Controlling Reperfusion Injury With Controlled Reperfusion: Historical Perspectives and New Paradigms

Abstract

Cardiac reperfusion injury is a well-established outcome following treatment of acute myocardial infarction and other types of ischemic heart conditions. Numerous cardioprotection protocols and therapies have been pursued with success in pre-clinical models. Unfortunately, there has been lack of successful large-scale clinical translation, perhaps in part due to the multiple pathways that reperfusion can contribute to cell death. The search continues for new cardioprotection protocols based on what has been learned from past results. One class of cardioprotection protocols that remain under active investigation is that of controlled reperfusion. This class consists of those approaches that modify, in a controlled manner, the content of the reperfusate or the mechanical properties of the reperfusate (e.g., pressure and flow). This review article first provides a basic overview of the primary pathways to cell death that have the potential to be addressed by various forms of controlled reperfusion, including no-reflow phenomenon, ion imbalances (particularly calcium overload), and oxidative stress. Descriptions of various controlled reperfusion approaches are described, along with summaries of both mechanistic and outcome-oriented studies at the pre-clinical and clinical phases. This review will constrain itself to approaches that modify endogenously-occurring blood components. These approaches include ischemic postconditioning, gentle reperfusion, controlled hypoxic reperfusion, controlled hyperoxic reperfusion, controlled acidotic reperfusion, and controlled ionic reperfusion. This review concludes with a discussion of the limitations of past approaches and how they point to potential directions of investigation for the future.

Introduction

In the United States, 43.8% of cardiovascular deaths are the result of coronary obstruction, with an estimated economic cost of $188B in 2015. ¹ Individuals dying from a myocardial infarction (MI) lose an estimated 16.6 years of life. ² The interruption of myocardial blood flow due to coronary obstruction, such as thromboembolism or atherosclerosis, is a significant source of morbidity and mortality. It has been demonstrated that morbidity and mortality associated with myocardial infarction (MI) are correlated with the cardiac infarct size. Furthermore, infarct size increases with the duration of ischemia. ^{3 -5} Therapies that quickly reperfuse tissue, such as balloon angioplasty and coronary stenting have significantly improved patient outcomes by reducing the duration of ischemia. ^6,7 Despite these advances, acute MI remains a leading cause of mortality overall. ^6,8 The mortality rate for MI patients is 14%-19%, equating to 150,000 deaths per year in the United States alone. ² Of those that do survive, 5%-20% develop heart failure. ^9,10 The rate of hospitalization for heart failure following ST-segment elevation myocardial infarction (STEMI) increases with increasing infarct size. ⁵ Thus the impetus remains for continued development of therapies to decrease infarct size.

One approach is to continue refining techniques that enable a reduced duration of ischemia (e.g., reducing door to balloon times). ^{11 -14} However, it is not the only approach. Figure 1 is a schematic of the amount of cell death that occurs due to ischemia with and without reperfusion and cardioprotective therapies. ¹⁵ The reduction in cell death due to a reperfusion therapy is called reperfusion salvage. Observed cell death after reperfusion is termed ischemia-reperfusion injury. A burst of cell death occurs immediately upon commencing reperfusion, indicating that it is not just an ischemic injury. The yellow shaded region of Figure 1 highlights the reperfusion injury. Up to 50% of volume of a myocardial infarct may be attributed to reperfusion injury. ^16,17 It should also be noted that following the acute phase (Figure 1), subsequent processes can occur during longer-term cardiac remodeling that can be either beneficial or detrimental, ¹⁸ a topic that will not be covered in this review.

Figure 1.

Schematic of ischemia-reperfusion cell death over time. At the start of an ischemic event, cells begin dying. If the ischemia is not relieved, cells continue dying; resulting in a progressively larger infarction (blue dashed line). If the tissue is reperfused (red dotted line), there is a reduction in the amount of cell death known as the reperfusion salvage. The cell death that does occur can be attributed to an ischemic injury and a separate reperfusion injury. ¹⁵ The yellow region highlights the reperfusion injury. Adapted from Garcia-Dorado et al. ¹⁹

An optimal set of therapies would eliminate the reperfusion injury. The 2010 NHLBI workshop on cardioprotection identified further studies of reperfusion injury therapies as a priority. ²⁰ This identification and call for novel therapies was reiterated in 2015 ⁷ and again in 2017 by the European Society of Cardiology. ²¹ A number of reperfusion injury therapies have been proposed based on addressing the multiple potential mediators of the injury, including oxidative stress, pH and calcium imbalances, mitochondrial dysfunction, and distal embolization. ²² Unfortunately, there has yet to be a large-scale clinical trial success. ^7,21,22

The objective of this review is to provide an overview on the role that controlled reperfusion could play in reducing reperfusion injury associated with myocardial infarction. For the purposes of this review, controlled reperfusion is defined as modifying the reperfusate in a controlled manner during the acute phase following ischemia. This may include changing mechanical properties of the reperfusing blood (e.g., blood pressure or blood flow rates as in postconditioning ^23,24 ) or biochemical properties (e.g., hyperoxic reperfusion ²⁵ ). Section 2 of this review briefly overviews pathways leading to reperfusion injury. The overview focuses on those pathways that may be inhibited, at least in part, by controlled reperfusion. Section 3 describes various approaches to achieving controlled reperfusion. Section 4 discusses some limitations to be considered for current controlled reperfusion therapies and potential future directions.

