Targeted antioxidant therapy reduces hyperglycemic exacerbation of myocardial ischemia/reperfusion injury

Abstract

Background:

Acute hyperglycemia (HG) enhances inflammatory and oxidative stress and exacerbates myocardial infarct size during ischemia/reperfusion injury (IRI) by activating splenic leukocytes. Formyl peptide receptor 1 (FPR1) on leukocytes is activated by and mediates myocardial IRI. We hypothesize that selective FPR1 antagonist cFLFLF (CF) or potent reducing agent tris (2-carboxyethyl) phosphine hydrochloride (TCEP) could abrogate hyperglycemic infarct exacerbation, both alone and synergistically via a novel cFLFLF-TCEP compound that would target leukocytes for antioxidative effect.

Materials and Methods:

Acute hyperglycemia (HG) was induced in wild type mice with an intraperitoneal dextrose injection followed by left coronary artery occlusion (30 min) and reperfusion (60 min). In treatment groups, CF (0.1 mg/kg or 1 mg/kg), TCEP (1 mg/kg or 20 mg/kg), or the CF-TCEP conjugate (0.1 mg/kg) was administered intravenously prior to reperfusion. The hearts were harvested to measure infarct size (IF).

Results:

HG resulted in >50% increase in IF compared to euglycemic mice (52.1±3.0 vs. 34.0±3.2%, p<0.05). Neither CF nor TCEP independently exerted an infarct-sparing effect at lower doses (46.2±2.1% or 50.9±4.1%, p>0.05 vs. HG control) but at high doses, significantly attenuated IF exacerbation (23.2±5.2% or 33.9±3.6%, p<0.05 vs. HG control). However, the low-dose CF-TCEP conjugate significantly reduced IF (39.1±1.7%, p<0.05 vs. HG control). IF was decreased to near euglycemic control levels (p>0.05).

Conclusions:

The CF-TECP conjugate synergistically attenuated HG infarct exacerbation at significantly lower respective doses of CF and TCEP. In addition to the intrinsic anti-inflammatory effect of blocking FPR1, CF is also a feasible tool for leukocyte-targeted therapy to treat IRI.

Introduction

Acute hyperglycemia (HG), or stress hyperglycemia, is common in patients who present with myocardial infarction (MI). About a quarter of MI patients present with acute or stress HG, of which nearly 30% do not have comorbid diabetes.^1–3 Moreover, HG is independently associated with larger infarct sizes, increased mortality both in-hospital and after discharge, and worse overall functional outcomes in both diabetic and non-diabetic populations.^{2, 4–11} The mechanisms underlying the hyperglycemic exacerbation of MI is an active field of investigation. Given the presentation in both diabetic and nondiabetic patients and worsened outcomes in those without diabetes, it is thought that stress hyperglycemia may be a marker of more severe disease¹². Furthermore, therapies adequately targeting the hyperglycemic exacerbation of MI remain lacking. The American Heart Association has highlighted the importance of further investigation to characterize the molecular mechanisms underlying this phenomenon and identify effective therapeutic targets for treatments to reduce infarct size and mortality stemming from acute hyperglycemia with MI.¹³

Acute hyperglycemia is known to exacerbate the inflammatory response ^{4, 14} and abolish any preconditioning from angina.¹⁵ Animal experiments have demonstrated that acute hyperglycemia exacerbates inflammatory responses and subsequent oxidative stress, worsening reperfusion injury and myocardial infarct size in response to elevations in blood glucose levels.^16–18 However, correction of this acute hyperglycemia with insulin has equivocal results in clinical trials, with potential functional benefit but limited mortality benefit. ^{11, 12, 19, 20} This may be due to the increased severity of disease in presentation of those with acute hyperglycemia. However, it may be in part due to the impact of initial hyperglycemia as mortality has been associated with admission glucose rather than glucose control. Initial hyperglycemia is thought to have already having triggered the inflammatory cascade prior to presentation, limiting the ability of subsequent glucose control to halt this pathway.¹² As such, insulin treatment is important to control further injury but alone will not be sufficient to halt this triggered cascade.

