Objective-We investigated whether red cell infiltration of atheromatous lesions promotes the later stages of atherosclerosis. Methods and Results-We find that oxidation of ferro (FeII) hemoglobin in ruptured advanced lesions occurs generating ferri (FeIII) hemoglobin and via more extensive oxidation ferrylhemoglobin (FeIII/FeIVϭO). The protein oxidation marker dityrosine accumulates in complicated lesions, accompanied by the formation of cross-linked hemoglobin, a hallmark of ferrylhemoglobin. Exposure of normal red cells to lipids derived from atheromatous lesions causes hemolysis and oxidation of liberated hemoglobin. In the interactions between hemoglobin and atheroma lipids, hemoglobin and heme promote further lipid oxidation and subsequently endothelial reactions such as upregulation of heme oxygenase-1 and cytotoxicity to endothelium. Oxidative scission of heme leads to release of iron and a feed-forward process of iron-driven plaque lipid oxidation. The inhibition of heme release from globin by haptoglobin and sequestration of heme by hemopexin suppress hemoglobin-mediated oxidation of lipids of atheromatous lesions and attenuate endothelial cytotoxicity. Conclusion-The interior of advanced atheromatous lesions is a prooxidant environment in which erythrocytes lyse, hemoglobin is oxidized to ferri-and ferrylhemoglobin, and released heme and iron promote further oxidation of lipids. Oxysterols and oxidation products of polyunsaturated fatty acids are present in human atheromatous lesions. 4,5 Atherosclerotic lesions are hazardous regions for nucleated cells, both endothelial cells and, quite probably, incoming macrophages. 6 The major cytotoxic species may be oxidation products of lipids, particularly lipid hydroperoxides (LOOHs), aldehydes, and carbonyls. 6,7 In artificial systems, oxidation of polyunsaturated fatty acids requires reactive transition metals such as iron and copper. Based on our earlier work, 6,8,9 the metal in atheromatous lesions might be iron derived from heme. Nonprotein-bound heme is a particularly deleterious species inasmuch as it is hydrophobic and easily able to enter cell membranes. 10 In previous studies, we found that endothelial cells exposed to oxidized low-density lipoprotein (LDL) upregulated both heme oxygenase-1 (HO-1) and ferritin, 8,9 presumably as a defense mechanism. 6,11-14 Upregulation of HO-1 15 and ferritin H chain 16 in endothelial cells has been reported in the early phase of progression of atherosclerotic lesions. Expression of HO-1 provides protection against atherosclerosis in several experimental models, 17,18 and HO-1 deficiency in humans has been associated with the appearance of vasculature fatty streaks and atheromatous plaques at the age of 6. 19 We tested the hypothesis that heme-iron may accumulate in atherosclerotic lesions by intrusion and lysis of erythrocytes. Liberated hemoglobin is oxidized, and released hemeiron-dependent oxidation of lipids is strongly favored, contributing to further plaque development. Methods Tissue SamplesSpecimens of ...

Hyperthyroidism elevates cardiovascular mortality by several mechanisms, including increased risk of ischemic heart disease. Therefore, therapeutic strategies, which enhance tolerance of heart to ischemia-reperfusion injury, may be particularly useful for hyperthyroid patients. One promising cardioprotective approach is use of agents that cause (directly or indirectly) A1 adenosine receptor (A1 receptor) activation, since A1 adenosinergic pathways initiate protective mechanisms such as ischemic preconditioning. However, previously we found great A1 receptor reserve for the direct negative inotropic effect of adenosine in isolated guinea pig atria. This phenomenon suggests that weakening of atria is a possible side effect of A1 adenosinergic stimulant agents. Thus, the goal of the present investigation was to explore this receptor reserve in hyperthyroidism. Our recently developed method was used that prevents the rapid intracellular elimination of adenosine, allowing sufficient time for exogenous adenosine administered for the generation of concentration-response curves to exert its effect. Our method also allowed correction for the bias caused by the consequent endogenous adenosine accumulation. Our results demonstrate that thyroxine treatment does not substantially affect the A1 receptor reserve for the direct negative inotropic effect of adenosine. Consequently, if an agent causing A1 receptor activation is administered for any indication, the most probable adverse effect affecting the heart may be a decrease of atrial contractility in both eu- and hyperthyroid conditions.

