Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during variousin vivo as well asin vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.     

Evidence of direct toxic effects of free radicals on the myocardium

The hypothesis that oxygen-derived free radicals do indeed play a role in myocardial ischemic and reperfusion injury has received a lot of support. Experimental results have shown that free radical scavengers can protect against certain aspects of myocardial ischemic injury and that on reperfusion the heart approaches a level that is more normal than those hearts not receiving additional scavenging agents. Superoxide dismutase, catalase, glutathione peroxidase, hydroxyl radical scavengers and iron chelators such as desferrioxamine have proven successful in providing an increased level of recovery. These results indicate, as would be expected, that superoxide, hydrogen peroxide and hydroxyl radicals may all, at some point, either contribute to the injury or be important in generating a subsequent radical which causes damage. In addition, solutions capable of generating free radicals have been shown to cause damage to myocardial cells and the vascular endothelium that is similar to the damage observed during myocardial ischemic and reperfusion injury. Alterations in function, structure, flow, and membrane biochemistry have been documented and compared to ischemic injury. The continued investigation of the role of free radicals in ischemic injury is warranted in the hope of further elucidating the mechanisms involved in free radical injury, the sources of their generation, and in defining a treatment that will provide significant protection against this particular aspect of ischemic damage.     

Myocardial gap junctions: targets for novel approaches in the prevention of life-threatening cardiac arrhythmias

Direct cell-to-cell communication in the heart is maintained via gap junction channels composed of proteins termed connexins. Connexin channels ensure molecular and electrical signals propagation and hence are crucial in myocardial synchronization and heart function. Disease-induced gap junctions remodeling and/or an impairment or even block of intercellular communication due to acute pathological conditions results in derangements of myocardial conduction and synchronization. This is critical in the development of both ventricular fibrillation, which is a major cause of sudden cardiac death and persistent atrial fibrillation, most common arrhythmia in clinical practice often resulting in stroke. Many studies suggest that alterations in topology (remodeling), expression, phosphorylation and particularly function of connexin channels due to age or disease are implicated in the development of these life-threatening arrhythmias. It seems therefore challenging to examine whether compounds that could prevent or attenuate gap junctions remodeling and connexin channels dysfunction can protect the heart against arrhythmias that cause sudden death in humans. This assumption is supported by very recent findings showing that an increase of gap junctional conductance by specific peptides can prevents atrial conduction slowing or re-entrant ventricular tachycardia in ischemic heart. Suppression of ischemia-induced dephosphorylation of connexin seems to be one of the mechanisms involved. Another approach for identifying novel treatments is based on the hypothesis that even non-antiarrhythmic drugs with antiarrhythmic ability can modulate gap junctional communication and hence attenuate arrhythmogenic substrates.     

Involvement of lipid peroxidation in regression of heart hypertrophy

The regression of hypertrophied heart presupposes disassembling of all cardiomyocyte components including membrane structures. The involvement of free radical oxidation of membrane phospholipids was studied in the cardiac regression. Altitude hypertrophy was developed in barocamera (7000 m, 6 hours daily). 3 weeks of periodical hypoxia leads to 1.5-fold increase of heart weight (right ventricle weight was 2-fold increased). In 7-10 days after adaptation the heart weight reduced to normal. Both, the development and regression of myocardial hypertrophy was accompanied by changes in Mb and Hb organ concentration proportional to weight changes. In lipid extraction of maximal hypertrophied heart, the 30% decrease of lipid peroxidation product (diene conjugates) regularly occurred. The rate of regression had negative correlation with peroxidation products accumulation. Intraperitoneal injection of free radical scavenger BHT attenuate the regression rate. The results suggest that unlike the common knowledge about the membrane injury effect, lipid peroxidation can play positive role in disassembling of superfluous cell membrane structures.     

Maintenance of left ventricular function (90%) after twenty-four-hour heart preservation with deferoxamine.

