Posters


Neuroscience

Next-generation neurological disease models: isogenic tools for investigating frontotemporal dementia, Alzheimer's and Parkinson's diseases

Alzheimer’s and Parkinson’s diseases are incurable and debilitating neurodegenerative conditions with strong links to age. Today Alzheimer’s disease alone accounts for 60-70% of dementia cases and Parkinson’s disease is expected to affect ~2% of individuals over 65 years. The human and economic impact of these conditions is expected to increase significantly with the increasingly aging global populations unless new therapeutic strategies can be developed. Currently, there are a lack of human cell-based models available in which to carry out such studies and further investigation is needed in order to elucidate disease mechanisms and determine the efficacy of novel drug compounds. iPSC-derived neural stem cells (NSCs) offer a virtually unlimited source of physiologically relevant isogenic lines for use in both disease modelling and drug discovery.

Combining the powerful tools of iPSC genome editing using CRISPR-Cas9 and directed differentiation, we have generated patient relevant NSC disease models carrying Alzheimer’s disease-associated microtubule-associated protein tau (MAPT) mutations, R406W, P301L and V337M and Parkinson’s disease-associated leucine-rich repeat kinase 2 (LRRK2) mutation, G2019S in both a heterozygous and homozygous manner. These clinically identified missense mutations in MAPT are thought to reduce the ability of tau to promote microtubule assembly and may contribute to neuronal death in Alzheimer’s disease. LRRK2 is thought to contribute to Parkinson’s disease via pathological mechanisms involving tau, oxidative stress, α-synuclein, and mitochondrial-synaptic-dysfunction.

Genome-edited iPSC lines were genotyped, karyotyped and subsequently differentiated using fully-defined, xeno-free neural induction conditions (Shi et al et al., 2012). Once the cells formed polarized neural tube-like rosette structures in monolayer culture, immunocytochemistry confirmed the expression of typical cerebral cortical NSC markers namely, PAX6 and FOXG1.

These genetically defined, functionally validated human iPSC-derived NSCs provide a renewable resource of disease- and biologically-relevant cells. These cells, carrying relevant mutations, offer a stable platform on which novel therapeutic agents can be screened and validated. Furthermore, these cells enable a direct comparison of the variant effect on cellular phenotype between isogenic lines cells and may therefore provide further insight into the pathology of these diseases.

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Functional phenotypic characterization of iPSC-neurons from Alzheimer’s disease patients carrying PS-1 mutation in drug screening and disease modeling

Adult cells from human individuals carrying disease-associated gene mutations can be reprogrammed into induced pluripotent stem cells (iPSCs) and can then be differentiated into a variety of cell types including human neural stem cells (hNSCs) and cerebral cortical neurons (hCCNs). Our aim was to phenotypically investigate patient iPSC-derived neurons carrying the presenilin-1 (PS-1) mutation (supplied by Axol Bioscience) and to compare them with cells from healthy controls (supplied by Axol Bioscience).

Transcriptome analysis revealed an up-regulation in the expression of neuronal genes and a decrease in pluripotency markers in hCCNs. Immunocytochemistry showed the appropriate neural cell morphology in hCCNs, with both cell types expressing markers typically associated with the corresponding developmental stage. Whole patch clamp and multi-electrode arrays (MEAs) successfully established electrical activity in these cells.

We differentiated these neural progenitor cells into spontaneously active neuronal networks using a xeno-free differentiation protocol and recorded spontaneous activity during neuronal differentiation using micro-electrode array (MEAs). Multi-parametric phenotypic analysis was used to identify specific differences of functional activity patterns during development into mature neuronal networks within 4-5 weeks. Moreover, we investigated the effects of neurotoxins on mutant and control neurons. We have identified a range of characteristics in the patient-derived and control- derived iPSC-neurons that establishes them as an ideal tool for use in numerous applications such as disease modeling, drug screening and toxicology and other assays.

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Characterization and potential applications of human iPSC-derived neural stem cells

Adult cells can be reprogrammed by defined factors, OCT3/4, KLF4, SOX2 and c-MYC, to generate induced pluripotent stem cells (iPSCs). These can then be differentiated into a variety of cell types including human neural stem cells (hNSCs) and cerebral cortical neurons (hCCNs). Reprogramming and differentiation can be carried out on cells from healthy donors and patients suffering from disease and could potentially be used as a model in the study of human neuronal development and disease. In order to determine the suitability of these iPSC-derived cells for neurobiological research, we conducted a series of e xperimental procedures to examine the functional characteristics of these cells and their progeny in vitro. Transcriptome analysis revealed an up-regulation in the expression of neuronal genes and a decrease in pluripotency markers in hNSCS. Immunocytochemistry showed the appropriate neural cell morphology in hNSCs and hCCNs, with both cell types expressing markers typically associated with the corresponding developmental stage. When cultured in 3D using a collagen matrix, both cell types formed common neural structures. hNSCs yielded the highest neurite length and branch point values when compared to a variety of other neural cell types. Whole patch clamp and multi-electrode arrays (MEAs) successfully established electrical activity in these cells. We have identified a range of characteristics in the hNSCs and hCCNs that establishes them as an ideal tool for use in numerous applications such as disease modelling, drug screening and toxicology and other assays.