Mechanisms Leading to Reperfusion Injury

Ischemia-reperfusion injury is multifaceted with the interplay between ischemia and reperfusion being complex. Discussion of the mechanisms of ischemic injury will be limited to their relationship with reperfusion injury. Furthermore, this section is not intended to be a complete overview of the biology and pathophysiology of cardiac reperfusion injury, for which many other excellent reviews exist. ^{26 -30}

No-Reflow Phenomenon and Microvascular Obstruction

Reperfusion injury, by its name, implies a reperfusion of tissue. No-reflow phenomenon is the development of patency in a previously obstructed coronary artery without achieving adequate microvascular reperfusion. ^{31 -37} Cardiac injury resulting from this phenomenon may fit more naturally within the confines of ischemic injury due to the inadequate blood flow. However, historically it has often been included in discussions of reperfusion injury due to its coincidence with the recanalization of the major artery. Preclinical studies from Kloner et al support the position that no-reflow does not contribute to the classical definition of reperfusion injury (i.e., injury that is primarily attributable to reperfusion of tissue). In canine models where coronary clamping was performed to control the timing of ischemia they found myocyte damage occurred before the microvascular damage that would lead to no-reflow, suggesting tissue regions with no-reflow were non-salvageable as a result of ischemia. ^31,38,39 A limitation of these studies is that collateral microvasculature circulation is significantly greater in canines than humans. ⁴⁰ However, subsequent studies have observed similar results with other animal models with a more anthropoid physiology. ^{41 -43} Another limitation in most pre-clinical studies is that ischemia is induced via clamping whereas no-reflow in the clinic can occur from fragments of the initial thrombus or atherosclerotic plaques that are disturbed via the surgical intervention to restore patency in the coronary artery. ^{36,44 -46}

Both pre-clinically and clinically, a mechanistic contribution to no-reflow is immune responses. ⁴⁷ With increasing ischemic duration, increasing neutrophil accumulation is observed in the vasculature, resulting in MVO. ⁴⁸ Additionally, monocytes and macrophages secrete pro-inflammatory signals that cause fibrin deposition that can form microemboli. ⁴⁹ The distal embolization risk is exacerbated by capillary endothelial cell swelling and immune-mediated tissue edema narrowing the vascular lumens. ^{43 -45} Finally, it should be noted that pre-clinical studies have found that cell death and no-reflow can increase over time, which could suggest reperfusion injury. ^41,50 Clinically, no-reflow has been found in some studies to correlate with poor prognosis. ^{34,46,51 -61} However, these effects could be explained by impaired cardiac healing that results in cardiac dysfunction and thus would not fit in the traditional definition of reperfusion injury. Although some uncertainty remains as to whether no-reflow contributes to reperfusion injury, the majority of the evidence in the literature suggests it does not.

Immune Response

Besides contributing to the no-reflow phenomenon, the immune response to ischemic injury can also play a role in reperfusion injury through multiple pathways. ²⁸ The full pathways have yet to be fully elucidated. The role of lymphocytes is outlined by Hofmann et al ⁶² and Linfert et al. ⁶³ Yang et al ⁶⁴ demonstrated that T-cells accumulate in the myocardium within 2 minutes of reperfusion and the use of knockout mice lacking mature lymphocytes showed less reperfusion injury. However, if the knockout mice receive T-cell transplantation the injury was restored. Although there is evidence of the role of immune cells in reperfusion injury, in vitro studies lacking any immune cells have also demonstrated ischemia-reperfusion injury and cardioprotection, indicating that non-immune mediated mechanisms also play a key role.

Oxidative Stress

A significant source of cell death in cardiac ischemia-reperfusion injury is due to oxidative stress resulting from the overwhelming production of reactive oxygen species (ROS). Multiple interlinking biological pathways are responsible for the ROS production. Primary among these pathways are: 1) dysfunction of the electron transport chain, 2) xanthine oxidase, 3) NADPH oxidase, and 4) uncoupled nitric oxide synthase. The relevance of each pathway and how they interlink is organ-dependent. These pathways, and others, are covered in detail elsewhere and thus only a brief overview is provided here. ^30,65 The ROS created through these pathways are directly cytotoxic ⁶⁶ and can initiate signaling pathways for apoptosis. ⁶⁷ However, it is important to note that ROS are also required as a part of several biochemical pathways that allow for cell survival in injury conditions (e.g., HIF1-α and PPAR-α pathways) and are therefore necessary in appropriate concentrations for ischemia-reperfusion injury recovery. ^68,69

Electron transport chain dysfunction

The means by which mitochondrial ion dysregulation occurs and causes dysfunction of the electron transport chain, specifically during ischemic conditions, has been well studied. ^{30,70 -72} In cardiac ischemia-reperfusion there is an influx of Ca²⁺ into the mitochondria, which facilitates mitochondrial permeability transition pore (MPTP) formation through a cyclophilin D-dependent mechanism. ^{73 -75} MPTP formation enables imbalance of the delicate ion homeostasis of the mitochondria. ^76,77 Notably, the severity and duration of ischemia, along with the presence of other molecules, such as ROS, modulate the amount of MPTP formation and therefore cell death. ^78,79 Even though ROS generation is limited in hypoxia due to the lack of O₂, the mechanisms are set for rapid ROS production during the reoxygenation that normally occurs during reperfusion. This cellular environment can be a key factor in tipping the scales to cell death during reperfusion.

MPTP-enabled imbalance in hydrogen ion homeostasis has detrimental effects on cells in hypoxic conditions as complexes I, III, and IV of the electron transport chain are all proton pumps. Short periods of ischemia cause dysfunction of complex I in the electron transport chain, whereas longer periods of ischemia cause dysfunction in complexes III and IV. ⁸⁰ Dysfunction of the electron-transport chain leads to buildup of reactive oxygen species.

Ischemia also limits the mitochondrial ability to perform aerobic respiration to produce sufficient ATP. ⁸¹ Therefore, anaerobic respiration ensues. During extended anaerobic conditions, other means of mitochondrial dysfunction and cell injury can occur. ^{82 -86} For example, the anaerobic generation of lactic acid exacerbates the proton imbalance thus decreasing the pH of the cell. ^87,88 Additionally, anaerobic respiration generates limited ATP, resulting in a paucity of ATP that reduces the production of antioxidant agents, making cells more susceptible to oxidative stress. ^82,89

At the time of reperfusion, reoxygenation not only enables the production of ROS as described, but it brings another set of damaging factors to the cardiac mitochondria. If initial ischemia lasts for long enough (30 minutes, according to several studies), then reperfusion exacerbates damage to complexes I and III of the electron transport chain, further decreasing mitochondrial function. ^90,91 The already damaged complexes are further damaged as oxidative respiration begins. A feed-forward loop of free oxygen radical production ensues, which results in loss of mitochondrial membrane potential. ^{92 -94} Additionally, due to changes in oxygen levels and energy demands, ETC uncoupling occurs and is exacerbated in reperfusion, leading to an imbalance in respiration substrates, thus decreasing glycolytic efficiency. Combined, these features lead to an increased propensity for further MPTP formation to occur as well, causing a second wave of cell death due to reperfusion injury.

