Ischemic Fibers Research Articles

Cardioprotective Effect of 2-Ethyl-3-Hydroxy-6-Methylpyridinium 2-Nitroxysuccinate Against Adrenaline/Hydrocortisone-Induced Myocardial Ischemia in Mice: Modulation of Free-Radical Processes in Biomembranes and Monoamine Oxidase A Activity.

Oxidative stress (OS) plays a key role in the development of cardiovascular diseases (CVD) in three major ways: reactive oxygen species (ROS)-induced reduction of nitric oxide (NO) bioavailability, ROS-induced inflammation and ROS-induced mitochondrial dysfunction. Oxidation of lipid molecules under the action of ROS leads to damage to membrane structures, changes the functioning of membrane-bound enzymes, and impairs membrane permeability and stability. An increase in OS results in the occurrence of endothelial dysfunction and drug tolerance, side effects, requiring discontinuation of drugs. All of these are significant problems of cardiotherapy. Therefore, the search for new alternative NO donors continues. The present research was aimed at studying the protective effect of 2-ethyl-3-hydroxy-6-methylpyridinium 2-nitroxysuccinate (NS) on the cardiovascular system on mouse myocardial ischemia (MI) model. The NS hybrid molecule includes a synthetic vitamin B6 analog 2-ethyl-3-hydroxy-6-methylpyridine (an antioxidant) and 2-nitroxysuccinic acid (a source of nitric oxide). Using the electron paramagnetic resonance (EPR) method and biochemical methods, we showed that the pronounced ability of NS to release NO is favorably combines with the capacity to prevent OS due to mechanisms such as suppression of the lipid peroxidation (LPO) process, antiradical activity and inhibition of the mitochondrial membrane-bound monoamine oxidase A (MAO-A). Using histological methods, we established that the administration of NS (10 mg/kg, i.p.) reduces the number of ischemic fibers and protects cardiomyocytes against ischemia injury. Thus, the complex protective effect allows us to consider NS as an alternative NO donor and a candidate for the development of a new pharmaceutical agent for the treatment of CVD.

Force relaxation and thin filament protein phosphorylation during acute myocardial ischemia

Ischemia impairs myocardial function and may contribute to the progression of heart failure. In this study, rats subjected to acute ischemia demonstrated reduced Ca(2+) -activated force as well as a decrease in myosin-binding protein-C, titin, and Ser23/24 phosphorylation of troponin I (TnI). All three proteins have been demonstrated to be downstream targets of β-adrenergic receptor activation (β-AR), leading to the hypothesis that decreased β-AR signaling during ischemia leads to reduced protein phosphorylation and reduced rate constants of force relaxation. To test this hypothesis, force relaxation transients were recorded from permeabilized perfused and ischemic rat heart fibers following photolysis of the caged chelator diazo-2. Relaxation transients were best fit by double exponential functions whereby the majority (>70%) of the force decline was described by the fast rate constant, which was ∼5 times faster than the slow rate constant. However, rate constants of relaxation between perfused and ischemic fibers were not different, despite significant decreases in sarcomeric protein phosphorylation in ischemic fibers. Treatment of perfused fibers with a cAMP analog increased Ser23/24 phosphorylation of TnI, yet the rate constants of relaxation remained unchanged. Interestingly, similar treatment of ischemic fibers did not impact TnI phosphorylation or force relaxation transients. Therefore, acute ischemia does not influence the rate constants of relaxation of permeabilized fibers. These results also suggest that the physiological level of sarcomeric protein phosphorylation is unlikely to be the primary driver of relaxation kinetics in permeabilized cardiac muscle fibers.

Regulation of Fibre Contraction in a Rat Model of Myocardial Ischemia

BackgroundThe changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.Methodology/Principal FindingsA rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and Ca2+ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca2+ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.ConclusionsIschemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation.

