A Method for A Method for Preserving Hearts Preserving Hearts

with Hydrogen with Hydrogen SulfideSulfide

Spring 2009 Proposal Spring 2009 Proposal DefenseDefense

Team Members (in alphabetical Team Members (in alphabetical order)order)

Elizabeth Chen Elizabeth Chen

Charles ChiangCharles Chiang

Elyse GeibelElyse Geibel

Steven Geng Steven Geng

Stevephen HungStevephen Hung

Kathy JeeKathy Jee

AAngela Leengela Lee

Christine LimChristine Lim

Sara Moghaddam-Sara Moghaddam-Taaheri Taaheri

Adam Pampori Adam Pampori

Kathy TangKathy Tang

Jessie TsaiJessie Tsai

Diana ZhongDiana Zhong

Organ Shortage: Societal Organ Shortage: Societal ProblemProblem

• Though 110,000 people Though 110,000 people are on organ donor are on organ donor lists, only 77 receive lists, only 77 receive transplants dailytransplants daily

• Storage time is limited Storage time is limited to 4 hoursto 4 hours

• Preservation-induced Preservation-induced injury is a major injury is a major contributing factor to contributing factor to early graft dysfunction early graft dysfunction in patientsin patients

Organ Storage TodayOrgan Storage Today

• Static Cold StorageStatic Cold Storage– University of Wisconsin SolutionUniversity of Wisconsin Solution– No significant improvements despite No significant improvements despite

two decades of researchtwo decades of research• Machine ReperfusionMachine Reperfusion

– Organ Care SystemOrgan Care System– Effective, but extremely expensiveEffective, but extremely expensive

Our Idea…Our Idea…

Overall ideaOverall idea: To modify the clinical : To modify the clinical cold storage procedure with Hcold storage procedure with H22SS

Global HypothesisGlobal Hypothesis: Controlled : Controlled delivery of Hdelivery of H22S throughout the heart S throughout the heart using gelatin microspheres will using gelatin microspheres will induce protective effects and a state induce protective effects and a state of hibernation that will prolong heart of hibernation that will prolong heart viability and reduce ischemia-viability and reduce ischemia-reperfusion injury in transplantsreperfusion injury in transplants

Cold Ischemia Leads to I/R Cold Ischemia Leads to I/R InjuryInjury

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

HH22S Protects Hearts S Protects Hearts During Ischemia from I/R During Ischemia from I/R

InjuryInjury

Mitochondria

K-ATP channels

K+

Calcium channel

H2S

H2S

Ca 2+

K-ATP channel opening•Hyperpolarizes membrane and reduces Ca 2+ influx

Suspended animation•Reduced metabolic rate•preserve energy stores•reduce byproducts

H2S

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

HH22S depletion and continuous S depletion and continuous releaserelease

• HH22S is depleted from solutionS is depleted from solution– NaHS releases HNaHS releases H22S quicklyS quickly– Tissue metabolism or escape from Tissue metabolism or escape from

solutionsolution– Limited time of protection after NaHS Limited time of protection after NaHS

depletiondepletion

• Continuous HContinuous H22S treatmentS treatment– Direct ROS-scavenging, K-ATP channel Direct ROS-scavenging, K-ATP channel

effect throughout ischemic periodeffect throughout ischemic period– Implications for suspended animation?Implications for suspended animation?

Controlled Drug DeliveryControlled Drug Delivery

• HydrogelsHydrogels– Synthetic or Synthetic or

NaturalNatural– GelatinGelatin

• CrosslinkingCrosslinking• Size of Size of

microspheresmicrospheres• Acidic vs. basicAcidic vs. basic

10 micron microspheres distribute evenly throughout the heart via antegrade injectionSource: Hoshino et. al (2006)

Specific Aim ISpecific Aim IHydrogen Sulfide Hydrogen Sulfide MetabolizationMetabolization

• Keeping NaHS concentration Keeping NaHS concentration constant in solution has proven to be constant in solution has proven to be a difficult taska difficult task

• Objective: Do cardiomyocytes Objective: Do cardiomyocytes metabolize Hmetabolize H22S?S?

• Methods:Methods:– After 24 hours incubation at 37°C, After 24 hours incubation at 37°C,

aqueous Haqueous H22S levels will be measured S levels will be measured using a Zinc Acetate assayusing a Zinc Acetate assay

Specific Aim ISpecific Aim INaHS Dosage TestNaHS Dosage Test

• Inconsistent reports of dosagesInconsistent reports of dosages• Objective: Objective: What is the most What is the most

effective concentration of NaHS effective concentration of NaHS for storage solutions?for storage solutions?

• Methods:Methods:– 0 to 150µM NaHS in UW solution0 to 150µM NaHS in UW solution– Biopsy LV at 2, 4, 6, 8 hoursBiopsy LV at 2, 4, 6, 8 hours

• ATP, Apoptosis, Creatine Kinase assaysATP, Apoptosis, Creatine Kinase assays• Langendorff Perfusion ColumnLangendorff Perfusion Column

Specific Aim II: Specific Aim II: Fabricating MicrospheresFabricating Microspheres

Microsphere Size Distribution

0

5

10

15

20

Diameter (microns)

Freq

uenc

y (n=

67)

A sample of microspheres with an average diameter of 6.8 ± 4 microns (n=67).

Objective:Objective: To determine if gelatin To determine if gelatin microspheres can release NaHS in a microspheres can release NaHS in a controlled fashioncontrolled fashion

Hypothesis: By varying cross-linkage, composition Hypothesis: By varying cross-linkage, composition of the microspheres, we will be able to control of the microspheres, we will be able to control the release of NaHSthe release of NaHS

• After NaHS loading, zinc acetate assay will be After NaHS loading, zinc acetate assay will be used at different time points to determine release used at different time points to determine release rate of NaHS from microspheresrate of NaHS from microspheres

Specific Aim III: Specific Aim III: NaHS in UW SolutionNaHS 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

UW solution

+ NaHSi

Specific Aim III: Specific Aim III: Continuous HContinuous H22S Treatment S 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

Specific Aim III: Specific Aim III: Gelatin Microspheres in Heart Gelatin Microspheres in Heart

VasculatureVasculature

• 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

Specific Aim III: Specific Aim III: Continuous HContinuous H22S Release from S Release from

Heart VasculatureHeart 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

In Conclusion…In Conclusion…

• Our new method for heart storage Our new method for heart storage which will: which will: – Reduce ischemia-reperfusion injury and Reduce ischemia-reperfusion injury and

radical oxidative species and improve radical oxidative species and improve heart functionheart function

– But will also be easily applicable to But will also be easily applicable to today’s organ transport methodstoday’s organ transport methods

Special Thanks: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?Any Questions?

How H2S works

Specific Aim II:Microsphere Fabrication

Organ StorageToday

Do Cells Metabolize

H2S?

Hydrogels as a Drug Delivery

Method

Specific Aim III:Does Controlled

Release of H2S improve heart function?

SpecificAim I

Effective concentration

of H2S for storage?

Team Organ

Varying ReleaseRate

Our Idea…