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Ann Thorac Surg 1996;61:1273-1280
© 1996 The Society of Thoracic Surgeons

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Current Review

Cardiac Surgical Implications of Calcium Dyshomeostasis in the Heart

Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado

	Abstract

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
The prevalence of coronary artery disease renders myocardial ischemia a leading cause of morbidity and mortality. Both cardiac bypass operations and cardiac transplantation cause myocardial ischemia and reperfusion injury. Intracellular calcium transport and regulation are of paramount importance in both normal and pathologic myocardial states. Calcium regulation is integral to nearly every myocyte function, from early development to senescence. Normal intracellular calcium-mediated excitation-contraction coupling and abnormal patterns of calcium regulation leading to systolic/diastolic dysfunction are now therapeutically accessible to the cardiac surgeon. Additionally, altered Ca²⁺ transport protein gene expression is a mechanism of myocardial dysfunction. Therapeutic strategies involve receptor-mediated transduction of signals to intracellular metabolic sites. Evidence implicates protein kinase C as well as a potential therapeutic role for Ca²⁺. The potential for pharmacologic access to this protective state has abundant clinical appeal. The protective state (cardiac ``preconditioning'') is transient but is amenable as therapy against operation-related ischemic events.

	Introduction

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Cardiac surgeons are now being asked to treat patients with progressive degrees of heart failure, hypertrophy, and age. Increased understanding of the etiologic mechanisms associated with these varieties of cardiac dysfunction should translate into more appropriately targeted therapy. Intramyocyte free calcium is pivotal to regulation of the rhythmic contraction/relaxation cycle in the heart. Recent ultrastructural evidence now convincingly indicates that the L-type calcium channels embedded in the transverse tubules are functionally and physically associated with the calcium ryanodine receptors/channels on the sarcoplasmic reticulum (SR) [1]. Intramembrane charge flux appears to open membrane calcium channels directly. The resultant small influx of calcium increases the calcium concentration in the microenvironment surrounding the ryanodine receptor/channel on the SR, evoking calcium release from the SR. This ``calcium-induced calcium release'' phenomenon increases cytosolic binding of calcium to troponin C, which serves as the fundamental basis of muscle contraction. Subsequent active uptake of cytosolic calcium by the SR provokes muscle relaxation. Calcium thus plays a critical role in excitation-contraction coupling and myocardial mechanical function.

With heart failure, patients exhibit both systolic and diastolic dysfunction [2, 3]. Potential contributing mechanisms for both systolic and diastolic dysfunction include (1) cell death (microinfarction and scar) [4], (2) dysfunctional cardiac metabolism (inadequate energy supply) [5, 6], (3) abnormalities of cytoskeletal and contractile proteins [7], (4) alterations in myocyte excitation-contraction coupling [8], (5) dysfunctional myocyte signal transduction [9–11], and (6) altered intracellular calcium transport and regulation [12–14]. Of these multiple mechanisms, the latter is the most dynamic and potentially reversible, and therefore the most treatable. Therapeutic control of calcium homeostasis is accessible to the cardiac surgeon. The purposes of this review are: (1) to explore normal intracellular calcium-mediated excitation-contraction coupling in cardiac myocytes; (2) to examine abnormal patterns of calcium regulation leading to systolic or diastolic dysfunction; (3) to focus on disordered intracellular calcium transport as a cause of heart failure; (4) to review evidence incriminating the altered expression of genes encoding calcium transport proteins as a mechanism of muscle dysfunction; and (5) to suggest strategies for constructive intervention into the intracellular regulation of calcium with application to cardiac surgical patients.

