Significant reduction of ischemia‐reperfusion cell death in mouse myocardial infarcts using the immediate‐acting PrC‐210 ROS‐scavenger

Abstract Managing myocardial infarction (MI) to reduce cardiac cell death relies primarily on timely reperfusion of the affected coronary site, but reperfusion itself induces cell death through a toxic, ROS‐mediated process. In this study, we determined whether the PrC‐210 aminothiol ROS‐scavenger could prevent ROS‐induced damage in post‐MI hearts. In a series of both in vitro and in vivo experiments, we show that: (a) in vitro, PrC‐210 was the most potent and effective ROS‐scavenger when functionally compared to eight of the most commonly studied antioxidants in the MI literature, (b) in vitro PrC‐210 ROS‐scavenging efficacy was both immediate (seconds) and long‐lasting (hours), which would make it effective in both (1) real‐time (seconds), as post‐MI or cardiac surgery hearts are reperfused with PrC‐210‐containing blood, and (2) long‐term (hours), as hearts are bathed with systemic PrC‐210 after MI or surgery, (c) systemic PrC‐210 caused a significant 36% reduction of mouse cardiac muscle death following a 45‐minute cardiac IR insult; in a striking coincidence, the PrC‐210 36% reduction in cardiac muscle death equals the 36% of the MI‐induced cardiac cell death estimated 6 years ago by Ovize and colleagues to result from “reperfusion injury,” (d) hearts in PrC‐210‐treated mice performed better than controls after heart attacks when functionally analyzed using echocardiography, and (e) the PrC‐210 ROS‐scavenging mechanism of action was corroborated by its ability to prevent >85% of the direct, H2O2‐induced killing of neonate cardiomyocytes in cell culture. PrC‐210 does not cause the nausea, emesis, nor hypotension that preclude clinical use of the WR‐1065/amifostine aminothiol. PrC‐210 is a highly effective ROS‐scavenger that significantly reduces IR injury‐associated cardiac cell death.


| INTRODUC TI ON
It is essential to re-establish blood flow (reperfusion) to the heart after blockage (ischemia) in a coronary artery. While reperfusion will ultimately reduce the overall size of the myocardial infarct, 20 the reperfused oxygenated blood, itself, significantly contributes to cardiac cell death. 14 Restoration of blood flow after ischemia results in the en masse formation of highly toxic reactive oxygen species (ROS) in myocardial cells, 32 which causes cardiomyocyte death and long-term reduction in myocardial function. 30 Bigger et al 1 reported that the prognosis for ischemic heart disease patients is inversely related to the extent of postmyocardial infarct tissue necrosis. The total annual cost of cardiovascular disease in the US is estimated at over $315 billion accounting for 15% of total health expenditures, more than any other disease. 12 At $315 billion/ year, any strategy that conferred a significant reduction in MI severity and tissue loss would have a significant impact to reduce health care costs, thus finding new ways to prevent MI-associated cardiac necrosis remains an important goal.
Animal and clinical research demonstrate that oxygen free radicals cause tissue damage during myocardial ischemia-reperfusion. 16,18 The bolus of oxygen that accompanies heart reperfusion generates ROS that simply exceed the cardiomyocyte detoxifying capability and reach levels that cause cardiomyocyte death. Davies et al 6 showed that the levels of several species of reduced oxygen are increased in myocardial tissue during postischemic reperfusion of the heart, both acutely (seconds) during reperfusion, and longer term, that is, hours/ days, from inflammatory cell-generated ROS at the post-MI site. 24 PrC-210 is a new direct-acting, actually immediate-acting, aminothiol ROS-scavenger 5,22 that can be administered IV, orally, or topically, and it has no measurable nausea/emesis nor hypotension side effects. 26 PrC-210 is unlike virtually all of the previously tested "antioxidants" in the cardiac ischemia-reperfusion literature. Earlier studied antioxidants either: (a) like Vitamin E, act indirectly over hours-days via NrF-2 to activate expression of protective genes, 28 (b) like IV-administered 50 kDa enzymes such as superoxide-dismutase or catalase, detoxify ROS but cannot impact millisecond half-life ROS generated inside reperfused cardiomyocytes, or (c) like adenosine or allopurinol, can possibly affect long-term oxygen metabolism in mammalian cells, but have no impact upon ROS generated in reperfused cardiomyocytes.
In this study, we first determined PrC-210 efficacy and potency to prevent an •OH insult, head-to-head in vitro against eight other "antioxidants" commonly studied in the cardiac ischemia-reperfusion literature. Because PrC-210 performed the best, we asked whether it could prevent ROS-induced cell death in mouse hearts using the standard mouse heart ischemia-reperfusion model. Finally, to corroborate the PrC-210 mechanism of action, we directly exposed primary neonate mouse cardiomyocytes to an H 2 O 2 insult and directly measured the PrC-210 time-and dose-dependency to prevent the H 2 O 2 -induced cardiomyocyte death.

