A Cell-Based Phenotypic Assay to Identify Cardioprotective AgentsNovelty and Significance
Rationale: Tissue ischemia/reperfusion (IR) injury underlies several leading causes of death such as heart-attack and stroke. The lack of clinical therapies for IR injury may be partly due to the difficulty of adapting IR injury models to high-throughput screening (HTS).
Objective: To develop a model of IR injury that is both physiologically relevant and amenable to HTS.
Methods and Results: A microplate-based respirometry apparatus was used. Controlling gas flow in the plate head space, coupled with the instrument's mechanical systems, yielded a 24-well model of IR injury in which H9c2 cardiomyocytes were transiently trapped in a small volume, rendering them ischemic. After initial validation with known protective molecules, the model was used to screen a 2000-molecule library, with post-IR cell death as an end point. Po2 and pH monitoring in each well also afforded metabolic data. Ten protective, detrimental, and inert molecules from the screen were subsequently tested in a Langendorff-perfused heart model of IR injury, revealing strong correlations between the screening end point and both recovery of cardiac function (negative, r2=0.66) and infarct size (positive, r2=0.62). Relationships between the effects of added molecules on cellular bioenergetics and protection against IR injury were also studied.
Conclusions: This novel cell-based assay can predict either protective or detrimental effects on IR injury in the intact heart. Its application may help identify therapeutic or harmful molecules.
Diseases of tissue ischemia/reperfusion (IR) injury are the leading causes of death in Western societies, with ≈1.6 million new incidences of stroke or myocardial infarction occurring annually in the United States alone.1 The underlying pathological event in IR injury is the blockade of blood flow, starving tissue of O2, and substrates and leading to accumulation of metabolites such as lactate. Uncontrolled, tissue reperfusion is also pathogenic, leading to cell death and infarct development.
To date, trial therapies for IR injury have targeted the mechanisms preceding cell death, or the signaling pathways invoked by endogenous protective mechanisms such as ischemic preconditioning (IPC).2 In the field of cardiac IR, pathological mechanisms targeted by candidate drugs include Na+ overload,3 Ca2+ overload,4 the mitochondrial permeability transition pore,5 and reactive oxygen species generation.6 Drugs designed to mimic IPC have included adenosine analogs,7 surface and mitochondrial K+ channel openers,8,9 nitric oxide donors,10 and vasodilators.8,11 To date however, no drug has been Food and Drug Administration–approved for reduction of myocardial infarct size.12 Similarly, there are no cell mechanism–based therapies for ischemic stroke, with available therapies limited mostly to clot-busters such as tissue plasminogen activator.
Unbiased high-throughput screening (HTS) is a mechanism-agnostic approach to drug discovery but has not been widely applied to the problem of IR injury. The complexity of IR pathology is such that simple cell-based IR models (eg, chemical ischemia, hypoxia alone, or O2/glucose deprivation) do not reproduce ischemic conditions well. In addition, these models often require media or gas exchanges that are incompatible with plate-reading devices. In contrast, more physiologically relevant IR models (eg, the ex vivo perfused heart13 or in vivo murine coronary artery occlusion14) are expensive, technically challenging, and low throughput.
The goal of this study was to overcome the trade-off between physiological relevance and ease of use to develop an IR injury model offering both accurate representation of IR conditions and high throughput. To accomplish this, a plate-based respirometry apparatus (Seahorse Bioscience XF-24) was used as a framework.15 The apparatus measures mitochondrial respiration (O2 consumption rate, OCR) and glycolysis (extracellular acidification rate, ECAR) by intact cells on a 24-well plate.15 Atop the cell plate rests a disposable cartridge with 24 plungers that travel in a vertical axis (Figure 1). Embedded in the plunger tips are fluorescent probes sensitive to Po2 and pH, which are interrogated by fiber optics. Lowering these plungers traps cells in a transient 7-μL microchamber, allowing measurement of changes in Po2 and pH in the extracellular space, and hence the calculation of rates.
