1. Introduction

Heart muscle is largely dependent on uninterrupted blood flow, which guarantees delivery of oxygen and substrates and washout of harmful products of metabolism. Ischemia, i.e. decrease or cessation of myocardial blood flow, leads to rapid changes in myocardial metabolism. The degree of these changes is highly dependent upon the severity of ischemia. Ischemia can be partial or total, and it can be regional or global. In general, ischemia is characterized by microscopic and macroscopic heterogeneity. Due to anatomical and physiological reasons contractile myocytes in endocardium are the most vulnerable cells. Ischemia is a dynamic process: with rapid reperfusion full recovery of myocardial metabolism occurs, but on the other hand, continuation of ischemia leads to total tissue necrosis in a few hours. Reperfusion, although generally considered beneficial, can cause tissue injury by several mechanisms, and thus affect the final recovery of the contractility. Endogenous protective mechanisms induced by short preconditioning ischemia and pre-existing collateral vessels can modify the changes caused by ischemia and reperfusion. In the following, the overall metabolic changes caused by myocardial ischemia and reperfusion are shortly described as well as their effects on myocardial contractility and cellular electrophysiology. In addition, the concepts of myocardial stunning, hibernation and ischemic preconditioning are shortly discussed.

2. Effects of Ischemia on Myocardial Metabolism

Total cessation of myocardial blood flow leads to rapid perturbations in myocardial metabolism. In a few seconds oxygen dissolved in cytoplasm or bound to myoglobin is consumed and thereby oxidative phosphorylation and mitochondrial ATP production are seriously disturbed. Levels of high energy phosphates, mainly creatine phosphate and ATP, are decreased, and the breakdown products of adenine nucleotides, such as inorganic phosphate and adenosine, accumulate. Adenosine acts as an endogenous protective agent: it is a powerful vasodilator, it reduces oxygen demand due to its negative chrono-, dromo- and inotropic effects on heart, and it also reduces aggregation of thrombocytes and adhesion of neutrophiles on endothelium.

Liberation of free fatty acids in lipolysis is stimulated in myocardial ischemia by increased circulating catecholamines, but fatty acid oxidation and tricarboxylic acid cycle are inhibited. This leads to cytosolic accumulation of free fatty acid CoA-esters and inhibition of adenine nucleotide translocase. Glycogenolysis and anaerobic glycolysis are stimulated leading to accumulation of lactate and H+-ions and intracellular acidosis. Finally the accumulation of protons, lactate and reduced form of nicotinamide adenine dinucleotide (NADH) lead to inhibition of glycolysis and anaerobic energy production through it [Opie et al., 1976; Stanley et al., 1997]. The energy-dependent transmembrane control is lost, with intracellular K+- and Mg2+-ions leaking out of the cells and with extracellular Na+- and Ca2+-ions entering the cells. Ca2+ accumulates also in mitochondria [Piper, 2000]. The redistribution of electrolytes leads to osmotic changes and cellular oedema. The degradation and synthesis of proteins are inhibited. In a time period of twenty minutes or so, ultrastructural changes appear. These include disappearance of glycogen granules, and oedema of mitochondria and sarcoplastic reticulum. Finally within hours myocardial contracture is developed, which microscopically is characterized by contraction band necrosis, distortion of Z-bands and breakdown of myofibrils. Mitochondria and sarcoplastic reticulum (SR) are disrupted and plasma membrane deteriorates. Intracellular macromolecules appear in interstitial fluid within an hour, and lysosomal enzymes are activated within one to four hours. Autolysis is initiated with intracellular material leaking exracellularly. Cell death and tissue necrosis finally develop, followed by inflammation and fibrosis. The time course of main metabolic changes taking place upon severe myocardial ischemia are depicted in Fig 1.

3. Effects of Ischemia on Contractile Function and Cellular Electrophysiology

Early diastolic relaxation is probably the earliest functional impairment appearing in a few seconds upon severe ischemia, but it is rapidly followed by a decrease in contractile function [Allen and Orchard, 1987; Serruys PW et al., 1984]. There are probably several mechanims being responsible for contractile dysfunction. Myocardial excitation is decreased and action potential shortened upon opening of K+-ATP-channels. Inorganic phosphate, lactate and H+-ions accumulate and are known to decrease myofilament Ca2+-sensitivity and maximum force. Depletion of high energy phosphates and collapse of intravascular pressure leading to shortening of perivascular sarcomeres will also affect systolic function. Possibly other mechanisms are involved, too.

