Review Paper: Pathophysiology of Myocardial Reperfusion Injury: The Role of Genetically Engineered Mouse Models

Abstract

Coronary heart disease is the leading cause of death worldwide, affecting millions of men and women each year. Following an acute myocardial infarction, early and successful reperfusion therapy with thrombolytic therapy or primary percutaneous coronary intervention plays an important role in minimizing tissue injury associated with cessation of blood flow. The process of restoring blood flow to the ischemic myocardium, however, can induce additional injury. This phenomenon, termed myocardial ischemia-reperfusion (MI-R) injury, can paradoxically reduce the beneficial effects of myocardial reperfusion. MI-R injury is characterized by the formation of oxygen radicals upon reintroduction of molecular oxygen to the ischemic tissue, resulting in widespread lipid and protein oxidative modifications, mitochondrial injury, and cell death. In addition, studies have shown that MI-R is characterized by an inappropriate immune response in the microcirculation, resulting in leukocyte-endothelial cell interactions mediated by the upregulation of both leukocyte and endothelial cell adhesion molecules. Furthermore, MI-R ameliorates the production of certain cardioprotective factors such as nitric oxide. Advances in the generation of genetically modified mouse models enable researchers to identify the functional importance of genes involved in these processes.

Myocardial Ischemia-Reperfusion and Inflammation

Coronary ischemia and subsequent reperfusion results in deleterious effects, one of the principal ones being vascular and myocardial inflammation. ³ A significant portion of the injury is attributed to leukocyte interactions with coronary vascular endothelial cells (EC), arterial walls, and cardiomyocytes. ⁹⁷ And a key component of myocardial ischemia reperfusion (MI-R) injury is the trafficking of polymorphonuclear leukocytes (PMNs). Microvascular injuries caused by PMNs results from both release of inflammatory mediators and progressive decrease in blood flow as a result of microvascular obstruction (“no-reflow” phenomenon). ²⁴

Leukocyte-endothelial cell adhesion molecules in myocardial I-R

Following reperfusion of the ischemic myocardium, leukocyte- endothelial cell interactions modulate the inflammatory response via enhanced endothelial cell adhesion molecule (ECAM) expression. ECAMs are upregulated during inflammation (e.g., infection by a bacterium) and aid in the trafficking of leukocytes along a chemotactic gradient to protect the host against invading bacteria. However, in the setting of MI-R, an inappropriate immune signal causes PMNs to localize to the ischemic myocardium. PMN-mediated injury is dependent on the interaction of the adhesion glycoproteins expressed on the surface of circulating neutrophils (L-selectin, Sialyl Lewis^X, P-selectin glycoprotein ligand-1 [PGSL-1] and CD11/CD18) with adhesion molecules expressed on the surface of the coronary endothelium (P-selectin, E-selectin, and intracellular adhesion molecule [ICAM-1]).

PMN-mediated MI-R injury is a sequential process involving 3 independent steps: neutrophil rolling, adhesion, and transmigration (or diapedesis). The 2 endothelial selectins, E-selectin and P-selectin, participate in rolling. ^{73, 82} Preformed pools of P-selectin are stored in intracellular Weibel-Palade (WB) bodies. On activation, the WB bodies quickly fuse with the luminal surface of the vascular EC and express P-selectin. ^{25, 67, 74, 98} Several hours following inflammatory stimulation (e.g., MI-R) de novo E-selectin and P-selectin become expressed on the surface of the endothelium. In addition to endothelial cells, leukocytes also express a selectin (L-selectin), which participates in low-affinity interactions with P-selectin and E-selectin during leukocyte rolling. Considering the importance of selectins in leukocyte-endothelial interactions, numerous studies have focused on their role in MI-R. In 1993, P-selectin deficient mice (SelptmlHyn ^−/−) were developed, which spawned many investigations into the various pathologic roles of P-selectin. ⁷³ Studies to determine importance on P-selectin in the pathogenesis of MI-R carried out on in vivo murine models of coronary artery ligation demonstrated that genetic absence of P-selectin confers cardioprotection in mice. ⁸² The importance on E-selectin has been demonstrated by the attenuation of neutrophil accumulation leading to a decrease in the myocardial infarct size in E-selectin deficient (Sele ^−/−) mice. ⁴⁸ Studies carried out on mouse models with multiple targeted deficiencies of E-selectin and P-selectin have revealed that during cytokine-induced inflammation, adhesion mechanisms independent of endothelial E-selectin and P-selectins mediate leukocyte rolling and adhesion, and L-selectin is responsible for a majority of these mechanisms. ^{49, 50, 86}

