HEART PERFUSION HEART PERFUSION EXAMINATIONSEXAMINATIONS

ADVANTAGES OF ADVANTAGES OF IN VITROIN VITRO HEART PERFUSION STUDIESHEART PERFUSION STUDIES

Can be studied quickly and in large numberCan be studied quickly and in large number Highly reproducibleHighly reproducible Enables biochemical, physiological, morphological studiesEnables biochemical, physiological, morphological studies Absence of confounding effect of organs, systemic Absence of confounding effect of organs, systemic

circulation, neurohumoral factorscirculation, neurohumoral factors Drugs, hormones can be added exogenously in a controlled Drugs, hormones can be added exogenously in a controlled

mannermanner Dose-response studiesDose-response studies Can be studied for several hours Can be studied for several hours Regional/global ischemia – anoxia/hypoxia - studiesRegional/global ischemia – anoxia/hypoxia - studies Induction of arrhythmiasInduction of arrhythmias ECG – mapping and ablation of conduction pathwaysECG – mapping and ablation of conduction pathways

SPECIES FOR PERFUSIONSPECIES FOR PERFUSION

Mammalian/non-mammalian hearts (frog, bird)Mammalian/non-mammalian hearts (frog, bird) Large animal hearts:Large animal hearts:

Pig, monkey, sheep, dogPig, monkey, sheep, dog High cost, greater variability, large volumes of perfusion High cost, greater variability, large volumes of perfusion

fluids, special equipmentfluids, special equipment Most frequently studied:Most frequently studied:

Rat, rabbit, guinea pig, hamster, ferret, mouseRat, rabbit, guinea pig, hamster, ferret, mouse Transgenic technology Transgenic technology Mouse hearts: small, high heart rateMouse hearts: small, high heart rate Rat: best characterized, most frequently used, ease of Rat: best characterized, most frequently used, ease of

handlinghandling Difficulties:Difficulties:

Rat: short action potentialRat: short action potential Rabbit: anesthesiaRabbit: anesthesia Guinea pig: collateralized vasculatureGuinea pig: collateralized vasculature

HEART PERFUSION HEART PERFUSION PREPARATIONSPREPARATIONS

LANGENDORFF HEART PERFUSION LANGENDORFF HEART PERFUSION SYSTEMSYSTEM

„„WORKING-HEART” PREPARATION WORKING-HEART” PREPARATION DESCRIBED BYDESCRIBED BY NEALY NEALY

LANGENDORFF HEART LANGENDORFF HEART PREPARATIONPREPARATION

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 LANGENDORFF PERFUSION OF RAT HEARTRAT HEART

PREPARATION I.PREPARATION I.

Anesthesia:Anesthesia: Inhalation of agents (ether, halothane or metoxyflurane)Inhalation of agents (ether, halothane or metoxyflurane) Injection: (i.p., i.v.) (pentobarbitone)Injection: (i.p., i.v.) (pentobarbitone)

Anticoagulation: Anticoagulation: Heparin Heparin

Excision of the heart from the donor Excision of the heart from the donor animalanimal

Immersion of heart in cold perfusion Immersion of heart in cold perfusion solution (4solution (400C)C)

18

Cannulation of Cannulation of the heartthe heart

! Air emboli! Coronary ostia! Aortic valves

LANGENDORFF PERFUSION OF LANGENDORFF PERFUSION OF RAT HEARTRAT HEARTPREPARATION II.PREPARATION II.

Washout period for 10 minutesWashout period for 10 minutes Instrumentation:Instrumentation:

Contractile function measurements: Contractile function measurements:

--Open tip pressure transducer with intraventricular ballOpen tip pressure transducer with intraventricular ballooonon ––intraventricular pressure, heart rate monitoringintraventricular pressure, heart rate monitoring

PacePace

--bipolar silver wire electrodebipolar silver wire electrode

EECCGG

--stainless steel cannulastainless steel cannula

„„Working-Working-heart” heart”

PreparationPreparation

Advantages:-filling pressures and afterload can be controlled

Used with hearts from rats, dogs, pigs

PERFUSION TEMPERATUREPERFUSION TEMPERATURE

Near or at the normal body temperatureNear or at the normal body temperature 37.0-37.537.0-37.500CC Temperature control:Temperature control:

-Thermostatically regulated cabinet in which -Thermostatically regulated cabinet in which warm air is circulatedwarm air is circulated

- Thermostatically controlled water-jacketed - Thermostatically controlled water-jacketed systemsystem

Avoid over-heating !Avoid over-heating !