Fiber Contractility In An In Vivo Model Of Myocardial Ischemia - Reperfusion

Altered blood flow to the heart, either transient or chronic, underscores the progression towards heart failure. Multiple models have suggested that alterations in Ca2+ handling and reduced energy reserves contribute to the reduction in cardiac muscle contractility. However, we have hypothesized that altered blood flow is also responsible for reversible, post-translational modifications to proteins of the contractile filaments, in turn limiting muscle contractility independent of available Ca2+ or ATP. Using an in vivo rat model, three experimental groups (perfused, ischemic, and reperfused) were established by limiting and re-establishing blood flow through the left anterior descending artery. Thin strips of the anterolateral papillary muscle were recovered and permeabilized with Triton-X100 to measure various contractile parameters. The maximum force and stiffness per cross-section (Fmax and Smax) of fibers from the three conditions were measured in pCa4 solution. The Fmax and Smax were significantly reduced in ischemic fibers (79% and 74% of perfused fibers), but restored to some extent in reperfused fibers (90% and 75% of perfused fibers). However, the Ca2+ sensitivity of contraction (EC50) was significantly shifted rightward only in ischemic fibers, with complete recovery in reperfused fibers. The reversible nature of the force decline and change in EC50 during ischemia suggests that the underlying changes in the contractile proteins were reversible, and most likely post-translational in nature. Additional experiments characterizing the altered contractility of ischemic fibers will be presented. Supported by NIH grant HL78845.

Fibrinogen: a diagnostic marker for early ischemia.

Although well established myocardial infarcts (MI) are not difficult to identify in routine hematoxylin and eosin stained sections, recent MI may present diagnostic problems. Fibrinogen is a useful marker for detecting early ischemic cell damage. Using immunofluorescence on frozen tissue, albumin, IgG and fibrinogen have been found throughout the sarcoplasm of ischemic fibers in human hearts. In this report, monoclonal antibodies to all three proteins were reacted using the avidin-biotin technique in formalin fixed, paraffin embedded sections from autopsy cases of sudden or intraoperative deaths with either subtle or no definite ischemic changes evident in routine sections. Strong staining of fibrinogen in the fibers associated with coagulative necrosis, contraction bands or wavy fibers, and in the fibers presumably associated with acute ischemia. Albumin and IgG staining was nonspecific. Fibrinogen is a reliable and reproducible marker for recognizing early ischemia. This method can be used to diagnose early sudden ischemic and intraoperative deaths due to coronary artery bypass graft and prosthesis-related complications and may be particularly useful for forensic autopsies.

A cytochemical method for the identification of ischemic and necrotic myocardial fibres.

The author studied the nickel-dimethylglioxim reaction in ischemic and necrotic myocardium fibres in dogs, following ligature of the left anterior descending coronary artery. The nickel-dimethylglioxim reaction was positive in ischemic fibers, but not in necrotic ones. The cytochemical reaction, observed in ischemic fibers, was localized in the intracellular compartment and in the mitochondria.

Actions of Volatile Anesthetics on Ischemic and Nonischemic Purkinje Fibers in the Infarcted Canine Heart

The effects of halothane, isoflurane, and enflurane on proximal (false tendon) and distal (apical) Purkinje fibers were measured in vitro in infarcted canine hearts to assess their effects on action potentials of fibers located within the nonischemic and ischemic regions, respectively. High- and low-dose anesthetic effects were evaluated in three groups of eight preparations and compared to changes occurring at identical times in eight infarcted control preparations. Under control conditions in all groups, the action potential duration at 90% repolarization (APD90, mean +/- SEM) of ischemic distal fibers (396 +/- 9 ms) was longer (P < or = 0.01) than that of nonischemic proximal fibers (344 +/- 5 ms) and the ischemic fibers exhibited (P < or = 0.05) reduced maximum diastolic potential, amplitude, and Vmax relative to nonischemic fibers. Halothane (0.25 and 0.6 mM), isoflurane (0.4 and 0.8 mM), and enflurane (0.8 and 1.6 mM) produced dose-dependent decreases of nonischemic fiber APD90 with less decrease (P < or = 0.01) of ischemic fiber APD90 and thereby accentuated (P < or = 0.05) regional differences of APD90 at high dose. The decreases of nonischemic fiber APD90 were greater (P < or = 0.01) for 0.8 mM (2.9 minimum alveolar anesthetic concentration [MAC]) isoflurane (-95 +/- 5 ms) and 1.6 mM (2.5 MAC) enflurane (-79 +/- 12 ms) than for 0.6 mM (2.2 MAC) halothane (-41 +/- 3 ms). Isoflurane increased the pathologic difference (ischemic > nonischemic) between the repolarization times (APD90) of Purkinje fibers in the infarcted heart more (P < or = 0.05) than halothane.(ABSTRACT TRUNCATED AT 250 WORDS)