As PMNs circulate through the ischemic reperfused myocardium, the selectins form loose transient bonds with PGSL-1 and Sialyl Lewis^X. These weak bonds initiate the second phase (firm adhesion) of neutrophil sequestration, adhesion of the neutrophil to the endothelium. This interaction involves endothelial-expressed ICAM-1 and neutrophil-expressed CD18. Studies carried out on ICAM-1 deficient (Icam1 ^−/−) mice, CD-18 deficient (Itgb2 ^−/−) mice, and junctional adhesion molecule-A (JAM-A) deficient mice (F11r ^−/−) ¹⁵ have demonstrated a significant reduction in myocardial necrosis and PMN infiltration after acute coronary artery ischemia and reperfusion. These data provide strong evidence that both of these cell adhesion molecules contribute to myocardial cell injury in the reperfused myocardium. ^{6, 76, 82, 93, 101} Individual antibody therapy against ICAM-1 and P-selectin or combined antibody therapy against both clearly demonstrates myocardial protection against ischemia-reperfusion injury in animal models. ²⁷

The last step during neutrophils sequestration involves diapedesis through EC-cell junctions via platelet EC adhesion molecule-1 (PECAM-1). PECAM-1, expressed on endothelial cells and neutrophils, has been targeted as a therapeutic target to diminish myocardial infarct size following reperfusion. Previous studies demonstrated reduction of myocardial infarct size using PECAM-1 neutralizing antibody in rats subjected to I-R. ^{31, 77}

Cytokine mutant mice and myocardial I-R

Increased production of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10, is an important component of myocardial I-R. Among the cytokines, IL-10 has been shown to diminish the inflammatory response in a number of disease states and is thought to be a beneficial cytokine. Interestingly, cytokines increase surface expression of endothelial cell adhesion molecules and promote leukocyte activation. ⁵⁵ A number of studies have investigated the potential role of cytokines in myocardial I-R injury using a variety of approaches, including pharmacologic cytokine inhibitors, monoclonal antibodies directed against cytokines, and various cytokine knockout mice. Studies of TNF-α deficient (Tnf ^−/−) mice have yielded conflicting reports in terms of myocardial injury following I-R injury. Several studies have provided clear evidence that genetic deficiency of TNF-α provides significant protection against myocardial reperfusion injury. ^{69, 94} In contrast, additional studies of TNF-α knockout mice have demonstrated that myocardial I-R promotes myocardial inflammation in a TNF-α independent manner, ⁸⁰ and that endogenous TNF-α actually protects the myocardium in the setting of acute myocardial infarction. ⁶⁰ A study of mice genetically deficient for IL-1 (Tlr2 ^−/−) receptors demonstrated significant attenuation of myocardial reperfusion injury. ⁹²