Parameters determined during Parameters determined during heart perfusion I.heart perfusion I.

MorMorphphology and vascular anatomyology and vascular anatomy Light/electron microscopyLight/electron microscopy MicrobiopsiesMicrobiopsies Fixation by perfusionFixation by perfusion

BiochemistryBiochemistry Arterio-venous differences in substrates, metabolites Arterio-venous differences in substrates, metabolites

(lactate, oxygen, proteins, enzymes)(lactate, oxygen, proteins, enzymes) Biopsies, NMR spectroscopy: on-line measurement of high Biopsies, NMR spectroscopy: on-line measurement of high

energy phosphates, metabolites, ionsenergy phosphates, metabolites, ions Microelectrodes: ions, pH, action potentialMicroelectrodes: ions, pH, action potential Delivery of vectors in gene transfer studiesDelivery of vectors in gene transfer studies

Cardiac rhythm and electrophysiologyCardiac rhythm and electrophysiology Conduction pathway mapping and selective ablationConduction pathway mapping and selective ablation

Parameters determined during Parameters determined during heart perfusion Iheart perfusion III..

Cardiac contractile functionCardiac contractile function Systolic, diastolic pressures, cardiac pump functionSystolic, diastolic pressures, cardiac pump function Echo techniques – pressure volume relationships, indices Echo techniques – pressure volume relationships, indices

of contractile functionof contractile function

PharmacologyPharmacology Various therapeutic agents, dose-response studiesVarious therapeutic agents, dose-response studies Great speed and reproducibility, drugs can be easily Great speed and reproducibility, drugs can be easily

washoutwashout

Vascular biologyVascular biology Vascular reactivity, endothelial and smooth muscle Vascular reactivity, endothelial and smooth muscle

functionfunction Interventions on coronary flow and its distributionInterventions on coronary flow and its distribution

COMPOSITION OF PERFUSION FLUIDCOMPOSITION OF PERFUSION FLUID

Krebs-HenseleitKrebs-Henseleit pH=7.4pH=7.4 NaCl:NaCl: 118.5 mM, 118.5 mM, NaHCONaHCO33: 25.0 mM, : 25.0 mM, KCl:KCl: 4.7 mM, 4.7 mM, MgSOMgSO44:: 1.2, 1.2,

KHKH22POPO44:: 1.2, 1.2, glglucoseucose:: 11.0 mM, 11.0 mM, CaClCaCl22:: 2.5 mM 2.5 mM Calcium?Calcium? Calcium and phosphate?Calcium and phosphate? Glucose?Glucose? Fatty acids?Fatty acids? Edema?Edema?

Filtration: 5 μm filterFiltration: 5 μm filter

OXYGEN DELIVERY DURING OXYGEN DELIVERY DURING PERFUSIONPERFUSION

Perfusion with asanguinous perfusion fluidsPerfusion with asanguinous perfusion fluids Perfusion with bloodPerfusion with blood Perfusion with oxygen-carrying hemoglobin Perfusion with oxygen-carrying hemoglobin

substitutessubstitutes

Gassed perfusion solution:Gassed perfusion solution:95% oxygen + 5% CO95% oxygen + 5% CO22

Required to the correct pHRequired to the correct pH In case of addition of fatty acids or proteins In case of addition of fatty acids or proteins

membrane oxigenator is recommendedmembrane oxigenator is recommended

Parabiotic preparation with support ratParabiotic preparation with support rat

BLOOD BLOOD PERFUSIONPERFUSION

-Decrease of hematocrit to 28-30% with Gelofusine solution-Ventilation of support rat with 95% oxygen-Control of body temperature, blood pressure, breathing

Less edemaStable heart functionAlmost physiological coronary flow rateBlood elements (neu)

Support animal?