As mentioned above, considerable ionic shifts occur upon ischemia including accumulation of extracellular K+, intra- and extracellular acidosis, accumulation of intracellular Na+ and Ca2+, and depletion of intracellular Mg2+ [Carmeliet, 1999]. Potassium loss during early ischemia occurs in three phases: a fast increase results in a plateau (10-20 mM) after 3-10 min and is followed (after 15-30 min) by a secondary and irreversible increase. Upon reperfusion before the irreversible phase, extracellular K+ recovers rapidly. Three main factors contribute to the rise: shrinking of the extracellular space, inhibition of active K+ influx, and an increase of passive K+ efflux.

During ischemia the fall in pH is caused by increased production and reduced removal of protons. Most plasma membrane channels, as well as gap junction channels, the SR Ca2+ release channel, and Na+ and Ca2+ exchange current are inhibited, some by extracellular and others by intracellular acidosis. This results mostly for a decrease in single-channel conductance and is seen as depolarization, prolongation of action potential and occurrence of early afterdepolarizations (EAD). During ischemia intracellular Na+ increases from 10 to 20 mM after 20-30 min. The mechanism is reduced active outward pumping and an increased inward leak through the Na+/H+ exchanger. During reperfusion these effects result in hyperpolarization and short action potentials combined with low extracellular K+. Dispersion is pronounced and is favorable to arrhythmias.

Cytosolic and mitochondrial Ca2+ levels increase upon ischemia and reperfusion (see later) due to less efficient removal to the extracellular space via the Na+/Ca2+ exchanger, a reduced uptake in the SR, an increased inward leak, and displacement by protons from binding sites [Piper, 2000]. An increase in cytosolic Ca2+ activates a number of channels, carriers, and enzymes and modulates others. The result is the occurrence of EAD, delayed afterdepolarizations (DAD) and arrhythmias.

During ischemia, the cytoplasmic free Mg2+ may increase ten-fold due to hydrolysis of ATP to which Mg2+ are bound, and partly to a deficient removal via a Mg2+-ATPase and the Na+/Mg2+ exchanger. The behaviour of channels and carriers is changed by Mg2+ via effects on phosphorylation, by blocking the pore (Na+ and Ca2+ channels), or by causing inward rectification in the case of K+ channels. At the multicellular level, Mg2+ may be considered to exert a stabilizing effect. During ischemia, amphiphiles, long-chain acylcarnitines (LCAC) and lysophosphoglyceride (LPG) and fatty acids including arachidonic acid and its metabolites accumulate in the plasma membrane, the gap junction, and the intracellular membranes of the SR and the mitochondrion. Amphiphiles and fatty acids may interact directly with channel proteins, with the phospholipids surrounding the channel proteins, or change the membrane fluidity. Amphiphiles increase inward current at the resting potential with simultaneous reduction of outward current through K+ channels. Carriers such as the Na+/Ca2+ exchanger and the Na+/K+ pump are inhibited. On the contrary, fatty acids activate outward currents and stimulate the Na+/Ca2+ exchanger. The simultaneous activation of inward and outward currents favors K+ loss and Ca2+ overload creating conditions that generate arrhythmias [Carmeliet, 1999].

Figure 1. Time course of metabolic changes in severe ischemia.

Oxidative stress during ischemia and especially upon reperfusion (see below) results from the excessive generation of radicals and the deficiency of protection by enzymes and scavengers [McCord, 1985; Opie 1989]. Radicals attack proteins and cause lipid peroxidation, resulting in block of most K+-channels, activation of the SR Ca2+ release channel, and eventually the mitochondrial megachannel. Electrophysiologically, upstroke velocity and conduction velocity of action potential are reduced, and the plateau is prolonged with the appearance of EAD and eventual depolarization to the plateau level. In a second stage, the cell may repolarize, again showing very short action potentials and finally the cell becomes inexcitable and goes into contracture.

During ischemia catecholamines are released. An immediate release (exocytotic) in the systemic circulation occurs after stimulation of pain receptors and afferent nerve fibers in the ischemic zone. Later at 10-15 min a second "metabolic" release phase starts that is much more important quantitatively. This is accompanied by a reversal of the Na+-dependent carrier that normally is responsible for the neurotransmitter reuptake at the presynaptic level. The effect of excessive release of catecholamines is amplified by increases in a- and b-receptor densities during ischemia favoring arrhythmias.