Halothane, Enflurane and Isoflurane on Abnormal Automaticity and Triggered Rhythmic Activity of Purkinje Fibers from 24-hour-old Infarcted Canine Hearts

The effects of inhalation anesthetics halothane, enflurane, and isoflurane on spontaneous impulse initiation (automaticity) and triggered sustained rhythmic activity were examined in Purkinje fibers derived from normal (n = 38) and 24-h-old infarcted canine hearts (n = 27) to further understanding of their influence on the cellular mechanisms underlying generation of cardiac arrhythmias. Purkinje fibers from normal or infarcted hearts were superfused with modified Krebs' solution (37 degrees C) with or without epinephrine (2 or 15 microM) and equilibrated with a 97% O2-3% CO2 gas mixture (control). Transmembrane action potentials were recorded using conventional microelectrode techniques, and Purkinje fibers were exposed to anesthetic concentrations equivalent to 2.0 MAC. Normal Purkinje fibers were not spontaneously active unless exposed to epinephrine. All anesthetics (enflurane greater than halothane, isoflurane; P less than 0.05) increased automaticity of normal Purkinje fibers exposed to either epinephrine concentration. Partially depolarized Purkinje fibers from infarcted hearts were either spontaneously active or were quiescent. For ischemic fibers that beat spontaneously, abnormal automaticity was sustained (duration greater than 300 s) or periodic (duration less than 300 s). Sustained abnormal automaticity was elicited by epinephrine (15 microM) in some quiescent partially depolarized fibers. None of the anesthetics affected the rate of sustained abnormal automaticity, regardless of whether the induction of such automaticity required epinephrine, nor did anesthetics significantly affect the duration of trains of periodic abnormal automaticity. Finally, quiescent, partially depolarized Purkinje fibers were tested for triggered rhythmic activity during pacing at a cycle length of 800 ms.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal automatic rhythms in ischemic Purkinje fibers are modulated by a specific alpha 1-adrenergic receptor subtype.

Recent advances in adrenergic pharmacology have made possible the identification of alpha 1-adrenergic receptor subtypes using the specific blockers chloroethylclonidine and WB 4101. In the present study, we used these two blockers to determine the mechanisms responsible for automatic rhythms occurring during simulated ischemia and reperfusion of isolated canine Purkinje fibers. Experiments were done in the presence of propranolol to minimize beta-adrenergic contributions to the rhythms studied. In the control situation, all fibers showed membrane potentials greater than -90 mV and normal automatic rhythms. During simulated ischemia, membrane potential depolarized to the -60 mV range. Abnormal automaticity was seen in 20% of fibers not treated with phenylephrine and in 50% of those superfused with 1 x 10(-7) M phenylephrine. The incidence of abnormal automaticity was reduced to 0% by WB 4101 (which blocks phosphoinositide metabolic effects of alpha 1-adrenergic stimulation in the heart) and was increased to 90% by chloroethylclonidine (which blocks Na-K pump-stimulating effects of alpha-agonists). Moreover, the ischemic fibers were significantly more hyperpolarized during superfusion with WB 4101 than with chloroethylclonidine. Triggered activity induced by delayed or early after depolarizations was not seen in any experiment. Automatic arrhythmias induced by alpha 1-adrenergic stimulation during simulated ischemia may be attributed to a specific alpha 1-adrenergic receptor subtype that is blocked by WB 4101. These results have important implications with respect to the induction of arrhythmias in the setting of ischemia and the means for their prevention or treatment.