During 24-h in vitro heart preservation and reperfusion, irreversible tissue damage occurs caused by reactive oxygen intermediates, such as superoxide radicals, singlet oxygen, hydrogen peroxide, hydroperoxyl, hydroxyl radicals, as well as the peroxynitrite radical. Reduction of the related oxidative damage of reperfused ischemic tissue by free radical scavengers and metal chelators is of primary importance in maintaining heart function. We assessed whether deferoxamine (DFR) added to a cardioplegia solution decreased free radical formation during 24-h cold (5 degrees C) heart preservation and normothermic reperfusion (37 degrees C) in the Langendorff isolated perfused rat heart. The deferoxamine treated hearts were significantly (p less than .001) better preserved than the control hearts after 24 h of preservation with regard to recovery of left ventricular diastolic pressure, contractility (+dP/dt), relaxation (-dP/dt), creatine kinase release, and lipid peroxidation. DFR preserved cell membrane integrity and maintained 93% of left ventricular contractility. The evidence suggests that DFR reduces lipid peroxidation damage by reducing free radical formation and thereby maintaining normal coronary perfusion flow and myocardial function.     

Oxygen radicals in cardiac surgery

Most cardiac surgical procedures require the use of prolonged induced myocardial ischemia. Experimental models of global myocardial ischemia which mimic cardiac surgical techniques have been developed to investigate the possibility of oxygen free radical development during prolonged myocardial ischemia or upon reperfusion. In such experiments, various free radical scavenging agents, including superoxide dismutase, catalase, and mannitol, have been shown to improve the tolerance of the heart to protracted global ischemia. Use of these agents has improved cardiac functional recovery and has attenuated the biochemical and structural changes which occur due to prolonged ischemia and reflow. In a recently developed porcine experimental model, the effects of preexisting regional myocardial ischemia with superimposed global ischemia and reperfusion have been studied, with free radical scavenging agents administered in an attempt to reduce myocardial infarction and improve regional functional recovery. In most such studies completed to date, free radical scavenging agents have resulted in better myocardial preservation, suggesting, at least indirectly, that there may be an oxygen free radical-mediated component of the ischemia-reperfusion injury seen in such models. Techniques for directly measuring myocardial oxygen free radical levels may allow for early clarification of the development of such toxic species in the clinical cardiac surgical setting.     

Free radical-mediated skeletal muscle dysfunction in inflammatory conditions.

Loss of functional capacity of skeletal muscle is a major cause of morbidity in patients with a number of acute and chronic clinical disorders, including sepsis, chronic obstructive pulmonary disease, heart failure, uremia, and cancer. Weakness in these patients can manifest as either severe limb muscle weakness (even to the point of virtual paralysis), respiratory muscle weakness requiring mechanical ventilatory support, and/or some combination of these phenomena. While factors such as nutritional deficiency and disuse may contribute to the development of muscle weakness in these conditions, systemic inflammation may be the major factor producing skeletal muscle dysfunction in these disorders. Importantly, studies conducted over the past 15 years indicate that free radical species (superoxide, hydroxyl radicals, nitric oxide, peroxynitrite, and the free radical-derived product hydrogen peroxide) play an key role in modulating inflammation and/or infection-induced alterations in skeletal muscle function. Substantial evidence exists indicating that several free radical species can directly alter contractile protein function, and evidence suggests that free radicals also have important effects on sarcoplasmic reticulum function, on mitochondrial function, and on sarcolemmal integrity. Free radicals also modulate activation of several proteolytic pathways, including proteosomally mediated protein degradation and, at least theoretically, may also influence pathways of protein synthesis. As a result, free radicals appear to play an important role in regulating a number of downstream processes that collectively act to impair muscle function and lead to reductions in muscle strength and mass in inflammatory conditions.     