In conclusion, arrest of coronary circulation causes depolarization of the resting Em, shortening of the action potential duration, but prolongation of the effective refractory period (ERP). Excitability may temporarily be increased but falls after 2-3 min. At the electrocardiographic level TQ and ST segments are shifted, and T waves are inverted [Kleber, 2000]. The increase in extracellular K+ and the existence of inward current are responsible for the depolarization. The shortening of the action potential is caused by increased outward current through K+ and Cl- channels. In the border zone, the shortening is induced by the occurrence of an injury current. Prolongation of ERP is due to slowed recovery from inactivation of the Na+ channels. Increased excitability followed by decrease is related to the extent of depolarization. Partial recovery of excitability has been explained by catecholamines. The conduction disturbance is due to a fall in Na+ conductance and later to increase in longitudinal resistance. The short APD during reperfusion despite the low extracellular K+ is explained by an excessive stimulation of the Na+ current. Action potential durations show an exaggerated dispersion, and Ca2+ overload may become pronounced [Carmeliet, 1999].

Examples of electrocardiograms and action potential recordings in ischemia and reperfusion are shown in Fig. 2.

Figure 2. Electrocardiograms and action potential recordings during ischemia and reperfusion

4. Reperfusion Injury

Early reperfusion is an absolute prerequisite for the survival of ischemic tissue. Reperfusion, although ultimately necessary for recovery is, however, considered a double-edged sword, and can lead to worsening of tissue injury by various mechanisms [Hearse 1977; Opie 1989; Ambrosio, 1999]. Except myocytes, the microvasculature and the endothelium, the conducting system and the connective tissue are all possible targets for reperfusion. If reperfusion is accomplished relatively quickly the ischemia induced ultrastructural changes disappear, but reperfusion followed by more sustained ischemia is accompanied by worsening of tissue ultrastructure, and even massive contracture, disruption of sarcolemma and myofibrils, cellular oedema and intramitochondrial deposits of calciumphosphate.

There is lot of evidence that Ca2+-overload and increased production of free radicals (especially oxygen) are the two most important mechanisms responsible for reperfusion injury. Ca2+-overload is caused by several mechanisms [Piper 2000]. During ischemia intracellular acidosis stimulates the Na+/H+ exchanger activity. Due to inhibition of the energy-dependent Na+/K+ -ATPase Na+ tends to accumulate, which on the other hand, increases intracellular Ca2+ through activation of the Na+/Ca2+ exchanger. The increase in the intracellular Ca2+ content activates phospholipases, which may alter membrane phospholipids. Cytosolic Ca2+ overload can result also from influx across the sarcolemmal cation channels or release from endogenous stores, in particular SR. The mechanisms responsible for excessive intracellular Ca2+ -overload upon ischemia and reperfusion are shown in Fig 3.

Figure 3. Mechanism of Ca²⁺ overload.

Ischemia followed by reperfusion favour free radical formation. The main mechanisms responsible for free radical production are xanthine oxidase reaction, mitochondrial respiratory chain (oxidation/reduction reactions), neutrophil activation and auto-oxidation of catecholamines. Free radicals are chemically very reactive and cause peroxidation of unsaturated fatty acids and phospholipids, disruption of mitochondrial and cellular membranes and inactivation of membrane enzymes.

It seems that Ca2+-overload, oxidative stress and energy depletion following ischemia and reperfusion also act as stimulators of apoptosis via permeability transition and opening of mega-channels in the mitochondrial membrane. This leads to efflux of mitochondrial constituents, such as cytochrome c, which is known to act as the messenger for caspase activation and cellular death through apoptosis.

Except cellular death, reperfusion injury may manifest clinically as reperfusion arrhythmias (for cellular mechanisms, see above) and postischemic contractile dysfunction or stunning of the myocardium [Bolli, 1990; Kloner et al., 1992]. Stunning is considered a reversible phenomenon lasting for hours to days. It can be limited to subendocardial layers of the myocardium only, and can be precipitated upon transiently elevated myocardial oxygen demands in the presence of partial coronary artery stenosis. Stunning is clinically encountered in various situations, such as coronary spasm, stable and unstable angina pectoris, acute myocardial infarction, coronary angioplasty, coronary bypass surgery and cardiac transplantation. The extent and duration of ischemia determine the degree of stunning. Stunning can manifest itself as diastolic or systolic dysfunction or both. Repetitive episodes of ischemia interrupted by reperfusion may cause cumulative depression of myocardial function. Stunned myocardium can be awakened and normal contractile function can be restored by means of inotropic stimulation.

