From myocyte isolation to data acquisition, analysis, and post-analysis plotting, join Dr. Michiel Helmes and Dr. Diederik Kuster as they demonstrate best practices and new techniques in high-content, higher throughput investigations of excitation-contraction coupling in isolated cardiomyocytes.
During this 60 minute live webinar, Michiel Helmes and Diederik Kuster will deliver a comprehensive how-to demonstration of higher throughput excitation-contraction coupling investigations with isolated cardiomyocytes.
Characterizing excitation-contraction coupling in isolated cardiac myocytes has been essential to our understanding of heart function. Historically these studies have been constrained by lower throughput data collection and limited sample sizes. Because isolated myocytes display a high degree of functional variability, acquiring data from more myocytes is required for greater accuracy and statistical confidence.
In this webinar, we will demonstrate important aspects of data collection from myocyte isolation to precision data acquisition, data analysis, and post-analysis interpretation. We will focus on how to get the most out of every isolation, how to collect quality data consistently, why statistical power matters, and how to get statistically meaningful data in hours.
Key Topics Include:
Get more from less: learn to maximize data from each animal
Quality and quantity: best practices for data acquisition
Knowledge is power: understand what your data means and how to interpret it
Go beyond numbers: see how to get automated, same-day post-analysis data plotting and processing
Call Girls in Mayapuri Delhi 💯Call Us 🔝9953322196🔝 💯Escort.
A Comprehensive How-To Demonstration of Higher Throughput Excitation-Contraction Coupling Investigations
1. A Comprehensive How-To
Demonstration of Higher
Throughput Excitation-Contraction
Coupling Investigations
Michiel Helmes, PhD
Co-CEO
IonOptix
Diederik Kuster, PhD
Assistant Professor
Department of Physiology
Amsterdam UMC
2. Experts present best practices and new techniques
in high-content, higher throughput investigations of
excitation-contraction coupling in isolated
cardiomyocytes
A Comprehensive How-To
Demonstration of Higher
Throughput Excitation-Contraction
Coupling Investigations
3. A Comprehensive How-To
Demonstration of Higher
Throughput Excitation-Contraction
Coupling Investigations
Michiel Helmes, PhD
Co-CEO
IonOptix
Copyright 2020 M. Helmes and InsideScientific. All Rights Reserved.
4. What 1 day of work can look like
Mouse N=1 ,n =25
5. Overview of the webinar
• Introduction
• Isolated cardiac myocytes as research model
• Importance and necessity of HTS in cardiac research
• Timeline of a typical full experiment
• Cardiomyocyte isolation
• Data acquisition
• Data analysis
• Simplified data export and plotting
• Example of published results
6. 1970s: first successful
isolation of calcium tolerant
intact myocytes.
For reference, see Brady, in
PHYSIOLOGICAL REVIEWS Vol.
71, No. 2, 1991
Powell ‘76
Cardiac myocytes: the smallest functional unit of the heart
7. What Contractility and Calcium tell us
about cardiac physiology
Interrogating several targetable elements involved in pathophysiological processes
Eisner et al 2017
8. The Technique Is Well-established But There
Are Pros And Cons
Pros
• It is a great assay to study whether drugs, disease, or mutations
influencing excitation contraction coupling
Cons
• Quality of cell isolation has a strong effect on results
• Cell numbers: Throughput is too low to detect subtle differences and
too low to screen compounds
9. • Conventional methods are time consuming
• Conflict between reducing the number of animals while needing to increase n
• Impactful with large animal (ex. porcine, canine models)
• Ability to screen a higher number of compounds or treatment combinations
• Reduce cost of testing compounds and refined pathway targeting
• Advanced Statistics
• Look at the distribution in a population, cluster analysis → Sikkel et al 2017