We hypothesized that on prolonged lowering of the plungers, cells would consume all available O2 in the microchamber, rendering an ischemic-like state. Similarly, raising the plungers would flood cells with bulk media, simulating reperfusion. To gain greater control over O2 levels in the media, the extracellular flux (XF) apparatus was adapted for argon gas flow in the head space of the cartridge (Online Figure I). These modifications afforded a 24-well model of IR injury, which was then used to screen a 2000-molecule library for protection against IR-induced cell death. Hits from the screen were validated with the use of a perfused heart model of IR injury. Furthermore, the measurement of cellular bioenergetic function throughout the IR procedure afforded novel insight into the relationship between IR injury and cell metabolism.
An expanded Methods section is available in the Online Data Supplement.
Reagents and Cell Culture
The Spectrum Collection chemical library was from MS-Discovery Inc (Gaylordsville, CT), supplied through the University of Rochester HTS core, and stored at −80°C on 96-well plates in 1 mmol/L in dimethylsulfoxide (DMSO). The cardiomyocyte-derived H9c2 cell line was obtained from ATCC (Manassas, VA) at passage 13 and maintained at subconfluence in Dulbecco Modified Eagle Medium (DMEM) with 25 mmol/L glucose, 1 mmol/L pyruvate, 4 mmol/L glutamine, 10% FBS and pen/strep, at 37°C with 5% CO2. Cells were used between passages 20 and 40, plated on XF-24 V7-PET plates at 15 to 30 000 cells/well, for 24–48 hours before testing. One hour before assay, media was replaced with 700 μL of assay media (DMEM with 25 mmol/L glucose, 1 mmol/L pyruvate, 4 mmol/L glutamine, no serum, no antibiotics, no bicarbonate, pH 7.4, at 37°C).
Adaptation of XF-24 for IR Injury
The Seahorse XF-24 measures OCR and ECAR by cells on a 24-well plate,15 using a disposable cartridge of moveable plungers embedded with fluorescent Po2 and pH probes (Figure 1). Holes were drilled in the cartridge to connect gas lines (Figure 1 and Online Figure I). Instrument sensors were disabled to permit removal of the cover to fit or remove gas lines.
IR Injury and End Points
Typical Po2 and pH traces during IR are shown in Figure 2. Preischemic measurements of OCR and ECAR were obtained both before and after injection of test molecules at 1.4 μmol/L. Flow of humidified argon was then initiated at 500 mL/min, followed 35 minutes later by lowering plungers for 60 minutes, modeling ischemia. After 1 hour of nominal ischemia with the plungers lowered, argon flow to the cartridge was replaced with room air, plungers were raised, and bulk media mixed for 5 minutes, modeling reperfusion. OCR and ECAR were measured again 1 hour later. Cell death was then assayed by the luminometric Cytotox-Glo assay (Promega, Madison WI) according to the manufacturer's protocol, on a fluorescent plate reader.
Method Workup and Screen Validation
Initial studies were undertaken with a variety of molecules known to protect the heart against IR injury: adenosine (50 μmol/L),16 diazoxide (10 μmol/L),17 carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 50 nmol/L),18 cyclosporine A (CsA, 0.2 μmol/L),5 and nitro-linoleic and nitro-oleic acids (1 μmol/L).19 Online Figure II shows that 5 of the 6 molecules resulted in significant reductions in post-IR cell death (P<0.05 by ANOVA), with CsA just failing to achieve significance (P=0.061). Because the CsA target protein cyclophilin D plays a role in necrotic but not apoptotic cell death,20 the mode of cell death in this model may be weighted toward apoptosis.