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Modelling neurological disease: in vitro gene editing and iPSC differentiation combine to create powerful new tools

Neurodegenerative diseases, such as Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and other age-related dementias are incurable and debilitating conditions, with Alzheimer’s disease alone accounting for ~60-70% of cases. With an increasingly ageing global population, the economic as well as human impact of these conditions is expected to increase unless novel therapeutics and care strategies can be developed. Induced pluripotent stem cells (iPSCs) and gene-editing technology, offers unprecedented biomedical potential for disease modelling, high-throughput drug screening and development of therapeutic strategies for such diseases.

We have generated stable human iPSC lines from normal human dermal fibroblasts and patient derived fibroblasts (e.g. Huntingdon’s and Alzheimer’s diseases). The fibroblasts were reprogrammed using a non-integrating episomal method coding for Yamanaka factors (license agreement with iPS Academia Japan) and then differentiated into neuronal stem cells (NSCs) and cortical neurons to provide a complete modelling solution in a dish. The iPSC lines derived from normal human dermal fibroblasts were stable with all the hallmarks of pluripotency and a normal karyotype for over 13 passages. These could be cultured as single cells, an essential prerequisite for efficient genome editing. Using the CRISPR-Cas9 genome–editing technology, we generated patient relevant disease models carrying microtubule-associated protein Tau (MAPT) mutations. Tau protein is normally associated with microtubules and is involved in their assembly and stabilization. In turn, microtubules are critical for cellular function, especially for neurons to facilitate the growth and integrity of axons and dendrites and transport between the cell body and distant dendrites. Clinically identified missense mutations reduce the ability of Tau to promote microtubule assembly, resulting in neuronal cell death and subsequent disease phenotype.

These renewable and biologically relevant resources will further enable investigation of the mechanisms of disease progression, with additional models relevant to Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease and epilepsy being generated to aid in the identification of novel drug discovery targets.

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Induction of plasticity phenomena in human induced pluripotent stem cell-derived cortical neurons

Long-term potentiation (LTP) and long-term potentiation depression (LTD) in neuronal networks has been analyzed using in vitro and in vivo techniques in simple animals to understand learning, memory, and development in brain function. Human induced pluripotent stem cell (hiPSC)- derived neurons may be effectively used for understanding the plasticity mechanism in human neuronal networks, thereby elucidating disease mechanisms and drug discoveries. In this study, we attempted the induction of LTP and LTD phenomena in a cultured hiPSC-derived cerebral cortical neuronal network using multi-electrode array (MEA) systems. High-frequency stimulation (HFS) produced a potentiated and depressed transmission in a neuronal circuit for 1 h in the evoked responses by test stimulus. The cross-correlation of responses revealed that spike patterns with specific timing were generated during LTP induction and disappeared during LTD induction and that the hiPSC-derived cortical neuronal network has the potential to repeatedly express the spike pattern with a precise timing change within 0.5 ms. We also detected the phenomenon for late-phase LTP (L-LTP) like plasticity and the effects for synchronized burst firing (SBF) in spontaneous firings by HFS. In conclusion, we detected the LTP and LTD phenomena in a hiPSC-derived neuronal network as the change of spike pattern. The studies of plasticity using hiPSC-derived neurons and a MEA system may be beneficial for clarifying the functions of human neuronal circuits and for applying to drug screening.

