In this webinar, Dr. Thao Nguyen discusses the exciting discoveries that her research team has made, debunks some common myths, and shares best-practices for data acquisition, analysis, and interpretation. Her in vivo studies of adult zebrafish cardiac electrophysiology rely on single-lead and multi-lead surface ECG in live anesthetized adult zebrafish. Zebrafish is a popular high-throughput vertebrate model to study human cardiac electrophysiology, arrhythmias, and myopathies. One reason for this popularity is the purported striking similarities between zebrafish and human electrocardiograms (ECGs). While human ECG, discovered 120 years ago, remains a standard technique in routine clinical practice, establishing similar standards for routine adult zebrafish cardiac research faces unique challenges. Yet, in vivo surface ECG offers the single most practical, economical, if not unique, solution to study adult zebrafish in vivo cardiac electrophysiology and arrhythmias.

1. Fishing for Insights from
Single-Lead and Multi-Lead
ECG of Live Adult Zebrafish
Thao P. Nguyen, MD, PhD
Associate Professor of Medicine,
David Geffen School of Medicine
University of California, Los Angeles

2. Dr. Thao P. Nguyen discusses the exciting
discoveries that her research team has made,
debunks some common myths, and shares
best-practices for data acquisition, analysis,
and interpretation.
Fishing for Insights from
Single-Lead and Multi-Lead
ECG of Live Adult Zebrafish

3. Fishing for Insights from
Single-Lead and Multi-Lead
ECG of Live Adult Zebrafish
Thao P. Nguyen, MD, PhD
Associate Professor of Medicine,
David Geffen School of Medicine
University of California, Los Angeles
Copyright 2021 T. Nguyen and InsideScientific. All Rights Reserved. @ThaoNguyenMDPhD

4. Can we really use zebrafish to study
human cardiac electrophysiology and arrhythmias?
4

5. Compared to human ECG,
zebrafish ECG looks strikingly similar
Human Zebrafish
Nguyen Lab, Cardiovasc Res 2020
Yan J et al, PLoS One 2020
Millan DJ et al, AJP Heart Circ Phys 2006
P
QRS
T
P
QRS
T
P
QRS
T
P
QRS
T
P
QRS
T
P
QRS
T
5

6. ECG was discovered 120 years ago
by Willem Einthoven
Wikipedia 6

7. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 7

8. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 8

9. Willem Einthoven was regarded as
the father of modern ECG
Historical Perspective
Wikimedia Wikimedia
9

10. Willem Einthoven invented
the 1st ECG recording device in 1903
Historical Perspective
Wikipedia 10

11. right arm left arm
left leg
-
+ +
lead I +
-
-
Einthoven’s triangle
Einthoven’s Law
II = I + III
Human Einthoven’s (bipolar) leads,
Einthoven’s triangle, and Einthoven’s law
Historical Perspective
11

12. right arm left arm
lead I
left leg
+
-
right arm left arm
left leg
-
+ +
-
12
Frontal electrical activity along the heart
short axis (lead I) vs long axis (leads II-III)
Historical Perspective

13. right arm left arm
left leg
+
lead I
Einthoven’s triangle
Human unipolar augmented limb leads
aVR, aVL, and aVF
Historical Perspective
aVR aVL
aVF
13

14. The Cabrera system is
a hexaxial reference system
Historical Perspective
+
lead I
aVR aVL
aVF
Cabrera system
0°
60°
180°
90°
-90°
lead I
120°
-30°
-150° aVR aVL
aVF
aVF
Q II Q I
Q III Q IV
14

15. Human Einthoven’s triangle defines
the main heart axis in Quadrant I (QI)
Right axis
Q II Q I
NORMAL AXIS
Q III
Extreme axis
Q IV
Left axis
-
+ +
lead I +
-
-
Historical Perspective
180°
90°
-90°
lead I
0°
15

16. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 16

17. Zebrafish
Human 4-chambered heart vs.
zebrafish 2-chambered heart
Anatomical Perspective
Human
Frank Netter
Zhao et al, JoVE 2019
Two atria
Two ventricles
17