Test molecules were diluted to 20 μmol/L in assay media on the day of assay, and 70 μL was loaded into cartridge injection ports (Figure 1) for a final injected concentration of 1.43 μmol/L (DMSO 0.57% vol). A standard multiplexing strategy was used,21 with 4 molecules per well in each of 20 test wells on the XF plate (80 molecules/plate×25 plates=2000). Two wells were used for background correction (media alone) and 2 for vehicle controls (no test molecules). Cell death in each well was expressed relative to the mean for the whole plate, including controls and test wells. This normalization requires that the molecule library is a random distribution of protective/inactive/detrimental molecules, as supported by Online Figure III, A.
Fifty-six wells (224 molecules) exhibiting the lowest post-IR cell death scores in round 1 were split up, and their components were tested at 1 molecule-per-well. Each plate comprised 16 test wells, 2 background correction wells, and 6 vehicle controls (16 molecules/plate×14 plates=224). This subset of the library was assumed biased toward protective molecules, thus precluding normalization of cell death to the plate average (see first round). Therefore, cell death in the second round was expressed relative to the 6 vehicle controls.
Although well assignments were randomized in both screening rounds, cell culture edge effects22 or variations in temperature or gas flow inside the instrument could result in hot spots of cell death on the plate. Thus, the average cell death in each well across all 39 plates was mapped (Online Figure III), and the map was used to apply a correction factor to cell death values, thus avoiding plate-position bias.
Secondary Validation and Development of Hits
A perfused rat heart model of IR injury was used, as previously described.13 Male Sprague-Dawley rats (Harlan, Indianapolis IN) weighing 200–250 g were maintained on a 12-hours light/dark cycle with food and water ad libitum, and all procedures were approved by the AAALAC accredited University Committee on Animal Resources, in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Hearts were perfused with Krebs-Henseleit buffer in constant flow mode (12 mL/min), with left ventricular pressure monitored by a balloon/transducer and digital data collection. Hearts were subjected to 35 minutes of global ischemia and 2 hours of reperfusion, followed by assessment of infarct size by tetrazolium chloride staining.14 Test compounds were administered at 1 μmol/L in DMSO (final DMSO <0.2%) for 20 minutes before ischemia through a port in the perfusion cannula. Tested compounds are listed in the Table and included beneficial, detrimental, and no-effect molecules.
Statistics and Correlations
All group-wise analyses were conducted with 5–7 animals per group. For comparison between groups, unless otherwise indicated, multiple-way ANOVA was used, with significance (P) set at 0.05. For correlations, linear regression curve fit was used.
A Seahorse XF-24 device was adapted for gas flow as shown in Figure 1 and Online Figure I. The mean Po2 and pH values for all wells on a plate during a typical experiment are shown in Figure 2. After 2 initial OCR/ECAR measurements, injection of test molecules, and 1 further OCR/ECAR measurement, argon delivery to the cell plate lowered the bulk media Po2 to <10 mm Hg within 35 minutes. During the subsequent 60-minute period of ischemia imposed by lowering the plungers, cell respiration brought the Po2 to <0.5 mm Hg within the first 10 minutes, where it remained for the duration. pH in the microchamber fell by ≈1 U during the nominal 1-hour ischemic period. On reperfusion (raising plungers and flushing the plate with room air), both Po2 and pH returned to normal levels. A final OCR/ECAR measurement was obtained at the end of the protocol, before plate ejection and a cell death assay.
Average OCR before test molecule(s) delivery was 172.3±0.01 pmol O2/min, and average ECAR was 14.8±0.01 mpH/min (mean±SEM). Delivery of test molecules caused an average 45.0±0.1% increase in ECAR and a 5.9±0.02% decrease in OCR. Thus, under these cell culture conditions, glycolysis (ECAR) appeared more susceptible to the effects of added molecules than did mitochondrial Ox-Phos (OCR). This observation is pursued further in the Discussion and Figure 6.