The role of IL-6 in myocardial I-R remains largely unresolved. A recent study utilizing IL-6 deficient mice (IL6 ^−/−) in a model of permanent coronary artery ligation resulting in severe myocardial infarction revealed that deficiency of IL-6 had no effect on survival, myocardial infarct size, or left ventricular remodeling. ²⁶ It has also been demonstrated that mice deficient in IL-10 (IL10 ^−/−) have an aggravated response to myocardial I-R, indicating that endogenous IL-10 is cardioprotective. ¹⁰⁶ A recent study of the adipocyte-derived cytokine adiponectin using adiponectin (Adipor1/r2) knockout mice demonstrated significant protection from MI-R. ⁹¹ Protection was derived through a dual mechanism of increased myocardial viability because of its antiapoptotic action and suppression of cardiac production of TNF-α. Another cytokine with a role in MI-R is the monocyte chemoattractant protein-1 (MCP-1), which belongs to the CC chemokine subfamily, and serves as a chemotactic and activating factor for the recruitment of monocytes. CC chemokine receptor 2 (CCR2) is a major receptor for MCP-1, and MCP-1 appears to bind solely to CCR2. ⁶⁸ Using MCP-1 knockout mice (Ccr2 ^−/−), it has been demonstrated that targeted deletion of the CCR2 gene attenuated myocardial ischemia-reperfusion injury via inhibition of macrophage-related oxidative stress and matrix metalloproteinase (MMP) activity. ^{34, 51}

Research efforts have also focused on the role of reactive oxygen species (ROS) in MI-R, which contribute significantly to myocardial injury. ⁶¹ Increased ROS following reestablishing blood flow to a previous ischemic myocardium results in irreversible damage to the vital cell components. One of the principal sources of ROS in the ischemic myocardium is the oxidation of coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) by NADPH oxidase. However studies in NADPH oxidase deficient (Ncf1 ^−/−) mice failed to show any improvement in myocardial necrosis, contractile function, or leukocyte endothelial cell interactions. ³⁸ Detoxification of ROS is catalyzed by enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Investigations using Copper-zinc SOD deficient (Cu/ZnSOD-KO) (Sod1 ^−/−), Cu/ZnSOD Tg, GPx-Tg (Gpx1), and manganese (Mn) SOD-TG (Sod2) mice have demonstrated that overexpression of MnSOD protects the myocardium from MI-R but neither GPx or Cu/ZnSOD appear to play a major role in determining the extent of injury. ⁴⁷

Nitric oxide and myocardial I-R

Nitric oxide (NO) is constitutively produced from L-arginine and oxygen in the vascular endothelium by endothelial NO synthase (eNOS). Though there are 2 other isoforms of the enzyme nitric oxide synthase (NOS), namely neuronal NOS (nNOS) and inducible NOS (iNOS), the principal source of NO in the vascular endothelial cells and cardiac myocytes is eNOS. Continuous release of NO from the endothelium is necessary for the maintenance of vascular homeostasis throughout the circulation and to protect against cardiovascular disease. The actions of NO include modulation of vasodilation, ^{28, 43, 44} regulating leukocyte–EC interaction, ⁵⁸ inhibiting platelet adhesion and aggregation, ^{45, 75} attenuating smooth muscle cell proliferation, ⁴³ and possible modulation of cardiac myocyte function. ⁵⁴ NO released from the endothelium has also been shown to inhibit surface expression of many ECAMs, including P-selectin, E-selectin, VCAM-1, and ICAM-1. NOS knockout mice, including eNOS (Nos3 ^−/−), iNOS (Nos2 ^−/−), and nNOS (Nos1 ^−/−), have been developed along with transgenic (TG) NOS overexpressors, including eNOS TG, iNOS TG, and a cardiac-specific eNOS TG (cs-eNOS TG), which has led to a better understanding of the exact role of each NOS. It has been demonstrated that eNOS overexpression in eNOS transgenic mice significantly attenuates the extent of myocardial infarct size following coronary artery ischemia and reperfusion. ⁴⁷

A large amount of evidence clearly demonstrates that biosynthesis of NO by constitutive NOS (endogenous NO) plays a critical role in alleviating the severity of myocardial cell injury during MI-R. Although there have been studies that show NO is detrimental, ^{17, 23, 71, 79, 83, 89, 100, 103} the majority of evidence has shown that NO is critical to preserve tissue function and viability during and ischemic insult and plays a fundamental cardioprotective role in myocardial reperfusion injury. ⁵