| Materials
Tissue culture media was from Gibco (ThermoFisher), fetal bovine serum was from Hyclone (Logan, UT), and culture-ware was from Falcon. Thirty percent H 2 O 2 was from Mallinckrodt. Synthesis of the PrC-210 HCl aminothiol is described separately. 4,9 PrC-210 HCl crystals are stored under a nitrogen atmosphere at −20°C, and even with routine thawing, use, and re-storage, crystalline PrC-210 is completely stable for >4 years by mass spectrometry analysis. Other chemical reagents were obtained from Sigma Aldrich (St. Louis, MO). C57 mice were from Jackson Laboratory (Bar Harbor, ME). Mice were maintained on 12-hour light/dark cycle and provided ad lib water and food. All animal procedures were conducted according to a protocol (#M05714) approved by the University of Wisconsin Institutional Animal Care and Use Committee. the left coronary ligation was released and removed, the lungs were re-expanded and the chest was closed. The animals were removed from the ventilator and allowed to recover on a heating pad.

| Myocardial infarct size
The myocardial infarct size was evaluated 24 hours after the 45-minute coronary artery ligation using Evan's Blue-triphenyltetrazolium chloride (TTC) double staining. Following the 24-hour reperfusion, 200 µL of a 2% solution of Evan's Blue dye was injected into the right jugular vein of the mouse. Five minutes after the injection, mouse hearts were rapidly removed, washed with a phosphate buffer solution and frozen at −20°C for at least 30 minutes. The entire heart was then cut into about five cross-sectional slices, and the sections were then incubated in 1% TTC at 37°C for 15 minutes; sections were then fixed in 10% formalin solution. The infarct area (white color) and area at risk (red and white color) were digitally imaged in each heart slice, and white, red, and blue areas in each slice were traced. JPG images of every heart section were analyzed using Image J software and areas (ie, Total slice area, white, red, and blue areas of each slice) were then summed and calculations of White Area/Total Red + White Area for each heart were determined. This gives the MI size as a percentage of the area at risk.

| Mouse heart echocardiography
In both control and PrC-210-treated mice, transthoracic echocardiography was conducted at 14 days and 28 days following 45-minute infarct to assess left ventricular morphology and function in vivo.
As previously described, mice were anesthetized with 5% isoflurane and then maintained with 1%-2% isoflurane and room air throughout the procedure; body temperature was maintained at 37°C using a heated platform. 11,13 Echocardiographic parameters were measured over at least three consecutive cardiac cycles and averaged.

| Mouse blood collection and troponin I ELISA
Twenty-four hours after release of the coronary artery ligation, 100-200 µL of blood was collected from mice via the jugular vein under isoflurane anesthesia; the blood was spun briefly in a 0.5 ml Eppendorf tube, and the supernatant was stored at −80°C prior to Troponin I assay. Troponin I levels were measured in mouse plasma and mouse cardiomyocyte tissue culture media using the Mouse Cardiac Troponin I ELISA Kit (Fisher #NC9402211). Absorbance (450 nm) in 96 wells was measured using a CLARIOStar plate reader.

| Neonate mouse primary cardiomyocyte cultures
Primary cultures of neonatal mouse cardiomyocytes from 3 to 4day-old C57 mice were prepared and cultured as described, 29

| ROS-induced naked DNA damage assay
The dose-dependent ability of the tested "antioxidants" to scavenge •OH was measured by their ability to prevent •OH-induced breaks in a plasmid DNA molecule. pGEM plasmid DNA was pu-  Figure 1 were obtained from Sigma-Aldrich, except PrC-210 which was synthesized as described above. Gels were stained with ethidium bromide, digitally imaged, and supercoiled vs nicked/•OH-damaged DNA band intensities were quantified using Image J software.

| Statistics
Data are expressed as means ± SEMs. One-way Student T tests were used to determine statistical difference and P values using Graphpad Prism software.

| PrC-210 reduces ROS-induced DNA damage to background
To compare both the potency and efficacy of PrC-210 against "antioxidants" previously tested for suppression of myocardial ischemia-reperfusion injury eg, 3,7,17,21,31 we used an established gel-based assay that uses naked plasmid DNA as a surrogate ROS target to score ROSinduced damage, that is, plasmid DNA breaks ( Figure 1A). This assay

| PrC-210 reduces myocardial infarct size and troponin I
With the above PrC-210 efficacy in scavenging ROS, we asked   Figure 3D).

| PrC-210 and functional performance of hearts following myocardial infarct
Cardiac function of post-MI hearts was evaluated by echocardiography. The data show that there was a trend toward improved cardiac function in mice that received PrC-210 (Table 1), but for most measured parameters the differences were not significantly different.