The model of IR injury was applied to screen a library of 2000 small molecules. As detailed in Methods, the first-round screen was multiplexed, with 4 molecules per well at 1.4 μmol/L each. The mean cell death across all 550 wells (500 test wells, 50 controls) was 17.8±0.02% (mean±SEM). Figure 3A shows normalized cell death scores for the 500 molecules tested, in rank order (Online Figure III, A, shows results in order of assay, and the full results are also in Online Table I). Scores <1.0 denote a protective effect, whereas those >1.0 denote a detrimental effect. Mean cell death was 1.0, and the standard deviation was ±0.27, indicated by the gray shading in Figure 3A. Sixty-three wells exhibited scores >1 SD below the mean (ie, <0.73), and 10 wells exhibited scores >2 SD below the mean (ie, <0.46, corresponding to z-score >2).
The 56 lowest-scoring wells were subjected to a second-round screen at 1 molecule per well (224 molecules) to reveal the active constituents. Results from the second round are in Online Table I, and Figure 3B shows normalized cell death scores in rank order. Mean cell death for the second round was 1.13, and the SD was ±0.29. A mean cell death value of >1 may seem counterintuitive, since the second round should be biased toward protective molecules (based on selection from the first round). However, if protection in the first round was conferred by only 1 of the 4 molecules in each well, then only one-fourth of second-round molecules should exhibit protection, with three-fourths exhibiting no or a damaging effect. Viewing the second-round results in assay order (Online Figure III, B) yielded an upward trend over time, consistent with the most protective wells from round 1 being screened first and less protective wells later.
In the second-round screen, 37 molecules exhibited scores >1 SD below the mean (ie, <0.84). These molecules are shown in Online Table II, ranked by score. The table also shows structures and chemical/biological properties. A number of structural motifs were common among the hits, including flavones, chalcones, sterols, terpenoids, and bile acids. Several pharmacological classes were also common, including antihelminthics, antibiotics, antihistamines, anticonvulsants, and antianginals.
Exclusion criteria were applied to the 37 hits (Online Table II), including whether compounds were commercially available in sufficient quantities, any known cardiovascular pharmacology issues, adherence to rule-of-5 filters for drug-like molecules,23 and exclusion of drugs already known to protect against IR injury. This resulted in a refined list of 10 candidates, of which 6 (Table) were chosen for further study in a physiologically relevant model of IR injury, the Langendorff-perfused rat heart. To further validate the screen, 3 molecules exhibiting the most detrimental effect on cell death, plus 1 molecule that exhibited no effect in the screen, were also tested in the perfused heart system.
The most protective molecule identified was the UV-blocking agent dioxybenzone, but preliminary studies revealed severe negative effects on cardiac contractility during initial infusion, precluding further investigation. As Figure 4 shows, the remaining 5 protective molecules—cloxyquin, methapyrilene, mevastatin, clorsulon, and 7-hydroxyflavone—at 1 μmol/L yielded either a significant improvement in the post-IR recovery of cardiac function (RPP=heart rate×pressure product), a significant reduction in myocardial infarct size, or both. Furthermore, Online Table III shows that these molecules also improved post-IR contractility (dP/dtMAX=contraction, dP/dtMIN=relaxation) and lowered the degree of hypercontracture experienced during ischemia.
Further validating the screen, Online Figure V shows that 3 detrimental molecules (apiin, allopregnanolone, and 2,4-dichlorophenoxybutyrate) all worsened the outcome of IR injury in the perfused heart system, whereas an ineffective molecule (propoxur) was without effect. Overall, for the 9 molecules tested, strong correlations (r2 >0.62) were observed between the primary end point of the screening assay (normalized cell-death score) and 2 outcome measures of IR injury in the intact heart (recovery of rate×pressure product and infarct size). These correlation coefficients (Figure 5) suggest that the screen has predictive power for ≈65% of the effect of a molecule in the intact heart.
Finally, an advantage of using the Seahorse XF apparatus in this model of IR injury was the ability to monitor O2 and pH in every well and thus to observe metabolic parameters (OCR and ECAR, see Figure 2) and the effects of test molecules or IR injury on metabolism. Figure 6 shows relationships between cell death, metabolism, and small molecule effects; these data are explored in detail in the Discussion.