A key part of myocardial ischemia-reperfusion injury (IRI) is the significant oxidative stress and changes in antioxidant defenses. This is in part facilitated by the activation of leukocytes and their mobilization to the injured myocardium. Formyl peptide receptor 1 (FPR1), a receptor overly expressed with high affinity in activated leukocytes, plays an important role in mediating IRI, particularly in recognizing and mobilizing leukocytes to the site of injury²¹ and subsequently via NADPH oxidase (NOX) activation and reactive oxygen species (ROS) production to exacerbate injury and infarct size.^22–24 Acute hyperglycemia is hypothesized to worsen this response via upregulation of FPR1 and also subsequent ROS production, in part by increasing NADPH production systemically via the hexose monophosphate shunt. This increase in NADPH substrate for NOX increases overall ROS and contributes to worsened outcomes in MI associated with acute hyperglycemia.²⁵

Antioxidant therapies to reduce the impact of ROS production have shown promise in pre-clinical studies but have not consistently translated in the clinical arena^26–28. Given this outcome, to elucidate potential treatments for hyperglycemic infarct exacerbation from increased ROS production, we investigated cinnamoyl-F-(D)L-F-(D)L-F) (CF), a FPR1 antagonist, as well as Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), a strong reducing agent. Given the central role of leukocytes in mediating oxidative stress during myocardial IR, we further synthesized a conjugate of TCEP to CF via an amide bond, to allow for targeted delivery of the reducing agent at the source of ROS production as well as the site of inflammation. We hypothesize that selective FPR1 antagonist CF or potent reducing agent TCEP alone can each abrogate hyperglycemic infarct exacerbation in a mouse model of myocardial IRI. Furthermore, we hypothesize that our CF-TCEP conjugate will synergistically reduce hyperglycemic infarct exacerbation at a significantly lower dosage via targeting leukocyte-mediated oxidative stress directly at its source. We then investigated the mechanism by which this is facilitated, investigating the role of the hexose monophosphate shunt via G6PD inhibition via 6-aminonicotinamide in these groups, NADPH production, ROS production in acute hyperglycemia with myocardial IRI.

Materials and Methods

This study complied with the Guide for the Care and Use of Laboratory Animals as recommended by the U.S. National Institutes of Health, ensuring that all animals received humane care. The University of Virginia Animal Care and Use Committee reviewed and approved the study protocol.

Animals and Materials

C57BL6 mice (male and female aged 9–12 weeks, purchased from Jackson Laboratory, Bay Harbour, ME) were used for this study. They were housed per institutional animal care and use committee protocols and were randomly chosen for each experimental group. Cinnamoyl-F-(D)L-F-(D)L-F (CF), a synthetic peptide and a selective FPR1 antagonist was utilized for FPR1 antagonism and targeting. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased from Sigma-Aldrich. CF was conjugated to TCEP via an amide bond to synthesize the CF-TCEP compound (Figure 1). 6-aminonicotinamide (6-AN), a potent glucose 6-phosphate dehydrogenase (G6PD) inhibitor,²⁹ was purchased from Sigma-Aldrich (St. Louis, MO).

Figure 1: Therapeutic drug conjugate structure.

Structure of cinnamoyl-F-(D)L-F-(D)L-F – Tris (2-carboxyethyl) phosphine hydrochloride (CF-TCEP) conjugate compound bound by amide bond utilized for FPR1-targeted antioxidant delivery.

Synthesis of CF-TCEP

CF was synthesized as described early by Zhang et al.^{30, 31} Briefly, CF was synthesized by standard solid phase chemistry. The high-performance liquid chromatography (HPLC)-purified CF was conjugated with TCEP in a stoichiometric ratio of 1:3 to form CF-TCEP. The CF-TCEP was purified again by HPLC to form the final drug.

In Vivo Myocardial Ischemia-Reperfusion Injury (IRI) Experiment

Treatment Groups

A total of 80 mice were assigned to eight groups. All mice underwent 30 minutes of left coronary artery (LCA) occlusion and 60 minutes of reperfusion (30’/60’). There were two control groups. Euglycemic mice underwent 30’/60’ IRI with preceding intraperitoneal (IP) injection of normal saline 10 minutes before LCA occlusion at a dose of 10 μl/g. For hyperglycemic mice, 20% dextrose (10 μl/g) was injected intraperitoneally 10 minutes prior to IRI to a target glucose of 300–450 mg/dl, based on protocol from prior experiments¹⁶.