Effects of clofilium on ischemic subendocardial Purkinje fibers 1 day postinfarction

One day after ligation of the canine anterior descending coronary artery, clofilium, a long-acting class III anti-arrhythmic agent, was studied for its effects on normal and ischemic Purkinje fibers. In normal Tyrode's solution (4 mMpotassium, 2.7 mMcalcium) clofilium (10−7to 10−5M) increased action potential duration. Although only 2 of 10 normal Purkinje fibers developed early afterdepolarizations and early afterdepolarization-initiated triggered activity, 10 of 11 ischemic Purkinje fibers developed these features. Consequently, action potentials in ischemic fibers were prolonged to durations >10 s. The triggered activity in the ischemic Purkinje fibers produced repetitive activity in adjacent normal ventricular muscle.In vivo, 3 days after ligation, the administration of 3 to 10 mg/kg clofilium induced grouped beating. Action potentials recorded subsequently from these same hearts in vitro showed early afterdepolarizations, triggered activity and a similar grouping of responses. Therefore, clofilium differentially produced early afterdepolarizations in ischemic Purkinje fibers. This is a mechanism by which clofilium could be arrhythmogenic in an ischemic heart.

Actions of halothane on the electrical activity of Purkinje fibers derived from normal and infarcted canine hearts.

The effects of 0.39 mM halothane (approx. 1.1 vol%) on the action potentials of proximal (false tendon) and distal (apical) left ventricular Purkinje fibers were compared in analogous in vitro preparations derived from normal dogs and animals surviving 1 day following acute myocardial infarction. In ten noninfarcted hearts, halothane reduced regional differences in repolarization by decreasing action potential duration (APD90, mean +/- SE) in proximal fibers from 300 +/- 7 to 277 +/- 6 msec (P less than or equal to 0.01) without decreasing APD90 in distal fibers (control 240 +/- 4 msec, halothane 249 +/- 5 msec). In ten infarcted hearts, halothane accentuated pathologic differences in repolarization by decreasing APD90 in the non-ischemic proximal fibers from 311 +/- 8 to 287 +/- 7 msec (P less than or equal to 0.01), while increasing APD90 in the ischemic distal fibers from 375 +/- 15 to 406 +/- 18 msec (P less than or equal to 0.01). Halothane also decreased the overshoot from 32.9 +/- 1.0 to 28.4 +/- 0.8 m V (P less than or equal to 0.01) and Vmax from 356 +/- 28 to 300 +/- 22 V/s (P less than or equal to 0.05) in ischemic fibers. In seven infarcts evaluated by extrastimulus techniques, halothane slowed the conduction of premature impulses and prolonged refractoriness, while, in five of the seven hearts, it reversibly increased the range of coupling intervals which induced probable reentrant responses. In a separate study of seven infarcts, halothane decreased the rate of spontaneous activity originating in the ischemic region. It is concluded that halothane facilitates the occurrence of re-entry while inhibiting the initiation of abnormal impulses in the in vitro canine infarction model.

Distribution of LDH-1 in normal, ischemic, and necrotic myocardium. An immunoperoxidase study.