Xanthine and NADPH oxidases

Xanthine oxidase (XO), in the presence of oxygen, converts xanthine to hypoxanthine. This reaction produces O₂ ^-. ⁸⁹ During hypoxia, the substrates for this ROS-generating reaction are formed (XO and xanthine). ^95,96 In turn hypoxanthine and O₂ ^- increase resulting in ischemia-reperfusion injury. ^{97 -99} NADPH Oxidase, or NOX, is a family of oxidases that produce NADH+ and O₂ ^-. Specifically, NOX2 and NOX4 are the source of significant ROS produced enzymatically during cardiac ischemia-reperfusion injury. ^69,100,101

Uncoupled nitric oxide synthase

Under physiologic conditions, nitric oxide synthase (NOS) produces nitric oxide (NO). In ischemic injury conditions and when under oxidative stress, the NOS substrate (L-arginine) and cofactor (BH4) availability are reduced. This causes the NOS to uncouple and begin to produce O₂ ^-. ^69,92,102 In addition, the uncoupling of NOS leads to reduced production of NO, a molecule which can be cardioprotective. ^103,104 Both the increase in ROS and decrease in NO production lead to cardiac injury in ischemia and reperfusion. The increase in ROS causes a feed forward loop of oxidative stress that causes further NOS uncoupling and more ROS production in reperfusion. ^102,103

Calcium overload

Shen and Jennings provided the first evidence of a correlation between the accumulation of intramitochondrial calcium and cardiomyocyte injury in the context of cardiac ischemia. ¹⁰⁵ As previously discussed, ischemia causes dysfunction of the electron transport chain, depleting ATP and causing intracellular acidosis. In response, the Na⁺/H⁺ exchanger is activated to remove hydrogen ions, at the expense of an influx of Na⁺ ions. The sudden influx of Na⁺ ions results in activation of the Na⁺/Ca²⁺ exchanger. As the Na+ are removed, an influx of Ca²⁺ into the cell occurs, which is exacerbated in reperfusion because extracellular pH drops, leading the Na⁺/H⁺ exchanger to increase its output, causing increased Na⁺ and therefore Ca²⁺ within the cell. ^{106 -108}

Additionally, the sarcoplasmic reticulum, where most myocardial calcium is stored, is disrupted as the sarcoplasmic ATPase, which normally brings Ca²⁺ into the sarcoplasmic reticulum, is rendered dysfunctional during ischemia. ¹⁰⁹ Because Ca²⁺ remains in the cytoplasm, the calcium-induced calcium release ryanodine receptor is activated, further releasing Ca²⁺ into the cytoplasm. ^107,110 This massive burst of calcium in the cytoplasm is then used by the cell to activate the troponin-tropomyosin complex, allowing myosin heads to bind to actin and hypercontracture to occur. ¹⁰⁹ Hypercontracture both triggers reperfusion-induced necrosis and can cause lethal mechanical disruption of membranes. ^111,112 A more detailed review calcium overload is provided by Chen. ¹¹³

Controlled Reperfusion

To inhibit or reduce the cellular death associated with the aforementioned reperfusion injury pathways, a number of approaches have been pursued. The focus of this section is to describe controlled reperfusion approaches. These include any methods that modify aspects of reperfusion in a controlled manner, including adjusting the overall flow rate, pressure, or the composition of the reperfusate, ¹¹⁴ sometimes referred to as gentle reperfusion. The latter covers modifying the concentration (either by increasing or decreasing) of naturally occurring molecules or compounds in the blood. The addition of non-naturally occurring molecules or compounds to the blood (such as pharmaceuticals) will not be covered, nor will hypothermic reperfusion. The interested reader is encouraged to read relevant review articles. ^{26,27,115 -118} Between 1986 and 1991, Buckberg and colleagues published an extensive set of “Studies of Controlled Reperfusion After Ischemia” in the Journal of Thoracic and Cardiovascular Surgery, some of which will be covered in this review but the interested reader is encouraged to review the entire series. ^{119 -142} This section is divided up into a review of studies on 1) ischemic conditioning, 2) gentle reperfusion, 3) controlled hypoxic reperfusion, 4) controlled hyperoxic reperfusion, 5) controlled acidotic reperfusion, and 6) control of other ions naturally occurring in blood. Figure 2 summaries the pathways of reperfusion injury and which pathways are impacted by the various controlled reperfusion approaches.

Figure 2.

Reperfusion injury pathways leading to cell death and cardiac dysfunction; points of intervention from controlled reperfusion therapies. An ischemic event, followed by normoreperfusion leads to cell death through a variety of pathways (black arrowed lines), with only those most relevant to cardiac ischemia-reperfusion injury identified here. No-reflow injury is more closely associated with ischemic injury, rather than reperfusion injury (black dashed arrowed line). For clarity, the interplay between the various pathways is only indicated (gray arrowed lines) for those most relevant to controlled reperfusion. Potential points of inhibition of the pathways via various controlled reperfusion therapies are indicated by the colored lines.

Ischemic Conditioning

Ischemic conditioning implements brief transient ischemic periods either before, during, or after the main ischemic insult. Ischemic preconditioning (i.e., transient ischemia before the main ischemic insult) has been robustly shown to reduce the magnitude of the reperfusion injury (Figure 1, yellow region) in many laboratories and animal models. ^16,82,143 However, preconditioning is not a feasible clinical therapy due to the spontaneous nature of MI.