18. Human heart normal compaction vs.
zebrafish heart normal non-compaction
Two ventricles
Fishbein, G for Nguyen Lab
Human abnormal
non-compaction (NC)
C
NC
Zebrafish normal
non-compaction (NC)
Nguyen Lab
Bulbus
arteriosus
Atrium
Ventricle
C
NC
Human normal
Compaction (C)
Frank Netter
Ramnani D, WebPathology.com
C
NC
Anatomical Perspective
18

19. Zebrafish pacemaker activity starts in
the SA node at the sinus venosus (SV)
Sedmera et al, AJP Heart 284(4) 2003
SA node
Anatomical Perspective
19

20. Zebrafish atrioventricular (AV) ring
acts like the human AV node
Sedmera et al, AJP Heart 284(4) 2003
SA node AV node
Anatomical Perspective
20

21. Sedmera et al, AJP Heart 284(4) 2003
SA node AV node
Zebrafish two main trabecular bands
act like the human His-Purkinje system
Anatomical Perspective
Two main trabecular bands
(~His-Purkinje system)
AV node
Apex
21

22. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 22

23. Human Zebrafish
Nguyen Lab, Cardiovasc Res 2020
P
QRS
T
Human & zebrafish ECGs look strikingly similar
but they were recorded from opposite leads!
P
QRS
T
rII
ECG Recording
23

24. Human lead II axis goes from right arm to left leg
at a +60o angle of orientation
right arm
left leg
-
+
Cabrera system
0°
60°
180°
+90°
-90°
Q IV
Q I
Q II
Q III
ECG Recording
24

25. Standard zebrafish ECG is recorded from lead
reverse II (rII) at a -120o angle of orientation
ADI Single-lead Bio Amp FE136
-
+
+
-
Cabrera system
0°
+60°
-120°
180°
+90°
-90°
Q IV
Q I
Q II
Q III
ECG Recording
25

26. The 4 basic steps of ECG recording ECG Recording
1. Setting up
2. Anesthesia Induction
3. ECG Lead Placement
4. ECG Recording
26

27. The 4 (+ 1) steps of single-lead ECG recording ECG Recording
27

28. STEP 1a: Setting up the test subjects ECG Recording
28

29. ECG Recording
STEP 1b: Setting up the equipment
https://www.adinstruments.com/research/animal/autonomic/ecg
PowerLab
Data acquisition
LabChart
Software
SINGLE Bio Amp FE 136
Amplifier
DUAL Bio Amp FE 232
OR
29

30. ECG Recording
STEP 2: Anesthesia induction
Choose the appropriate anesthetics
Only tricaine (MS-222) is FDA-approved
Determine the minimal concentration needed
Capitalize on synergistic potency
Know anesthesia goals (level 4 of anesthesia)
Consult IACUC veterinarian for additional
guidance
Determine the route of administration
30

31. ECG Recording
STEP 3a: Single-lead placement (e.g., rII)
31
ADI Single Bio Amp FE 136

32. ECG Recording
The more leads, the better
if the test subject can afford chest real-estate
32

33. 33
ECG Recording
STEP 3b: Dual-lead placement (e.g., I & II)
to construct the 1st zebrafish Einthoven’s triangle
Zebrafish
lead I
+
-
-
+
tail
right left
Human
right arm left arm
lead I
left leg
-
+
+
+
-
-
Einthoven’s Law
II = I + III

34. 34
ECG Recording
A 6-leads for 2 deal!
Zebrafish concurrent recording
from leads I-II
DUAL Bio Amp FE 232 III
II
aVR
aVL
aVF
I

35. Human lead II
right arm
left leg
-
+
35
ECG Recording
STEP 3b: Dual-lead placement for lead II, then…
Zebrafish lead II
-
+
right
tail
ADI Dual Bio Amp FE 232
Zebrafish lead rII
ADI Single Bio Amp FE 136

36. Zebrafish
ADI Dual Bio Amp FE 232
lead I
+
-
right left
Human
right arm left arm
lead I +
-
36
ECG Recording
STEP 3b: Dual-lead placement for lead I

37. 37
ECG Recording
STEP 4: ECG recording for
single lead vs. dual leads
Single-lead Dual-lead

38. 38
ECG Recording
STEP 4: Remember to identify your lead(s)

39. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 39

40. 40
ECG Interpretation
ECG interpretation should be done live
to optimize quality
III
II
aVR
aVL
aVF
I