	Intracellular Ca2+ Homeostasis in the Resting State

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
In the resting state, cardiac myocyte cytosolic calcium ion concentration ([Ca²⁺]_i) is maintained at less than 200 nmol/L [15]. The control of [Ca²⁺]_i is accomplished by coordinated regulation of a number of Ca²⁺ transport systems. These systems include regulation of the extracellular calcium source ([Ca²⁺]_o) against a 5,000-fold concentration gradient across the sarcolemma. The regulation of extracellular calcium entry is mediated by voltage-gated and receptor-mediated Ca²⁺ channels, which remain closed until electrophysiologic activation of the cell occurs. The regulation of intracellular to extracellular Ca²⁺ export is accomplished by an adenosine triphosphate (ATP) driven sarcolemmal Ca²⁺ pump (sarcolemmal Ca²⁺-ATPase) and the Na⁺-Ca²⁺ exchanger. In the resting myocardium, the sarcolemmal Ca²⁺-ATPase extrudes Ca²⁺ to compensate for Ca²⁺ leak and to maintain [Ca²⁺]_i. The Na⁺-Ca²⁺ exchanger moves Ca²⁺ either into or out of the cytosol, depending on the electrochemical gradients of both Ca²⁺ and Na⁺. Intracellular Ca²⁺ sources include the SR, the mitochondria, and the cytosolic binding proteins parvalbumin and calmodulin, which can rapidly buffer cytosolic calcium. Thus, multiple redundant systems have evolved to guarantee intracellular Ca²⁺ homeostasis in the healthy myocyte.

	The Cardiac Action Potential

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Cardiac action potentials are more complex than those of skeletal muscle or nerve. The cardiac action potential lasts more than 300 milliseconds, compared with a few milliseconds for skeletal muscle and nerve. Moreover, the action potential of cardiac myocytes consists of five phases (Fig 1), compared with the biphasic action potential of muscle and nerve. Figure 1 illustrates a typical action potential of a cardiac myocyte, the ionic mechanisms responsible for each phase, and correlation with the surface electrocardiogram. The resting transmembrane potential is -80 to -90 mV. After stimulation, there is an extremely rapid depolarization (phase 0), which is characterized by rapid influx of Na⁺ through the voltage-gated Na⁺ channels (Fig 1A). Phase 0 lasts 1 to 2 milliseconds, during which the transmembrane potential becomes positive by 15 to 30 mV. Depolarization is followed by a brief repolarization (phase 1) caused by increased Cl^- permeability and decreased Na⁺ permeability (Fig 1B). Phase 2 of the action potential is caused by a rapid rise in Ca²⁺ permeability with a prolonged fall to baseline (Fig 1C). The plateau phase is followed by repolarization caused by a decrease in Ca²⁺ permeability (phase 3). Pacemaker cells are capable of progressive slow depolarization caused by slow influx of Na⁺ during phase 4 (Fig 1D). The QRS complex from the surface electrocardiogram corresponds to phases 0 and 1 of the single cell electrogram, the duration of which is approximately 100 milliseconds (Figs 1E, 1F). Calcium entry during depolarization (see Fig 1C) leads to Ca²⁺-induced Ca²⁺ release from the SR (Figs 2A, 2B).

View larger version (27K): [in this window] [in a new window]   Fig 1. . Cardiac action potential. Typical action potential of a cardiac myocyte, the ionic shifts responsible for each phase, and correlation with the surface ECG. (A) Phase 0: rapid depolarization, characterized by rapid influx of Na+ through the voltage-gated Na+ channels. (B) Phase 1: brief repolarization, characterized by transient influx of Cl-. (C) Phase 2: plateau phase, characterized by a rapid rise in Ca2+ permeability through L-type Ca2+ channels. Phase 3: repolarization with K+ exiting the cell. (D) Slow depolarization of pacemaker cells caused by slow influx of Na+. (ECG = electrocardiogram.)

View larger version (31K): [in this window] [in a new window]   Fig 2. . Cardiac myocyte Ca2+ transport systems. (A) Transverse T-tubule and L-type Ca2+ channel. (B) Ca2+-induced Ca2+ release through the sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channel. (C) Ca2+ binding to troponin C induces a conformational change in this inhibitory complex, allowing actin and myosin to interact. (D) Sarcoplasmic reticulum Ca2+-ATPase sequesters Ca2+ after excitation-contraction coupling. (E) Calsequestrin and calreticulin bind intra-SR Ca2+, forming a stable matrix. (F) Na+/H+ exchanger. (G) Na+/Ca2+ exchanger. (H) Beta-adrenergic-mediated upregulation of cyclic adenosine monophosphate (cAMP). (I) Select protein kinase C (PKC) isoforms activate phospholamban after activation by cAMP. (J) Phospholamban mediates sarcoendoplasmic reticulum calcium ATPase (SERCA) 2a activity. (K) 1,4,5-inositol triphosphate (IP3) receptor-mediated SR Ca2+ channel. (DHP = dihydropyridine.)