Among the 6 acute HG treatment groups, the treatment drug was given 5 minutes prior to reperfusion intravenously via the external jugular vein. Two groups received CF intravenously at either a low dose, 0.1 mg/kg, or high dose, 0.5 mg/kg. Another two groups were treated with TCEP at either 1 mg/kg, the low dose group, or 20 mg/kg, the high dose group. In the last 2 groups, one group was treated with the CF-TCEP conjugate at a low dose of 0.1 mg/kg. The last group was treated with 6-AN at a dose of 10 μg/kg (Figure 2).

Figure 2: Experimental Protocol.

C57BL6 mice underwent 30 min of LCA occlusion followed by 60 min of reperfusion before infarct size was evaluated by TTC-Phthalo blue staining. Hyperglycemia (HG) was induced 10 min before LCA occlusion. Treatment with low or high dose CF or TCEP, CF-TCEP conjugate, or 6- aminonicotinamide (6-AN) was given IV 5 minutes prior to reperfusion. CF – cinnamoyl-F-(D)L-F-(D)L-F); TCEP = Tris (2-carboxyethyl) phosphine hydrochloride; LCA – left coronary artery; ‘-min.

Induction of myocardial IRI

Mice were subjected to 30-min left coronary artery (LCA) ligation followed by 60 min of reperfusion as described previously^{16, 32, 33}. Briefly, the mice were anesthetized with Avertin (Tribromoethanol), IP, at a dose of 250 mg/kg (redosed at 100 mg/kg every 30 min) and intubated with PE-60 tubing. They were then ventilated at 120 breaths/min, with a tidal volume of 6 cc/kg with 21% FiO2. The chest skin was then incised, and the heart was exposed via left thoracotomy, by cutting the 3^rd and 4^th rib of the left chest. After exposing the left ventricle, the left coronary artery (LCA) was identified under microscope and an 8–0 prolene suture was passed under the LCA at the level of the left atrium. It was tied against a section of PE-50 tubing to occlude the LCA to induce myocardial ischemia. Successful LCA occlusion was confirmed by color change of the affected region. After 30 min of occlusion, the tube was removed to allow for reperfusion of the LCA for a 60 min period. After the reperfusion period was completed, a midline sternotomy was made, and blood was collected. The heart was subsequently harvested for staining to measure infarct size.

Determination of Infarct Size

After the hearts were harvested, the aorta was cannulated with a 23-gauge needle and 3 ml of 37°C 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS, pH=7.4, Gibco, ThermoFisher, Waltham, CT) was injected into the heart. The suture encircling the LAD was then tied to ligate the LCA and 100–200 μl of 2% Phthalo Blue (Heubach Ltd, Fairless Hills, PA) was then used to stain the perfused region of the heart. The heart was then frozen, and the atria and right ventricle were removed. The left ventricle was then divided into 5–7 transverse slices and fixed in 10% neutral buffered formalin solution. The basal and apical sides of each slice were then imaged. The infarcted area, identified as tan-white after staining, the risk region (RR), identified as red after staining, and non-ischemic areas, identified as blue stained with phthalo blue, were calculated as a percentage of the corresponding slice multiplied by the weight of the slice. Further calculations determined the infarct size as a percentage of the RR as well as the RR as a percentage of the left ventricle (LV)^{16, 32, 33}.

In Vivo Experiment Without IRI

In a parallel group of mice without IRI, the mice were assigned into sham control, hyperglycemic control, and treatment groups. Sham mice received an intraperitoneal injection of 10 μl/g of PBS. The hyperglycemic control mice received an intraperitoneal injection of 10 μl/g of 20% dextrose. The treatment group first received intraperitoneal 10 μl/g of 20% dextrose followed by intravenous injection of 6-AN (10 μg/kg) 10 minutes after dextrose injection. After 60 minutes, the blood glucose was measured (Quintet AC blood glucose monitoring system, PSS World Medical, Jacksonville, FL), and the spleen, blood, and heart were harvested.