Myocardial Apoptosis in I-R

Myocardial cell apoptosis has been shown to play a major role in several cardiac disease states, including myocardial infarction and congestive heart failure. ^{22, 29, 66} Cell death in MI-R is believed to occur through apoptosis and necrosis. ^{16, 33} Apoptosis occurs via 3 main pathways involving the caspase family of enzymes, which are the major executioners of apoptosis. ^{8, 35, 84} The extrinsic pathway is mediated by the death receptor Fas/FasL and involves activation of caspase-8. ^{39, 72, 96, 99, 105} The intrinsic pathway involves mitochondrial dysfunction, cytochrome-c release, and activation of caspase-9. ^{59, 63, 95} The third pathway is activated by endoplasmic reticulum stress and involves caspase-12. ⁷⁸ Novel mouse models have been developed lacking key genes, which encode proteins constituting the core apoptotic cascade. ^{40, 42, 81, 102, 107, 108, 109}

Inhibition of the apoptotic process was shown to decrease the loss of contractile cells and therefore is an additional approach to attenuating injury caused by MI-R. Apoptosis has been shown to be positively and negatively regulated by the Bcl-2 family of proteins. ^{20, 65} Proapoptotic proteins include Bax, Bak, Bcl-XS, Bad, Bid, Bik, Bim, Hrk, and Bok, whereas antiapoptotic proteins include Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1/Bfl-1. ^{7, 30, 85} The Bcl-2 family plays a major role in ischemia and oxidative stress. ^{4, 37, 70} Transgenic mice overexpressing the antiapoptotic Bcl-2 protein have shown superior recovery following MI-R. ^{12, 110} Similarly studies involving knockout mice (Bax ^−/−) for the Bax proapoptotic protein ^{57, 56} have shown that suppression of the Bax gene improves myocardial tolerance to MI-R injury. ³⁶

Apoptosis has also shown to be inhibited by another family of proteins called the inhibitor of apoptosis proteins (IAPs). ^{18, 21, 88} IAPs are thought to inhibit apoptosis by blocking the activation of various caspases. ^{19, 87} A recent study using transgenic mice overexpressing IAP-2 (Birc3) from the mouse α-myosin heavy chain promoter (B6C3) has also shown cardioprotection. ¹⁴ Furthermore, studies have shown that IAPs inhibition of apoptosis may involve mechanisms other than just caspase inhibition, such as a result of protein degradation or regulation of signaling pathways. ^{13, 41} Though the precise mechanism is unclear, the findings show that overexpression of IAPs does prolong cardiomyocyte survival after MI-R.

Cardiovascular disease risk factors

To date, the majority of investigations into the pathophysiology of MI-R have been performed using animal models lacking any of the risk factors associated with cardiovascular disease. Although all of these studies provided meaningful results, considering the overwhelming prevalence of cardiovascular risk factors such as obesity, diabetes, hypertension, and hypercholesterolemia in patients, the relevance of these findings in patient populations most susceptible to MI-R remains unclear. The investigation of mice with either spontaneous or engineered genetic mutations has facilitated important investigations into the interplay of various risk factors and their role on MI-R.