A1 adenosine receptors (A1 receptors) are widely expressed in mammalian tissues; therefore attaining proper tissue selectivity is a cornerstone of drug development. The fact that partial agonists chiefly act on tissues with great receptor reserve can be exploited to achieve an appropriate degree of tissue selectivity. To the best of our knowledge, the A1 receptor reserve has not been yet quantified for the atrial contractility. A1 receptor reserve was determined for the direct negative inotropic effect of three A1 receptor full agonists (NECA, CPA and CHA) in isolated, paced guinea pig left atria, with the use of FSCPX, an irreversible A1 receptor antagonist. FSCPX caused an apparently pure dextral displacement of the concentration-response curves of A1 receptor agonists. Accordingly, the atrial A1 receptor function converging to inotropy showed a considerably great, approximately 80-92 % of receptor reserve for a near maximal (about 91-96 %) effect, which is greater than historical atrial A1 receptor reserve data for any effects other than inotropy. Consequently, the guinea pig atrial contractility is very sensitive to A1 receptor stimulation. Thus, it is worthwhile considering that even partial A1 receptor agonists, given in any indication, might decrease the atrial contractile force, as an undesirable side effect, in humans.

This study sought to characterize the relation between the oxidation of protein sulfhydryl (SH) groups and Ca2+-activated force production in the human myocardium. Triton-permeabilized left ventricular cardiomyocytes from donor hearts were exposed to an oxidative (2,2'-dithiodipyridine, DTDP) agent in vitro, and the changes in isometric force, its Ca2+ sensitivity, the cross-bridge-sensitive rate constant of force redevelopment at saturating [Ca2+] (k(tr,max)), and protein SH oxidation were monitored. DTDP (0.1-10 mM for 2 min) oxidized the myocardial proteins and diminished the Ca2+-activated force with different concentration dependences (EC(50,SH) = 0.17 +/- 0.02 mM and EC(50,force) = 2.46 +/- 0.22 mM; mean +/- SEM). The application of 2.5 mM DTDP decreased the maximal Ca2+-activated force (to 64%), its Ca2+ sensitivity (DeltapCa(50) = 0.22 +/- 0.02), and the steepness of the Ca2+-force relation (n(Hill), from 2.01 +/- 0.08 to 1.76 +/- 0.08). These changes were paralleled by reductions in the free SH content of the proteins (to 15%) and in k(tr,max) (to 75%). SH-specific labeling identified SH oxidation of myosin light chain 1 and actin at DTDP concentrations at which the changes in the contractile parameters occurred. Our data suggest that SH oxidation in selected myofilament proteins diminishes the Ca2+-activated force and its Ca2+ sensitivity through an impaired Ca2+ regulation of the actin-myosin cycle in the human heart.