	Ca2+-Mediated Excitation-Contraction Coupling in Cardiac Myocytes

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

Depolarization of the cell opens both the voltage-gated Na⁺ channel and the voltage-gated Ca²⁺ channel. The initial upstroke of the cardiac action potential is caused by Na⁺ influx, whereas the subsequent inward Ca²⁺ current through L-type Ca²⁺ channels maintains the plateau phase of the action potential [16]. During the early part of the cardiac action potential, influx of Ca²⁺ through the voltage-gated L-type Ca²⁺ channel triggers the release of Ca²⁺ from the SR ryanodine receptor channel (Fig 2B). Sham [1] reported a quantitative comparison between the rate of Ca²⁺ release from the SR ryanodine Ca²⁺ channel and the sarcolemma Na⁺/Ca²⁺ exchanger. Sham observed a subcellular differential in the onset and rate of Ca²⁺ release between these ports of Ca²⁺ entry, with the SR ryanodine Ca²⁺ channel being more rapid than the Na⁺/Ca²⁺ exchanger (Figs 2F, 2G). They concluded that the L-type and the SR ryanodine Ca²⁺ channels are linked and that the subcellular local increases in [Ca²⁺]_i required for contraction are the result of Ca²⁺ release from SR ryanodine Ca²⁺ channels in intimate association with the L-type Ca²⁺ channels. This calcium-induced calcium release results in an increase in [Ca²⁺]_i. Cytosolic binding of Ca²⁺ to troponin C, which is part of the myofilament regulatory complex, produces a conformational change in this inhibitory complex that allows interaction of actin and myosin, formation of cross bridges, and activation of the contractile apparatus (Fig 2C). Relaxation occurs when Ca²⁺ dissociates from the contractile apparatus. Subsequent SR sequestration of Ca²⁺, accomplished predominantly by the SR Ca²⁺-ATPase and provoked by a Ca²⁺ threshold, ends the cycle (Figs 2D, 2E). Thus, initial phase 0 myocyte depolarization is Na⁺ driven, whereas both contraction and relaxation are governed almost entirely by Ca²⁺.

	Role of Ca2+ in Myocardial Ischemic Injury

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Coordinated and precise regulation of the oscillating extremes of [Ca²⁺]_i mediates systolic contraction, diastolic relaxation, enzymatic activity, and mitochondrial function. Disruption of the scheme outlined earlier may lead to dysfunctional excitation-contraction coupling and, therefore, systolic or diastolic dysfunction. Therefore, Ca²⁺ dyshomeostasis as the pathophysiologic basis of cardiac ischemic injury and myocardial pump failure has seemed logical. In fact, the association between irreversible myocellular ischemic injury and massive Ca²⁺ influx was first demonstrated by Shen and Jennings [17]. Both postischemic mechanical dysfunction of reversibly injured myocytes (stunning) and lethal injury (infarction) have been related to elevated [Ca²⁺]_i [18]. Cardiac myocyte Ca²⁺ handling can be assessed using several objective indices. First, the cardiac contractile state can be assessed as developed force or pressure. Second, the function of the sarcolemma is reflected in the action potential. Third, SR function is demonstrated by the Ca²⁺ transient. Fourth, the myofilament-regulatory complex is exhibited by the association between the Ca²⁺ transient and the force of contraction.

