Using Stem Cells in Drug Discovery for Heart Attacks

A loss of cardiomyocytes replaced by non-contractile scar tissue characterizes myocardial infarction (MI).

Cell damage is compounded during reperfusion through an increase in inflammation and free radicals, which induce cell death. Scar formation would be reduced if cardiomyocyte death could be reduced. This, in turn, could improve cardiac function and reduce the risk of heart failure.

Celastrol is a modulator of HSP90 activity and a compound isolated from an oriental medicinal plant. It has been previously shown to activate HSF1, limit infarct size, and preserve heart function in a model of permanent ischemic myocardium. Overexpression of HSPs is induced by Celastrol, including Heme-Oxygenase 1 (HO-1, or HSP32), which is a potent cardiac antioxidant factor.

Animals where pre-treated with Celastrol or DMSO (Control), then subjected to permanent ischemia for 2 weeks.  A) Celastrol reduces fibrosis following 2 weeks of cardiac permanent ischemia (Masson’s trichrome coloration). B) Celastrol pre-treatment preserves cardiac function after 2weeks of permanent ischemia. (Short axis, 2D images taken before and after 2 weeks of cardiac ischemia).

Animals where pre-treated with Celastrol or DMSO (Control), then subjected to permanent ischemia for 2 weeks.  A) Celastrol reduces fibrosis following 2 weeks of cardiac permanent ischemia (Masson’s trichrome coloration). B) Celastrol pre-treatment preserves cardiac function after 2weeks of permanent ischemia. (Short axis, 2D images taken before and after 2 weeks of cardiac ischemia). Image Credit: Axol Bioscience

The purpose of this research was to validate Celastrol and expand the search for cardioprotective drugs in a clinically relevant model of ischemia/reperfusion (I/R) stress. This was carried out in appropriate cell lines (iPSC-derived human mature cardiomyocytes) using high throughput screening equipment and validation.

Image Credit: Axol Bioscience

Method

Primary Screening in H9c2 cells (Viability following 2 components of I/R damage)

Image Credit: Axol Bioscience

Validation in H9c2 and iPSC-derived human mature cardiomyocytes

Image Credit: Axol Bioscience

Results

Primary screening: H9c2 cells subjected to 48h Ischemia or 1h oxidative stress

Image Credit: Axol Bioscience

Some compounds, in comparison to control, protect from 48h ischemia (N=6) and oxidative stress (N=6). This includes the commercially available Celastrol.

Image Credit: Axol Bioscience

A microscopic image was taken using the high content screen Operetta system. In this image, alive cells are shown in green and dead cells in red.

Reporter assay and western blot confirmation

Image Credit: Axol Bioscience

When compared to controls, some compounds (including Celastrol) also induce the activity of the heat shock element (HSE) and the antioxidant response element (ARE), as measured by the expression of inducible luciferase in transfected H9c2 cells (N=6).

Image Credit: Axol Bioscience

HO-1 expression was induced by all of the tested compounds. However, HSP70 expression and/or ERK or Akt activity was also induced by protective compounds.

Validation:

Image Credit: Axol Bioscience

Celastrol and some of the other selected compounds showed a protective effect on H9c2 cells subjected to I/R Stress in comparison to controls (N=6).

Image Credit: Axol Bioscience

Celastrol pre-treatment was also shown to induce resistance to MPTP opening in H9c2 cells following ionomycin 5 nM challenge. *p<0,02.

Image Credit: Axol Bioscience

Celastrol was shown to be protective in preliminary results (N=2) of screening (I/R in vitro) using induced human induced pluripotent stem cell-derived mature beating cardiomyocytes.

Conclusions

Celastrol was identified and validated as a novel infarct sparing agent and was found, along with other analog compounds, to have superior potency. In a first step towards developing a novel drug, these candidate compounds are tested and compared to Celastrol ex vivo using a Langendorff preparation (see Can J Cardiol, Vol.32, Issue 10, S192-3). These would be designed as a first-line medication for the treatment of MI and adjunct therapy to reperfusion procedures.

Image Credit: Axol Bioscience

Acknowledgments

Produced from materials originally authored by H Aceros, S Der Sarkissian, M Borie, L Stevens, S Mansour, and N Noiseux from Université de Montréal, Montréal, Canada, and CRCHUM, Montréal, Canada.

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Feb 18, 2020 at 11:26 AM

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