Remote ischemic conditioning induces transient controlled ischemia in tissue beds located distal to the main ischemic event. Przyklenk et al first demonstrated this in the heart. ¹⁴⁴ Transient ischemia has been induced in limbs and can occur before, ¹⁴⁴ during, ¹⁴⁵ or after ^23,146 the ischemic event in order to significantly reduce reperfusion injury in animal models. ^{147 -150} Both humoral and neural mechanisms are being investigated. ^{147,149,151,152} Humoral mechanisms, in particular, may result in modification of the blood via either removal of detrimental components or increased cardioprotective components, resulting in a modified reperfusate. Outcomes reported from small clinical trials of remote ischemic conditioning have been positive or neutral, while large trial outcomes have been neutral. ^{153 -155} Additionally, remote ischemic conditioning patient protocols have limited clinical applicability for the approximately 22%-42% of patients who also have peripheral artery disease. ^{156 -158}

An alternative ischemic conditioning therapy is postconditioning, where reperfusion is modified in a controlled manner to include (30-60 s) interruptions of blood flow in the recanalized vessel immediately after the ischemic event. As first demonstrated by Zhao et al, postconditioning has reduced the infarct volume up to 50% in an animal model ²³ and has been observed by many laboratories using a variety of animal models. ¹⁵⁹ Staat et al translated postconditioning to myocardial infarction patients by cyclically inflating and deflating the angioplasty balloon post stent deployment and observing a reduction in creatine kinase release, a biomarker of infarct size. ¹⁶⁰ The protective mechanisms of ischemic preconditioning and postconditioning have been reported to share activation of certain pathways, such as the reperfusion-injury salvage kinase (RISK) pathway, and inhibition of MPTP formation. ^{161 -164} Endothelial dependent vasodilation function was observed to improve in patients treated with postconditioning relative to those without. ¹⁶⁵ It has also been reported that postconditioning inhibits inflammatory responses, potentially through reduced tissue necrosis factor alpha (TNFα) and interleukin-6 (IL-6). ¹⁶⁶ However, in vitro and ex vivo studies of postconditioning resulting in reduced infarction demonstrate that cardioprotective effects can be obtained independent of immune mediators. ¹⁵⁹ Reduction of ROS is also observed with ischemic postconditioning. ¹⁶⁷

Unfortunately, although several small clinical trials using postconditioning demonstrated improved outcomes, the same is not true for large clinical trials. ^{24,168 -170} One hypothesized cause of these clinical trial failures is the mechanical stress on the diseased coronary vessel caused by multiple balloon catheter inflations. The mechanical stress may result in the release of pro-inflammatory and thrombogenic material. ¹⁷¹ An alternative hypothesis for the lack of successful clinical translation is that the timing protocols for the cessations of flow were not optimized. ²⁴ This hypothesis is consistent with the meta-analysis of pre-clinical animal experiments performed by Skyschally et al. ¹⁵⁹ The timing protocols include how soon after initial reperfusion the postconditioning controlled reperfusion protocol is initiated (typically within 60 s), the duration of each reocclusion-reperfusion cycle (typically 60-120 s) and the number of reocclusion-reperfusion cycles (typically 2-10 cycles). ^{24,159,160,172 -175} Kin et al ¹⁷⁶ and Yang et al ¹⁶² both demonstrated in animal models that the cardioprotective effects of ischemic postconditioning are lost if the start of postconditioning was delayed by 60 s or more after the end of the main ischemic event. Additionally, Granfeldt et al note that the optimal postconditioning controlled reperfusion parameters vary by species. ¹⁷⁷ A variety of postconditioning protocols have been performed clinically. ¹⁶⁸ The failed translation of ischemic postconditioning in large clinical trials may be due to the varying protocols, the appropriateness of a single protocol for heterogenous patient populations, or potentially inappropriate patient selection. ^{24,178 -180}

Gentle Reperfusion

Although postconditioning has been largely abandoned as a potential clinical tool, the proof-of-principle preclinical experiments teach us that there is value in transiently modifying the reperfusion flow rate. In fact, well before ischemic postconditioning was attempted, the concept of “gentle reperfusion” (occasionally called gradual, modified, or staged reperfusion) was investigated. ^128,181 Whereas postconditioning via balloon angioplasty is like a switch either allowing unrestricted flow or no flow, gentle reperfusion via an extracorporeal pump is like a dial, allowing for variable flow rates. The reperfusion may be dictated by flow rate, perfusion pressure, or another reperfusion-related parameter and can be gradually increased to normal physiologic values. ¹⁶⁸

Gentle reperfusion has been investigated extensively in the context of coronary ischemia, ^{128,182 -186} cardioplegia and cardiopulmonary bypass, ^{187 -190} and cerebral ischemia. ¹⁹¹ It has consistently been shown to reduce reperfusion injury relative to standard reperfusion.

Okamoto et al, as part of the Buckberg series of “Studies of Controlled Reperfusion after Ischemia,” compared sudden reperfusion and gentle reperfusion in an open-chest left anterior descending coronary ischemia canine model. ¹²⁸ Gentle reperfusion was achieved by using a vented bypass to infuse blood with a roller pump at a flow rate of 25 to 30 mL/min for 20 min, after which physiologic flow was allowed. It was estimated that the gentle reperfusion approach resulted in a perfusion pressure of less than 50 mmHg while sudden reperfusion corresponded to a perfusion pressure of 80 mmHg. Gentle reperfusion resulted in a reduced infarct area relative to area at risk and reduced edema. Yamazaki et al performed a similar study in canines, but also included a 3-week follow-up period wherein the controlled reperfusion group exhibited faster improvement in cardiac function and fewer premature ventricular complexes than sudden (i.e., uncontrolled) reperfusion. ¹⁹² As compared to both of these studies, which step-wise increased the reperfusion pressure, Vinten-Johansen et al ramped the perfusion pressure from 0 mmHg to the mean arterial pressure in a linear fashion over the first 30 minutes of reperfusion and likewise saw improvements in infarct size. ¹⁸² They also observed that studies with a relatively short periods of controlled reperfusion (15 min versus 60 or 120 min) did not show as robust of a benefit.