Splenocyte Cell Culture

The spleen was harvested from naïve intact wild type mice. It was then dissociated into a single-cell suspension with PBS supplemented with 10% fetal bovine serum (Gibco, ThermoFisher, Waltham, CT) in GentleMACs tubes (Miltenyi Biotec). Red blood cells were lysed by re-suspending splenic cells in ammonium chloride-Tris buffer and incubating them for 8 minutes at room temperature. After centrifugation, leukocytes were collected and washed twice in PBS. The splenic leukocytes were resuspended in PBS, enriched to 2 × 10⁶/μl and incubated in 6-well plates (BD Company) at a concentration of 8.2×10⁶ cells in 1.2 ml of culture media (Dulbecco’s Modified Eagle Medium, Gibco, ThermoFisher, Waltham, CT) per well. For the sham media, dextrose was added to a 5.5 mM concentration. In subsequent groups, dextrose (60mM) or dextrose (60 mM) with 6-AN (250 μg/ml) were added to 4‐well (n=4/group) cell culture plates and incubated for 4 hours. Following the incubation period, supernatant was collected and G6PD activity and NADPH levels were measured with ELISA kits (Abcam, Waltham, CT). Splenocyte thiobarbituric acid reactive substances (TBARS, BioAssay Systems, ThermoFisher, Waltham, CT) was also measured using ELISA after 4-hour incubation in sham media, media with dextrose (60mM), dextrose (60 mM) with 6-AN (500 μg/ml), or dextrose (60 mM) with CF-TCEP (20 μg/ml).

Statistical Analysis

Continuous data were evaluated to have normal distribution using the Shapiro-Wilk Test. All groups were confirmed to have normal distribution. Comparisons between groups were performed with one-way analysis of variance with Bonferroni’s correction for multiple comparisons and unpaired Student’s t-test. Prism 10 (GraphPad Software Inc., La Jolla, CA) was used to perform statistical calculations. Groups were not blinded. Data are presented as mean±standard deviation of the mean, with a p-value of 0.05 indicating statistical significance.

Results

Infarct size with low dose therapy

In the HG mice, glucose levels were more than doubled compared to euglycemic (EG) mice (320.9±59.7 vs. 152 ± 32.3, p<0.001). Ischemic risk region (RR, as a percentage of LV mass) was comparable for all experimental groups (p>0.05). Ischemic size (IF) was measured as a percentage of RR.

In HG controls, IF was 55.7±10.3%, an over 50% increase in IF from EG controls (34.0±9.5% EG control, p<0.001). Neither CF nor TCEP independently exerted an infarct-sparing effect at lower doses (46.2±5.9% or 50.9±11.5% vs. HG control, p=0.06 and p=0.38). However, the low-dose CF-TCEP conjugate significantly attenuated IF exacerbation (39.1±4.5%, p=0.001 vs. HG control, Figure 3). The CF-TCEP IF was significantly lower compared to all low dose hyperglycemic groups as well (p=0.02 vs. low CF and p= 0.02 vs. low TCEP).

Figure 3: Effect of low dose treatments on infarct size.

Risk Region (RR) as a percent of left ventricle (LV) and infarct size as a percent of RR across euglycemic (EG), hyperglycemic (HG), low dose CF-treated, low dose TCEP-treated and CF-TCEP conjugate-treated mice. CF - cinnamoyl-F-(D)L-F-(D)L-F); TCEP - Tris (2-carboxyethyl) phosphine hydrochloride

Infarct size with high dose therapy

High-dose TCEP, at a dosage 20 times that of low dose TCEP, independently and significantly attenuated IF exacerbation (33.9±9.5% vs. HG control, p<0.001). High-dose CF, five-fold greater than its low dosage, was also able to independently decrease hyperglycemic IF exacerbation to 23.2±13.66% (p<0.001). After treatment with either the CF-TCEP conjugate, high dose TCEP, or high dose CF in HG mice, IF was reduced to a level similar to that in euglycemic control mice (p=0.27, p=0.99, and p=0.08 vs. EG control, respectively Figure 4). The treatment groups were not significantly different from each other.

Figure 4: Effect of high dose treatments on infarct size.

Risk Region (RR) as a percent of left ventricle (LV) and infarct size as a percent of RR across euglycemic (EG), hyperglycemic (HG), 6-aminonicotinamide (6-AN)-treated, high dose CF-treated, high dose TCEP-treated and CF-TCEP conjugate-treated mice. CF - cinnamoyl-F-(D)L-F-(D)L-F); TCEP - Tris (2-carboxyethyl) phosphine hydrochloride

HG mice treated with 6-AN, inhibiting G6PD and the hexose monophosphate shunt, had significant attenuation of hyperglycemic infarct exacerbation (26.8%±15.4% vs. HG control, p<0.001), to EG control levels (p=0.27, Figure 4).