Diabetes and obesity are metabolic disease states characterized by a high concentration of inflammatory mediators such as leptin, IL-6, and TNF-α. ² Studies have also shown that eNOS-derived NO release and eNOS function are significantly impaired in diabetics. ^{9, 10} Several murine models are currently being used in the study of type-2 diabetes. Of particular interest to us are the ob/ob (Lep ^−/−) mouse and the db/db (Lepr ^−/−) mouse, which suffer from spontaneous genetic mutations in the leptin pathway. The ob/ob mice lack functional leptin. They are grossly overweight and hyperphagic, particularly at young ages, and develop severe insulin resistance. The ob/ob mouse has been used as a model for obesity and for studies on initial aspects of metabolic disturbances leading to type 2 diabetes. ⁶⁴ The db/db mouse suffers from a leptin receptor mutation resulting in the inability to regulate metabolism and therefore also resulting in obesity, elevated plasma glucose and insulin levels, and increased plasma free fatty acids. ^{1, 11, 90} Studies carried out on the db/db mouse have shown that the extent of myocardial infarction is significantly increased following ischemia-reperfusion injury when compared with age-matched nondiabetic controls. ^{46, 32} The db/db mouse has played an important role in demonstrating that HMG-CoA reductase inhibitors (i.e., statins) reduce injury in the diabetic heart following MI-R by increasing the bioavailability of NO, independent of reductions in serum cholesterol levels. ^{62, 104}

Similar to diabetes, hypercholesterolemia is a primary risk factor for myocardial infarction. Prospective studies have established that the risk of cardiac morbidity and mortality is directly related to the concentration of plasma cholesterol. ^{52, 53} Despite the development of several agents to reduce serum cholesterol levels, coronary heart disease and subsequent myocardial infarction still represent a major health concern in our society. The development of several transgenic mice has paved the way to a number of important investigations into the roles of specific receptors and enzymes involved in hypercholesterolemia. Studies of atherosclerosis, using mouse models, have mainly involved different inbred strains of mice fed a high-fat, high-cholesterol, cholate-containing diet. A potential drawback to using mice is that the diet itself may prove inflammatory. However, gene targeting and transgenic technology have provided mutant mouse models of hypercholesterolemia and atherosclerosis that do not require feeding high-fat, high-cholesterol, cholate-containing diets. Among these, the most widely used mouse models are apolipoprotein E (ApoE)–deficient mice (Apoe ^−/−) ⁴⁸ , in which targeted deletion of the Apoe gene leads to severe hypercholesterolemia and spontaneous atherosclerosis. A newly emerging model is the ApoE∗3Leiden (E3L) transgenic mouse, in which a mutated form of the human apoE3 gene has been introduced; E3L mice have a hyperlipidemic phenotype, develop atherosclerosis on being fed cholesterol, and are more sensitive to lipid-lowering drugs than ApoE^−/− and low density lipoprotein receptor (LDLr) (Ldlr ^−/−) deficient mice. The above mentioned and other models such as the LDL receptor–deficient mice, ⁶ in which atherosclerosis develops, especially when fed a lipid-rich diet, have been valuable models in the investigation of the effects of hypercholesterolemia on MI-R.

Summary and Future Directions

The development of novel gene-targeted mice has provided researchers with the necessary tools to begin to define the cellular and molecular mechanisms involved in the pathogenesis of acute myocardial infarction. Gene targeted animals have provided a wealth of novel insights into the major pathologic events that occur in the coronary circulation and in the cardiac myocytes following ischemia and reperfusion. The insights gained from transgenic mice should be viewed with caution since the permanent overexpression or deletion of a single gene can result in molecular compensations that complicate the interpretation of the findings. In addition, the chronic and robust overexpression of various genes can also result in highly artificial physiologic conditions that may not provide clinically relevant information.

The more recent development of mice with conditional and tissue-specific genetic manipulations should provide additional and highly useful information regarding pathologic mechanisms related to myocardial I-R injury. Furthermore, scientists will likely develop new gene-targeted animals with precise molecular defects in specific cell-signaling pathways that will allow for more molecular insights into cardiovascular disease states. It has also become clear that cardiovascular risk factors, including obesity and diabetes, are highly prevalent among the human population around the world and newer, more clinically relevant, animal models for the study of obesity and diabetes are required. A number of research labs are currently developing new gene-targeted animal models to more closely mimic metabolic syndrome and type 2 diabetes in humans. The development of these animals may significantly extend our current understanding of how obesity and diabetes exacerbate myocardial I-R injury and cardiovascular diseases in general.