Although the association between massive Ca²⁺ influx and myocellular ischemic injury has been established, the source of the elevated [Ca²⁺]_i remains controversial and may have therapeutic importance. The most likely scenarios involve either ineffective sarcolemmal Ca²⁺ extrusion or inadequate SR Ca²⁺ sequestration. Either seems plausible because both exhibit energy-dependent kinetics [15]; that is, after ischemia, the ATP-hungry sarcolemmal Ca²⁺-ATPase or the SR Ca²⁺-ATPase would be unable to bring [Ca²⁺]_i back to the basal levels required for muscle relaxation [5, 6]. This, in turn, would decrease muscle shortening during a contraction, leading to both systolic and diastolic dysfunction. Indeed, both mechanisms of Ca²⁺ dyshomeostasis appear to be involved [17, 19]; however, the enormous concentration differential between [Ca²⁺]_o and [Ca²⁺]_i makes a breakdown in [Ca²⁺]_o handling appear more ominous. An interesting mechanism of Ca²⁺ mishandling has been proposed involving the second messenger cyclic adenosine monophosphate (cAMP) [20]. Cyclic AMP is a ß-adrenergic-stimulated second messenger that is known to be important in modulating [Ca²⁺]_i through several protein kinases (Fig 2H). These protein kinases have the ability to turn on and off processes at important cellular sites by phosphorylating proteins at the sarcolemma (Ca²⁺ channels, ion exchangers) and the SR (SR Ca²⁺-ATPase), as well as the myofilaments (regulatory complex). In support of this hypothesis, Trautwein and Heschler [21] demonstrated that phosphorylation of the sarcolemmal L-type Ca²⁺ channels increases the influx of calcium with each depolarization. By this mechanism, it is supposed that ischemia leads to ß-adrenergic stimulation, which in turn increases cAMP, resulting in successive increases in [Ca²⁺]_i. Although this may in part explain the increased [Ca²⁺]_i, cAMP phosphorylates other proteins that control Ca²⁺ after ischemia. For instance, cAMP-regulated phosphorylation of phospholamban (Fig 2I), the regulatory subunit of the SR Ca²⁺ pump (Fig 2J), actually enhances SR sequestration of Ca²⁺ [22]. Indeed, in patients with heart failure, isolated myocardium does not respond well to ß-adrenergic agonists; however, inotropic stimulation can be preserved with forskolin, an adenylate cyclase activator [23]. Thus, [Ca²⁺]_i is increased after myocardial ischemic injury; the disruption in the normal handling of [Ca²⁺]_i may be related to a breakdown of intracellular signaling. The two most likely mechanisms relate to decreased ATP availability to the energy-dependent sarcolemmal and SR Ca²⁺ pumps. Indeed, the SR is exquisitely sensitive to ischemic insult. Krause and Hess [24] demonstrated a markedly reduced SR Ca²⁺ uptake and depressed SR Ca²⁺-ATPase activity after brief ischemia of canine myocardium. Furthermore, Xu and co-workers [25] demonstrated functional coupling between glycolysis and SR Ca²⁺ transport. Recent studies, however, suggested that the L-type Ca²⁺ channel is not the principal site of Ca²⁺ entry after ischemia and reperfusion (I/R). Elegant studies by Chiamvimont and associates [26] demonstrated that transfection of the pore-forming subunit (alpha) of the L-type Ca²⁺ channels into various cell types requires sulfhydryl oxidation before opening. The redox state of the cellular environment after ischemia does not favor sulfhydryl oxidation. Indeed, when subjected to hypoxic conditions, cells transfected with the L-type Ca²⁺ channel alpha subunit did not undergo sulfhydryl modification, the L-type Ca²⁺ channel did not open, and an increase in [Ca²⁺]_i was not observed. In contrast, sulfhydryl modification of the alpha subunit did open the channels as well as increase [Ca²⁺]_i. Unlike cardiac myocytes, the cell types transfected did not contain the Ca²⁺-rich SR; therefore, these authors concluded that the source of increased myocyte [Ca²⁺]_i is most likely the SR or another cardiac myocyte organelle. They further concluded that this response to ischemia is adaptive; that is, ischemia favors a redox state, which closes L-type Ca²⁺ channels. To summarize, there are two major ways in which calcium alters the contractile state of the heart: 1) by changing calcium availability to the myofilaments, and 2) by changing the responsiveness of the myofilaments to calcium.

Although these findings provide evidence that strongly associates abnormal elevations in [Ca²⁺]_i and decreased force of contraction through alterations in myofilament Ca²⁺ sensitivity, there are other potential deleterious effects of pathologic Ca²⁺ levels. Activation of proteases, ATPases, and lipases, as well as mitochondrial dysfunction and free radical production, all occur in the presence of excess Ca²⁺. Whatever the mechanism, there is considerable evidence that Ca²⁺ overload contributes to myocardial dysfunction after I/R. Because [Ca²⁺]_i remains relatively constant during ischemia, but rises rapidly with reperfusion, several investigators have advocated the use of low [Ca²⁺] perfusate solutions [24, 25]. Indeed, reperfusion with low [Ca²⁺] perfusate solutions does improve myocardial function after sustained ischemia [27–29].

