Dan Atar

Cardiac Troponin I Is Modified in the Myocardium of Bypass Patients

Background—Selective proteolysis of cardiac troponin I (cTnI) is a proposed mechanism of contractile dysfunction in stunned myocardium, and the presence of cTnI degradation products in serum may reflect the functional state of the remaining viable myocardium. However, recent swine and canine studies have not demonstrated stunning-dependent cTnI degradation. Methods and Results—To address the universality of cTnI modification, myocardial biopsy samples were obtained from coronary artery bypass patients (n=37) before and 10 minutes after removal of cross-clamp. Analysis of biopsy samples for cTnI by Western blotting revealed a spectrum of modified cTnI products in myocardium both before and after cross-clamp, including degradation products (7 products resulting from differential N- and C-terminal processing) and covalent complexes (3 products). In particular, a 22-kDa cTnI degradation product with C-terminal proteolysis was identified, which may represent an initial ischemia-dependent cTnI modification, similar to cTnI1–193 observed in stunned rat myocardium. Although no systematic change in amount of modified cTnI was observed, subgroups of patients displayed an increase (n=10, 85±5% of cTnI remaining intact before cross-clamp versus 75±5% after) or a decrease (n=12, 67±5% before versus 78±5% after). Electron microscopy demonstrated normal ultrastructure in biopsy samples, which suggests no necrosis was present. In addition, cTnI modification products were observed in serum through a modified SDS-PAGE methodology. Conclusions—cTnI modification, in particular proteolysis, occurs in myocardium of bypass patients and may play a key role in stunning in some bypass patients.

Aspirin inhibits surface glycoprotein IIb/IIIa, P-selectin, CD63, and CD107a receptor expression on human platelets

&NA; Platelet inhibition after aspirin therapy reduces the risk for the development of acute coronary syndromes. However, the mechanism by which aspirin affect platelets other than by prostaglandin blockade is unclear. We sought to determine the in vitro effects of aspirin on the surface expression of nine platelet receptors using whole blood flow cytometry. Blood from 24 healthy volunteers was incubated for 30 min with 1.8 and 7.2 mg/l phosphatebuffered saline‐diluted acetylsalicylic acid in the presence or absence of apyrase. Platelet serotonin release, and the surface expression of platelet receptors with or without apyrase were determined using the following monoclonal antibodies: anit‐CD41 [glycoprotein (GP)IIb/IIIa], CD42b (GPIb), CD62p (P‐selectin), CD51/CD61 (vitronectin receptor), CD31 [platelet/endothelial cellular adhesion molecule‐1 (PECAM‐1)], CD107a [lysosomal associated membrane protein (LAMP)‐1], CD107b (LAMP‐2), CD63 (LIMP or LAMP‐3), and CD151 (PETA‐3). Samples were then immediately fixed with 2% paraformaldehyde, and run on the flow cytometer within 48 h. Aspirin does not affect serotonin release from human platelets. Dose‐dependent inhibition of GPIIb/IIIa, P‐selectin, CD63, and CD107a receptor expression was observed in the aspirin‐treated whole‐blood samples. Apyrase potentiates the effects of aspirin, and independently inhibits PECAM‐1. In addition to the known effect of irreversibly inhibiting platelet cyclooxygenase‐1, thereby blocking thromboxane A2 synthesis, it appears that aspirin exhibits direct effects on selective major platelet receptors. Blood Coagul Fibrinolysis 14:249‐253 & 2003 Lippincott Williams & Wilkins.

Increased soluble platelet / endothelial cellular adhesion molecule-1 and osteonectin levels in patients with severe congestive heart failure. Independence of disease etiology, and antecedent aspirin therapy

Platelet–endothelial interactions modulated by adhesion molecules, may play an important role in the pathogenesis of congestive heart failure (CHF). Soluble levels of these molecules and platelet‐derived substances are reportedly elevated in patients with CHF. However, no data are available on the plasma levels of Platelet / Endothelial Cell Adhesion Molecule‐1 (PECAM‐1), and platelet‐derived osteonectin in this growing population.

Whole blood impedance aggregometry for the assessment of platelet function in patients with congestive heart failure (EPCOT Trial).

Data from small studies have shown the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of the whole blood aggregometry (WBA) in a random outpatient CHF population.

Lack of nuclear apoptosis in cardiomyocytes and increased endothelin-1 levels in a rat heart model of myocardial stunning.