The mechanisms by which gentle reperfusion provide cardioprotection remain to be fully elucidated. Unlike ischemic postconditioning, it does not activate the reperfusion-injury salvage kinase (RISK) pathway in a swine model. ¹⁸³ However, it can improve endothelial function ¹⁸⁴ and potentially reduce MPTP opening by inhibiting ROS production. ^193,194 Takeo et al observed in isolated perfused rat hearts that gentle reperfusion improved LVDP recovery following ischemia by attenuating the increase in myocardial calcium and sodium. ¹⁹⁵

Ferrera et al demonstrated that controlled low-pressure reperfusion (LPR) can reduce lethal myocardial reperfusion injury, even when performed up to 20 minutes after the onset of reperfusion. ¹⁸⁵ This result suggested that the benefits from controlled LPR, unlike other approaches, was not associated with ameliorating the initial burst of oxidative stress upon standard reperfusion. To obtain a protective effect, the duration of controlled LPR has to be increased in comparison to ischemic postconditioning, with a minimum of 10 minutes, ¹⁹⁶ further suggesting that mechanism of cardioprotection is different from postconditioning. Pantsios et al investigated the impact of high-pressure reperfusion in vivo by partially cross-clamping the aorta during reperfusion of the heart after a transient occlusion of the anterior descending coronary artery. When aortic pressure was increased by ˜20% over baseline, both the infarct area and the area of no-reflow were increased relative to control experiments. ¹⁹⁷ Takeo et al observed that an optimal reduced perfusion flow rate to maximize left ventricular function could be found, though the translational value of the precise flow rate is limited as the work was performed in isolated rat hearts. ¹⁹⁵ Similarly working in an isolated rat heart model, Nemlin et al found an optimal pressure for LPR of 51 mmHg (whereas normal pressure was 73 mmHg). ¹⁹⁸ Although optimal pressure and flow are reported, it should be noted that these 2 parameters cannot be independently adjusted due to their interplay with respect to vascular resistance.

Besides modifying the perfusion flow/pressure, it is also possible to elicit beneficial effects by modifying the chemical composition of the reperfusate. Vinten-Johansen et al stated that “the value of controlling reperfusate composition without simultaneous control of reperfusion conditions is limited.” ¹²¹ Julia et al reported that including blood within the controlled reperfusate was beneficial, potentially due to the ROS scavenging activity of red blood cells. ¹⁴² Techniques to evaluate different blood components in ex vivo systems have also been developed. ¹⁹⁹ These studies in controlled reperfusion point to the importance of modifying oxygen content too, which will be covered in the following sections.

Controlled Hypoxic Reperfusion

Reoxygenation, as described in Section 2, plays a key role in reperfusion injury. This observation leads to the so-called oxygen paradox: oxygenation is necessary to alleviate an ischemic injury but it can simultaneously cause reperfusion injury via conversion into oxygen radicals and creation of reactive oxygen species (ROS) during reoxygenation. ^{121,200 -204} Therefore, limiting ROS represents a potential therapeutic pathway. Although not a form of controlled reperfusion, pre-clinical studies administering antioxidants to reduce ROS and thereby reduce cellular death and infarct size demonstrates the potential of this therapeutic pathway. ^{89,205 -208} Translation of antioxidant therapy to the clinic has provided mixed results with a need for larger randomized clinical trials measuring more robust clinical outcomes being needed. ²⁰⁹

An alternate treatment approach is to reduce the production of ROS. ROS production has a first-order dependence on the concentration of O₂. ²¹⁰ Therefore, reducing the O₂ bioavailablity to cells is a potential therapeutic pathway. This strategy is supported by experimental results demonstrating a reduction in cell injury following ischemia via reperfusion with an anoxic perfusate containing glucose. ^211,212 Gradual reoxygenation in an in vivo porcine model of cardiopulmonary bypass showed improved ventricular function and reduced biomarkers of oxidative stress. ^187,188 Postconditioning, which also reduces O₂ concentration via transient interruptions in flow, decreases the generation of ROS and increases cell viability in a cell culture model. ²¹³ Reducing O₂ availability minimizes ROS production by inhibiting the electron transport chain process for hyperpolarized mitochondria ²¹⁴ and oxidase enzyme systems, such as xanthine. ⁶⁶ Because this oxidative stress burst occurs only during the first several minutes (<10 min) of reperfusion, controlled hypoxic reperfusion is likely only necessitated on a similar time-scale.

Serviddio et al reported data supporting this hypothesis by exposing isolated perfused rat hearts to complete global ischemia, followed by 40 minutes of normoxic reperfusion (600 mmHg) or 3 minutes of hypoxic reperfusion (150 mmHg) followed by 37 minutes of normoxic reperfusion. ²¹⁵ Multiple measures of oxidative stress were reduced in the controlled hypoxic reperfusion group relative to the normoxic group. Additionally, the controlled hypoxic reperfusion group had improved left ventricular function over the 40 min of reperfusion. Hearse et al found in an isolated heart model that creatine kinase, as a marker of cardiac injury, increased monotonically with increasing the oxygen content in the reperfusate buffer. ²⁰⁰ Angelos et al performed a detailed study measuring ROS production in an isolated perfused rat heart where the first 5 minutes of reperfusion were carried out with buffer saturated with either 95%, 20%, or 2% O₂. ²¹⁶ In contrast to Serviddio et al, Angelos et al reported more ROS as measured by electron paramagnetic resonance spectroscopy with increasing hypoxia. Notably, the duration of global ischemia was shorter in the study by Angelos et al (20 min versus 40 min), which could impact the initial tissue oxygen tension at the time of reperfusion and thus the various pathways producing ROS. Angelos et al also performed experimental arms with allopurinol (a xanthine oxidase inhibitor), diphenyleneiodonium (an NADPH oxidase inhibitor), and Tiron (a superoxide scavenger) and found that all 3 reduced ROS production, suggesting that multiple pathways of ROS production are active in ischemia-reperfusion injury with both normoxic and hypoxic controlled reperfusion.