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Functional maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture

The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20–30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

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Pharmacological responses in cultured human iPSC-derived cortical neurons using multi-electrode array

The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20–30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

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In vitro electrophysiological drug testing using human induced-pluripotent stem cells

Human induced pluripotent stem cell (hiPSC)-derived neurons may be effectively used for drug discovery and cell-based therapy. We here used a multi-electrode array (MEA) system to investigate the functional characteristics of hiPSC-derived neurons on their long-term spontaneous activity and drug responsiveness over 300 days culture. We demonstrated that hiPSC-derived neurons allowed the culture to be maintained over 10 months with long-term spontaneous activity. After 70 days of culture, we observed synchronous burst firing activity due to synapse transmission within neuronal networks. Addition of the synapse agonist and antagonists kainic acid, bicuculline, CNQX and AP5 induced significant changes of the firing rate in spontaneous firings and electrical evoked responses. Furthermore, we demonstrated that epilepsy phenomenon was evoked by administration of pentylentetrazole (PTZ) and was inhibited by anti-epilepsy drug phenytoin and sodium valproate (VPA). High frequency synchronized bursts were evoked over PTZ 100 μM. These bursts were gradually decreased with the increasing the dose of anti-epilepsy drug, and disappeared over phenytoin 100μM or VPA 1 mM respectively. These results suggested that long-term electrophysiological measurements in hiPSC-derived neurons using a MEA system may be beneficial for drug screening applications.

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In vitro pain responses of dorsal root ganglion neurons using multi-electrode arrays

Dorsal root ganglion (DRG) sensory neurons are pain-related neurons and have a variety of sensory receptors that are activated by chemical, thermal, and mechanical stimuli. Establishment of pharmacological assay in pain research and drug screening is important issue. Here, we used the multi-electrode array (MEA) system to detect the electrophysiological responses by chemical and thermal stimuli in cultured DRG neurons. After 2 days of culture on the MEA, we observed spontaneous activities and chemical responses. Addition of the capsaicin, menthol and wasabi induced significant changes of the firing rate and concentration-dependent responses. Furthermore, temperature elevation increased the number of firings and it showed the largest increase at 43 degrees. We also detected the responses to temperature and capsaicin in hiPSC derived sensory neurons at 14 DIV. We confirmed that the typical response of DRG neurons can be easily obtained using MEA system. These results suggested that electrophysiological measurements in DRG neurons using a MEA system may be beneficial for clarifying the functions of DRG neurons and human iPSC derived sensory neurons in pain research and for drug screening applications.

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Implementation of high throughput screening on iPS cells

Induced pluripotent stem cells (iPSCs) belong to a growing list of stem cell populations that hold great potential for use in cell-based assay development and screening. iPSC-derived cells like cardiomyocytes and neuronal cells represent a reliable source of cellular material with robust assay performance and scalability which is essential to any HTS campaign and subsequent follow-up. Here we demonstrate the impact of iPSC-derived neurons in modern drug discovery. We present examples of assay automation and miniaturization of cellular assays based on high content imaging using the Operetta and Ca-flux using the FLIPR instruments. Our examples show the opportunities and benefits that iPSC-derived cells may provide in the search for new chemical starting points for drug discovery in the near future.

20 Qualified Hit List reports to the target owners. The over 40 HTS campaigns performed to date cover a wide range of target classes including more demanding cellular targets like ion channels and GPCR’s. Cellular HTS assays have been successfully miniaturized to 384- and 1536-well format in order to perform them in a cost-efficient manner. Induced pluripotent stem cells (iPSCs) belong to a growing list of stem cell populations that hold great potential for use in cell-based assay development and screening. iPSC-derived cells like cardiomyocytes and neuronal cells represent a reliable source of cellular material with robust assay performance and scalability which is essential to any HTS campaign and subsequent follow-up. Here we demonstrate the impact of iPSC-derived neurons in modern drug discovery. We present examples of assay automation and miniaturization of cellular assays based on high content imaging using the Operetta and Ca-flux using the FLIPR instruments. Our examples show the opportunities and benefits that iPSC-derived cells may provide in the search for new chemical starting points for drug discovery in the near future.

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Using iPSC-derived neural stem cells as a CNS model to study neuronal behaviour in development and neurodegernation

Induced pluripotent stem cell (iPSC)-derived neural cells provide a powerful tool that can be used to model neuronal behaviour and disease pathology. The increased use of these cells in drug discovery promises to help accelerate current drug screening processes and reduce the use of in vivo models used at the earliest stages of testing. Importantly, the production of specific populations, such as cortical and dopaminergic neurons, has allowed researchers to investigate the activity of neural networks from particular regions of the brain. We developed a number of endpoint assays using human iPSC-derived neural stem cells to determine the functionality of these cells and their response to toxins or disease-relevant biomarkers in both Alzheimer’s disease and epilepsy. We have also manipulated the cells using Lentivirus and have demonstrated long-term expression of over 9 months. The methods developed offer a platform to facilitate our understanding of normal physiological functions and the causes of central nervous system (CNS) pathology.

