A Method for Preserving Hearts with Hydrogen Sulfide Spring 2009 Proposal Defense Team Members (in alphabetical order) Elizabeth Chen Charles Chiang Elyse Geibel Steven Geng Stevephen Hung Kathy Jee Angela Lee Christine Lim Sara Moghaddam-Taaheri Adam Pampori Kathy Tang Jessie Tsai Diana Zhong

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

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

Our Idea… Overall idea: To modify the clinical cold storage procedure with H 2 S Global Hypothesis: Controlled delivery of H 2 S 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

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

H 2 S Protects Hearts During Ischemia from I/R Injury Mitochondria K-ATP channels K+K+ Calcium channel H2SH2S H2SH2S Ca 2+ K-ATP channel opening Hyperpolarizes membrane and reduces Ca 2+ influx Suspended animation Reduced metabolic rate preserve energy stores reduce byproducts H2SH2S ROS oxygen ROS-scavenging Directly neutralizes oxygen free-radicals Upregulates anti-oxidant defenses Adapted from: Elrod et. al 2007, Hu et. al 2007, Johansen et. al 2006

H 2 S depletion and continuous release H 2 S is depleted from solutionH 2 S is depleted from solution –NaHS releases H 2 S quickly –Tissue metabolism or escape from solution –Limited time of protection after NaHS depletion Continuous H 2 S treatmentContinuous H 2 S treatment –Direct ROS-scavenging, K-ATP channel effect throughout ischemic period –Implications for suspended animation?

Controlled Drug Delivery HydrogelsHydrogels –Synthetic or Natural –Gelatin CrosslinkingCrosslinking Size of microspheresSize of microspheres Acidic vs. basicAcidic vs. basic 10 micron microspheres distribute evenly throughout the heart via antegrade injection Source: Hoshino et. al (2006)

Heart Drug Delivery Gelatin is biocompatibleGelatin is biocompatible –Used in various applications Intracoronary vs. Intramyocardial injectionIntracoronary vs. Intramyocardial injection Antegrade vs. Retrograde Coronary injectionAntegrade vs. Retrograde Coronary injection Retrograde via aortaRetrograde via aorta –Langendorff

Specific Aim I Hydrogen Sulfide Metabolization Keeping NaHS concentration constant in solution has proven to be a difficult taskKeeping NaHS concentration constant in solution has proven to be a difficult task Objective: Do cardiomyocytes metabolize H 2 S?Objective: Do cardiomyocytes metabolize H 2 S? Methods:Methods: –After 24 hours incubation at 37°C, aqueous H 2 S levels will be measured using a Zinc Acetate assay

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

Specific Aim II: Fabricating Microspheres A sample of microspheres with an average diameter of 6.8 ± 4 microns (n=67). 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 microspheresAfter NaHS loading, zinc acetate assay will be used at different time points to determine release rate of NaHS from microspheres

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 H 2 S protective mechanisms described before Methods: – –Stored at 4 o C for eight hours, and reperfused for 30 min – –LVDP recovery, ATP content, apoptosis, CK, and H 2 S will be measured UW solution + NaHS i

Specific Aim III: Continuous H 2 S Treatment Objective: To determine how hearts stored in solution with continuous H 2 S treatment compare with hearts stored in a solution where H 2 S is depleted Hypothesis: Continuous H 2 S treatment will better preserve hearts UW solution + NaHS i UW solution + NaHS i NaHS-loaded microspheres

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 + NaHS i UW solution + NaHS i PBS-loaded microspheres

Specific Aim III: Continuous H 2 S 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 H 2 S more uniformly compared to submersion in NaHS solution – –Both continuous H 2 S treatments will better preserve hearts compared to submersion in solution where H 2 S is depleted UW solution +NaHS i UW solution + NaHS i NaHS-loaded microspheres

In Conclusion… Our new method for heart storage which will: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

Special Thanks: Dr. John FisherDr. John Fisher Dr. Agnes AzimzadehDr. Agnes Azimzadeh Dr. Lars BurdorfDr. Lars Burdorf Tom HarrodTom Harrod Dr. James WallaceDr. James Wallace Dr. Rebecca ThomasDr. Rebecca Thomas Courtenay BarrettCourtenay Barrett

Any Questions? How H2S works Specific Aim II: Microsphere Fabrication Organ Storage Today Do Cells Metabolize H 2 S? Hydrogels as a Drug Delivery Method Specific Aim III: Does Controlled Release of H2S improve heart function? Specific Aim I Effective concentration of H 2 S for storage? Team Organ Varying Release Rate Our Idea…