Introduction
Randomization, blinding, power analysis and statistics
Study design, randomization, blinding, power analysis, statistics
Exploratory vs. prospective studies
Study design
Selection of variables/endpoints
Number of experimental groups
Inclusion and exclusion criteria
Effect size and sample size
Randomization
Data analysis
Parametric vs. non-parametric statistics
Missing values and outliers
Data presentation and statistical analysis
Use of statistical analyses in experimental and clinical cardioprotection studies
Specific guidance for power calculations and statistics in clinical myocardial infarction trials
Specific guidance for power calculations and statistics in clinical cardiac surgery trials
Isolated cardiomyocytes
Protocol for the isolation and culture of adult rat or mouse ventricular cardiomyocytes
General aspects
Preparation of media and reagents for the isolation
Powell medium (1 L)
Calcium chloride stock (CaCl2)
Media
Materials
Cell isolation
Cultivation of cardiomyocytes
Some specific experimental cardiomyocyte models
Membrane-permeabilized cardiomyocytes
End-to-end connected cardiomyocytes
Use of isolated cardiomyocytes
Isolated cardiomyocytes for pathophysiological studies
Mitochondrial de- and re-energization during ischemia/reperfusion
Pelleted freshly isolated cardiomyocytes
Modes of cell death and cellular viability assessment
Cardiomyocyte necrosis
Other forms of cell death
Limitations of the cardiomyocyte cell model
Future perspectives of isolated cardiomyocyte models
Involvement of mitochondria in cardiomyocyte injury
Isolation of subsarcolemmal and interfibrillar mitochondria from cardiac tissue
Isolation of subsarcolemmal mitochondria (SSM) only
Isolation of both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria
Mitochondrial parameters and experimental models
Preparation | Parameter |
---|---|
Isolated mitochondria | Oxygen consumption |
ATP synthesis | |
Redox changes (NAD and FAD) | |
ROS formation | |
ΔΨm (quantitative) | |
Matrix volume | |
Ion movements | |
Isolated cells | Oxygen consumption |
Redox changes (NAD and FAD) | |
ROS formation | |
ΔΨm (semiquantitative) | |
Matrix volume | |
Ion movements (reliable only with genetically encoded probes) | |
Whole Hearts | Oxygen consumption |
(severe) mitochondrial ROS formation | |
ATP content |
Preparation | Parameter |
---|---|
Isolated mitochondria (or permeabilized cells) | Swelling |
Ca2+ retention capacity (CRC) | |
Permeability to solutes | |
CsA inhibitable changes | |
Isolated cells | Calcein redistribution |
Swelling | |
CsA inhibitable changes | |
Whole hearts | Mitochondrial NAD depletion |
Mitochondrial accumulation of tritiated deoxyglucose | |
CsA inhibitable changes |
Major methods for evaluating mitochondrial function in vivo
Oxygen consumption
Mitochondrial membrane potential
Mitochondrial calcium homeostasis
Permeability transition
Calcein release
Mitochondrial reactive oxygen species
Use of genetically encoded fluorescent probes, exemplified by HyPer
Major methods for evaluating mitochondrial function in vitro
Oxygen consumption
Mitochondrial swelling
Calcium retention capacity
Measurement of reactive oxygen species by Amplex Red
Measurement of mPTP opening in whole hearts
Issues and limitations in evaluating mitochondrial parameters
Cell types other than cardiomyocytes
HL-1 cardiomyocytes
H9c2 cells
Stem cells
Endothelial cells
Quantitation of infarct size by morphology, biomarkers and imaging
Common key issues for infarct size measurement
Why measure infarct size?
