Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium

a novel function beyond muscle contraction

Yi Shuai Zhang, Bin Liu, Xiu Ju Luo, Ting Bo Li, Jie Jie Zhang, Jing Jie Peng, Xiao Jie Zhang, Qi Lin Ma, Chang Ping Hu, Yuan Jian Li, Jun Peng, Qingjie Li

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia–reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H<inf>2</inf>O<inf>2</inf> and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H<inf>2</inf>O<inf>2</inf> levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

Original languageEnglish (US)
JournalBasic Research in Cardiology
Volume110
Issue number4
DOIs
StatePublished - Jul 18 2015

Fingerprint

Cardiac Myosins
NADPH Oxidase
Muscle Contraction
Myocardium
Myosin-Light-Chain Kinase
Phosphorylation
Wounds and Injuries
Transcription Factor TFIIB
myosin light chain 2
Cell Hypoxia
RNA Polymerase II
Chromatin Immunoprecipitation
Consensus Sequence
Myosins
Reperfusion Injury
Luciferases
Reporter Genes
Immunoprecipitation
Genes
Ligation

Keywords

  • Cardiac myosin light chain 2
  • Ischemia
  • NADPH oxidase 2
  • Reperfusion
  • Transcription

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)
  • Physiology

Cite this

Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium : a novel function beyond muscle contraction. / Zhang, Yi Shuai; Liu, Bin; Luo, Xiu Ju; Li, Ting Bo; Zhang, Jie Jie; Peng, Jing Jie; Zhang, Xiao Jie; Ma, Qi Lin; Hu, Chang Ping; Li, Yuan Jian; Peng, Jun; Li, Qingjie.

In: Basic Research in Cardiology, Vol. 110, No. 4, 18.07.2015.

Research output: Contribution to journalArticle

Zhang, Yi Shuai ; Liu, Bin ; Luo, Xiu Ju ; Li, Ting Bo ; Zhang, Jie Jie ; Peng, Jing Jie ; Zhang, Xiao Jie ; Ma, Qi Lin ; Hu, Chang Ping ; Li, Yuan Jian ; Peng, Jun ; Li, Qingjie. / Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium : a novel function beyond muscle contraction. In: Basic Research in Cardiology. 2015 ; Vol. 110, No. 4.
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abstract = "Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia–reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.",
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AU - Zhang, Yi Shuai

AU - Liu, Bin

AU - Luo, Xiu Ju

AU - Li, Ting Bo

AU - Zhang, Jie Jie

AU - Peng, Jing Jie

AU - Zhang, Xiao Jie

AU - Ma, Qi Lin

AU - Hu, Chang Ping

AU - Li, Yuan Jian

AU - Peng, Jun

AU - Li, Qingjie

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AB - Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia–reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

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KW - Reperfusion

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