Importance and necessity of increased throughput in
cardiac myocyte research
10. Effect of Automation on a project
• Improved throughput
• Computers don’t get tired
• Rapid repeated time points
• Taking out experimenter bias
• Blinded experiments by default
• Automated analysis
• Rapid feedback allowing to correct a
project
11. Data acquisition using the MultiCell HTS
• Fast measurements at 250 Hz
• Sarcomere and Fura-2 AM
• Real time image processing: WYSIWYG
• Fast movement of objective not of sample
• No moving solutions, 50ms to next field of view
• Light tight and temperature controlled
• Fully automated cell finding
• >80% good measurements
• Can be adapted to several different dyes
12. Simultaneous High-throughput Ca2+, Voltage
And Contractile Measurements
Wistar rats, Rhod-2/AM + FluoVolt
Full case study available at ionoptix.com
Data courtesy of Patrick Schönleitner, IonOptix
13. A project in a day
Monday Tuesday Wednesday Thursday Friday Saturday
Morning Planning experiments 1st Isolation 2nd Isolation
Analysis, graphing and
writing
Travel Conference
Afternoon Preparing solutions MultiCell MultiCell
Finishing touches and
printing
14. Timeline of a typical experiment
• 9:00 isolation
• Preparation of solutions, setting up equipment, isolation proper
• 11:30 Cell Plating
• Setting up MultiCell and Pump System
• 12:30 Cells Ready for Loading
• Preparing the system, perfusion and dye loading
• 13:15 Dish 1: poor loading, redo the loading dish 2
• 14:00 Dish 2: still not great but…
• Measure 25 cells with manual selection →12 min
• Add isoproterenol incubate 1min
• Measure again → 5 min
• 15:00 – 17:00 Dish 3 & 4 cells start to deteriorate
• 17:00 – 17:30 Data analysis and graphing
15. Timeline of a typical experiment, again
• 9:00 isolation
• Preparation of solutions, setting up equipment, isolation proper
• 11:30 Cell Plating
• Setting up MultiCell and Pump System
• 12:30 Cells Ready for Loading
• Preparing the system, perfusion and dye loading
• 13:15 Dish 1: poor loading
• 14:00 Dish 2: somewhat better loading
• 14:30 Dish 3: victory, good loading (Fura-4)
• 15:15 Dish 4: good loading again, Fura-2 LR
• 16:00 – 16:30 Data analysis and graphing
25. What the real time measurement looks like
Manually find and record 25 cells + background = 12 min Re-record = 3 min
Add Iso 50nM
2 1/2 min incubationPause in recording
28. Can we do >1000 myocytes/day?
• Non-repeated measures, decent quality cells, proper density plating, 5
seconds measurements: 18 seconds per cell (on average 13 sec for cell
finding)
• Theoretically 200 cells per hour
• Overhead (dish changes, compound addition, etc) reduces this number
• In practice 125-150 cells per hour
• For murine myocytes, no calcium, we now average 1000 cells/day
29. High-throughput Ca2+ And Contractile Measurements
In Large Animals
Full case study available at ionoptix.com
Large Animal (N=2, n=91), Fura-2/AM
Multicell Lite =
Classical C&C
+ motorized stage
+ same software as MultiCell
30. Juni et al 2019 Effect of Empagliflozin on Endothelial Cell-Cardiomyocyte Interaction
1st Published Results Using High-Throughput System
31. %ShortBaseline Contra V Relax V T to Peak 50% relax Tau
Large-Scale Contractility Measurements
Reveal Large Atrioventricular and Subtle
Interventricular Differences in Cultured
Unloaded Rat Cardiomyocytes
32. 2.4 mM Omecamtiv Mecarbil
Approaches to High-Throughput
Analysis of Cardiomyocyte Contractility
33. • HTS increases the yield per animal
• Reduces number of animals needed
• Allows new experimental strategies, compound testing
• Rapid feedback on project progress
• Cell preparation is still the main limiting step requiring expertise and
experience
Summary
34. Next…
• Support for multiwell dishes
• Full climate control
• iPSC derived cardiomyocytes
• Cloud based version of analysis
36. Application of MultiCell
Measurements
Copyright 2020 D. Kuster and InsideScientific. All Rights Reserved.