To study the distribution of lactate dehydrogenase (LDH-1) (H4) in normal, ischemic, and necrotic myocardium using the peroxidase-antiperoxidase technic, the authors studied formalin-fixed paraffin-embedded sections of human (n = 11) and canine (n = 28) myocardium. All normal control myocardium showed positive immunostaining for LDH-1 (H4). In infarcts 10 hours or more old, the histologically necrotic myocardium (by triphenyl tetrazolium chloride staining) (TTC) showed markedly diminished immunostaining. In 24-dogs ischemia was induced in a closed-chest model using a balloon-tipped catheter inflated in the left anterior descending coronary artery. In dogs with 3 hours or more of occlusion, myocardium that was necrotic by TTC staining, light and/or electron microscopy, showed diminished staining for LDH-1, while normal, control myocardium stained intensely. In four dogs, ischemia was induced by a controlled perfusion apparatus by which left main coronary flow was reduced by 50%. Ischemia without necrosis was documented by demonstration of glycogen loss with no light or electron microscopic evidence of necrosis. These ischemic fibers stained intensely for LDH-1, as did controls. Thus, by immunoperoxidase staining, LDH-1 can be demonstrated in normal human and canine myocardium. In experimental models of ischemia in dogs, tissue that was ischemic but not necrotic showed no diminished staining. LDH-1 loss can be detected in necrotic myocardium as early as 3 hours after coronary artery occlusion.

Effect of tetrodotoxin and ethmozine on arrhythmias of the dog's heart isolated in the late stage of experimental myocardial infarction

Dog hearts with ventricular extrasystole that developed 24 hours after coronary artery occlusion were isolated and perfused with blood from support dogs. After heart isolation the rhythm disturbances persisted regardless the decreased frequency of the ventricular beats. Administration of tetrodotoxin (4 X 10-8--10-7 g/ml) and ethmozine (3--5X X10-5 g/ml) abolished ventricular arrhythmias and restored the sinus rhythm. Potential mechanisms of the increased susceptibility of ischemic myocardial fibers to tetrodotoxin and antiarrhythmic drugs are discussed.

Effects of lidocaine on the electrophysiological properties of subendocardial Purkinje fibers surviving acute myocardial infarction

An electrophysiologic study was conducted in vitro on the surviving subendocardial Purkinje fibers of the canine left ventricle 20 to 24 h after coronary occlusion. The surviving Purkinje cells displayed a low maximum diastolic potential, reduced action potential amplitude, depressed maximal rate of upstroke and a prolonged action potential duration. Spontaneous discharges of high frequency and induced repetitive activity are consistently observed in the infarcted zone but are rarely seen in the normal tissue. Superfusion of lidocaine hydrochloride in concentrations of 1 × 10 −5 to 1 × 10 −4 m slowed or abolished spontaneous and induced firings. The effective refractory period and effective refractory period relative to the total action potential duration were consistently prolonged by lidocaine in the ischemic fibers. Membrane response curves of ischemic fibers were shifted farther to the right than those of normal fibers. Lidocaine exhibited a differential effect on maximul rate of upstroke ( dV dt ) of ischemic Purkinje fibers while causing minor change in the normal fibers. It is suggested that lidocaine exerts its antiarrhythmic effect by selectively depressing the already depressed conducting fibers in the infarcted heart.

Isoproterenol-induced 45ca uptake into myocardium of rats.

Myocardial lesions induced by isoproterenol (ISP) are similar to those of human "coagulative mycocytolysis" or "myofibrillar degeneration". The localization of tritiated ISP in the damaged myocardium of rats has been recently described. Since H3-ISP was noted in "grooves" along the sarcolemma of ischemic and necrotic fibers, an action on the membrane with exaggerated calcium inflow was suggested. For this reason, the quantity of 45Ca in rats treated with ISP was studied. Thirty rats were given 5 mu Ci of 45Ca. Twenty (Group B) of them were also given ISP 10 mg/kg. Animals were killed at 1 h after the injections. The hearts were sectioned transversally and autoradiography was performed. Serial sections were examined to localize the 45Ca, and its topographic distribution. In Group A (injected only with 45Ca) the myocardium showed + of 45Ca (600 dots in a field x 400) inside the fibers. In Group B (injected also with ISP) 45Ca was deposited +++ (more than 1,200 dots) and related to extensive myocytolysis. These findings confirm that the crucial point in the ISP-induced lesions is the increase of calcium inflow, and if the pathogenesis of human myocytolysis is related to catecholamine effects pharmacological measures may be adopted to prevent these myocardial lesions.