The goal of this study was to develop an HTS method for testing the protective efficacy of small molecules in a cell model of IR injury. The model recapitulates several physiological features of IR, including low Po2 and acidic pH, both of which were independently monitored in each well of the screen. Prevalidation tests indicated that the screen can pick up known cardioprotective agents (Online Figure II), and postvalidation tests in a perfused heart model of IR indicate that the screen has predictive power (r2 ≈0.65) for the beneficial or detrimental effect of molecules in the intact heart.
The protective hits identified by the screen are structurally and pharmacologically diverse (see Table), and their bioactivities warrant some discussion. Cloxyquin (Figure 4A) is an 8-hydroxyquinoline, and its close relative clioquinol is currently under investigation as an Alzheimer disease therapeutic.24 Clioquinol stimulates autophagy,25 and it is thought that autophagy plays an important role in cardioprotection triggered by IPC.26 Thus, it is possible that IR protection by cloxyquin and related molecules may proceed through this mechanism.
Methapyrilene (Figure 4B) is an H1 histamine receptor antagonist, and, although histamine is known to confer detrimental effects in IR injury,27 it is unclear whether H1 antagonists in general may be cardioprotective. In a previous study, we demonstrated cardioprotection by the H1 antagonist meclizine but not by pheniramine or pyrilamine.28
Long-term treatment with mevastatin (Figure 4C) was previously shown to afford protection in a rat model of neuronal IR injury.29 Another statin (simvastatin) is also known to be cardioprotective in vivo.30 The current investigation administered mevastatin acutely, and in this regard it is known that mevastatin can protect neonatal cardiomyocytes against reoxygenation injury by an Akt/GSK-3β signaling mechanism.31 Similarly, simvastatin,32,33 atorvastatin,34 and rosuvastatin35 all protected perfused hearts from IR injury when delivered acutely.
Clorsulon (Figure 4D) is a veterinary antihelminthic. Its proposed mechanism of action is glycolytic inhibition at phosphoglycerate kinase,36 although we observed no effect of clorsulon on ECAR in the XF system. Notably, the antihelminthics clorsulon, albendazole, and diethylcarbamazine were all in the top 37 hits, and all are routinely used in combination with ivermectin. Ivermectin enhances P2X4 purinergic receptor channel activity,37 and these channels are known to improve cardiac contractility.38 Thus, the cardioprotective effects of ivermectin in combination with its veterinary cohorts may be worthy of investigation.
7-Hydroxyflavone (Figure 4E) is a vasorelaxant,39 an effect mediated in part by calcium activated potassium (KCa) channels.39 The latter are implicated in the mechanism of IR protection afforded by volatile anesthetic preconditioning,40 and bona-fide KCa channel openers are known to protect the heart and brain against IR injury.41
The 4 remaining molecules from the refined list of 10 protective hits are also interesting from a cardioprotection standpoint. Phenacemide and Beclamide are anticonvulsants, with good cardiovascular safety records. Phenacemide has been shown to inhibit aldose reductase, an important player in the polyol pathway of oxidative stress.42 Although no effects of Beclamide on the heart are known, it is metabolized to 3-chloropropionate, an agonist of the GHB receptor,43 which is expressed on cardiomyocytes and drives a tachycardic response. 3,4′-Dimethoxyflavone is an antagonist of the aryl hydrocarbon receptor (AhR),44 and the known interplay between the AhR and hypoxia-inducible factor signaling may have implications for ischemic tolerance. Albendazole inhibits fumarate reductase,45 the invertebrate analog of succinate dehydrogenase. The latter is linked to activation of mitochondrial KATP channels, which are important in IPC.46 3βHydroxy-23,24-bisnorchol-5-enic acid is a bile acid, and many similar molecules have been shown to be protective against oxidative stress.
