Dr. David Barefield gives an in-depth discussion on his research investigating the role of the MyBP-HL protein in atrial dysfunction. Myosin binding protein H-like (MyBP-HL) is an atrial myofilament protein that establishes a stoichiometry with cardiac myosin binding protein-C (cMyBP-C). Loss of MyBP-HL leads to atrial dilation, arrhythmia, and dilated cardiomyopathy. Several human MYBPHL nonsense variants are reported in the gnomAD database. The Barefield Lab asked whether nonsense variants prevent MyBP-HL incorporation into the myofilament, or if truncation variants result in alternative protein localization within the cardiomyocyte. Neonatal rat cardiomyocytes were isolated and transfected with wild-type mouse Mybphl or mouse Mybphl that had human MYBPHL nonsense mutations. Immunofluorescence confocal microscopy showed wild-type mouse MyBP-HL colocalized with cMyBP-C. The W54X, R133X, W158X, W192X, K250X, and R255X variants all failed to co-localize. As the MYBPHL stop variants prevented myofilament binding, the Barefield Lab performed biophysical experiments on single myofibrils isolated from Mybphl null mice. Mybphl null myofibrils showed significantly faster phase of linear relaxation. These data show that MYBPHL premature stop variants do not encode functional proteins and loss of MyBP-HL causes biophysical defects in atrial myofibrils. Key Topics Include: - Myosin binding protein H-like competes for thick filament binding with cardiac myosin binding protein-C. - Nonsense variants in MYBPHL result in truncated proteins that do not incorporate into the atrial sarcomere. - Mice lacking myosin binding protein H-like have an accelerated linear phase of myofibril relaxation.

1. Copyright 2022. All Rights Reserved. Contact Presenter for Permission
Investigating a Novel Regulator of
Atrial Contractility
David Barefield, PhD
Assistant Professor
Cell and Molecular Physiology
Loyola University Chicago

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3. David Barefield, PhD
Loyola University Chicago
InsideScientific Webinar Series
October 19th, 2022
Atrial myofilament function
in genetic cardiomyopathies

4. Cardiomyopathy can be caused by genetic mutations
and modified by other genes and the environment
McNally, Barefield, Puckelwartz, 2015. Cell Metabolism

5. Mutations in MYH7 and MYBPC3 account for ~80% of HCM
Thick
Filament
Thin
Filament
Barefield, Sadayappan, 2010. JMCC

6. Genes that cause DCM are involved in a range of cardiomyocyte functions
McNally, Golbus, Puckelwartz, 2013. JCI

7. Diverse genetic defects cause cardiomyopathies; current genetic panel
testing is ~50% sensitive
McNally, Barefield, Puckelwartz, 2015. Cell Metabolism

8. In addition to impaired contractility and relaxation, cardiomyopathy is associated
with atrial fibrillation and ventricular arrhythmia
Ho et al., 2018. Circulation

9. New Cardiomyopathy Gene Discovery

10. Whole-genome sequencing identified a MYBPHL R255Stop
variant in a family with DCM and arrhythmias
Barefield et al., 2017. Circulation

11. Welikson and Fischman. 2002 Journal of Cell Science
MYBPHL encodes myosin binding protein H-like, a member
of the myosin binding protein family
Barefield et al. 2017 Circulation

12. Truncations in myosin binding protein-C prevent myofilament
binding and result in protein degradation
WT cMyBP-C
Truncated cMyBP-C

13. MyBP-HL is myofilament associated and the R255X mutation
prevents sarcomere incorporation
MyBP-HL
Barefield et al., 2017. Circulation

14. NRVM
Alvarez-Arce, Barefield et al., Unpublished

15. Human iPSC-cardiomyocytes with the R255X variant do not
express the mutant allele
Control R255X Het
Control
R255X Het
Control
R255X Het
Barefield et al., 2017. Circulation
Control
R255X Het
Control
R255X Het

16. Mybphl null mouse
Barefield et al., 2017. Circulation

17. MyBP-HL is found in mouse atria and in a subset of the ventricle conduction system
25µm
Barefield et al., 2022. JMCC

18. Heterozygous and homozygous Mybphl mice develop LV
dysfunction and atrial enlargement
Barefield et al., 2017. Circulation

19. Loss of Mybphl causes electrophysiological abnormalities and
increased occurrence of arrhythmias
Barefield et al. 2017 Circulation
Barefield et al., 2017. Circulation

20. Barefield et al., 2022. JMCC
Loss of Mybphl causes electrophysiological abnormalities and
increased occurrence of arrhythmias

