Hsp70 as a Molecular Mechanism of Cardioprotection: In Vitro Validation Anne Roessler,1 Kristin Luther,1 Xiaoping Ren,1,2 Yang Wang,1 Ruben Mestril,3 WK Jones.1

1 Loyola University Chicago, Department of Molecular Pharmacology and Therapeutics, 2 Harabin Medical, 3 Loyola University Chicago, Department of Cell and Molecular Physiology

Introduction Results

Hypothesis & Objective

References

Conclusions & Future Directions

Methods

Hypothesis: Pharmacological upregulation of Hsp70 is cardioprotective in an in vitro model of ischemia and reperfusion injury.

Objective: To evaluate the molecular mechanisms of cardioprotection at the cellular level preliminary to in vivo studies.

Cells: H9C2 cells were treated with 17-DMAG, radicicol, or a vehicle control 18 hours prior to protein collection. H9C2 cells were treated with Hsp90 inhibitors 4 hours prior to switching to ischemia mimetic media without the drug (4) or treated with Hsp90 inhibitors 9 hours prior to switching to ischemia mimetic media with the drug (9). For both survival studies, a separate group of H9C2 cells were treated with AdipoRon as a positive control.

Oxygen Glucose Deprivation (OGD): Cells were plated with an ischemia mimetic solution (in mM: 125 NaCl, 8 KCl, 20 2-deoxyglucose, 0.5 Na-Lactate, 1.25 MgSO4, 1.25 CaCl, 1.2 KH2PO4, 6.25 NaHCO3, 20 HEPES (pH 7.4), pH 6.8) and maintained at 37°C with 5% CO2 and 0.05% O2 for an 18 hour incubation.

Cell Survival: Propidium iodide (PI) and Hoechst staining measured cell viability before and after OGD. Cell viability was calculated as all cells (Hoechst positive) minus dead cells (PI positive). Cell survival was calculated as cell viability after OGD as a percentage of cell viability before OGD.

Heat Shock: Cells were placed at 42°C for 1 hour followed by an 8 hour recovery period before OGD or a 17 hour recovery period before protein collection.

Western blotting was used to assess the changes in protein levels. Blots were quantified with ImageJ.

Figure 4: 17-DMAG increases Hsp70 proteins levels. H9C2 cells were treated with different doses of 17-DMAG for 18 hours prior to protein collection. Western blotting was performed on cell lysates and densitometry was quantified by Image J. Protein levels were normalized to β-actin and expressed as fold change vs. vehicle (n=3-18).

• In 1986, the discovery of ischemic preconditioning (IPC) demonstrated the existence of endogenous protective mechanisms against ischemia and reperfusion injury after myocardial infarction.1

• Nociceptor-induced conditioning (NIC), a phenomenon whereby stimulation of skin nociception initiates protection, offers a more practical way to invoke cardioprotection. 2

• The mechanisms underlying different forms of cardioprotection are similar but not identical.

• NF-κB-dependent transcription of heat shock proteins (Hsps) is necessary for late IPC cardioprotection.3 Hsps act in unity to assist in protein folding and repair following myocardial injury.

• The small molecule Hsp90 inhibitors, 17-DMAG and radicicol, release HSF1 from Hsp90, allowing it to become transcriptionally active and upregulate heat shock proteins. Other Hsp90 inhibitors, such as geldanamycin and herbimycin A, work in a similar manner and have been shown to pharmacologically upregulate Hsp70 and confer cardioprotection in neonatal rat cardiomyocytes.4,5,6

• The role of Hsp70 and Hsp90 in NIC remains to be determined and is the focus of this study.

•17-DMAG and radicicol increase Hsp70 levels but are not associated with protection in this model.

•Hsp90 inhibition may be protective in neonatal rat cardiomyocytes but detrimental in H9C2 cells.6 Phenotypic differences between cell lines may implicate different molecular mechanisms of cardioprotection.

•The complex choreography of heat shock protein interactions during ischemia and reperfusion injury is still under investigation.

•Further studies will evaluate Hsp90 inhibitors as pharmacological tools for cardioprotective studies. We hope to gain insight into the role of Hsp90 and its client proteins.

Figure 3: Heat shock protects H9C2 cells from oxygen glucose deprivation. Cells were heat shocked at 42°C for 1 hour followed by an 8 hour recovery period prior to OGD. Hoechst and propidium iodide staining was performed on H9C2 cells before and after OGD. Cell survival after OGD was normalized to cell viability before OGD (n=6). *P≤0.05 relative to vehicle.

Figure 1: Potential model of Hsp90 inhibitors on the heat shock response.6

Figure 5: Radicicol increases Hsp70 proteins levels. H9C2 cells were treated with different doses of radicicol for 18 hours prior to protein collection. Western blotting was performed on cell lysates and densitometry was quantified by Image J. Protein levels were normalized to β-actin and expressed as fold change vs. vehicle (n=3-6).

Figure 6: Hsp90 inhibitor treatment does not protect H9C2 cells from oxygen glucose deprivation. H9C2 cells were treated with 17-DMAG (0.01 μM), radicicol (5 μM), or vehicle 4 hours prior to OGD, then switched to ischemia mimetic media without the drug for the duration of OGD. AdipoRon (2.7 nM) was included as a positive control. Hoechst and propidium iodide staining was performed on H9C2 cells before and after OGD. Cell survival after OGD was normalized to cell viability before OGD (n=14-16). *P≤0.05 relative to vehicle.

Figure 2: Hsp70 protein levels after heat shock. A. Cells were heat shocked at 42°C for 1 hour. Protein was collected 18 hours after the initiation of heat shock. Western blotting was performed on cell lysates. Densitometry was quantified by Image J, and protein levels were normalized to β-actin and expressed as fold change vs. vehicle. B. Quantification (n=6). *P≤0.05 relative to vehicle.

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Figure 7: Hsp90 inhibitor treatment does not protect H9C2 cells from oxygen glucose deprivation. H9C2 cells were treated with 17-DMAG (0.01 μM), radicicol (5 μM), or vehicle 9 hours prior to OGD, then switched to ischemia mimetic media containing the drug for the duration of OGD. AdipoRon (2.7 nM) was included as a positive control. Hoechst and propidium iodide staining was performed on H9C2 cells before and after OGD. Cell survival after OGD was normalized to cell viability before OGD (n=14-16). *P≤0.05 relative to vehicle.

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