	Altered Ca2+ Transport Protein Gene Expression

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Recombinant DNA technology has changed the practice of medicine and will undoubtedly influence the future practice of cardiac surgery. Changes in gene expression in both adaptive and maladaptive states are well established. Delineating alterations in gene expression is of paramount importance to understanding the basis of myocardial disease. Further defining the molecular alterations that occur in myocardial disease states will allow more specifically targeted therapy [13]. For example, cells may change at the gene level to acquire and display a more adaptive phenotype. Induction of such protective changes before an anticipated insult, such as an operation, has been termed ``cardiac preconditioning'' and may allow us to provoke endogenous protective systems. In this regard, the cardiac ryanodine receptor and type 1 1,4,5,-inositol triphosphate (IP3) receptor (both of which regulate SR Ca²⁺ discharge), the SR Ca²⁺-ATPase, phospholamban, the K⁺ channel, and the L-type Ca²⁺ channel have each been cloned [30–36]. Changes in cardiac Ca²⁺ channel gene expression have been demonstrated during development, aging, and heart failure. For the purposes of this review, we have focused on the changes in cardiac gene expression during heart development, senescence, and heart failure that relate specifically to mechanisms of cardiac myocyte Ca²⁺ handling and are of interest to the cardiovascular surgeon.

Changes in Gene Expression During Ontogenic Development and Aging
Developmental and age-related changes in contractility result from alterations in SR properties. The aging mammal has a prolonged rate of contraction and a slower rate of relaxation. These changes have been related to slower SR Ca²⁺ uptake [37]. It is interesting that the rate of Ca²⁺ uptake and Ca²⁺-ATPase activity are also lower in the fetus than in the young adult [38]. The fetal heart has a sparse SR network, which matures by birth [39]. Changes in SR Ca²⁺ pump mRNA expression during development and senescence may explain the alterations in SR Ca²⁺ regulation observed at each end of the developmental spectrum. There are types 1, 2, and 3 sarcoendoplasmic reticulum calcium ATPase (SERCA) gene transcripts in the heart, with a relative abundance of SERCA 2. In situ hybridization studies indicate that both the SERCA 2a and the SERCA 3 mRNA isoforms are present in the heart tube during early development [37]. As development proceeds, SERCA 3 disappears from cardiac myocytes and is found only in the endothelial layer of coronary arteries after birth [40]; SERCA 2a then remains as the almost exclusive SERCA mRNA isoform present in cardiac myocytes. Interestingly, this change in SERCA isoform expression may directly influence SR function, in that the SR becomes functional only after birth [41]. The level of SERCA 2a mRNA is increased at birth [42] and decreased during senescence [43]. There is a concomitant increase in SERCA 2a and major histocompatibility complex mRNA during development, suggesting a common mechanism for induction of the two genes [42]. It is possible that thyroid hormone, which is thought to be responsible for the induction of both the major histocompatibility complex and the SERCA genes, explains the common induction mechanism. There is a relative decrease in SERCA 2a mRNA during senescence [37], suggesting that the decrease in SERCA 2a mRNA is not due to decreased myocyte transcriptional activity or the loss of myocytes with aging. These findings further implicate an imbalance of SR Ca²⁺ handling in the contraction/relaxation alterations observed in the senescent heart.

Moorman and colleagues [41] extended these observations concerning the relative expression of SERCA 2 and phospholamban mRNAs during development and related them to functional changes with age. During embryonic development, there is a sequential complex remodeling of the heart, likely produced by genetic reprograming. The atrium is characterized by rapid, brief contractions and is relaxed during the majority of the cardiac cycle. The ventricle, however, has rapid, long contractions. Because the major factor in the contractile status of myocytes is the SR-controlled [Ca²⁺]_i, myocytes with an abundance of SERCA 2, such as atrial myocytes, would clear free intracellular Ca²⁺ rapidly and have a shorter contraction phase and a longer relaxation phase. Conversely, the ventricle has less SERCA 2, and therefore slower Ca²⁺ uptake and a more sustained contraction. Note that these differences in SERCA 2 and phospholamban mRNA expression are only observed during embryonic development, which suggests that the embryonal heart has devised a means to compartmentalize contraction in the absence of a formal conduction system.