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Modelling Alzheimer's disease: Development of a scalable, high-throughput-compatible assay to detect tau aggregates using iPSC-derived cortical neurons maintained in a 3D-culture format

We describe a robust, scalable and disease relevant model of tau aggregation using iPSC- derived cortical neurons that can be applied to drug discovery programs in neurodegeneration.

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Cardiovascular

In vitro assays and screening platforms for exploring ventricular and atrial phenotypes in iPSC-derived cardiomyocytes

Existing cardiac safety testing regimes have successfully prevented new drugs coming to market with pro-arrhythmic risk, but they are expensive (e.g. TQT clinical studies ICH-E14) and the reliance on preclinical hERG screening (ICH-S7B) is undermined by the fact that many new chemical scaffolds have been excluded from further drug development while older drugs that inhibit hERG are not associated with arrhythmia. This has prompted the FDA to implement the Comprehensive in vitro Pro Arrhythmia initiative (CiPA) which involves 3 parts: 1) High quality in vitro cardiac ion channel assays, 2) Comprehensive in silico action potential (AP) models and 3) Predictive assays using stem cell-derived cardiomyocytes.

Our presentation will focus on recent electrophysiological ion channel profiling undertaken at Metrion using human induced pluripotent stem (iPSC)-derived cardiomyocytes from Axol Bioscience. Action potentials and ionic currents were monitored using current- and voltage-clamp manual patch electrophysiology recordings, respectively. Pharmacology was assessed using representative compounds from the CiPA validation toolbox, as well as compounds selectively targeting ventricular and atrial ion channels.  Molecular and biophysical properties of these cells were assessed to explore the relative composition of functional and interdependent cardiac cell phenotypes. Phenotypic measurements of impedance (contraction) and field potential (excitability) were also undertaken using the CardioExcyte96 screening platform. Profiling of ion channel expression with a variety of pharmacological tools and techniques enabled us to ascertain the physiology of iPSC-derived cardiomyocytes and assess their potential utility in more efficacious drug screening and toxicology campaigns.

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Serum-free human iPSC-derived cardiomyocytes for in vitro testing

Using defined factors OCT3/4, KLF4, SOX2 and c-MYC, adult cells from healthy and patient donors can be reprogrammed to generate induced pluripotent stem cells (iPSCs). Subsequently, these can be differentiated into a variety of cell types including cardiomyocytes. Human iPSC-derived cardiomyocytes (iPSC-CMs) can be cultured in vitro under serum-free conditions and as such, offer a platform investigate the effect of growth factors, cytokines and drugs on the development and functionality of human cardiomyocytes in vitro. Following differentiation, spontaneously beating iPS-CM’s were evaluated in 2D and 3D culture. Expression profiling by immunohistochemistry confirms the expression of cardiomyocyte selective markers including α-actinin, myosin heavy chain, atrial and ventricular myosin light chains, troponin-T and -I, β-catenin, vimentin, L-type calcium channels, connexin-40 and -43, telethonin and ankyrin repeat domain-1 (ANKRD1). Immunohistochemistry findings were validated by Western Blot for α-actinin and cardiac troponin-T expression. Additional analyses conducted, include bi-nucleate cell counts, cell form factor measurements and a comparison of plating efficiencies across a variety of substrates. The electrical activity of the iPSC-CMs was confirmed using a multi-electrode array (MEA), and the calcium dye Fluo4. One application of these cells is drug toxicity testing. To show proof of principle that this can be undertaken in a contactless manner using only genetically encoded tools, which offers several advantages compared to low throughput contact based methods with chemical dyes, we developed a simultaneous optical control/calcium imaging approach to replace the need for electrode stimulation and dyes. We are able to control the beat frequency of iPSC- CMs across the physiological range (0.3Hz – 2Hz) and can observe the anticipated effects of compounds such as Dofetilide, a known hERG inhibitor. Here, we have identified a range of characteristics in these human iPSC-CMs that confirms their ability to function as a highly-pure population of single beating human cardiomyocytes in vitro and presented evidence of a technically simple and scalable platform for cardiotoxicity screening assays.

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Effects of transient hypoxia/ischemia on inducible human pluripotent stem cell (iPSC)-derived cardiomyocytes

Hypoxia/ischemia is a central elicitor of ischemia/reperfusion (I/R) injury in the human heart. However, there is an apparent lack of suitable in ­vitro models for the investigation of I/R ­induced cellular mechanisms in human cardiac cells. We investigate whether inducible human pluripotent stem cell (iPSC)-­derived cardiomyocytes are susceptible to physiologically defined transient hypoxic/ischemic conditions.