G6PD and NADPH in HG mice

Levels of G6PD and NADPH were measured in the plasma and spleen in EG sham mice, HG controls, and 6-AN-treated HG mice. HG increased G6PD activity both in the spleen (493±18 mU/mg vs. 383±11mU/ml EG sham, p<0.001) and the plasma (32.1±6.7 mU/ml vs. 23.3±1.5 mU/ml EG sham, p=0.04). This increase was significantly attenuated when the mice were treated with 6-AN after hyperglycemia in both the spleen (437±33 mU/mg spleen vs. HG control, p=0.02) and the plasma (15.8±1.1 mU/ml vs. HG control, p<0.001). HG also increased NADPH levels in the plasma by over 40% (21.1±5.0 pmol/μl vs. 14.7±0.7pmol/μl, p=0.04) and 6-AN reduced this rise to sham levels (13.9±0.4 pmol/μl vs. HG control, p=0.02). Within the spleen, NADPH levels trended down after HG (2.38±0.1 pmol/μg vs. 2.54±0.09 pmol/μg sham, p=0.06), but did not quite reach significance. These levels trended further downward with G6PD inhibition with 6-AN, but not with significance (2.19±0.29 pmol/μg vs. HG control, p=0.27, Figure 5).

Figure 5: Effect of hyperglycemia in the spleen and plasma.

(A) Spleen and (B) Plasma glucose 6 phosphate dehydrogenase (G6PD) activity and NADPH levels in sham, hyperglycemic (HG), and 6-aminonicotinamide (6-AN) treated HG mice without ischemia reperfusion injury.

G6PD, NADPH, and TBARS in cultured splenocytes

Splenocytes were cultured in euglycemic media, high dextrose (HG), high dextrose with 6-AN (HG+6-AN) or dextrose with CF-TCEP (HG+CF-TCEP) media. Similar to our in vivo studies, G6PD activity was increased in splenocytes when cultured in high dextrose media (10±0.8 mU/ml vs. 7.5±0.9 mU/ml, p=0.02). Addition of 6-AN to the high dextrose culture media significantly reduced G6PD activity (8.3±0.5 mU/ml, vs. HG p=0.04, Figure 6A). NADPH levels in the splenocytes followed a similar pattern, with significant increase in the high dextrose media (7.5±0.1 pmol/μl vs. 6.3±0.2 pmol/μl, p<0.001) that was attenuated with the addition of 6-AN (5.7±0.8 pmol/μl vs. HG, p=0.02, Figure 6B) to sham levels.

Figure 6: Effect of hyperglycemia in splenocytes.

(A) Glucose-6-phosphate activity, (B) NADPH levels, and (C) TBARS concentration in mice splenocytes cultured in euglycemic (EG) medium, high dextrose medium (HG), HG medium with 6-aminonicotinamide (6-AN), or HG medium with CF-TCEP conjugate. CF - cinnamoyl-F-(D)L-F-(D)L-F); TCEP - Tris (2-carboxyethyl) phosphine hydrochloride; TBARS - Thiobarbituric acid reactive substances, a measure of lipid peroxidation to represent reactive oxygen species production

To measure oxidative stress, lipid peroxidation products were measured via TBARS. There was a marked increase in TBARS concentration with high dextrose media (6.6±0.3 μM vs 1.0±0.04 μM, p<0.001). This rise was significantly attenuated by both 6-AN (4.7±0.2 μM vs HG, p<0.001) and CF-TCEP (3.8±0.5 μM vs HG, p<0.001, Figure 6C).

Discussion

With myocardial infarction prevalent and cardiac disease a leading cause of morbidity and mortality across the world, adjuncts to preserve myocardium are vital to conjunction with revascularization procedures such as PCI or CABG. These can enhance overall outcomes in both short and long term outcomes but remain limited. In investigation of acute hyperglycemia with MI, a selected more vulnerable population with worse outcomes, using medical treatment to enhance surgical outcomes is a promising strategy.

In the current study to investigate such options, we found that hyperglycemia increased infarct size significantly by 50%. Treatment with low dose CF-TCEP allowed for synergistic attenuation of HG infarct exacerbation at significantly lower respective doses of CF and TCEP, with a 42% decrease in IF with treatment (Figure 3). Similar effect was only seen if CF or TCEP were given at doses 5 times higher for CF and 200 times higher for TCEP than present in the conjugate (Figure 4) and was ineffective at equivalent low doses of individual components. Thus, the CF-TCEP conjugate allowed for infarct mitigation via localization and directed delivery of the reducing agent, TCEP.