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2ϩ sensitivity of isometric force development along with sarcomere length (SL) is considered as the basis of the FrankStarling law of the heart, possibly involving the regulation of crossbridge turnover kinetics. Therefore, the Ca 2ϩ dependencies of isometric force production and of the cross-bridge-sensitive rate constant of force redevelopment (k tr) were determined at different SLs (1.9 and 2.3 m) in isolated human, murine, and porcine permeabilized cardiomyocytes. k tr was also determined in the presence of 10 mM inorganic phosphate (Pi), which interfered with the force-generating cross-bridge transitions. The increases in Ca 2ϩ sensitivities of force with SL were very similar in human, murine, and porcine cardiomyocytes (⌬pCa 50: ϳ0.11). ktr was higher (P Ͻ 0.05) in mice than in humans or pigs at all Ca 2ϩ concentrations ([Ca 2ϩ ]) [maximum ktr (ktr,max) at a SL of 1.9 m and pCa 4.75: 1.33 Ϯ 0.11, 7.44 Ϯ 0.15, and 1.02 Ϯ 0.05 s Ϫ1 , in humans, mice, and pigs, respectively] but ktr did not depend on SL in any species. Moreover, when the ktr values for each species were expressed relative to their respective maxima, similar Ca 2ϩ dependencies were obtained. Ten millimolar Pi decreased force to ϳ60 -65% and left ⌬pCa 50 unaltered in all three species. P i increased ktr,max by a factor of ϳ1.6 in humans and pigs and by a factor of ϳ3 in mice, independent of SL. In conclusion, species differences exert a major influence on k tr, but SL does not appear to modulate the cross-bridge turnover rates in human, murine, and porcine hearts. mouse; pig; heart; skinned muscle; myofilament length-dependent activation; rate of tension redevelopment; calcium THE LENGTH-DEPENDENT INCREASE in myofilament Ca 2ϩ sensitivity is considered to be the explanation for the improved cardiac systolic performance following an increase in end-diastolic ventricular volume, i.e., for the Frank-Starling mechanism. Despite recent advances in the understanding of various aspects of the Frank-Starling mechanism (for a recent review, see Ref. 12), the molecular mechanism of the length-dependent Ca 2ϩ sensitization remains obscure. Ca 2ϩ regulates force production through changes in cross-bridge turnover kinetics in both the skeletal (4) and the cardiac (2, 44) muscle; accordingly, length-dependent alterations of cross-bridge transitions may well underlie the Frank-Starling law of the heart.Using rabbit skeletal muscle fibers, Zhao and Kawai (45) observed alterations in the rates of cross-bridge transitions during osmotic compression. These results were consistent with the length-dependent decrease in cross-bridge turnover and cross-bridge detachment at higher sarcomere length (SL), where lattice spacing was also reduced (45). However, in rat slow-twitch and rabbit fast-twitch skeletal muscle fibers the rate of tension redevelopment (k tr ), a measure of the rate of cross-bridge cycling, was reduced at short SL compared with a longer SL (24). In skinned rat cardiac trabeculae, simultaneous measurements of isometric force and rates of ATP consump...

Adenosine is a ubiquitous, endogenous purine involved in a variety of physiological and pathophysiological regulatory mechanisms. Adenosine has been proposed as an endogenous antiarrhythmic substance to prevent hypoxia/ischemia-induced arrhythmias. Adenosine (and its precursor, ATP) has been used in the therapy of various cardiac arrhythmias over the past six decades. Its primary indication is treatment of paroxysmal supraventricular tachycardia, but it can be effective in other forms of supraventricular and ventricular arrhythmias, like sinus node reentry based tachycardia, triggered atrial tachycardia, atrioventricular nodal reentry tachycardia, or ventricular tachycardia based on a cAMP-mediated triggered activity. The main advantage is the rapid onset and the short half life (1- 10 sec). Adenosine exerts its antiarrhythmic actions by activation of A1 adenosine receptors located in the sinoatrial and atrioventricular nodes, as well as in activated ventricular myocardium. However, adenosine can also elicit A2A, A2B and A3 adenosine receptor-mediated global side reactions (flushing, dyspnea, chest discomfort), but it may display also proarrhythmic actions mediated by primarily A1 adenosine receptors (e.g. bradyarrhythmia or atrial fibrillation). To avoid the non-specific global adverse reactions, A1 adenosine receptor- selective full agonists (tecadenoson, selodenoson, trabodenoson) have been developed, which agents are currently under clinical trial. During long-term administration with orthosteric agonists, adenosine receptors can be internalized and desensitized. To avoid desensitization, proarrhythmic actions, or global adverse reactions, partial A1 adenosine receptor agonists, like CVT-2759, were developed. In addition, the pharmacologically "silent" site- and event specific adenosinergic drugs, such as adenosine regulating agents and allosteric modulators, might provide attractive opportunity to increase the effectiveness of beneficial actions of adenosine and avoid the adverse reactions.

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