More recently, Farine et al have observed that controlled hypoxic reperfusion for 2 minutes is beneficial in a rat working heart global ischemia model, reporting improvements in cardiac function, including left ventricular work, left ventricular developed pressure, and maximum and minimum first derivatives of left ventricular pressure. ¹¹⁷ Studies from our own laboratory focusing on measurement of infarct size also demonstrate the effectiveness of controlled hypoxic reperfusion. Isolated buffer-perfused rat hearts in a Langendorff apparatus (studies approved by our local Institutional Animal Care and Use Committee) were subjected to 30 min global ischemia followed by 120 min reperfusion. In the control cohort, reoxygenation was achieved by re-administration of buffer bubbled with 95% oxygen (O₂) while, in hearts that received controlled hypoxic reperfusion, global ischemia was followed by 1 min of reperfusion at a low oxygen partial pressure (21%), 1 min normal oxygen partial pressure (95%), 1 min low oxygen partial pressure (21%), and 117 min of normal oxygen partial pressure (95%). Infarct size (delineated by triphenyl tetrazolium chloride (TTC) staining) was reduced (Figure 3) in the controlled hypoxic reperfusion group relative to the control (Figure 3).

Figure 3.

Improved ventricular function and reduced infarct size with controlled hypoxic reperfusion. Langendorff prepared rat hearts were exposed to ischemia-reperfusion either without treatment (control, n = 8) or with controlled hypoxic reperfusion (CHR, n = 9). TTC staining was used to denote viable (red) and infarcted tissue (white) for (A) a control heart and (B) a CHR heart. A statistically significant reduction in infarct size (C) was observed with controlled hypoxic reperfusion as denoted by the line. The asterisk denotes an outlier based on Tukey’s method.

We are exploring a potentially translationally relevant approach to reducing O₂ concentration during acute reperfusion using ultrasound-mediated oxygen scavenging. ^217,218 In vitro cell culture studies have found that HL-1 cardiomyocyte viability at 24 h is increased when simulated reperfusion is carried out with culture media where ultrasound-mediated oxygen scavenging has been performed. ²¹⁹ However, this area is ripe for the exploration of translationally-relevant approaches to achieving controlled hypoxic reperfusion following myocardial infarction.

Controlled Hyperoxic Reperfusion

Although the oxygen paradox and controlled hypoxic reperfusion data described indicates that reduced oxygen levels during acute reperfusion may be beneficial by reducing ROS production, hypoxia in tissue during ischemia is known to be detrimental. Thus some investigators have been motivated to study the potential for hyperoxia to reduce infarct size in cardiac ischemia-reperfusion injury. Sterling et al used an open chest rabbit left coronary artery ligation model of myocardial infarction to demonstrate reduced infarct size when the animal was placed in an oxygenated hyperbaric chamber (100% O₂, 2.5 atm) during ischemia, reperfusion, or ischemia and reperfusion relative to an animal exposed to non-hyperbaric oxygen (100% O₂, 1 atm) or a moderately oxygenated environment (40% O₂, 2.5 atm). ²²⁰

Hyperbaric oxygen therapy is not practical in typical clinical settings for the treatment of MI. An alternate O₂-supersaturated crystalloid solution that could be mixed with blood (termed aqueous oxygen (AO)) has been developed as a means of increasing the oxygen content of the reperfusate in a manner that could be undertaken via an endovascular approach. ^221,222 Spears et al tested AO in a canine model by withdrawing blood from the femoral artery, mixing it with the O₂-supersaturated crystalloid solution to form AO, and then infusing it back into the left coronary artery with a roller pump after 90 min of balloon-induced ischemia and 30 min of autoreperfusion (physiologic, passive reperfusion with normoxemic blood). ²²³ This delayed controlled hyperoxic reperfusion demonstrated improved left ventricular ejection fraction, fractional shortening, and ST-segment depression relative to autoreperfusion alone or autoreperfusion for 30 min followed by left coronary roller pump infusion of normoxemic blood (not mixed with O₂-supersaturated crystalloid solution). A similar study in swine was performed, but the intracoronary AO perfusion was performed 24 h after autoreperfusion rather than 30 min. Infarct size and left ventricular ejection fraction were observed to improve. ²⁵

Concurrent with these pre-clinical studies, pilot studies were performed in patients with acute myocardial infarction. Improved left ventricular function was observed both immediately and up to 3 months post infarction with no adverse events during treatment. ^{224 -226} As with the animal models, recanalization was observed as indicated by a TIMI score of 2 or 3 before AO was administered. Larger follow-up trials, AMIHOT-I, ²²⁷ AMIHOT-II, ²²⁸ and IC-HOT ²²⁹ were performed with patients with anterior ST-elevated MI (STEMI). In AMIHOT-I, the subgroup of patients who were treated with AO within 6 h of symptom onset demonstrated improvements in wall motion, but no clinical improvements were observed in the full group analysis. A pooled analysis of patients enrolled in AMIHOT-I and AMIHOT-II that were treated with AO within 6 h observed improvements in infarct size. ²²⁸ However more frequent hemorrhagic complications occurred in the AO treatment group and non-significant trends for more stent thrombosis and death were observed. The IC-HOT trial was performed with modifications made to the protocol with a goal of improved safety, which was observed relative to the AMIHOT-II observations. ²²⁹ Overall net adverse clinical events occurred at a rate of 7.1%, which was lower than the pre-study threshold of 10.7% that was established in concert with the US FDA.

Timing of AO therapy is critical to understanding the apparent paradox between positive pre-clinical results for controlled hypoxic reperfusion and controlled hyperoxic reperfusion. The aforementioned controlled hypoxic reperfusion studies were designed such that the initial phase of reperfusion was hypoxic. Although the time between balloon angioplasty and stenting was not reported in controlled hyperoxic reperfusion, it is not unreasonable to presume based on common clinical practices and the fact that recanalization was confirmed angiographically before AO administration, that AO therapy did not occur within the first several minutes of reperfusion and therefore did not contribute to increased ROS. Even if hyperoxia does contribute to some increase in oxidative stress, the benefits for endothelial health or ameliorating the tissue hypoxia during no-reflow may be more important. ^220,230 It is feasible to envision a dual hypoxia/hyperoxia therapy with the hypoxemic treatment occurring in the early phase of reperfusion, followed by hyperoxemia during later phases of reperfusion.