The pathophysiology of stunning has been studied extensively in experimental animals. During ischemia high-energy phosphates get depleted, and resynthesis may be delayed upon reperfusion, but this is probably not the only mechanism. It has been postulated that stunning would be caused by Ca2+ overload and decreased sensitivity of the myofilaments to Ca2+, impaired sympathetic neural responsiveness, heterogenous impairment of regional perfusion, loss of creatinine kinase activity and impaired utilization of energy to the myofibrils, damage to the extracellular collagen matrix, and leucocyte activation. Free radicals (see above) are considered as important triggers for postischemic stunning (Fig. 4.) [Bolli, 1990].

Figure 4. Pathogenesis of postischemic myocardial dysfunction = stunning.

5. Endogenous Protection Against Myocardial Ischemia

5.1. Preconditioning of the Myocardium

It was found over ten years ago that brief episodes of myocardial ischemia render the heart more resistant to subsequent ischemic episodes [Murry et al., 1986; Murry et al., 1991]. The phenomenon termed as ischemic preconditioning has been extensively studied since the first observation was made. Preconditioning ischemia induces endogenous protective mechanisms in heart muscle, and in fact is the most effective means for myocardial protection. This endogenous protection seems to be functioning in all animals studied so far including man, although the exact cellular mechanisms may not be the same in all species. There are many proposed triggers for preconditioning-induced protection such as adenosine, bradykinin, morphine, catecholamines (a1) and acetylcholine, which bind to specific receptors. According to the prevailing hypothesis receptor activation leads to activation of protein kinase C and phosphorylation of ATP-sensitive K+ -channels in sarcolemmal or mitochondrial membranes. Inhibition of the mitochondrial F1F0-ATPase has also been suggested for one mechanism [Vuorinen et al., 1997]. In preconditioned myocardium intracellular acidosis is less severe, high energy phosphates and ionic homeostasis are better preserved and finally ischemic injury is less severe than in nonpreconditioned hearts. Reperfusion-induced arrhythmias are also less common in preconditioned myocardium.

The protection induced by short preconditioning ischemia periods disappears within 1-2 hours, but reappears after 24-72 hours. This "second-window protection" probably utilizes different cellular mechanisms as "classical preconditioning" and requires protein synthesis. It has been proposed that synthesis of antioxidant proteins or heat shock proteins play a role.

There is evidence that ischemic preconditioning also functions in man [Kloner and Yellon, 1994], although results obtained in studies of coronary angioplasty and bypass surgery are somewhat controversial. Patients with preinfarction angina, however, seem to recover from their myocardial infarctions better than those with no preinfarction angina.

5.2. Hibernation of the Myocardium

Hibernating myocardium refers to the presence of persistent myocardial and left ventricular dysfunction at rest, associated with conditions of severely reduced coronary blood flow. This left ventricular dysfunction probably represents an adaptive mechanism preventing irreversible myocardial cell damage, since left ventricular dysfunction in hibernating myocardium improve following the restoration of coronary blood flow. Hibernation can be acute, subacute or chronic in nature. It is not accompanied by classical signs of myocardial ischemia such as lactate production or ECG changes. Hibernating myocardium is viable and retains its metabolic activity. It has been suspected that hibernation might be caused by repetitive episodes of ischemia/reperfusion induced stunning. The mechanisms behind hibernation have remained unclear, and this is partly because there are no good animal models for hibernation. Positron emission tomography (PET) has revealed glucose utilization to be increased in areas of reduced perfusion (=hibernating myocardium), and it has been suggested that increased anaerobic glycolysis and ATP production would maintain cellular integrity in these hearts.

Left ventricular dysfunction in hibernating myocardium is thought to be caused by acidosis and accumulation of inorganic phosphate, which inhibit release of Ca2+ from SR or directly affect the contractile system. Another alternative is adenosine, which stimulates opening of ATP-sensitive K+ -channels and inhibits entry of Ca2+ in the cells. The same mechanism has actually been suggested to be responsible for ischemic preconditioning.

The viability of hibernating myocardium can be verified either by demonstrating functional improvement upon pharmacological stimulation (dobutamine eg) or regional blood flow and ongoing active metabolism (thallium/fluorodeoxyglucose and PET). In principle, identification of hibernating myocardium is important, since such myocardium shows a return of function after revascularization, and long-term survival can often be improved with revascularization [Kloner et al., 1992].

 

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