FPR1 is a G protein-coupled pattern recognition receptor with several roles in myocardial infarction. As a differentially expressed gene in MI, it is highly expressed on leukocytes and increases neutrophil chemotaxis to the site of injury. Moreover, activation enhances multiple intracellular signaling pathways, including MAPK leading to increased apoptosis and inflammation and NOX activation with subsequent ROS production.²² Prior studies have demonstrated increased FPR1 expression with myocardial IRI^{23, 32} and that silencing FPR1 in mice reduced inflammation, apoptosis, and infarct within injured myocardium.³⁴ Moreover, prior studies from our lab have demonstrated localization of CF to activated leukocytes in inflammatory states³⁵ and that specific to MI, the increase in circulating neutrophils and myocardial neutrophil infiltration driven by DAMPs and factors released due to ischemia were mitigated with CF treatment via FPR1 antagonism.³³ Thus, these prior findings were concordant with the decrease in infarct size with CF, but only when treated with high CF dosages. Given that FPR1, while preferential to leukocytes in inflammatory states, is present in other organs, having a lower effective treatment dose as a conjugate mitigates potential off-target effects.

Our previous studies have also underscored the role of oxidative stress and NOX in acute hyperglycemic exacerbation of MI.^{16, 29} Acute hyperglycemia led to increases in infarct size that were directly reduced when treated with an antioxidant or pretreated with apocynin to reduce ROS production from NOX. Moreover, NOX2 isoforms have been implicated greatly in reperfusion injury and are strongly expressed in inflammatory cells such as neutrophils that are key mediators of reperfusion injury.^36–38 With hyperglycemia, increased shunting to the hexose monophosphate shunt increases NADPH production and subsequent substrate for consumption by NOX at activated leukocytes with increased oxidative burden (Figure 7). This was evident in our experiment when 6-AN was used to block the hexose monophosphate shunt via G6PD antagonism; the infarct exacerbation by hyperglycemia was eliminated, reducing IF by over 50% (Figure 4). Thus, the production of NADPH, by providing excess substrate for NOX, is a vital part of increased infarct size, likely from increasing the oxidative stress produced by NOX.

Figure 7: Mechanism of increased inflammatory and oxidative stress with hyperglycemia and targets of action for CF, TCEP, and 6-aminonicotimamide (6-AN).

Increased glucose leads to increased shunting from glycolysis to through the hexose monophosphate shunt, producing more NADPH from NADP⁺. This provides increased substrate for NADPH oxidase in leukocytes, a primary producer of reactive oxygen species (ROS), which leads to increased ROS produced at the site of injury, due to leukocyte chemotaxes to injured myocardium. CF targets leukocytes via upregulated formyl peptide receptor 1 (FPR) and TCEP targets ROS itself. 6-AN targets glucose 6 phosphate dehydrogenase (G6PD) to inhibit shunting through the hexose monophosphate shunt. CF - cinnamoyl-F-(D)L-F-(D)L-F); TCEP - Tris (2-carboxyethyl) phosphine hydrochloride

Moreover, acute hyperglycemia independently increased both G6PD activity in the plasma and spleen, reflecting increased shunting through the hexose monophosphate shunt. Treatment with 6-AN reduced this increase in G6PD activity, as expected, in both the spleen and plasma. Similarly, NADPH levels were also increased with acute hyperglycemia and reduced by 6-AN in the plasma, consistent with hyperglycemia causing increased shunting through the hexose monophosphate shunt with subsequently increased NADPH production. In the spleen, NADPH levels trended downwards with hyperglycemia and further after treatment 6-AN (Figure 5). This decreasing trend with hyperglycemia is likely a reflection of the NADPH produced being utilized within the spleen by the NOX splenic leukocytes present therein and is a consumptive reduction rather than a reduction in production, when taking the increased G6PD activity into consideration.

This was further seen in in-vitro splenocyte cultures. When cultured with high dextrose, G6PD activity, NADPH levels and lipid peroxidation end products (TBARS) were all increased. With addition of 6-AN in the culture media, a significant reduction in all three of these substances were seen. This indicates reduction in shunting through the hexose monophosphate shunt leading to decreased NADPH production and resultingly, reduced oxidative stress produced with splenic leukocytes. When the CF-TCEP conjugate was added to the high dextrose culture medium, TBARS levels were reduced in splenocytes as well, acting on this pathway.