Controlled hyperoxic reperfusion studies have consisted of a mix of blood with an oxygenated buffer. In contrast, controlled hypoxic reperfusion studies have been performed both with and without blood. Morita et al, reported that the inclusion of blood in the reperfusate conferred additional protective benefit. ¹⁸⁹ This work suggests that in addition to modifying the oxygen content of reperfusate, a benefit is also derived from modifying other aspects of the reperfusate.

Controlled Acidotic Reperfusion

As described in section 2, H+ concentration plays a role in multiple pathways of ischemia-reperfusion injury. The earliest studies (before reperfusion injury was a well-defined concept) focused on the impact of pH during ischemia, particularly acidosis using multiple ex vivo models (e.g., isolated whole heart and isolated papillary muscle), timing of the introduction of a modified pH and hypoxia, and means of modifying the buffering solution pH (e.g., HCl, NaOH, or CO₂). ^{231 -236} A majority of studies observed the benefit of mild acidosis (pH in a range of approximately 6.8 to 7.0), however more severe acidosis (6.6) was not effective.

Upon the recognition of the independent role of reperfusion injury, studies focused on the use of an acidic reperfusate to investigate its potential role in attenuating the deleterious effects of calcium overload and dysfunction in complexes I, III, and IV of the electron transport chain. Kitakaze et al explicitly noted that because acidosis is an antagonist for calcium influx, hypercontracture could be attenuated by acidosis, observing that intracellular acidosis was effective in recovering left ventricular developed pressure. ²³⁷ Whereas Kitakaze et al, used HCl to modify pH, Hori et al investigated the impact of controlled acidotic reperfusion by noting that in gradual reperfusion, the pH of the tissue naturally remains acidotic. ²³⁸ It was observed that in gradual reperfusion (termed staged reperfusion by the authors) fractional shortening improved relative to normal abrupt reperfusion and relative to gradual reperfusion where NaHCO₃ was introduced to obtain a pH of 7.3 to 7.4 during the entirety of reperfusion. Additionally, the use of HCl to obtain controlled acidotic reperfusion with abrupt reperfusion (rather than gradual reperfusion) demonstrated similar improvements in fractional shortening. Taken together, the work supports a hypothesis that gradual reperfusion is beneficial, in part, due to controlled acidotic reperfusion.

As described earlier, gradual reperfusion and ischemic postconditioning can achieve similar results. Several studies were reported looking at acidosis and ischemic postconditioning. Cohen et al and Fujita et al both observed that postconditioning maintained mild acidosis during early reperfusion and improved cardiac function and reduced infarct area relative to abrupt reperfusion and postconditioning with buffer designed to increase the tissue pH. ^239,240 Fujita et al demonstrated that improvement in cardiac recovery occurred via activation of the RISK pathways. ²⁴⁰ Cohen et al demonstrated that this improvement occurs in part due to attenuated MPTP formation and furthermore was dependent on the timing of the postconditioning and acidosis. ²³⁹ Others also observed that controlled acidotic reperfusion attenuates MPTP formation. ^241,242 Interaction between controlled acidotic reperfusion and ROS was observed by Penna et al, where the benefits of acidosis were slightly (but not entirely) blunted by the inclusion of antioxidant enzymes. ²⁴³ The severity of ischemia may also play a role as Farine et al did not observe cardioprotective benefits from controlled acidotic reperfusion in their model that used a shorter ischemic period. ¹¹⁷ These studies were all carried out using ex vivo tissue.

The benefit of controlled acidotic reperfusion was also demonstrated using an in vivo canine myocardial infarction model where autologous acidotic blood (achieved both via HCl and increased pCO₂) was infused for the first 30-60 minutes of reperfusion through a bypass in the left anterior descending coronary artery. ^244,245 Both Kitakaze et al and Preckel et al demonstrated reduced infarct size. ^244,245 White et al used a cardiac transplant model, where donor pigs were anesthetized, extubated, and the heart excised after circulatory arrest had occurred for 15 minutes. ²⁴⁶ The excised heart was perfused with a buffer at a pH of 6.4, 6.9, 7.4, or 7.9. Cardiac output, coronary vascular resistance, and myocardial oxygen consumption were all most improved at the end of recovery for a buffer pH of 6.9, consistent with the studies discussed earlier. Follette et al used an in vivo open-chest cardiac bypass model of ischemia-reperfusion with a cardioplegic buffered to a pH 7.8, which demonstrated better left ventricular recovery than a cardioplegic buffered to a pH of 7.4. A key difference in these controlled alkalotic reperfusion studies however was the heart was arrested during ischemia via topical ventricular hypothermia (16°C). Hypothermia reduces metabolism and the state of the heart at reperfusion would be substantially different than a heart maintained at normothermic temperatures.

Controlled Hypocalcemia, Hypernatremia, Hyperkalemia, and Hyperosmolarity

Given the early positive results for controlled acidosis to attenuate reperfusion injury, with data supporting an impact on attenuated calcium overload, multiple groups performed studies modifying not just pH but also other ions, including sodium and calcium. ^246,247 In fact, the means of modifying pH could also contribute to a change in Na⁺ and osmolarity. ^231,248 Harada et al observed that in addition to improved recovery of left ventricular developed pressure via controlled acidosis, using hypernatremic blood in an isolated blood-perfused canine heart also achieved improvements in left ventricular developed pressure and reductions in calcium overload that were consistent with modification of sodium-hydrogen and sodium-calcium pump function. ²⁴⁷

Osmolarity

Modification of solute concentration affects osmolarity. Additionally, edema is commonly observed in the myocardium following ischemia-reperfusion. This provides motivation to use a hyperosmotic reperfusate to reduce the drive of fluid from the vascular compartment to the myocardial tissue. Follette et al observed that edema was reduced with a hyperosmotic reperfusate. ²⁴⁹ Although controlled hyperosmotic reperfusion did improve perfusion, it did not significantly improve ventricular performance relative to normoreperfusion. Okamoto et al observed similar results of increased perfusion but not ventricular function (unless the reperfusate was also hyperglycemic). ¹²² Shen et al investigated a hyperosmotic reperfusion via increased sodium chloride and found that coronary resistance and injury were reduced and contractility improved relative to a control Krebs-Henseleit buffer. ²⁵⁰ Commensurate with the changes in contractility were reduced oxidative stress, though the optimal sodium chloride hyperosmotic concentrations for improving flow and reducing in injury were not consistent.