41. 41
ECG Interpretation
ECG interpretation of
depolarization vs repolarization directions
Richard Klabunde, PhD. Cardiovascular Physiology Concepts
https://www.cvphysiology.com/Arrhythmias/A014
Maximal ECG voltage: electrical activity is parallel with lead axis
Flat line: electrical activity (if any) is perpendicular to lead axis
(+) ECG voltage: depolarization travels toward (+) electrode
repolarization travels away from (+) electrode
(-) ECG voltage: depolarization travels away from (+) electrode
repolarization travels toward (+) electrode

42. 42
ECG Interpretation
Post-recording ECG interpretation: ECG settings

43. 43
ECG Interpretation
Heart rhythm and heart rate
Software misidentifies
the P wave and R wave
Manual corrections
Lead rII

44. 44
ECG Interpretation
Fishing for insights from heart rate:
Heart rate variability (HRV)
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Lead rII

45. 45
ECG Interpretation
Amplitudes and durations

46. 46
ECG Interpretation
Fishing for insights from polarized amplitudes:
Direction of wave propagation
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Lead rII

47. 47
ECG Interpretation
ECG predictions based on polarized amplitudes
were confirmed by voltage optical mapping
Baseline
Basoapical repolarization
P
R
S
T
base
apex
Apicobasal activation
Zhao Y et al. Cardiovasc Res. 2020 Jul
31
H2O2-mediated Oxidative Stress
Late QRS inversion
Apicobasal repolarization
Early T wave inversion
Basoapical activation
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31

48. 48
ECG Interpretation
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Fishing for insights from durations:
Perturbation of wave propagation
Lead rII

49. 49
ECG Interpretation
QRS prolongation predicts
impaired ventricular activation in optical maps
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Baseline H2O2
Activation
Map

50. 50
ECG Interpretation
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Baseline H2O2
Repolarization
Map
QTc prolongation predicts
impaired ventricular repolarization on optical maps
Apex
Base

51. 51
ECG Interpretation
ST prolongation predicts
APD prolongation in optical maps
Baseline H2O2
APD
Map
Apex
Base
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31

52. Comparing human and zebrafish
Einthoven limb leads I-III
Zebrafish
ADI Dual Bio Amp FE232
Human
52
ECG Interpretation
P T
QRS
P T
QRS
P
T
QRS
P T
QRS
I
P T
QRS
P T
QRS
II
III

53. I
Like humans,
zebrafish manifests intra-lead polarity concordance
Human Zebrafish
+
+
+
+
+
II
III
+
+
+
+
+
+
-
- -
-
- -
53
ECG Interpretation
+

54. Like humans,
zebrafish show positive concordance in lead I
+
-
isoelectric
54
ECG Interpretation
+
+
+
I
+
+
+
P T
QRS P T
QRS
100
0
(%)
100
0
(%)
Human lead I (n = 24) Zebrafish lead I (n = 30)

55. The concordance in leads II and III is
positive for humans, but negative for zebrafish
Human Zebrafish
55
ECG Interpretation
II
III
+
+
+
+
+
+
-
- -
-
- -

56. What is the implication of human and zebrafish
opposite concordance in leads II and III?
+
-
isoelectric
56
ECG Interpretation
+
+
+
II
-
- -
P T
QRS P T
QRS
100
0
(%)
100
0
(%)
Human lead II (n = 24) Zebrafish lead II (n = 30)

57. 57
ECG Interpretation
Fishing for insights from dual-lead ECG:
Prediction of the three frontal axes between 0 and -120o
0°
-120°
lead I

58. 58
ECG Interpretation
Fishing for insights from dual-lead ECG:
Accurate determination of the main heart (QRS) axis in Q IV
I
II
III
aVR
aVL
aVF
Main heart axis
in Q IV
I
II
III
aVR aVL
aVF

59. In healthy adult humans,
all three heart axes are in Q I
Humans (n =24)
P axis
+46o
Q I
T axis
+39o
Q I
QRS axis
+51o
Q I 59
ECG Interpretation