Objective. Reperfusion injury may affect the cardiac NO and endothelin production. We investigated whether 20 min of total ischemia followed by 40 min of reperfusion can induce apoptosis in a Langendorff model of retrogradely perfused rat hearts (37°C; paced at 300/’), and we attempted to correlate these findings with measured tissue NO and ET-1 levels. Methods. An apoptosis detection system was utilized which catalytically incorporates fluorescein-12-dUTP at the 3’-OH DNA ends using the principle of the TUNEL assay, with direct visualization of the labeled DNA. ET-1 was measured by radioimmunoassay and NO3/NO2 by ion pairing HPLC on C18 reverse phase columns. Results. None of the postischemic (n = 6) nor of the control perfused (90 min, n = 6) hearts showed signs of apoptosis, while those exposed to longer ischemia (40 min) and reperfusion (2 h) confirmed the presence of apoptotic cells. Myocardial ET-1 concentrations were 4.8±1.0 versus 8.3±2.5 pg/100 mg (control vs. ischemic hearts, respectively; mean ±SD; p < 0.05). Myocardial NO contents showed no differences. Conclusion. These data suggest that the time window of apoptosis with detectable DNA fragmentation exceeds 20 min of global total ischemia and 40 min of reperfusion, a model frequently used for inducing myocardial stunning. While NO was not increased in postischemic hearts, increased ET-1 levels indirectly argue for a role of ET-1 as inducer of apoptosis, but only at a later stage of reperfusion.

Comparison of methods of fractional area change for detection of regional left ventricular dysfunction

Three methods for assessment of fractional area change (FAC) and conventional versus cross-sectional segmentation were compared under conditions known to occur frequently during stress echocardiography. Quantitative analysis of 80 echocardiograms obtained from healthy subjects, patients with left ventricular (LV) dysfunction and after coronary artery bypass grafting included segmental and cross-sectional FACs by the centroid method with fixed and floating reference and a method with floating external reference. All segmental and cross-sectional FACs were equally sensitive to LV dysfunction, and segmental FACs failed to accurately predict the location of coronary lesions. The centroid method with floating reference and cross-sectional FACs were the least affected by surgery induced intrathoracic heart motion. In moderate to severe LV dysfunction FAC by the centroid method with floating reference and cross sections were rarely within normal limits. Cross-sectional FACs may prove to be useful in stress echocardiography. For viability studies segmental FAC by fixed reference appears to be the method of choice.

Carnitine — from cellular mechanisms to potential clinical applications in heart disease

This review deals with cellular metabolic actions of carnitine and its potential role as a drug investigated in a number of clinical settings. It is not the aim of the present work to provide a comprehensive overview over the details of cellular metabolism or of all potential clinical applications, but rather to highlight the involvement in major metabolic pathways potentially relevant to clinical benefit. L-Carnitine is an organic acid molecule consisting of seven carbons (Fig. 1) [1]. It acts as an essential cofactor in cellular metabolism. Analogues of carnitine mostly used in clinical settings are the derivatives, acetylLcarnitine and propionylL carnitine [2]. Within the scope of this minireview, they will all be termed carnitine. Figure 2 summarizes the cellular processes in which carnitine is involved. Its main function is within fatty acid metabolism [3]. The highest intracellular concentration of carnitine is in the cytosol. Whenever long-chain fatty acids are to be used for energy generation, their binding to carnitine is mandatory for their translocation into the mitochondria (Fig. 2). This happens by means of a translocase enzyme, the carnitine acetyl transferase [4]. Degradation of fatty acids within the citric acid cycle subsequently liberates free energy by lipid oxidation [1]. While long-chain fatty acids are the main substrate for energy generation in muscle, short-chain fatty acids are theoretically used as well. However, they can enter the mitochondrial matrix without the carnitine carrier system. The overall body content of carnitine is in the range of 25 g. Carnitine is mainly synthesized by the liver; the only other organ with carnitine synthesis capacity is the kidney [3]. Precursors of carnitine are the amino acids lysine and methionine. The two compartments of the body with the highest energy demands from fatty acid metabolism are the skeletal and cardiac muscle [5]. Interestingly, these two tissue compartments do not synthesize carnitine by themselves, and yet they are characterized by some of the highest tissue concentrations of carnitine [3,6]. Dietary sources of carnitine do supplement the overall body content. These are predominantly meat and diary products [3,7]. The mechanism of intestinal uptake of carnitine is not yet completely understood, but it seems to be a slow saturable process. Supplementation of orally administered carnitine results in increased serum concentrations within hours, while intravenously administered carnitine leads to a serum peak within minutes [8]. However, serum carnitine concentrations after intravenous injection return to baseline after 12 h, suggesting a twoor three-compartment distribution within the body. Furthermore, a substantial fraction, i.e. 70–80%, of carnitine is cleared by the kidney as acylcarnitine within 24 h. Hepatic carnitine concentration is increased shortly after supplementation, which is not the case with myocardial or skeletal carnitine content. Myocardial uptake occurs slowly by a specific carrier protein [9]. In normal individuals, myocardial tissue carnitine levels greatly exceed plasma levels, i.e. uptake occurs against a 60-fold serum concentration gradient [10].

