High performance automated patch-clamp characterisation ofmammalian atrial cardiomyocytes
Fitzwilliam Seibertz1,2, Markus Rapedius3, Funsho Fakuade1,2, Philipp Tomsits4,5,6, Lukas Cyganek2,7,Nadine Becker3, Sebastian Clauß4,5,6, Niels Fertig3, Niels Voigt1,2,8
1Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Georg-August University Göttingen, Germany. 2DZHK (German Center for Cardiovascular Research), partner site Göttingen, Germany. 3Nanion Technologies GmbH,Munich, Germany. 4Department of Medicine I, University Hospital, LMU Munich, Munich, Germany. 5Institute of Surgical Research at the Walter-Brendel-Centre of Experimental Medicine, University Hospital, LMU Munich, Munich, Germany. 6DZHK(German Centre for Cardiovascular Research), Partner Site Munich, Munich Heart Alliance (MHA), Munich, Germany. 7Stem Cell Unit, Clinic for Cardiology and Pneumology, University Medical Center Göttingen, Georg-August University Göttingen,Germany. 8Cluster of Excellence "Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells" (MBExC), Georg-August University Göttingen, Germany

BACKGROUND
Automated patch-clamp (APC) technology is emerging as a promising
method to increase the throughput of functional electrophysiologicalmeasurements. Typical APC devices use microfluidic cell handlingchannels, which has historically limited this method to the study ofsmaller cellular constructs such as expression systems.
The newly-designed APC device, Syncropatch 384 (NanionTechnologies), operates using a fixed well mechanism, with nomicrofluidics present. We aim to test this device using induced
pluripotent stem cell derived cardiomyoctes (iPSC-CM) and primaryatrial cardiomyoctes (CM) to assess the extent to which APC cancontribute in the future study of atrial fibrillation associated remodelling.
This could be essential for potential therapeutic strategy development,
which to date remains substantially scarce.

Figure 1. Automated Patch Clamp (APC) overview. A, Traditional patch clamp (upper) and fixedwell APC (lower). B, Syncropatch 384. C, Consumable 384-well chip (upper) with a software
screenshot during an example experiment with a representative trace highlighted (lower).

RESULTS

Baseline

EIM (pA/pF)

50-5+10 mV-1 0-40 mV-80 mV
100 ms

-1 5-20Atrial-15-10-5Vent.

AtrialFigure 2. iPSC-CM assays using APC. A, Directed differentiation using induced
pluripotent stem cells (iPSC) generated from healthy donors. Atrial differentiation isachieved through retinoic acid. B, Action potentials measured from ventricular (left) andatrial (right) iPSC-CM. C, Mean±SEM APD90. D, L-type Ca2+ currents (ICa,L) measured
from ventricular (left) and atrial (right) iPSC-CM. E, Mean±SEM ICa,L.*P<0.05 vs. Vent.

ANativeatrial CM-2405 ms

1 nA

VM (mV)0-4 0-8 00-2+60 mV-4-6-8100 ms-40 mV-80 mV
100 ms

ICa,L (pA/pF)

E

2IM (pA/pF)

D

100 ms-20-15A-5-425 nM Nif-65 µM Nif (Full block)
20 ms

C

IM (pA/pF)

Response

IM (pA/pF)

5 nM Nif

200 s

0 .5

Ventricular (n=39)
IC50 = 5.9 nM-8-70Vent.

Atrial10 ms-30 mV

100 Seal >250MΩ
8060
40
2077

iPSCCM

NativeCM

AP252520151050ICa,L

77

iPSCCM

NativeCM252015105077iPSCCMNativeCMIK1201510507NativeCM

Humanpatient

Right atrialappendage

BICa,LINa

Human atrial CM

2-2 0+10 mV-4 0-6 0-80 mV-8 010 ms

0-2-4-6-8-120 mV
30 ms+60 mV

20 ms-40 mV-80 mV
100 msFigure 7. Successful recording of human atrial CM using APC. A, Isolated atrial CM
from a right atrial appendage procured during open heart surgery. B, Representative
peak sodium current (INa; left) and ICa,L (right) recording using APC.
Atrial(n=94)
IC50 = 7.6 nM-960 s

Atrial1 .00 .0-1 0-10

VM = -90 mV

01nM Nif-2-8BBaseline

0-20

BSeal
formation-20 mVAVent.

-30

BREAKING NEWS APRIL 2022

Figure 3. Native CM assays using APC. A, CM origin and isolation. B, Action potentials
measured from ventricular (left) and atrial (right) CM. C, Mean±SEM APD90. D, ICa,L
measured from ventricular (left) and atrial (right) CM. E, Mean±SEM ICa,L.*P<0.05 vs. Vent.

A-1.0**-100-40

Atrial50Vent.

-0.5

AtrialFigure 6. Preliminary success rates. A, Seal formation example (left) and percentageof cells with a successful seal per APC run in iPSC-CM and native CM (right). B,Absolute success rate of robust recording detection for action potential (AP; left), ICa,L(middle) and IK1(right) per APC run. n=Number of APC runs.

1000-1 2 0Vent.

Figure 5. Native CM inward rectifier assays using APC. A, Inward rectifier currents inventricular (left) and atrial (right) CM before and after Carbachol (CCh) application. Acurrent increase after CCh indicates the presence of acetylcholine activated inward
rectifiers (IK,ACh). B, Mean±SEM inward component of IK1. C, Time course of a single
APC plate for both ventricular (left; n=11) and atrial (right; n=10) CM measured at-90mV. D, Mean±SEM inward component of IK,ACh. *P<0.05 vs Vent.

0150

0BaCl20VM = -90 mV

60 s

200

APD90 (ms)

VentricularC

AtrialCarbachol-1.5-3Nativeventricular CMBControl

BaCl2-1*0100 ms

Carbachol

0Vent.

-20

Success (%)

DICa,L (pA/pF)

100 ms-40D

200-120-80 mV

CControl02000 ms*-60

10pA/pF

IM (pA/pF)-80-100 mV

Success (%)-40

400+40 mV

IM (pA/pF)

0*

CCh, 2 µM

IK,AChIM (nA)

5 ms

1 nA

VM (mV)

40600

Baseline

CCh, 2 µM

Success (%)

Atrial-80 -60 -40 -20 0 20 40

10pA/pF

IM (nA)

VentricularC
APD90 (ms)B

-80

IK1-80 -60 -40 -20 0 20 40

IK1B

AtrialVentricular

IK1 (pA/pF)

A

IK,ACh (pA/pF)

A

Success (%)

Atrial / Ventricular
iPSC-CM-6lg [Nifedipine] (M)Figure. 4, Pharmacological modulation of ICa,L in native CM. A, Representative atrial
ICa,L with increasing Nifedipine (Nif) concentrations. B, Time course of a single atrial APC
plate (n=10). C, Normalised concentration response curve. n=Number of CM.

CONCLUSION AND OUTLOOK• For the first time, APC has successfully recorded currents and action
potentials from native cardiomyocyte material.
• Sensitive discrimination of typical atrial phenotypes shows promisefor future studies of atrial fibrillation.
• Optimisation of overall success rates will further increase throughput.
www.molecular-pharmacology.de
will.seibertz@med.uni-goettingen.deNo conflicts of interest to declare