Changes in Gene Expression During Cardiac Hypertrophy and Failure
Cardiac hypertrophy is recognized as an adaptive process that precedes heart failure. Cardiac hypertrophy and failure are both characterized by alterations in myocardial relaxation. Abnormal Ca²⁺ handling has been implicated in both of these states, and in vitro studies performed on microsomes have implicated dysfunctional SR as the cause [44]. There is decreased SR Ca²⁺ uptake in cardiac hypertrophy and a further decrease with cardiac failure [44]. This decrease is not associated with a decrease in the rate of Ca²⁺ uptake by existing pumps, but rather to a decrease in SERCA gene expression in both hypertrophy and failure [45]. In humans, there is a direct correlation between the relative decrease in SERCA mRNA levels and the degree of clinical heart failure [46]. In addition, the expression of other genes encoding proteins that form the SR membrane is also depressed during hypertrophy and failure, including phospholamban [47], which suggests a global disruption of the SR membrane. It is notable that exercise-induced cardiac hypertrophy is characterized by an upregulation of SERCA 2 mRNA levels [48]. To summarize, hypertrophy associated with failure is characterized by decreased SERCA mRNA levels, whereas hypertrophy associated with exercise is characterized by increased SERCA mRNA levels. Furthermore, SERCA 2 mRNA levels are positively correlated with cardiac norepinephrine levels [48], which implicates mechanisms of cardiac endogenous adaptive/protective responses.

The structures of the SR Ca²⁺ release channels have also been determined by cDNA cloning [30]. The ryanodine receptor and the IP3 receptor channels have been cloned and are members of a gene family that comprises the largest channel structures yet identified, consisting of four 565-kD and four 313-kD subunits, respectively. In accordance with the observed decrease in SERCA mRNA levels, there is a similar decrease in ryanodine receptor SR release channels in the myopathic left ventricle [49]. Go and associates [30] demonstrated a distinct and opposite regulation of the IP3 receptor (Fig 2K). In contrast to gene expression of all other Ca²⁺ channels determined to date, the IP3 receptor gene is upregulated in patients with end-stage cardiomyopathy. Indeed, pharmacologic therapy designed to enhance the upregulated IP3 receptor may prove effective in failing hearts.

	Therapeutic Strategies

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Despite advances in myocardial preservation during routine cardiac operations and heart transplantation, the obligate I/R invariably leads to some ventricular dysfunction. Most current efforts to minimize the effects of I/R during cardiac operations focus on optimizing metabolic fuels and decreasing myocardial oxygen demand. The argument that Ca²⁺ dyshomeostasis contributes to myocardial dysfunction after I/R is persuasive. Therapeutic strategies to enhance cardiac myocyte Ca²⁺ handling will be explored.

Calcium Channel Antagonists
Calcium channel antagonists are the intuitive answer to the problem of Ca²⁺-induced I/R injury. However, unlike other cell types in which [Ca²⁺]_o is the important source of Ca²⁺ overload after I/R [50], the primary route of calcium entry upon reperfusion is through sarcolemmal Na⁺-Ca²⁺ exchange channels and the SR [51]. Although beneficial effects of Ca²⁺ channel blockers have been reported, these agents have predictably proved disappointing. Preischemic delivery is required to limit the infarct size after transient coronary occlusion [52]. Beneficial effects on myocardial stunning have also been reported when Ca²⁺ channel blockers were administered either before or after reperfusion [52]. Because Ca²⁺ channel blockers have broad hemodynamic effects, including decreased afterload, decreased heart rate, and platelet antagonism, it is likely that their mechanism of action is not due to a reduction in myocyte Ca²⁺ overload. Furthermore, human trials in which Ca²⁺ channel blockers were administered in conjunction with thrombolysis in the setting of acute myocardial infarction have failed to demonstrate any additional efficacy over thrombolysis alone [53].