In addition to the IR protective molecules identified herein, the validity of this screen is further established by the observation that several other known IR protective molecules yielded protective scores in the second round, including amlodipine, perhexilene, captopril, acadesine, rotenone, amiloride, and digoxin (Online Table I). Although many known protective molecules did not proceed beyond the first round screen (including meclizine, pravastatin, ranolazine, nitroprusside, adenosine, quinidine, warfarin, cyclosporine, clopidogrel, carvedilol, diazoxide, and resveratrol), this probably was due to their not being present at appropriate concentrations to afford protection, since prevalidation tests (Online Figure II) confirmed protection by a number of known cardioprotective agents in this model system. Achieving the correct concentration is a common problem in HTS experiments; to rerun the entire screen at several different concentrations would be prohibitive, whereas choosing a single concentration will inevitably result in some molecules being outside of their effective concentration range.
Alternatively, the protective effects of these molecules in round 1 may have been cancelled out by detrimental effects of other molecules in the same well. Such generation of false-negatives is an acknowledged limitation of pooling approaches in HTS.21 One approach that can mitigate this is an orthogonal pooling strategy, wherein molecules are pooled differently in multiple screening rounds, such that each molecule is tested several times in the presence of different compounds. However, orthogonal pooling is known to generate a higher rate of false-positives (eg, an inactive compound tested alongside positive compounds in all rounds would falsely register as positive) and requires a larger number of plates or wells and sophisticated data decoding algorithms.21 Notably, it is important to note that the pooling method used herein does not generate false-positives; that is, any protective scores in round 1 that resulted from drug interactions or synergy would have been eliminated in the round-2 screen at 1 molecule per well.
Among the detrimental and no-effect molecules that were validated in the perfused heart model (the insecticide propoxur, the herbicide 2,4-dichlorophenoxybutyrate, the neurosteroid allopregnanolone, and the flavone glycoside apiin), none to date has been linked with adverse cardiovascular effects. A number of molecules with known detrimental effects in IR injury were seen to have a detrimental effect in round 1 (eg, the aldehyde dehydrogenase 2 inhibitor disulfiram47), but, due to the nature of the selection process from round 1 to round 2 (ie, a focus on the protective end of the spectrum), these molecules were not pursued further. Given the current findings, it may be worthwhile to further investigate these and other detrimental molecules from the screen to determine if they represent cardiac safety concerns.
A major advantage to the use of XF apparatus in this model is the measurement of metabolic parameters throughout the protocol. Figure 6A shows the relationship between the effect of test molecules on OCR (Ox-Phos) versus ECAR (glycolysis), revealing 2 interesting results: First, although an inverse relationship between these parameters was expected (ie, Ox-Phos inhibition should correlate with glycolytic stimulation and vice versa), no correlation was observed. Second, glycolysis appeared more plastic than Ox-Phos. This may simply reflect the metabolism of H9c2 cells in this system but may also have implications for drug targeting, suggesting that glycolysis is more amenable to pharmacological manipulation than is Ox-Phos.
Many Ox-Phos inhibitors are known to protect against IR injury,48 and a previous screen for molecules that alter cell metabolism identified an Ox-Phos inhibitor (meclizine) that protected against IR injury in heart and brain.28 However, in the current screen of 224 molecules, a negative correlation was seen between cell death and OCR/ECAR (Figure 6B), suggesting that molecules that shift metabolism away from Ox-Phos toward glycolysis are detrimental. Breaking this correlation down further into its component phenomena reveals that the bulk of the effect lies within Ox-Phos (Figure 6C and 6D). Thus, whereas Ox-Phos is not as amenable to pharmacological manipulation as glycolysis (see Figure 6A), altering Ox-Phos appears to have more impact on cell death in IR injury.