Failure of Platelet Parameters and Biomarkers to Correlate Platelet Function to Severity and Etiology of Heart Failure in Patients Enrolled in the EPCOT Trial With Special Reference to the Hemodyne ® Hemostatic Analyzer

Data from small studies have suggested the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of different platelet parameters and platelet-endothelial biomarkers in a random outpatient CHF population investigated in the EPCOT (‘Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure’) Trial. Blood samples were obtained for measurement of platelet contractile force (PCF), whole blood aggregation, shear-induced closure time, expression of glycoprotein (GP) IIb/IIIa, and P-selectin in 100 consecutive patients with CHF. Substantial interindividual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were grouped according to incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Aspirin use did not affect instrument readings either. PCF correlates very poorly with whole blood aggregometry (r2 = 0.023), closure time (r2 = 0.028), platelet GP IIb/IIIa (r2 = 0.0028), and P-selectin (r2 = 0.002) expression. Furthermore, there was no correlation with brain natriuretic peptide concentrations, a marker of severity and prognosis in heart failure reflecting the neurohumoral status. Patients with heart failure enrolled in the EPCOT Trial exhibited a marginal, sometimes oppositely directed change in platelet function, challenging the diagnostic utility of these platelet parameters and biomarkers to serve as useful tools for the identification of platelet abnormalities, for predicting clinical outcomes, or for monitoring antiplatelet strategies in this population. The usefulness of these measurements for assessing platelets in the different clinical settings remains to be explored. Taken together, opposite to our expectations, major clinical characteristics of heart failure did not correlate well with the platelet characteristics investigated in this study.

Between observer variation is not eliminated by standardised analysis of dobutamine–atropine stress echocardiography

Aims: The conventional analysis of dobutamine–atropine stress echocardiography (DASE) is poorly defined and subject to considerable variation. The aim of this study was to investigate the reproducibility of strictly standardised qualitative analysis in DASE. Methods and results: Strict criteria for standardised DASE interpretation were defined through logistic regression analysis on categorical parameters obtained from 20 patients with coronary artery disease (CAD) and 20 healthy controls subjected to DASE. Three expert echocardiographers analysed DASE recordings from 100 consecutive patients referred for coronary angiography. Specificity for CAD and for predicting significant stenosis of a major coronary artery was 94% (95% CI: 83–100%) and 79% (95% CI: 63–96%), whereas sensitivity was 49% in both cases (95% CI: 38–60% and 37–61%). Within and between observer reproducibility was moderate to fair (κ = 0.56 and 0.38; 95% CI: 0.40–0.72 and 0.24–0.52). In patients without prior myocardial infarction and in echogenic patients within observer reproducibility was good (κ = 0.72 and 0.74; 95% CI: 0.52–0.92 and 0.56–0.92). Conclusions: Observer variation was not eliminated in standardised qualitative DASE interpretation based on criteria that predicted the presence of CAD with a high specificity and reproducibility was good only in certain subgroups of patients.

Platelets and Thrombolysis: Cooperation or Contrariety?

Fibrinolytic therapy is the established treatment for the management of patients with ST elevation acute myocardial infarction (AMI). Present thrombolytic regimens have a number of limitations, including the failure to produce early and sustained reperfusion, as well as an inability to prevent reocclusion in at least some patients. Platelets play an important role in coronary thrombosis responsible for AMI. The effect of coronary thrombolysis on platelets has been extensively debated in the literature, with controversial evidence of both platelet activation and inhibition. Among fibrinolytic agents, tissue plasminogen activator (t-PA) is considered to be the cornerstone in the treatment of acute coronary occlusion. The native t-PA molecule has been modified in an attempt to achieve improved lytic characteristics with less risk of bleeding events. Extensive research has led to a group of mutant t-PA variants referred to as third-generation plasminogen activators. TNK-t-PA is one bioengineered variant of t-PA; another is reteplase (r-PA). They have been developed to establish more rapid, complete and stable coronary artery patency, thus promising reduced mortality. Both r-PA and TNK-t-PA are effective when given as bolus therapy, a feature that may facilitate earlier treatment initiation as well as lower treatment costs. New acute coronary treatment regimens include potent antiplatelet agents on top of thrombolysis that may improve sustained reperfusion. This review summarizes the latest and often contradictory data on the interaction between fibrinolytic therapy and platelets in certain in vitro, animal and clinical scenarios.