Molecular Mechanisms of Inherited Arrhythmias

Inherited arrhythmias and conduction system diseases are known causes of sudden cardiac death and are responsible for significant mortality and morbidity in patients with congenital heart disease and electrical disorders. Knowledge derived from human genetics and studies in animal models have led to the discovery of multiple molecular defects responsible for arrhythmogenesis. This review summarizes the molecular basis of inherited arrhythmias in structurally normal and altered hearts.On the cellular and molecular levels, minor disturbances can provoke severe arrhythmias. Ion channels are responsible for the initiation and propagation of the action potential within the cardiomyocyte. Structural heart diseases, such as hypertrophic or dilated cardiomyopathies, increase the likelihood of cardiac electrical abnormalities. Ion channels can also be up- or down-regulated in congenital heart disease, altering action potential cellular properties and therefore triggering arrhythmias. Conduction velocities may be inhomogeneously altered if connexin function, density or distribution changes. Another important group of electrophysiologic diseases is the heterogeneous category of inherited arrhythmias in the structurally normal heart, with a propensity to sudden cardiac death. There have been many recent relevant discoveries that help explain the molecular and functional mechanisms of long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia, and other electrical myopathies. Identification of molecular pathways allows the identification of new therapeutic targets, for both disease palliation and cure. As more disease-causing mutations are identified and genotypic-phenotypic correlation is defined, families can be screened prior to symptom-onset and patients may potentially be treated in a genotype-specific manner, opening the doors of cardiac electrophysiology to the emerging field of pharmacogenomics.     

Membrane Effects of the n-3 Fish Oil Fatty Acids,which Prevent Fatal Ventricular Arrhythmias

Fish oil fatty acids are known to exert beneficial effects on the heart and vascular systems. We have studied the membrane effects on ion channel conductance by the n-3 fish oil fatty acids that account for these beneficial effects. We have confirmed that these fatty acids prevent fatal cardiac arrhythmias in a reliable dog model of sudden cardiac death. This finding was followed by experiments indicating that the n-3 fatty acids electrically stabilize heart cells and do so largely through modulation of the fast voltage-dependent Na⁺ currents and the L-type Ca²⁺ channels in a manner, which makes the heart cells resistant to arrhythmias. Others and we have demonstrated that these membrane effects on the heart can prevent fatal cardiac arrhythmias in humans.     

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.     

Mitochondrial criticality: a new concept at the turning point of life or death

A variety of stressors can cause the collapse of mitochondrial membrane potential (DeltaPsi(m)), but the events leading up to this catastrophic cellular event are not well understood at the mechanistic level. Based on our recent studies of oscillations in mitochondrial energetics, we have coined the term "mitochondrial criticality" to describe the state in which the mitochondrial network of cardiomyocytes becomes very sensitive to small perturbations in reactive oxygen species (ROS), resulting in the scaling of local mitochondrial uncoupling and DeltaPsi(m) loss to the whole cell, and the myocardial syncytium. At the point of criticality, the dynamics of the mitochondrial network bifurcate to oscillatory behavior. These energetic changes are translated into effects on the electrical excitability of the cell, inducing dramatic changes in the morphology and the threshold for activating an action potential. Emerging evidence suggests that this mechanism, by creating spatial and temporal heterogeneity of excitability in the heart during ischemia and reperfusion, underlies the genesis of potentially lethal cardiac arrhythmias.     

Regulation of cation channels in cardiac and smooth muscle cells by intracellular magnesium

Magnesium regulates various ion channels in many tissues, including those of the cardiovascular system. General mechanisms by which intracellular Mg(2+) (Mg(i)(2+)) regulates channels are presented. These involve either a direct interaction with the channel, or an indirect modification of channel function via other proteins, such as enzymes or G proteins, or via membrane surface charges and phospholipids. To provide an insight into the role of Mg(i)(2+) in the cardiovascular system, effects of Mg(i)(2+) on major channels in cardiac and smooth muscle cells and the underlying mechanisms are then reviewed. Although Mg(i)(2+) concentrations are known to be stable, conditions under which they may change exist, such as following stimulation of beta-adrenergic receptors and of insulin receptors, or during pathophysiological conditions such as ischemia, heart failure or hypertension. Modifications of cardiovascular electrical or mechanical function, possibly resulting in arrhythmias or hypertension, may result from such changes of Mg(i)(2+) and their effects on cation channels.     

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.     

Chronic cardiac insufficiency, a disease of adaptation]

Cardiac hypertrophy due to a mechanical overload is not a disease per se but the physiological reaction of the heart to a disease which is usually arterial hypertension and/or coronary insufficiency. It results from the put into play of several changes in gene expression, frequently species-specific, which explain the thermodynamic improvement of the cardiac function and the physiological adaptation of the heart to the new environmental requirements. Some of these modifications can have detrimental consequences and explain the modifications of ventricular compliance and the high incidence of arrhythmias in ventricular hypertrophy. The most important changes in the genetic expression which have been reported so far after pressure overload are located on contractile proteins and on membrane proteins.     

