Transcript A method for preserving hearts with hydrogen sulfide

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 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
• 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…
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
Continued metabolism
•Accumulation of metabolic waste products
•ATP depletion
Na+
Continued cell
processes
Na channels
Ionic balance disruption
•Decrease in ATP leads to less
active ionic pumps
•Na+ and Ca 2+ accumulate
•Cell swelling
ROS
oxygen
Calcium channel
Ca 2+
Adapted from: Di Lisa et. al 2007, Jamieson et. al 2008
ATP
Lactate, protons,
hypoxanthine
Mitochondria
ROS production
•Inefficiencies in electron transport
chain lead to ROS
H2S Protects Hearts
During Ischemia from I/R Injury
ROS-scavenging
•Directly neutralizes
oxygen free-radicals
•Upregulates anti-oxidant
defenses
K+
K-ATP
channels
H 2S
H 2S
K-ATP channel opening
•Hyperpolarizes
membrane and reduces
Ca 2+ influx
ROS
H 2S
oxygen
Mitochondria
Calcium channel
Ca 2+
Adapted from: Elrod et. al 2007, Hu et. al 2007, Johansen et. al 2006
Suspended animation
•Reduced metabolic rate
•preserve energy stores
•reduce byproducts
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
• 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
• 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
• 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
Frequency (n=67)
Objective: To determine
if gelatin
microspheres can release
Microsphere
Size Distribution
NaHS in a controlled fashion
Hypothesis:
20 By varying cross-linkage, composition of the
microspheres, we will be able to control the release of NaHS
15
• After NaHS
loading, zinc acetate assay will be used at different
time points
to determine release rate of NaHS from
10
microspheres
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0
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Diameter (microns)
A sample of microspheres with an average
diameter of 6.8 ± 4 microns (n=67).
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Specific Aim III:
NaHS in UW Solution
UW
solution
UW solution
+ NaHSi
• 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
Specific Aim III:
Continuous H2S Treatment
UW solution
UW solution
+ NaHSi
+ NaHSi
NaHS-loaded
microspheres
• 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
Specific Aim III:
Gelatin Microspheres in Heart
Vasculature
UW solution
UW solution
+ NaHSi
+ NaHSi
PBS-loaded
microspheres
• 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
Specific Aim III:
Continuous H2S Release from
Heart Vasculature
UW
solution
+NaHSi
UW solution
+ NaHSi
NaHS-loaded
microspheres
• 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
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:
• Dr. John Fisher
• Dr. Agnes Azimzadeh
• Dr. Lars Burdorf
• Tom Harrod
• Dr. James Wallace
• Dr. Rebecca Thomas
• Courtenay Barrett
Any Questions?
Team Organ
Organ Storage
Today
How H2S
works
Specific
Aim I
Do Cells
Metabolize
H2S?
Effective
concentration
of H2S for
storage?
Our Idea…
Hydrogels as a
Drug Delivery
Method
Varying Release
Rate
Specific Aim II:
Microsphere
Fabrication
Specific Aim III:
Does Controlled
Release of H2S improve
heart function?