P H Y S I O LO G Y
Diederik Kuster, PhD
Assistant Professor
Department of Physiology
Amsterdam UMC
37. Application of the MultiCell Measurements
• Parthenolide treatment in hypertrophic cardiomyopathy (traditional
approach)
• Contractile differences between CMs from different regions from the
heart (initial foray into large scale measurements)
• Unbiased approach to identify modulators of contraction/relaxation
(compound screening)
38. Hypertrophic cardiomyopathy
• HCM has a prevalence of 1:200-500
• Hypertrophy AND diastolic dysfunction
• Caused by mutations in sarcomere proteins but pathophysiology is
poorly understood
• Approach: proteomics on tissue from large patient cohort
39. Results
• Microtubule remodeling was one of
the key hits
• Large upregulation of detyrosinated
tubulin
• DetyrTub can contribute to
increased myofilament stiffness
(Prosser lab, Ward lab)
40. Parthenolide treatment
• PTL blocks tubulin detyrosination
• Approach: incubate CMs isolated from HCM mouse model
(MYBPC3c.2737INSG) with PTL and measure contractility and CaT
41. PTL treatment improves relaxation, but does not change Ca-reuptake
N = 4 WT (n = 191 DMSO, 99 PTL)
N = 6 HCM (n = 169 DMSO, 123 PTL)
Data from: Schuldt M, .., Kuster. Circulation HF 2020 in press
42. Interventricular differences in contraction/relaxation of CMs from healthy rats
• Goal: use MultiCell measurements to establish if LV, RV, IVS and atrial
cardiomyocytes are inherently different
• Approach: Langendorff digestion (n=5) with liberase, separate into
different regions before tritutation. Plate in separate laminin coated
35 mm dishes
• Pacing at 2Hz
47. Identify modulators of contraction/relaxation
• Goal: identify novel modulators of contraction/relaxation using
unbiased approach
• Approach: use compound library (160 kinase inhibitors) to test effect
on adult mouse cardiomyocytes
• Each compound is initially tested in 2 animals (aim for 30
cells/compound/animal). Positive hits are screened further
48. Technical challenges to overcome
• Activation of wide array of signaling molecules
• How many control measurements to include?
• Run down during a measurement day?
49. Technical approach
• Use two 24 well plate (laminin coated) per day (coat half the wells)
• Plate cells in plating medium (containing 5% FBS)
• Exchange to tyrode after ≥1 hour
• Measure at 37°C ± 0.5°
• Measure max 15 minutes per well
• Randomize compounds (use blinding)
50. Activation of signaling cascades
• Testing agonists is clear-cut, but antagonists need activated signaling
• Solution: use 2% serum
Control Isoproterenol Propanolol
0
5
10
15
Fractionalshortening(%)
Control Isoproterenol Propranolol
0
5
10
15
Fractionalshortening(%)
In tyrode + 2% FBS
51. Data from 1 measurement day
(1056 cells measured, 783 cells included in analysis)
1
C
on
1
2
3
C
on
2
4
5
C
on
3
6
7
C
on
4
8
9
C
on
5
10
11
C
on
6
12
13
C
on
7
14
15
C
on
8
16
0
2
4
6
8
Fractionalshortening(%)
Plate 1 Plate 2
Solution: normalize to neighboring control
53. Conclusions
• Large scale measurements are feasible
• 24 well format has considerable advantages
• First large scale compound screening for contraction/relaxation is in
progress
• Pay attention to experimental artefacts and curate your data well
54. VU Medical Center:
• Maike Schuldt
• Prof. Jolanda van der Velden
• Valentijn Jansen
Acknowledgements
IonOptix:
• Tom Udale
• Sander de Wit
• Patrick Schoenleitner
• Emmy Manders
• Adam Veteto
• Johny Pires
55. Thank you for participating!
Michiel Helmes, PhD
Co-CEO
IonOptix
Diederik Kuster, PhD
Assistant Professor
Department of Physiology
Amsterdam UMC
CLICK HERE to learn more and
watch the webinar
56. Thank you for participating!
Before you go…
1. Complete the Survey – we’d love to get
your feedback
2. Still have questions? Use the Ask a
Question panel
3. Interested in learning more? Explore the
Resources or visit www.ionoptix.com