60. Q I Q I
Q I
In healthy adult humans,
all three heart axes are in Q I
Humans (n =24) Zebrafish (n = 30)
P axis T axis
QRS axis
-70o -69o -51o
Q IV Q IV
Q IV
P axis
+46o
T axis
+39o
QRS axis
+51o
In healthy adult zebrafish,
all three heart axes are in Q IV
60
ECG Interpretation
Fishing for insights from dual-lead ECG:
First determination of zebrafish three frontal axes in Q IV

61. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 61

62. Clinical relevance of zebrafish in the
diagnosis of arrhythmias
62
Applications
Sinus tachy-bradyarrhythmia
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
Sinus bradyarrhythmia
Sinus arrest rescued by
ventricular escape rhythm

63. P
Clinical relevance of zebrafish in the
diagnosis of conduction blocks
63
Applications
Second-degree AV block type I
(Mobitz I)
Zhao Y, James N, et al. Cardiovasc Res. 2020 Jul 31
First-degree AV block
Third-degree AV block
(complete heart block)
Second-degree AV block type II
(Mobitz II)
P
R
R
R
R
R
R
P P
P P P P P
P
P
R
P
R
P
P
P
P
R R
R R R R
R
P P P P P P P

64. Source: Arthur Haines
Digitalis (Foxglove) plant
Clinical relevance of zebrafish Einthoven triangle for
in-vivo screening of ‘digitalis effect’ & toxicities
Source: wikipedia 64
Applications

65. Zebrafish recapitulates human ‘digitalis effect’
Sagging ST depression
Lead II
Lead I
Zebrafish
https://litfl.com/digoxin-effect-ecg-library
Baseline
STD
STE
Digitoxin
Human
Lead II
STD
65
Applications

66. Lead II
Lead I
https://litfl.com/digoxin
-
+
-
+
-
Zebrafish recapitulates human ‘digitalis effect’
T-wave inversion
66
Applications
Zebrafish
Baseline
Digitoxin
Human

67. Zebrafish recapitulates human ‘digitalis effect’
PR prolongation (first-degree AV block)
Lead II
Lead I
Digoxin levels of 4.7 mg/L
(therapeutic range 1–2 mg/L)
Lead II
Lead I
67
Applications
Zebrafish
Baseline
Digitoxin
Human

68. STD STD STD
Q
Q
R
Q
STE STD STD
Lead II
Lead I
S
Dual-lead beats single-lead zebrafish ECG,
such as in screening for ‘digitalis effect’
68
Applications

69. 69
Applications
Zebrafish recapitulates human
suppressant ‘digitalis cardiotoxicities’
Mobitz I
Complete
heart block
Mobitz II
P P P P P P P P P P P P P
P P P P P P P P P P P P
R R R
R R R
R R
P P
P
8:20 8:25
R R R R R R R R
R R R R
P P
P

70. 70
Zebrafish recapitulates human
excitatory ‘digitalis cardiotoxicities’
Atrial tachycardia
Atrial fibrillation
Ectopic
atrial activity
Atrial flutter
Applications

71. 71
Zebrafish recapitulates human
hyperkalemic ‘digitalis cardiotoxicities’
Petrov DB. N Engl J Med 2012; 366:1824
Wide complex rhythm with sine wave morphology
(loss of P waves, QRS widening, and fused T waves)
Zebrafish
Human
I I
II
II
Ko 9.4 mmol/L
Applications

72. A scientific preview
Perspectives
Understand the historical perspective
Appreciate the anatomical perspective
ECG Recording
Describe the four steps of ECG recording
Single-lead vs. dual-lead ECG recording
ECG Interpretation
Critique ECG recording quality
Interpret single-lead vs. dual-lead ECG
Applications
Diagnose arrhythmias and conduction blocks
Diagnose drug-induced cardiotoxicities 72

73. Acknowledgements
NGUYEN LAB
Zebrafish Project
Yali Zhao, PhD
Binh Nguyen, MD
Nicholas James
Ashraf Beshay
Connie Chen
Morgan Yun
Andrew Lin
Thomas Issa
Michelle Tran
Faiza Bashar
Abram Wassily
Francia Lopez
Francia
Lopez
Morgan
Yun
Yali
Zhao
Michelle
Tran
Nicholas
James
Ashraf
Beshay
Faiza
Bashar
Abram
Wassily
73

74. Thao P. Nguyen, MD, PhD
Associate Professor of Medicine,
David Geffen School of Medicine
University of California, Los Angeles
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