Sodium

Sodium can impact the intracellular calcium concentration, ²⁵¹ and thus heart contracture, through the sodium-calcium exchange. Elevated perfusate sodium concentrations prior to reperfusion causes contractile dysfunction. ^252,253 Tani and Neely observed in an isolated perfused heart model that at the end of ischemia and the first 2 minutes of reperfusion, there is a linear correlation between intracellular sodium concentration, calcium uptake, and subsequent reduction of ventricular function investigated. ²⁵⁴ Low flow perfusion during ischemia with a hypernatremic solution lowers the intracellular sodium concentration. ²⁵⁵

Calcium

As already described, calcium plays a central role in cardiomyocyte contracture and thus is important in understanding the pathophysiology of ischemia-reperfusion injury. ¹¹³ Intracellular calcium concentration can be indirectly modified through various means of controlled reperfusion. Limiting calcium overload can also be achieved directly by 1) restricting the amount of calcium using controlled hypocalcemic reperfusion, 2) employing a calcium chelation agent such as citrate, or 3) blocking calcium channels directly (or indirectly). The latter 2 approaches are outside the scope of this review but are approaches that have provided significant insight into the understanding of ischemia-reperfusion injury. ^{127,135,248,256,257} The calcium paradox sets a lower bound on the calcium concentration used for controlled hypocalcemia reperfusion. ^{200,246,256,258 -260}

Throughout the 1980s numerous studies were carried out demonstrating that by nearly all metrics, controlled hypocalcemic reperfusion was advantageous for reducing cardiac injury following reperfusion in a variety of ex vivo and in vivo models. ^246,256 By the 1990s there was evidence that a hypocalcemic reperfusate provides clinical benefit in the context of cardioplegics in cardiac surgery. ^261,262 In the laboratory setting, reduced intracellular and extracellular calcium concentration have been observed to be beneficial for cardiac recovery. ²⁵⁴ The reperfusate calcium concentration that maximizes recovery varies by study model and is also impacted by the sodium, potassium, and magnesium concentrations in the reperfusate. ²⁶³

Potassium

Controlled hyperkalemic reperfusion has been investigated to reduce reperfusion injury through 2 mechanisms. Tani and Neely observed that during the first 10 minutes of reperfusion with a hypocalcemic solution, intracellular sodium concentration remained elevated and upon introduction of a normocalcemic reperfusate, Ca²⁺ overload developed, likely through the Na⁺/Ca²⁺ exchange. However, if a high concentration of potassium was included in the hypocalcemic reperfusate, intracellular sodium dropped and when normocalcemic reperfusion was implemented there was reduced calcium load. ²⁶⁴ Follette et al investigated the use of potassium in a cardiac bypass reperfusion model. They observed that increased potassium concentration in the reperfusate helped maintain heart arrest, thereby reducing the metabolic activity of the heart, which reduced edema. ²⁴⁹

Current Limitations in Controlled Reperfusion

Most investigations of the effects of controlled composition of the ion content in the blood reperfusate have been in the context of cardiac bypass. Normal management in the context of cardiac bypass enables highly controlled and localized modification of the reperfusate through the bypass ports. Achieving this in the context of current standard of care for acute myocardial infarction, particularly percutaneous coronary intervention, remains a challenge. ²⁶⁵ However, the technologies developed to clinically implement supersaturated oxygen therapy ²²² could potentially be used for modifying more than just the oxygen content of the reperfusate. Device development is needed to affect the preclinical successes observed in controlled reperfusion. The importance of this is further emphasized in the context of applying these therapies as personalized medicine.

The number of failed trials in the broader field of cardioprotection is disheartening. The proper selection of patients may play a role, as the comorbidities, nature of the tissue at risk of ischemia-reperfusion injury, and other individual factors are all important. Improved methods of rapidly assessing patients, both through biochemical assays and imaging assays could be critical for identifying patients that may be responsive to controlled reperfusion. An example of predicting (and thus guiding therapeutic decision making) outcome originates from the DEFUSE study, which used perfusion-weighted MRI to identify the patients that would be most likely to respond to thrombolytic therapy. ^266,267

The lack of translation is not solely based on the diversity inherent in epidemiology but is also a result of the methodological weaknesses of some studies. Heusch and Rassaf describe heterogeneity that has contributed to the current uncertainty on the efficacy of ischemic conditioning procedures in clinical practice. ¹⁶⁸ They also emphasize that there are several consensus papers that highlight these problems with the existing studies on ischemic conditioning and make detailed recommendations on how an ideal study should be conducted. ^{15,19,173,268,269} Although these recommendations are primarily in the context of ischemic conditioning, many of them should be judiciously applied in other forms of controlled reperfusion being pursued.

Another aspect that may be limiting successful clinical translation is that ischemia-reperfusion injury occurs along multiple pathways (Figure 2). As such, a therapeutic protocol that can be broadly successful across a large patient population may require a multitarget strategy. ^164,249 Fortunately, many of the controlled reperfusion approaches described in this review are compatible and could be implemented in a single protocol.

Summary

Although not all of the biological aspects of cardiac ischemia-reperfusion injury have been completely identified, and their relevance varies depending on the nature of the model used, there is sufficient understanding to guide the development of potential therapies. Controlled reperfusion, defined as the orderly modification of the composition of the physiologically occurring components of the reperfusate or modification of the fluid mechanical properties of the reperfusate, is a well-studied and promising therapeutic approach. These modifications include flow rates, perfusion pressure, O₂ content, pH, and ion content. Critical needs remain around defining treatment protocols that translate from animals to humans, identifying approaches to personalizing treatment based on patient-specific factors, and developing appropriate devices to induce controlled reperfusion.