatment or therapy with tempol attenuated these increases. While APT increased RV + dP/dt (max), tempol infusions had no effects. APT increased RV MMP-9 (but not MMP-2) levels. In line with these findings, APT increased RV MMP activities, and this finding was confirmed by in situ zymography. APT increased the RV ROS levels and tempol infusion, before or after APT, and blunted APT-induced increases in MMP-9 levels, MMP activities, in situ MMP activities, and ROS levels in the RV. cTI concentrations increased after APT, and tempol attenuated these increases. RV oxidative stress after APT increases the RV MMP activities, leading to the degradation of sarcomeric proteins, including cTI. Antioxidant treatment may prevent MMP activation and protect against cardiomyocyte injury after APT.

Late remote ischemic preconditioning in children undergoing cardiopulmonary bypass: A randomized controlled trial

Objective: Cardiopulmonary bypass is associated with ischemia-reperfusion injury to multiple organs. We aimed to evaluate whether remote ischemic preconditioning performed the day before surgery for congenital heart disease with cardiopulmonary bypass attenuates the postoperative inflammatory response and myocardial dysfunction. Methods: This was a prospective, randomized, single-blind, controlled trial. Children allocated to remote ischemic preconditioning underwent 4 periods of 5 minutes of lower limb ischemia by a blood pressure cuff intercalated with 5 minutes of reperfusion. Blood samples were collected 4, 12, 24, and 48 hours after cardiopulmonary bypass to evaluate nuclear factor kappa B activation in leukocytes by quantification of mRNA of I kappa B alpha by real-time quantitative polymerase chain reaction and for interleukin-8 and 10 plasma concentration measurements by enzyme-linked immunosorbent assay. Myocardial dysfunction was assessed by N-terminal pro-B-type natriuretic peptide and cardiac troponin I plasma concentrations, measured by chemiluminescence, and clinical parameters of low cardiac output syndrome. Results: Twelve children were allocated to remote ischemic preconditioning, and 10 children were allocated to the control group. Demographic data and Risk Adjustment for Congenital Heart Surgery 1 classification were comparable in both groups. Remote ischemic preconditioning group had lower postoperative values of N-terminal pro-B-type natriuretic peptide, but cardiac troponin I levels were not significantly different between groups. Interleukin-8 and 10 concentrations and I kappa B alpha gene expression were similar in both groups. Postoperative morbidity was similar in both groups; there were no postoperative deaths in either group. Conclusions: Late remote ischemic preconditioning did not provide clinically relevant cardioprotection to children undergoing cardiopulmonary bypass. (J Thorac Cardiovasc Surg 2012;144:178-83)

Minimal extracorporeal circulation is a promising technique for coronary artery bypass grafting

BACKGROUND: Minimal extracorporeal circulation (MECC) is a promising perfusion technology, taking the advantage of an ECC while having a significantly reduced priming volume. We analyzed the actual possible benefits of using MECC in patients undergoing CABG procedures and compared the results with conventional extracorporeal circulation (CECC). METHODS: One thousand fifty-three consecutive patients underwent CABG surgery using the MECC perfusion technique. Subgroup analyses focused on perioperative myocardial markers (cardiac troponin I [cTnI]), incidence of atrial fibrillation (AF), and perioperative evaluation of inflammatory markers and data were compared with those of patients who underwent CABG using CECC. A propensity score analysis was performed. RESULTS: Patient characteristics and distribution of EuroSCORE risk were similar in both groups. Severity of coronary artery disease and extent of revascularization were also comparable in both groups (number of distal anastomoses: 3.2 +/- 1.1 in CECC vs 3.2 +/- 0.9 in MECC; p = not significant [ns]). The cTnI was significantly lower in the MECC group (11.0 +/- 10.8 microg/L in MECC vs 24.7 +/- 25.3 microg/L in CECC; p < 0.05). Incidence of AF was 11.1% in MECC and 39.0% in CECC (p < 0.05). Inflammatory markers (interleukin-6, SC5b-9) were lower in MECC patients (p < 0.05). Propensity score analysis confirmed faster recovery in MECC patients and lower incidence of AF. CONCLUSIONS: Minimal extracorporeal circulation is a safe perfusion technique for CABG and may therefore concurrence OPCAB and traditional CABG under CECC.