A Method for Preserving Hearts with Hydrogen Sulfide

1. A Method for Preserving Hearts with Hydrogen Sulfide Spring 2009 Proposal Defense

2. Organ Shortage: Societal Problem Though 110,000 people are on organ donor lists, only 77 receive transplants daily Storage time is limited to 4 hours Preservation-induced injury is a major contributing factor to early graft dysfunction in patients

3. Organ Storage Today Static Cold Storage University of Wisconsin Solution No significant improvements despite two decades of research Machine Reperfusion Organ Care System Effective, but extremely expensive

4. Our Idea… Overall idea: To modify the clinical cold storage procedure with H2S Global Hypothesis: Controlled delivery of H2S throughout the heart using gelatin microspheres will induce protective effects and a state of hibernation that will prolong heart viability and reduce ischemia-reperfusion injury in transplants

5. Cold Ischemia Leads to I/R Injury • Continued metabolism • Accumulation of metabolic waste products • ATP depletion Na+ Continued cell processes ATP Na channels Lactate, protons, hypoxanthine • Ionic balance disruption • Decrease in ATP leads to less active ionic pumps • Na+ and Ca 2+ accumulate • Cell swelling ROS oxygen Mitochondria • ROS production • Inefficiencies in electron transport chain lead to ROS Calcium channel Ca 2+ Adapted from: Di Lisa et. al 2007, Jamieson et. al 2008

6. H2S Protects Hearts During Ischemia from I/R Injury • ROS-scavenging • Directly neutralizes oxygen free-radicals • Upregulates anti-oxidant defenses K+ K-ATP channels H2S H2S • K-ATP channel opening • Hyperpolarizes membrane and reduces Ca 2+ influx ROS H2S oxygen Mitochondria • Suspended animation • Reduced metabolic rate • preserve energy stores • reduce byproducts Calcium channel Ca 2+ Adapted from: Elrod et. al 2007, Hu et. al 2007, Johansen et. al 2006

7. H2S depletion and continuous release • H2S is depleted from solution • NaHS releases H2S quickly • Tissue metabolism or escape from solution • Limited time of protection after NaHS depletion • Continuous H2S treatment • Direct ROS-scavenging, K-ATP channel effect throughout ischemic period • Implications for suspended animation?

8. Controlled Drug Delivery • Hydrogels • Synthetic or Natural • Gelatin • Crosslinking • Size of microspheres • Acidic vs. basic 10 micron microspheres distribute evenly throughout the heart via antegrade injection Source: Hoshino et. al (2006)

9. Specific Aim IHydrogen Sulfide Metabolization • Keeping NaHS concentration constant in solution has proven to be a difficult task • Objective: Do cardiomyocytes metabolize H2S? • Methods: • After 24 hours incubation at 37°C, aqueous H2S levels will be measured using a Zinc Acetate assay

10. Specific Aim INaHS Dosage Test • Inconsistent reports of dosages • Objective: What is the most effective concentration of NaHS for storage solutions? • Methods: • 0 to 150µM NaHS in UW solution • Biopsy LV at 2, 4, 6, 8 hours • ATP, Apoptosis, Creatine Kinase assays • Langendorff Perfusion Column

11. Specific Aim II: Fabricating Microspheres Objective:To determine if gelatin microspheres can release NaHS in a controlled fashion Hypothesis: By varying cross-linkage, composition of the microspheres, we will be able to control the release of NaHS After NaHS loading, zinc acetate assay will be used at different time points to determine release rate of NaHS from microspheres A sample of microspheres with an average diameter of 6.8 ± 4 microns (n=67).

12. Specific Aim III: NaHS in UW Solution • Objective: To determine what is the effect of NaHS in conjunction with UW solution on the cold storage of hearts • Hypothesis: NaHS with UW will improve the preservation of hearts through H2S protective mechanisms described before • Methods: • Stored at 4oC for eight hours, and reperfused for 30 min • LVDP recovery, ATP content, apoptosis, CK, and H2S will be measured UW solution + NaHSi UW solution

13. Specific Aim III: Continuous H2S Treatment • Objective: To determine how hearts stored in solution with continuous H2S treatment compare with hearts stored in a solution where H2S is depleted • Hypothesis: Continuous H2S treatment will better preserve hearts UW solution + NaHSi UW solution + NaHSi NaHS-loaded microspheres

14. Specific Aim III: Gelatin Microspheres in Heart Vasculature • Objective: To determine the effect of gelatin microspheres alone on heart preservation when administered to the heart vasculature • Hypothesis: Gelatin microspheres alone will have negligible effect, as their safety has been demonstrated in previous applications UW solution + NaHSi UW solution + NaHSi PBS-loaded microspheres

15. Specific Aim III: Continuous H2S Release from Heart Vasculature • Objective: To determine whether NaHS-loaded microspheres administered to the heart vasculature preserve hearts better than submersion in [UW + NaHSi] or [UW + NaHSi + NaHS microspheres in solution] • Hypothesis: • Gelatin microspheres administered to the vasculature will improve preservation by delivering H2S more uniformly compared to submersion in NaHS solution • Both continuous H2S treatments will better preserve hearts compared to submersion in solution where H2S is depleted UW solution +NaHSi UW solution + NaHSi NaHS-loaded microspheres

16. In Conclusion… • Our new method for heart storage which will: • Reduce ischemia-reperfusion injury and radical oxidative species and improve heart function • But will also be easily applicable to today’s organ transport methods

17. Special Thanks: Dr. John Fisher Dr. Agnes Azimzadeh Dr. Lars Burdorf Tom Harrod Dr. James Wallace Dr. Rebecca Thomas Courtenay Barrett

18. Any Questions? Team Organ Organ Storage Today Our Idea… Hydrogels as a Drug Delivery Method Varying Release Rate How H2S works Specific Aim II: Microsphere Fabrication Specific Aim I Do Cells Metabolize H2S? Effective concentration of H2S for storage? Specific Aim III: Does Controlled Release of H2S improve heart function?