Gap junction protein beta 4 plays an important role in cardiac function in humans, rodents, and zebrafish

PLoS One. 2020 Oct 13;15(10):e0240129. doi: 10.1371/journal.pone.0240129. eCollection 2020.


Aims: GJB4 encodes a transmembrane connexin protein (Cx30.3) that is a component of gap junctions. This study investigated whether GJB4 plays an important role in human heart disease and function.

Methods and results: We examined a patient and her older brother who both presented with complicated severe hypertrophic cardiomyopathy (HCM) and whose parents are healthy married cousins. The gene exome analysis showed 340 single nucleotide polymorphisms (SNPs) that caused amino acid changes for which the patient was homozygous and both parents were heterozygous. After excluding all known common (>10%) SNP gene mutations, the gene for GJB4 was the only identified gene that is possibly associated with cardiac muscle. The resultant E204A substitution exists in the 4th transmembrane domain. GJB4-E204A impaired the binding with gap junction protein A1 (GJA1) compared with GJB4-WT. The expression of GJB4 was induced in rat disease models of left and right ventricle hypertrophy and mouse disease models of adriamycin-induced cardiomyopathy and myocardial infarction, while it was not detected at all in control. An immunohistochemical study was performed for autopsied human hearts and the explanted heart of the patient. GJB4 was expressed and colocalized with GJA1 in intercalated discs in human diseased hearts, which was extensively enhanced in the explanted heart of the patient. The abnormal expression and localization of GJB4 were observed in beating spheres of patient's induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). We generated knockout zebrafish of GJB4 by CRISPR/Cas9 and the endodiastolic volume and the ventricular ejection fraction were significantly lower in GJB4-deficient than in wild-type zebrafish at five days post-fertilization.

Conclusions: These results indicate both that GJB4 is defined as a new connexin in diseased hearts, of which mutation can cause a familial form of HCM, and that GJB4 may be a new target for the treatment of cardiac hypertrophy and dysfunction.

Publication types

  • Case Reports
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Amino Acid Substitution
  • Angiotensin II / toxicity
  • Animals
  • Animals, Genetically Modified
  • COS Cells
  • Cardiomyopathy, Hypertrophic, Familial / diagnosis
  • Cardiomyopathy, Hypertrophic, Familial / genetics*
  • Cardiomyopathy, Hypertrophic, Familial / pathology
  • Cardiomyopathy, Hypertrophic, Familial / surgery
  • Child
  • Chlorocebus aethiops
  • Connexin 43 / metabolism
  • Connexins / genetics*
  • Connexins / metabolism
  • DNA Mutational Analysis
  • Disease Models, Animal
  • Doxorubicin / toxicity
  • Female
  • Gap Junctions / pathology
  • Gene Knockout Techniques
  • Genetic Testing
  • Heart Transplantation
  • Humans
  • Induced Pluripotent Stem Cells
  • Male
  • Mice
  • Myocardial Infarction / etiology
  • Myocardial Infarction / pathology*
  • Myocardium / cytology
  • Myocardium / pathology*
  • Myocytes, Cardiac
  • Pedigree
  • Primary Cell Culture
  • Protein Domains / genetics
  • Rats
  • Zebrafish
  • Zebrafish Proteins / genetics
  • Zebrafish Proteins / metabolism


  • Connexin 43
  • Connexins
  • Zebrafish Proteins
  • Angiotensin II
  • connexin 30.3
  • Doxorubicin

Grant support

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture, Japan, and the Mie Medical Foundation (19K08578 to R.O. and M.I.). R.O. was supported by grants from the Okasankato Culture Shinkozaidan Foundation and the Mie medical association. The Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, received research grants from Bristol-Myers Squibb, MSD K.K., Pfizer Japan Inc., Takeda Pharmaceutical Co., Ltd., Astellas Pharma Inc., Daiichi Sankyo Pharmaceutical Co., Ltd., Genzyme Japan, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Mitsubishi Tanabe Corporation, Otsuka Pharmaceutical Co., Ltd., Bayer Yakuhin, Ltd., AstraZeneca K.K., and Boehringer Ingelheim Co., Ltd. Masaaki Ito received lecture fees from Daiichi Sankyo Pharmaceutical Co., Ltd., Mitsubishi Tanabe Corporation, Bayer Yakuhin, Ltd. and Takeda Pharmaceutical Co., Ltd. Yoshinoi Yoshida owns stock in iPS Portal, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.