ERYTHROCYTE ERYTHROCYTE PERFUSIONPERFUSION

Washed red blood cellsWashed red blood cells Membrane oxygenatorMembrane oxygenator Hematocrit of 25-40% Hematocrit of 25-40% Blood cells from Blood cells from

different species – sheepdifferent species – sheep

StabilityLess edemaImmunological reactions

Ischemia – blood supply is less than the required amountReperfusion – restoration of blood flow after coronary occlusionIschemic preconditioning – short repeated ischemic episodes evokes the preconditioning of the heart (that means cardioprotection during the next longer ischemic period )

3131P NMRP NMR SPECTROSCOPY/ SPECTROSCOPY/LANGENDORFF HEART PERFUSIONLANGENDORFF HEART PERFUSION

ENERGY METABOLISM IN THE STRIATED AND HEART ENERGY METABOLISM IN THE STRIATED AND HEART MUSCLEMUSCLE

Striated muscleStriated muscle Heart muscleHeart muscleMitochondriaMitochondria ++++ +++++ +++++

Basic act.Basic act. FFA (adipose tissue)FFA (adipose tissue)

keton bodies (liver)keton bodies (liver)

Medium act.Medium act. FFAFFA FFA +++FFA +++

keton bodies keton bodies blood glucose +blood glucose +

blood glucoseblood glucose keton bodies +keton bodies +

Max. act.Max. act. +fermentation of glycogen+fermentation of glycogen + creatine-phosphate + creatine-phosphate +creatine-phosphate+creatine-phosphate

Energy metabolism.Energy metabolism. mostly aerobicmostly aerobic fully aerobicfully aerobic

max. act. - anaerobicmax. act. - anaerobic

Energy poolsEnergy pools glycogenglycogen creatine-phosphate creatine-phosphate

creatine-phosphate creatine-phosphate (glycogen(glycogen ))

CREATINE/PHOSPHOCREATINE CREATINE/PHOSPHOCREATINE TRANSFORMATIONTRANSFORMATION

PiPCr

γ-P α-Pβ-PATP

IR+G

IR

I

N

REPRESENTATIVE REPRESENTATIVE 3131P NMR P NMR SPECTUMS OF HIGH ENERGY SPECTUMS OF HIGH ENERGY

PHOSPHATESPHOSPHATES

0

20

40

60

80

100

120

3 6 9 3 15 30 3 6 9 12 15

perfúziós idő (perc)

CrP

(nor

mox

iás é

rték

%-a)

Kontroll IR IR+L-2286 10 µM IR+L-2286 20 µM

RECOVERY OF CREATINE PHOSPHATE RECOVERY OF CREATINE PHOSPHATE AFTER ISCHEMIA-REPERFUSION IN AFTER ISCHEMIA-REPERFUSION IN LANGENDORFF PERFUSED HEARTLANGENDORFF PERFUSED HEART

RECOVERY OF CREATINE PHOSPHATE RECOVERY OF CREATINE PHOSPHATE AFTER ISCHEMIA-REPERFUSION IN AFTER ISCHEMIA-REPERFUSION IN LANGENDORFF PERFUSED HEARTLANGENDORFF PERFUSED HEART

DETERMINATION OF HEART FUNCTIONDETERMINATION OF HEART FUNCTION

Insertion of a latex balloon into the left ventInsertion of a latex balloon into the left ventrricleicle End-diastolic pressure 8-12 End-diastolic pressure 8-12 mmmmHgHg Selection of hearts: on the basis of the stability of Selection of hearts: on the basis of the stability of

high-energy phosphates (assessed by NMR)high-energy phosphates (assessed by NMR) Normoxia 15min, ischemia 25 min, reperfusion 45 Normoxia 15min, ischemia 25 min, reperfusion 45

minmin Functional data:Functional data:

LVEDP=left ventricular end-diastolic pressureLVEDP=left ventricular end-diastolic pressure LVDP=levt ventricular developed pressureLVDP=levt ventricular developed pressure RPP=rate pressure productRPP=rate pressure product HR=heart rateHR=heart rate dP/dtdP/dt

9.25 Hgmm

89.25 Hgmm

0 msec 1000 msec

8.5 Hgmm

25.5 Hgmm

0 msec 1000 msec

LVDP=levt LVDP=levt ventricular ventricular developed pressuredeveloped pressure

LVEDP=left ventricular LVEDP=left ventricular end-diastolic pressureend-diastolic pressure

HR=heart rateHR=heart rate

RPP=rate RPP=rate pressure pressure productproduct

dP/dtdP/dt

DOXORUBICIN-INDUCED DETERIORATION OF HEART FUNCTION