Cardiac Preconditioning
The heart has intrinsic defense mechanisms to I/R injury that can be elicited by brief periods of ischemia. This protective phenomenon is termed ``ischemic cardiac preconditioning'' [54]. Two forms of preconditioning exist: One is induced within minutes, is brief, and is independent of gene expression and protein synthesis; the other is sustained and apparently lies dormant within the myocardial genome [9]. There is increasing evidence that ischemic stress hormones, adenosine and norepinephrine, are involved in the early phase of preconditioning, acting synergistically through the activation of protein kinase C (PKC) isoforms [10, 11, 55]. Subsequent phosphorylation and synthesis of stress proteins are the endogenous strategies that improve cardiac contractility after reperfusion and promote resistance to reperfusion arrhythmias (Fig 3). The activated hydrolysis of phosphatidyl-4,5-bisphosphate by phospholipase C produces IP3 and diacylglycerol, the intracellular targets of which have been identified as PKC and calcium-storage organelles [11]. Calcium homeostasis is maintained by many signaling systems. In most cells, physiologic responses persist longer than it takes Ca²⁺ concentrations to return to basal levels. Activation of cell surface receptors often results in regular oscillations of [Ca²⁺]_i. The extent, localization, and spatial relation of Ca²⁺ release, as well as the physiologic importance during cardiac preconditioning, remain unknown. It is known, however, that as [Ca²⁺]_i increases, the phospholipid hydrolysis required to activate some PKC isoforms diminishes. Calcium oscillations may therefore be involved in inducing, mediating, or sustaining the effects of preconditioning. Sustained activation of cardiac preconditioning before predictable ischemic events such as cardiac transplantation and cardiopulmonary bypass has abundant clinical appeal. Manipulating Ca²⁺ oscillations during cardiac preconditioning may allow amplification of the cardiac preconditioning response. Pharmacologic cardiac preconditioning is a Ca²⁺-mediated strategy of myocardial protection that is accessible to cardiac surgeons.

View larger version (28K): [in this window] [in a new window]   Fig 3. . Cardiac preconditioning: stimulants, second messengers, and effectors. Transient ischemia causes the release of the ischemic stress hormones adenosine and norepinephrine. These two hormones act synergistically through phospholipase C (PLC) to produce diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP3). Adenosine stimulates PLC by a G-protein (G)-mediated mechanism. Diacylglycerol stimulates protein kinase C (PKC), which phosphorylates (upregulates) stress proteins; IP3 stimulates sarcoplasmic reticulum (SR) Ca2+ release. Increased intracellular Ca2+ activates calcium-dependent protein kinase C (cPKC) which, in turn, can also activate PKC. Furthermore, the stimulus required to activate some PKC isoforms diminishes when intracellular Ca2+ is elevated.

Several beneficial effects of adenosine on the ischemic myocardium have been proposed [56]. Protective effects of adenosine during hypoperfusion include coronary artery vasodilation, which increases oxygen supply; and negative inotropy, which decreases myocardial oxygen demand [57]. Ashraf and colleagues [58] suggested that adenosine A₁-stimulated preconditioning may be mediated by reducing the Ca²⁺ load that occurs during I/R injury. They proposed that adenosine A₁ activation opens K⁺ channels, thereby hyperpolarizing the myocyte and reducing Ca²⁺ influx through the L-type Ca²⁺ channels. Although there is no direct evidence to support this hypothesis, Ashraf and colleagues [58] demonstrated a beneficial effect of adenosine A₁ agonists on myocardial ultrastructure and viability following the Ca²⁺ paradox (see below, Ca²⁺ Preconditioning and the Ca²⁺ Paradox).