| PrC-210 reduces ROS-induced death of neonate mouse cardiomyocytes
To corroborate that PrC-210 protected cardiac cells by directly scavenging ROS, neonate mouse cardiomyocyte cultures were directly exposed to H 2 O 2 with or without PrC-210 present. This model also allowed us to directly measure both the time-and dose-dependency of PrC-210 effect. Collagenase dispersion of neonate mouse F I G U R E 2 Agarose gel separation of supercoiled and nicked/•OH-damaged forms of plasmid DNA after exposure of plasmid DNA to a 60 sec pulse of an •OH generator (H 2 O 2 + UV light 10 . Supercoiled DNA was incubated with buffer (lane A) or buffer + 20 mmol L −1 PrC-210 (lanes B-G) for the indicated times and then exposed for 1 min to the •OH generator. Aliquots of each reaction were immediately electrophoresed, stained with EtBr and digitally imaged. Three replicate reactions and gels were done, and band intensities were quantified using Image J software

| D ISCUSS I ON
Ischemia-reperfusion injury during heart attack remains a profound problem, and more broadly, IR-injury also contributes considerable morbidity to many surgical subspecialties, including cardiac surgery. A PubMed search of "cardiac ischemia-reperfusion injury AND antioxidants" resulted in 7159 citations. These "antioxidant" reports primarily studied "antioxidants" (eg, Vitamin E) and nonantioxidant molecules (eg, adenosine) which have no direct effect upon ROS, and most commonly saw no suppression of IR injury eg. 3,7,17,21,31 These earlier reports are important for having provided proof of concept for ROS-suppression strategies, however, prolonged time to a protective effect and/or severe side effects of a known ROS scavenger (ie, amifostine/WR-1065) prevented any of these molecules from becoming treatments in heart attack.
Traditional "antioxidants" have zero ROS-scavenging capability, rather they are Michael acceptor activators of NrF-2 and the downstream "protective" Phase 2 gene products activated by NrF-2. This gene activation process requires hours to days to achieve protective effect, and the protection, if any, in a post-MI heart arrives long after the cardiac reperfusion ROS bolus. Other published strategies have tested post-MI inflammatory mediators, growth factors, and stem cells in an effort to decrease IR injury following cardiac ischemia-reperfusion, for example. 19 Although the above strategies may have some effect, they do not address the primary IR insult, which is the bolus of ROS that is generated within seconds to minutes following re-establishment of blood flow to the previously infarcted heart tissue. PrC-210 could well be this direct-acting, ROS-scavenger.
Ovize and colleagues 20 calculated that 36% of cardiac cell death was due to ROS-induced "reperfusion injury" (see our Figure 6B).
In a striking coincidence, in our first test of PrC-210 effect against cardiac ischemia-reperfusion, we observed a 36% suppression of cardiac cell death (see Figure 3A); that is the same percentage that Ovize estimated 6 years before.
PrC-210's direct mechanism of action as an ROS-scavenger means it confers protection within seconds ( Figure 2). Importantly, at time points ranging from 1 hour to 30 second before •OH insult, PrC-210 prevented 100% of the ROS-induced damage. DNA protection was not seen when PrC-210 was added 1 minute after •OH exposure; therefore, PrC-210 confers protection by directly scavenging shortlived (milliseconds) ROS, before DNA damage occurs. These findings indicate that PrC-210 has both rapid onset and a lasting effect when administered prior to the generation of ROS. This essentially "immediate" onset of action is a key characteristic that distinguishes PrC-210 from all previously studied cardiac "antioxidants." Finally, the ability of PrC-210 to confer 100% protection to DNA against ROS, regardless of when administered prior to ROS insult, combined with its small molecular weight (MW = 148), would allow PrC-210 to be used in a variety of MI and cardiac surgery settings to reduce IR injury. For example, PrC-210 could be: (a) administered IV to MI patients, both in ambulances (for "leakage" past cardiac blockages) and in the minutes before the angioplasty balloon is inflated so that PrC-210 blood levels are "therapeutic" or even "super-therapeutic" when the oxygenated blood re-enters the ischemic cardiac muscle, (2) added to cardioplegia heart preservation solutions flushed through hearts before cardiac surgeries, (3) flushed directly into hearts immediately before implantation into heart transplant recipients, or (4) be given intravenously to a patient post-MI or postopen heart surgery to suppress inflammatory cell-induced ROS damage.
Importantly, the nature of PrC-210 as a direct-acting, highly effective ROS scavenger would allow it to be used in any environment in which blood flow is stopped and restarted ( Figure 6A).
Ischemia-reperfusion injury in heart attack remains a profound problem with significant implications for cardiac cell death and health care resource utilization in the MI aftermath. Therefore, it is imperative that improved strategies to prevent cardiac IR injury are developed. Here, we have shown PrC-210 to be a highly effective ROS-scavenger that significantly reduces IR injury-associated cardiac cell death.

ACK N OWLED G EM ENTS
Supported by Grant #R03CA176799 to William Fahl.