A method for preserving hearts with hydrogen sulfide
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A Method for Preserving Hearts with Hydrogen Sulfide. Spring 2009 Proposal Defense. 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

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A Method for Preserving Hearts with Hydrogen Sulfide

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A method for preserving hearts with hydrogen sulfide

A Method for Preserving Hearts with Hydrogen Sulfide

Spring 2009 Proposal Defense


Organ shortage societal problem

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


Organ storage today

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


Our idea

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


Cold ischemia leads to i r injury

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


H 2 s protects hearts during ischemia from i r injury

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


H 2 s depletion and continuous release

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?


Controlled drug delivery

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)


Specific aim i hydrogen sulfide metabolization

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


Specific aim i nahs dosage test

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


Specific aim ii fabricating microspheres

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).


Specific aim iii nahs in uw solution

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


Specific aim iii continuous h 2 s treatment

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


Specific aim iii gelatin microspheres in heart vasculature

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


Specific aim iii continuous h 2 s release from heart vasculature

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


In conclusion

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


Special thanks

Special Thanks:

Dr. John Fisher

Dr. Agnes Azimzadeh

Dr. Lars Burdorf

Tom Harrod

Dr. James Wallace

Dr. Rebecca Thomas

Courtenay Barrett


Any questions

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?


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