Spontaneously contracting iPSC-­derived cardiomyocytes were subjected to enzymatically induced hypoxic/ischemic conditions by using glucose oxidase (GO, 2U/ml) and catalase (CAT, 120U/ml). Morphological assessment and measurements of LDH activity released from damaged cells (Cytotoxicity Detection Kit; Roche, Mannheim, Germany) were used for evaluating and quantifying hypoxia/ischemia induced cytotoxicity (Figure 1).

Hypoxic/ischemic conditions were rapidly established after the addition of GO/CAT and resulted in pO2 levels <10mmHg after 30 minutes (lasting for at least 4 hours), a gradual decrease of glucose concentration (from 4g/l to <1g/l after 4 hours) and a decline of pH from 7.65 to 6.98. iPSC­derived cardiomyocytes showed spontaneous contractions after 10 days in culture with a beating frequency of 15­20 contractions/min under normoxia as well as under hypoxic/ischemic conditions (normoxia: 18.40±3.41 bpm; hypoxia/ischemia: 15.50±2.60bpm; low resolution video file). Directly after replacing the hypoxic/ischemic medium by normoxic culture medium, frequency of contractions increased by 2­fold in the hypoxia/ischemia group (normoxia: 16.72±0.93bpm; hypoxia/ischemia: 31.58±0.75bpm). 24 hours after the 4 hour hypoxia period, iPSC-­derived cardiomyocytes showed clear morphological signs of cell damage such as cell rounding, swelling and detachment from the growth surface. In cultures that were subjected to 4 hours of hypoxia, LDH release as a marker of cell damage was increased 3­fold while iPSC-­derived cardiomyocytes that were cultured under normoxic conditions did not reveal morphological changes or increased LDH release after 24 hours (normoxia: 0.10±0.00au; hypoxia/ischemia: 0.29±0.02au).

Inducible human pluripotent stem cell-­derived cardiomyocytes are susceptible to transient hypoxia/ischemia in ­vitro. The described culture system closely resembles hypoxic/ischemic conditions in­vivo and may help to elucidate cellular/molecular mechanisms of ischemia/reperfusion injury as well as cardioprotective strategies.

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A drug discovery platform for the identification of novel infarct sparing agents for treatment of ischemic heart disease

Any intervention aiming to protect the myocardium by reducing cardiomyocyte death following myocardial infarction (MI) would benefit cardiac repair and functional recovery. Therefore, identification of drugs that may protect the heart during an ongoing MI or reperfusion of the culprit coronary is highly relevant in the clinical setting. We have shown that the natural compound Celastrol protects the heart from permanent ischemia-induced death in vivo in a rat model of MI and in vitro in H9c2 rat cardiomyoblasts. Based on these findings, we set to validate Celastrol and expand our search for cardioprotective drugs through a series of clinically relevant tests (ischemia/reperfusion (I/R)), using high throughput screening equipment and validation in appropriate cell lines (iPSC-derived human mature cardiomyocytes).

Compounds are screened in viability assays using pre-treated H9c2 placed under hypoxic (<1% O2; 48hr) and oxidative stress (<4mM H2O2, 60min). Protective compounds are then subjected to an I/R stress assay (<1% O2, low serum/glucose 18h followed by 6h normal culture). Results are validated using iPSC-derived cardiomyocytes (Axol Bioscience). Viability is quantified using the live/dead kit with results analyzed by automated high content screening (Operetta). Next, a secondary reporter assay is used to quantitate the protective heat shock and antioxidant cellular responses (HSE and ARE reporter assays; SABiosciences). Compounds are assayed for their potency to induce protective proteins, activate survival kinases and tested in their ability to inactivate the opening of the mitochondrial permeability transition pore (mPTP assay).

Celastrol (10-6M) pre-treatment increases H9c2 viability submitted to hypoxic stress by 6.7%, oxidative stress by 14.5% and I/R stress by 18% (p<0.05); reduces troponin content by 28.8% in I/R culture media, and increases viability by 7% in iPSC-derived cardiomyocytes submitted to I/R. Celastrol (10-6M) induces a 35.8 and 3.1-fold increase in HSE and ARE reporters (p<0.001), increases protective HO1 (22.5 fold) and HSP70 (11.9 fold) protein expression, as well as pERK (2 fold) and pAKT (8.6 fold) activation. Celastrol prevents the opening of the mPTP pores in ionomycin-challenged H9c2.

We have identified and validated Celastrol as a novel infarct sparing agent and identified as well other analog compounds with superior potency. These will be tested and compared in vivo as a first step towards developing a novel drug designed as a first line medication for the treatment of MI and adjunct therapy to reperfusion procedures.

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