Infarct size according to models
Animal models
Cell death after hypoxia reoxygenation
Clinical settings
Measuring reperfusion infarction: a critical unsolved issue
Infarct size as a function of area at risk
Reference techniques
Histology
Imaging
Biomarkers
Practical summary: your best way to measure infarct size
Isolated perfused hearts
Species and strain
The Langendorff apparatus
Harvesting and cannulation of the heart
Perfusate buffer
Instrumentation
Coronary perfusion
Constant perfusion pressure model
Constant coronary flow model
Working heart model
Temperature monitoring and control
Electrocardiogram (ECG)
Pacing
Ischemia/reperfusion
Stabilization
Ischemia
Global ischemia
Regional ischemia
Reperfusion
Exclusion criteria
Parameter | Mouse heart | Rat heart (250–400 g bw) |
---|---|---|
Baseline (prior to any intervention, e.g. IPC) | ||
Time to perfusion | > 3 min | > 3 min |
Coronary flow | < 2 or > 5.5 ml/min | < 10 or > 28 ml/min |
Arrhythmias | > 10 ectopics during 10 min baseline recordings (VT or VF should not occur) | > 10 ectopics during 10 min baseline recordings (VT or VF should not occur) |
Heart rate | < 300 bpm | < 200 or > 400 bpm |
Left ventricular end-diastolic pressure | < 5 or > 10 mmHg | < 5 or > 10 mmHg |
Left ventricular developed pressure | < 60 or > 140 mmHg | < 70 or > 140 mmHg |
Temperature | 37 ± 0.5 °C (< 36 °C or > 38 °C for > 1 min) | 37 ± 0.5 °C (< 36 °C or > 38 °C for > 1 min) |
Reperfusion | ||
Coronary flow | ≤ ischemic flow | ≤ ischemic flow |
Arrhythmia duration (ventricular tachycardia or fibrillation) | > 2 min (intervene immediately—flicking, cold buffer or KCl) | > 2 min (intervene immediately—flicking, cold buffer or KCl) |
Heart rate | < 150 bpm (irrelevant if pacing) | |
Infarct criteria | ||
Area at risk | n/a for global ischemia | n/a for global ischemia; for regional ischemia: < 40 or > 70% of ventricular tissue |
Measurement of injury by contractile function
Measurement of infarct size by TTC staining
Measurement of infarct size by necrotic marker proteins in perfusate
Harvesting of left ventricular tissue for western blot analysis
Hybrid studies using a cross-species approach
Transfer of circulating cardioprotective mediators from humans to rabbit hearts
Transfer of circulating cardioprotective mediators from humans to mouse hearts
Transfer of extracellular vesicles between animals
Small animal hearts in situ
Species (mouse, rat, rabbit)
Strain, sex, age, weight
Mouse
Rat
Rabbit
Housing and feeding conditions
Mouse
Rat
Rabbit
Pre-operative preparation
Mouse
Rat
Rabbit
Anesthesia
Mouse
Rat
Surgery
Tracheotomy, intubation and temperature
Mouse
Rat
Rabbit
Thoracotomy, induction of ischemia and reperfusion
Mouse
Rats
Rabbit
Duration of ischemia and reperfusion
Mouse
Rat
Rabbit
Endpoints
Determination of infarct size
Mouse
Rat
Rabbit
Larger mammal hearts in situ
General considerations
Animals
Species and strains
Age and sex
Pre-medication and anesthesia
Experimental preparation and monitoring
Induction of myocardial ischemia: closed vs. open-chest models
Hemodynamics, myocardial blood flow and regional myocardial function
Blood samples, microdialysis, tissue samples/biopsies
Imaging
Ischemic conditioning
Pharmacologic pre-, per- or postconditioning
Area at risk and areas of no reflow
Euthanasia
Infarct size
Exclusion criteria
The human right atrial trabeculae model of simulated ischemia and reperfusion
Outline of model
Sample collection and transport
Dissection of atrial trabeculae
Simulation of ischemia and reperfusion
Recovery of function
Critical analysis of the model
Unbiased “omics” technologies for mechanistic studies and identification of molecular targets
Genomics and epigenomics
Transcriptomics and epitranscriptomics
Sampling and storage
RNA isolation and quality check
Data evaluation
Validation of transcriptomic data
Proteomics
Metabolomics
Multiomics
Proof-of-concept clinical phase I/II trials with surrogate endpoints
Surrogate endpoints: peripheral circulation
Surrogate endpoints: coronary circulation
ST segment elevation myocardial infarction
Patients
Age |
Gender |
Risk factors |
Smoking |
Comorbidities |
Dyslipidemia |
Hypertension |
Diabetes mellitus |
History of coronary artery disease |
Previous MI |
Preinfarction angina within 1 week before the index event |
Infarct-related artery |
Left anterior descending artery |
Circumflex artery |
Right coronary artery |
Not identifiable |
Number of other vessels with clinically significant disease |
Symptom-to-balloon time |
TIMI flow grade at admission |
0/I/II/III |
Stenting of culprit lesion by pPCI |
TIMI flow grade after procedure |
0/I/II/III |
History of medication |
Beta-blockers |
Statins |
ACE-inhibitors |
AT-blockers |
Calcium channel blockers |
Long acting nitrates |
Metformin |
Insulin |
Other antidiabetic drugs |
Drugs given at time of pPCI |
Heparin |
Aspirin |
P2Y12-inhibitors |