HEART PERFUSION
EXAMINATIONS
ADVANTAGES OF IN VITRO
HEART PERFUSION STUDIES
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Can be studied quickly and in large number
Highly reproducible
Enables biochemical, physiological, morphological studies
Absence of confounding effect of organs, systemic circulation,
neurohumoral factors
Drugs, hormones can be added exogenously in a controlled
manner
Dose-response studies
Can be studied for several hours
Regional/global ischemia – anoxia/hypoxia - studies
Induction of arrhythmias
ECG – mapping and ablation of conduction pathways
SPECIES FOR PERFUSION
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Mammalian/non-mammalian hearts (frog, bird)
Large animal hearts:
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Most frequently studied:
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Pig, monkey, sheep, dog
High cost, greater variability, large volumes of perfusion
fluids, special equipment
Rat, rabbit, guinea pig, hamster, ferret, mouse
Transgenic technology
Mouse hearts: small, high heart rate
Rat: best characterized, most frequently used, ease of
handling
Difficulties:
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Rat: short action potential
Rabbit: anesthesia
Guinea pig: collateralized vasculature
HEART PERFUSION
PREPARATIONS
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LANGENDORFF HEART PERFUSION
SYSTEM
„WORKING-HEART” PREPARATION
DESCRIBED BY NEALY
LANGENDORFF HEART
PREPARATION
MODES OF PERFUSATE DELIVERY:
-constant flow rate
-constant hydrostatic pressure
-Shattock electrical feedback system
Used with hearts from mice,
rats, guinea pigs, rabbits
LANGENDORFF PERFUSION OF
RAT HEART
PREPARATION I.
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Anesthesia:
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Inhalation of agents (ether, halothane or metoxyflurane)
Injection: (i.p., i.v.) (pentobarbitone)
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Anticoagulation:
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Excision of the heart from the donor animal
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Immersion of heart in cold perfusion solution
(40C)
Heparin
18
Cannulation of
the heart
! Air emboli
! Coronary ostia
! Aortic valves
LANGENDORFF PERFUSION OF
RAT HEART
PREPARATION II.
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Washout period for 10 minutes
Instrumentation:
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Contractile function measurements:
-Open tip pressure transducer with intraventricular balloon –
intraventricular pressure, heart rate monitoring
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Pace
-bipolar silver wire electrode
ECG
-stainless steel cannula
„Workingheart”
Preparation
Advantages:
-filling pressures and
afterload can be controlled
Used with hearts from rats,
dogs, pigs
PERFUSION TEMPERATURE
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Near or at the normal body temperature
37.0-37.50C
Temperature control:
-Thermostatically regulated cabinet in which warm air
is circulated
- Thermostatically controlled water-jacketed system
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Avoid over-heating !
Parameters determined during
heart perfusion I.
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Morphology and vascular anatomy
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Biochemistry
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Arterio-venous differences in substrates, metabolites
(lactate, oxygen, proteins, enzymes)
Biopsies, NMR spectroscopy: on-line measurement of high
energy phosphates, metabolites, ions
Microelectrodes: ions, pH, action potential
Delivery of vectors in gene transfer studies
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Conduction pathway mapping and selective ablation
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Light/electron microscopy
Microbiopsies
Fixation by perfusion
Cardiac rhythm and electrophysiology
Parameters determined during
heart perfusion II.
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Cardiac contractile function
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Pharmacology
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Systolic, diastolic pressures, cardiac pump function
Echo techniques – pressure volume relationships, indices of
contractile function
Various therapeutic agents, dose-response studies
Great speed and reproducibility, drugs can be easily washout
Vascular biology
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Vascular reactivity, endothelial and smooth muscle function
Interventions on coronary flow and its distribution
COMPOSITION OF PERFUSION FLUID
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Krebs-Henseleit
pH=7.4
NaCl: 118.5 mM, NaHCO3: 25.0 mM, KCl: 4.7 mM, MgSO4: 1.2,
KH2PO4: 1.2, glucose: 11.0 mM, CaCl2: 2.5 mM
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Calcium?
Calcium and phosphate?
Glucose?
Fatty acids?
Edema?
Filtration: 5 μm filter
OXYGEN DELIVERY DURING
PERFUSION
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Gassed perfusion solution:
95% oxygen + 5% CO2
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Perfusion with asanguinous perfusion fluids
Perfusion with blood
Perfusion with oxygen-carrying hemoglobin
substitutes
Required to the correct pH
In case of addition of fatty acids or proteins
membrane oxigenator is recommended
Parabiotic preparation with support rat
BLOOD
PERFUSION
-Decrease of hematocrit to 2830% with Gelofusine solution
-Ventilation of support rat with
95% oxygen
-Control of body temperature,
blood pressure, breathing
Less edema
Stable heart function
Almost physiological coronary
flow rate
Blood elements (neu)
Support animal?
ERYTHROCYTE
PERFUSION
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Washed red blood cells
Membrane oxygenator
Hematocrit of 25-40%
Blood cells from
different species – sheep
Stability
Less edema
Immunological reactions
31P
NMR SPECTROSCOPY/
LANGENDORFF HEART PERFUSION
Ischemia – blood supply is less than the required amount
Reperfusion – restoration of blood flow after coronary occlusion
Ischemic preconditioning – short repeated ischemic episodes
evokes the preconditioning of the heart (that means
cardioprotection during the next longer ischemic period )
ENERGY METABOLISM IN THE STRIATED AND HEART
MUSCLE
Striated muscle
Mitochondria
Basic act.
++
Heart muscle
+++++
FFA (adipose tissue)
keton bodies (liver)
Medium act.
FFA
FFA +++
keton bodies
blood glucose
blood glucose +
keton bodies +
Max. act.
+fermentation of glycogen
+creatine-phosphate
+ creatine-phosphate
Energy metabolism.
mostly aerobic
fully aerobic
max. act. - anaerobic
Energy pools
glycogen
creatine-phosphate
creatine-phosphate
(glycogen )
CREATINE/PHOSPHOCREATINE
TRANSFORMATION
REPRESENTATIVE 31P NMR
SPECTUMS OF HIGH ENERGY
PHOSPHATES
Pi
PCr
γ-P
α-P
β-P
ATP
IR+G
IR
I
N
CrP (norm oxiás é rté k %-a)
RECOVERY OF CREATINE PHOSPHATE
AFTER ISCHEMIA-REPERFUSION IN
LANGENDORFF PERFUSED HEART
120
100
80
60
40
20
0
3
6
9
3
15
30
3
6
9
perfúziós idő (perc)
Kontroll IR
IR+L-2286 10 µM
IR+L-2286 20 µM
12
15
DETERMINATION OF HEART
FUNCTION
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Insertion of a latex balloon into the left ventricle
End-diastolic pressure 8-12 mmHg
Selection of hearts: on the basis of the stability of
high-energy phosphates (assessed by NMR)
Normoxia 15min, ischemia 25 min, reperfusion 45 min
Functional data:
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LVEDP=left ventricular end-diastolic pressure
LVDP=levt ventricular developed pressure
RPP=rate pressure product
HR=heart rate
dP/dt
LVDP=levt
ventricular
developed pressure
89.25 Hgmm
HR=heart rate
RPP=rate
pressure
product
dP/dt
9.25 Hgmm
0 msec
1000 msec
25.5 Hgmm
8.5 Hgmm
0 msec
1000 msec
LVEDP=left ventricular
end-diastolic pressure
DOXORUBICIN-INDUCED
DETERIORATION OF
HEART FUNCTION