Hyperoxia causes oxygen free radical-mediated membrane injury and alters myocardial function and hemodynamics in the newborn

Newborn children can be exposed to high oxygen levels (hyperoxia) for hours to days during their medical and/or surgical management, and they also can have poor myocardial function and hemodynamics. Whether hyperoxia alone can compromise myocardial function and hemodynamics in the newborn and whether this is associated with oxygen free radical release that overwhelms naturally occurring antioxidant enzymes leading to myocardial membrane injury was the focus of this study. Yorkshire piglets were anesthetized with pentobarbital sodium (65 mg/kg), intubated, and ventilated to normoxia. Once normal blood gases were confirmed, animals were randomly allocated to either 5 h of normoxia [arterial Po(2) (Pa(O(2))) = 83 +/- 5 mmHg, n = 4] or hyperoxia (Pa(O(2)) = 422 +/- 33 mmHg, n = 6), and myocardial functional and hemodynamic assessments were made hourly. Left ventricular (LV) biopsies were taken for measurements of antioxidant enzyme activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)] and malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) as an indicator of oxygen free radical-mediated membrane injury. Hyperoxic piglets suffered significant reductions in contractility (P < 0.05), systolic blood pressure (P < 0.03), and mean arterial blood pressure (P < 0.05). Significant increases were seen in heart rate (P < 0.05), whereas a significant 11% (P < 0.05) and 61% (P < 0.001) reduction was seen in LV SOD and GPx activities, respectively, after 5 h of hyperoxia. Finally, MDA and 4-HNE levels were significantly elevated by 45% and 38% (P < 0.001 and P = 0.02), respectively, in piglets exposed to hyperoxia. Thus, in the newborn, hyperoxia triggers oxygen free radical-mediated membrane injury together with an inability of the newborn heart to upregulate its antioxidant enzyme defenses while impairing myocardial function and hemodynamics.     

Mechano-electric feedback and arrhythmias

The mechanical state of the heart feeds back to modify cardiac rate and rhythm. Mechanical stretch of myocardial tissue causes immediate and chronic responses that lead to the common end point of arrhythmia. This review provides a brief summary of the author's personal choice of contributions that she considers have fostered our understanding of the role of mechano-electric feedback in arrhythmogenesis.

Acute mechanical stretch reversibly depolarises the cell membrane and shortens the action potential duration. These electrophysiological changes are related to the activation of mechano-sensitive ion channels. Several different ion channels are involved in the sensing of stretch, among them K⁺-selective, Cl⁻-selective, non-selective, and ATP-sensitive K⁺ channels. Sodium and Ca²⁺ entering the cells via non-selective ion channels are thought to contribute to the genesis of stretch-induced arrhythmia. Mechano-sensitive channels have been cloned from non-vertebrate and vertebrate species.

Chronic stress on the heart activates gene expression in cardiomyocytes and non-myocytes. The signal transduction involves atrial natriuretic peptides and growth factors that initiate remodelling processes leading to hypertrophy which in turn may contribute to the electrical instability of the heart by increasing the responsiveness of mechano-sensitive channels. Selective block of these channels could provide some new form of treatment of mechanically induced arrhythmias, although at present there are no drugs available with sufficient selectivity. Detailed understanding of how mechanical strain on myocardial cells is translated into channel activation will allow to identify new targets for putative antiarrhythmic drugs.     

Reperfusion-induced arrhythmias: do free radicals play a critical role?

This article assesses whether oxygen-derived free radicals are one of the molecular causes of life-threatening arrhythmias that arise upon reperfusion of the ischemic myocardium. Evidence supporting this proposition has been obtained from studies of the effects of free radical scavengers and antioxidants, free radical generating systems, inhibition of various sources of free radicals and studies investigating the formation of free radicals and their products during early reperfusion. It has been hypothesized that free radical formation causes localised membrane damage to the sarcolemma that results in focal alterations in transmembrane ionic fluxes, particularly potassium. These changes in ionic fluxes may then lead to electrophysiological abnormalities that culminate in ventricular arrhythmias.