While certain studies may suggest that the increased production of NADPH can be protective in ischemic states, such as stroke, as that involves scavenging ROS to convert NADP⁺ to NADPH,³⁹ this occurs at locations distant from the site of insult. As a result, when migrating leukocytes arrived during reperfusion at injured myocardium and the abundant NOX enzymes have ample substrate to utilize at the site of injury to produce deleterious ROS. We have shown previously that hyperglycemia leads to NOX activation to worsen infarct size in myocardial IRI.¹⁶ Investigation into antioxidants as potential therapies has not consistently translated to the clinical arena^26–28. One potential reason for lack of translation may be the dose or delivery-based failures. In our study, low dose TCEP itself is not effective, though high doses, with increased risk of toxicity, can mitigate hyperglycemic exacerbation of MI acutely. However, with targeted therapy and directed delivery of reducing agents such as TCEP to mitigate the overall harmful impact of the oxidative burst, the impact of the drug is likely more effective in attenuating infarct size.

Our study does have some limitations. We did not study the effect of acute hyperglycemia on cardiomyocytes; we presume that HG may also increase ROS production via HMS inside cardiomyocytes themselves that are then vulnerable to inflammatory injury. While the characterization and effect of hyperglycemia on infarct can be seen with 30 minutes, this shorter ischemic period does not fully represent the entire ischemic region. However, it was used to allow for better visualization of infarct exacerbation with hyperglycemia as there is then adequate myocardium for infarct evolution to occur. Moreover, when used in comparison, it is still efficacious in noting differences with hyperglycemic insult and benefits from treatment. Our experiment did not investigate the impact of low dose CF and low dose TCEP given together without linkage. While this would be a good comparison for future studies, to ensure that synergistic effect is co-localized with co-delivery rather than the drugs working in sync. Given the limited effect of both drugs at low doses however, our conjugate remains promising as a delivery-therapeutic combination. While our targeted drug conjugate demonstrated promising results, long term or off target effects were not investigated. For instance, while FPR1 is preferentially expressed in activated leukocytes, it may be present in other cell types, with potential for off target effects that require further investigation³⁵. Similar studies may also be needed for the use of TCEP, given its risk of moderate toxicity, or if a different linked antioxidant drug may be a safer option. Future studies investigating these drugs as well as further investigation of safety in larger animal models are warranted to confirm potential clinical translation. Despite these limitations, our study demonstrates effective reduction of the infarct exacerbation caused by an acute hyperglycemic insult with targeted FPR1 inhibition and delivery of an antioxidant to the main site of antioxidant production. Moreover, this therapy may have other effects that may be useful for future investigations, including potential use in ischemia/reperfusion insults inherent in cardiac bypass and CABG procedures that may be useful to investigate further.

Conclusion

In conclusion, acute hyperglycemia exacerbates myocardial infarct size, in part by increasing G6PD and NADPH production via the HMS, resulting in increased oxidative stress and worsened injury. Using CF as a synergistic vehicle to target FPR1 on leukocytes, integral effectors of oxidative stress in MI, to deliver the antioxidant TCEP to the source of ROS production and site of injury effectively reduces hyperglycemic infarct exacerbation. Via targeted treatment, CF-TCEP is effective at over 5 times lower dose of CF and 200 times lower dose of TCEP, reducing potential deleterious side effects. CF acts as a feasible tool for leukocyte-targeted therapy in IRI and the mechanism underlying CF-TCEP’s cardioprotective effective warrants further investigation.

List of abbreviations

HG

Hyperglycemia

IRI

Ischemia-reperfusion injury

FPR1

Formyl Peptide Receptor 1

CF

Cinnamoyl-F-(D)L-F-(D)L-F

TCEP

Tris (2-carboxyethyl) phosphine hydrochloride

MI

Myocardial infarction

EG

Euglycemia

IF

Infarct size

IP

Intraperitoneal

RR

Risk region

LV

Left ventricle

LCA

Left coronary artery

HMS

Hexose monophosphate shunt

NADPH

Nicotinamide adenine dinucleotide phosphate

NOX

NADPH Oxidase

ROS

Reactive oxygen species

TTC

2,3,5-triphenyltetrazolium chloride

PBS

Phosphate buffered saline