The importance of cell metabolism during recovery from IR injury was also examined. Contrary to what might be predicted, no correlation was seen between recovery of Ox-Phos and cell death (Figure 6E). Conversely, however, a significant correlation was seen between recovery of glycolysis and cell death (Figure 6F). One reason why excessive glycolysis during reperfusion may be detrimental is the removal of lactic acid via the sodium-hydrogen exchanger, which drives cytosolic Na+ overload.49
Despite several advantages, there are also limitations to the current model of IR injury; for example, it does not reproduce the substrate deprivation seen in ischemia. Based on measured glycolysis rates during ischemia, estimated anaerobic glucose consumption is 18 pmol · min−1 · well−1. Thus, the 175 nmol of glucose in the 7-μL microchamber is sufficient for 162 hours of metabolism. A nonphysiological glucose level (150 μmol/L) would be required to ensure depletion during 1 hour of ischemia. Another caveat is the use of the cardiomyocyte-derived H9c2 cell line. Although primary cardiomyocytes would be more physiologically relevant, such preparations may not be uniform enough for high-throughput approaches. Finally, the term “high-throughput” is used somewhat casually in reference to the current 24-well system, whereas clearly, a 96-well XF instrument would yield a faster screen.
Finally, an important limitation of this screen was the choice to add small molecules before ischemia. This effectively limits the clinical applicability of any identified hits to a prophylactic model. From the perspective of therapeutics for acute myocardial infarction, it would be far more desirable to deliver molecules at reperfusion (eg, during balloon angioplasty in a percutaneous coronary intervention setting). Thus, future iterations of this screen, delivering small molecules at reperfusion, may yield more clinically applicable hits. Furthermore, it has been suggested that the signaling mechanisms of ischemic preconditioning and ischemic postconditioning overlap significantly.50 Therefore, comparison of the cardioprotective efficacy of a large chemical library applied before versus after ischemia could provide novel insight into shared signaling mechanisms between these protective paradigms.
In summary, we have adapted a readily available apparatus to afford a 24-well model of IR injury and screened 2000 molecules for their effects. The screen was validated through the use of a perfused heart model of IR, resulting in identification of 5 new protective molecules. This screening method is applicable to IR in other tissues and cell types, to enhance the discovery of therapeutics for human disease, and may also find use in the identification of molecules with a detrimental effect on cardioprotection.
Sources of Funding
This work was funded by a grant from the US National Institutes of Health (HL-071158) to P.S.B.
We thank Alan Smrcka and the University of Rochester HTS Core for technical assistance and critique of the manuscript.
In January 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.88 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.263715/-/DC1.
Non-standard Abbreviations and Acronyms
- cyclosporine A
- Dulbecco Modified Eagle Medium
- extracellular acidification rate
- high-throughput screening
- ischemic preconditioning
- oxygen consumption rate
- rate×pressure product
- extracellular flux
- Received January 5, 2012.
- Revision received February 21, 2012.
- Accepted February 24, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
There is a need for new drugs to treat cardiac ischemia-reperfusion (IR) injury associated with acute myocardial infarction.
Testing for such drugs in current animal and whole-organ models is time-consuming and suboptimal for high-throughput analysis.
What New Information Does This Article Contribute?
We have developed a method to expose cells growing in a 24-well plate to IR injury.
Using this new method, we screened 2000 small drug-like molecules and identified several new candidates that protect cells from IR injury.
By comparing the effects of molecules in our screen, with an established intact heart model of IR injury, we validated that the screen has predictive power for cardioprotection.
The current models of cardiac IR injury are either very complicated and slow (eg, perfused hearts or open heart surgery on mice) or they are overly simple and do not model the conditions of ischemia well (eg, hypoxia in cell culture). Therefore, we developed a method to expose cells in a 24-well plate to IR injury. We used this method to screen a library of 2000 small molecules and identified numerous hits that protected cells from IR. We then validated these molecules in more traditional and physiologically relevant models of IR. The results suggest that this screening method can be used to predict whether a given molecule (or group of molecules) will protect the heart against IR injury. We anticipate that this method will be a useful tool in the search for cardioprotective therapeutics. In addition, the application of this screening method to other types of libraries (eg, siRNA) may provide new information regarding the involvement of various genes and proteins in the pathology of IR injury.