21. MYBPHL was identified in a GWAS screen of individuals
with P-R prolongation
92,340 individuals of European descent
Van Settern et al., 2018 Nature Communications

22. MyBP-HL in the ventricular
conduction system

23. Monteiro et al., 2017. Regenerative Medicine
The cardiac conduction system is comprised of specialized cardiomyocytes

24. MyBP-HL is found in mouse atria and in a subset of the ventricle conduction system
25µm
Barefield et al., 2022. JMCC

25. Reduction in MyBP-HL-expressing ventricular cardiomyocytes
in Mybphl heterozygous ventricles
25µm
Barefield et al., 2022. JMCC

26. Lightsheet microscopy reveals localization of
ventricular MyBP-HL foci
WT p5 Heart
MyBP-HL
Cntn2

27. Mybphl expressing ventricular cells are enriched <100 µm from the conduction system
Barefield et al., 2022. JMCC

28. Goodyear et al. Circ Res 2019
The extent of cardiomyocyte heterogeneity is beginning to be understood
Goodyer et al. 2019. Circ Res

29. MyBP-HL localization
within the sarcomere

30. Sarcomere nomenclature
Myofilament nomenclature (1970’s)
• Myosin protein preparation contaminants
isolated and named
• Named alphabetically in order of
molecular weight from myosin preps
EM nomenclature (1950’s-60’s)
• Anisotropic/Isotropic – A/I band
• H-zone, from the German word
heller, meaning lighter
• C-zone named later for MyBP-C
localization
Starr & Offer 1973

31. MyBP-HL localizes in the A-band of atrial sarcomeres
Barefield & McNally. Unpublished

32. Myosin binding proteins have preferential localization along the A-band
MyBP-H, Psoas muscle
Slow skeletal MyBP-C, Soleus muscle
Bennett et al. 1986 J Muscle Res and Cell Motility

33. Barefield & McNally. Unpublished
MyBP-HL and cMyBP-C localize to the C-zone;
MyBP-HL is closer to the M-line

34. Normal atria have less cMyBP-C than ventricle
Human Tissue
Mouse Tissue
Barefield & McNally. Unpublished

35. Myosin binding proteins maintain a stoichiometry in the atria
Barefield & McNally. Unpublished

36. Myosin binding proteins maintain a stoichiometry in the atria
Barefield & McNally. Unpublished

37. Acute competitive binding between MyBP-HL and MyBP-C in NRVMs
(X, Y) = (HL+/Nucleus),(cMyBP-C HL+/cMyBP-C HL-)
Araujo, Barefield. Unpublished
cMyBP-C
MyBP-HL
Transfect: RFP Transfect: MyBP-HL

38. Immuno-EM reveals MyBP-HL binding 161 nm from the M-line
Barefield & McNally. Unpublished
500 nm

39. Previous work measured MyBP-H’s major stripe 160 nm from the center of the M-line
MyBP-H
Psoas muscle
160 nm: M-line to stripe 2
Bennett et al. 1986 J Muscle Res and Cell Motility

40. Immuno-EM reveals MyBP-HL binding 161 nm from the M-line
Barefield & McNally. Unpublished
Slow skeletal MyBP-C, Soleus muscle
Bennett et al. 1986 J Muscle Res and Cell Motility

41. Myosin binding proteins C and HL compete for binding sites in the atria
Barefield & McNally. Unpublished

42. Isometric force and calcium sensitivity of force development is not
altered in Mybphl null atrial cardiomyocytes
Barefield & McNally. Unpublished

43. Loss of Mybphl reduces time and rate of the linear
relaxation phase
Barefield & McNally. Unpublished

44. • MyBP-HL is associated with atrial and ventricular dilation and
arrhythmia in humans and mice
• MyBP-HL is expressed throughout the atria, but only found in
specific foci in the ventricle
• MyBP-HL and cMyBP-C maintain a strict stoichiometry in the atria

45. University of Vermont
Michael Previs
New York University
Glenn Fishman
Northwestern University
Beth McNally
Meg Puckelwartz
Lisa Wilsbacher
Andy Wasserstrom
Center for Advanced Microscopy
University of Arizona
Henk Granzier
Paola Tonino
R01 HL128075 (McNally)
R00 HL141698 (Barefield)
R56 HL165137 (Barefield)
University of Colorado
KC Woulfe
Barefield Lab
Kelly Araujo
Alejandro Alvarez-Arce
Hannah Cizauskas
Geena Fritzman
Alex Pena
Lucas Wittenkeller
@BarefieldD
BarefieldLab.Org
Loyola University Chicago
Jonathan Kirk

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