₁-ADRENERGIC STIMULATION.
A mechanistic approach to preconditioning that implicates ₁-adrenergic stimulation has been demonstrated recently in our laboratory [9–11]. We postulated that transient ischemia is sensed by the myocardium, promoting the elaboration of an endogenous mediator(s) that transduces alterations in myocardial metabolism, thereby implementing protection against I/R. This hypothesis is based on observations that skeletal muscle and gut have greater tolerance to ischemia than do the heart and brain. During systemic shock, catecholamine release promotes vasoconstriction in these vascular beds to preserve perfusion of the heart and brain. In previous work, we demonstrated that catecholamines transduce beneficial metabolic changes in the bowel and liver during stress [59]. Therefore, we postulated that adrenergic receptors may mediate favorable metabolic responses in myocardial tissue, which may underlie ischemic preconditioning. The role of ₁-adrenergic stimulation by catecholamines in cardiac preconditioning is supported by the following observations [10, 11]: (1) Brief ischemia promotes the release of norepinephrine from myocardial adrenergic neurons; (2) the beneficial effects of ischemic preconditioning are completely eliminated by previous reserpine-induced depletion of neuronal norepinephrine stores; (3) exogenous administration of norepinephrine simulates the protective effects of transient ischemia; and (4) preconditioning is completely eliminated when selective ₁-adrenergic blockade is introduced before or after transient ischemia. Similarly, selective ₁-adrenergic blockade also eliminates the protective effect of exogenously administered norepinephrine. Finally, selective ₁-adrenergic stimulation by phenylephrine infused before sustained ischemia also induces protection equivalent to ischemic preconditioning.

Both adenosine A₁ and ₁-adrenergic stimulation are external stimuli that exert the beneficial ischemic preconditioning effects through the transduction of intracellular signals to an intracellular regulatory site. Although there are clear species differences in the receptor activation of preconditioning, it is likely that common distal effectors exist. Intracellular Ca²⁺ is one of the important intracellular signals involved in ₁-adrenergic preconditioning. There are several mechanisms by which ₁-adrenergic stimulation increases [Ca²⁺]_i. As [Ca²⁺]_i rises, the phospholipid hydrolysis required to activate some isoforms of PKC, an important intracellular mediator of preconditioning [11], diminishes.

PROTEIN KINASE C.
The quick onset and waning nature of myocardial protection induced by preconditioning suggest that a rapid transient metabolic or ionic alteration is involved. Rapid, reversible protein phosphorylation is temporally consistent with the early phase of preconditioning. Furthermore, many receptors activate protein kinases downstream in their transduction cascades. Protein kinase C is a ubiquitous regulatory enzyme with many isoforms activated by numerous effectors, growth factors, hormones, and neurotransmitters [60]. Work in our laboratory has implicated PKC in preconditioning by both transient ischemia and exogenous ₁-adrenergic stimulation [11]. There are several Ca²⁺-dependent PKC isoforms that require less activation stimulus, and sustain activity longer, in the presence of elevated [Ca²⁺]_i. Brief exposure to exogenous Ca²⁺ itself can induce cardiac preconditioning against the Ca²⁺ paradox, strongly implicating a Ca²⁺-dependent mechanism in cardiac preconditioning.

Ca²⁺ PRECONDITIONING AND THE Ca²⁺ PARADOX.
Calcium paradox refers to the paradoxic destruction of myocytes upon Ca²⁺ repletion of ca²⁺-depleted hearts [58]. calcium paradox injury indeed approximates the damage inflicted during i/r injury. there is a rapid and severe loss of structural integrity of the sarcolemma, mitochondrial calcification, contracture necrosis, atp depletion, creatine kinase release, and a rise in [ca²⁺]_i [61]. after ca²⁺ depletion, myocytes are ultrastructurally preserved; it is not until ca²⁺ repletion that these deleterious effects are observed. calcium paradox induces such severe myocardial injury that most attempted interventions have proven impotent. ashraf and associates [58] postulated that if the ca²⁺ paradox approximates the effects of severe ischemia, then very transient cyclic exposure to ca²⁺ depletion and ca²⁺ repletion might simulate the effects of ischemic preconditioning. indeed, five transient (1 minute) cyclic episodes of ca²⁺ depletion and repletion protect against a subsequent 10-minute ca²⁺ paradox insult [58, 61]. it is therefore logical to speculate that ca²⁺ preconditioning may also be protective against a global ischemic insult. this, as well as the mechanism of the protective effects of ca²⁺ preconditioning, remains to be determined. indeed, a common pathway converging on PKC is a distinct possibility.

	Footnotes

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 
Address reprint requests to Dr Harken, Department of Surgery, University of Colorado Health Sciences Center, 4200 E Ninth Ave, C-305, Denver, CO 80262.

	References

Top Footnotes Abstract Introduction Intracellular Ca2+ Homeostasis... The Cardiac Action Potential Ca2+-Mediated Excitation... Role of Ca2+ in... Altered Ca2+ Transport Protein... Therapeutic Strategies References

 

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