Calnexin silencing in mouse neonatal cardiomyocytes induces Ca2+ cycling defects, ER stress, and apoptosis

J Cell Physiol. 2014 Mar;229(3):374-83. doi: 10.1002/jcp.24459.

Abstract

Calnexin (CNX) is an endoplasmic reticulum (ER) quality control chaperone that has been implicated in ER stress. ER stress is a prominent pathological feature of various pathologic conditions, including cardiovascular diseases. However, the role of CNX and ER stress has not been studied in the heart. In the present study, we aimed to characterize the role of CNX in cardiomyocyte physiology with respect to ER stress, apoptosis, and cardiomyocyte Ca(2+) cycling. We demonstrated significantly decreased CNX mRNA and protein levels by LentiVector mediated transduction of targeting shRNAs. CNX silenced cardiomyocytes exhibited ER stress as evidenced by increased GRP78 and ATF6 protein levels, increased levels of spliced XBP1 mRNA, ASK-1, ERO1a, and CHOP mRNA levels. CNX silencing also led to significant activation of caspases-3 and -9. This activation of caspases was associated with hallmark morphological features of apoptosis including loss of sarcomeric organization and nuclear integrity. Ca(2+) imaging in live cells showed that CNX silencing resulted in Ca(2+) transients with significantly larger amplitudes but decreased frequency and Ca(2+) uptake rates in the basal state. Interestingly, 5 mM caffeine stimulated Ca(2+) transients were similar between control and CNX silenced cardiomyocytes. Finally, we demonstrated that CNX silencing induced the expression of the L-type voltage dependent calcium channel (CAV1.2) but reduced the expression of the sarcoplasmic reticulum ATPase (SERCA2a). In conclusion, this is the first study to demonstrate CNX has a specific role in cardiomyocyte viability and Ca(2+) cycling through its effects on ER stress, apoptosis and Ca(2+) channel expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 6 / genetics
  • Activating Transcription Factor 6 / metabolism
  • Animals
  • Animals, Newborn
  • Apoptosis* / drug effects
  • Caffeine / pharmacology
  • Calcium Channels, L-Type / genetics
  • Calcium Channels, L-Type / metabolism
  • Calcium Signaling* / drug effects
  • Calnexin / genetics
  • Calnexin / metabolism*
  • Caspase 3 / metabolism
  • Caspase 9 / metabolism
  • Cell Survival
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / pathology
  • Endoplasmic Reticulum Stress* / drug effects
  • Genetic Vectors
  • Glycoproteins / genetics
  • Glycoproteins / metabolism
  • HEK293 Cells
  • Heat-Shock Proteins / genetics
  • Heat-Shock Proteins / metabolism
  • Humans
  • Lentivirus / genetics
  • MAP Kinase Kinase Kinase 5 / genetics
  • MAP Kinase Kinase Kinase 5 / metabolism
  • Mice
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Oxidoreductases
  • RNA Interference*
  • RNA, Messenger / metabolism
  • Regulatory Factor X Transcription Factors
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / genetics
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Time Factors
  • Transcription Factor CHOP / genetics
  • Transcription Factor CHOP / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transduction, Genetic
  • Transfection
  • X-Box Binding Protein 1

Substances

  • Activating Transcription Factor 6
  • Atf6 protein, mouse
  • CACNA1C protein, mouse
  • Calcium Channels, L-Type
  • DNA-Binding Proteins
  • Ddit3 protein, mouse
  • Glycoproteins
  • Heat-Shock Proteins
  • RNA, Messenger
  • Regulatory Factor X Transcription Factors
  • Transcription Factors
  • X-Box Binding Protein 1
  • XBP1 protein, human
  • Xbp1 protein, mouse
  • Calnexin
  • Transcription Factor CHOP
  • Caffeine
  • Ero1l protein, mouse
  • Oxidoreductases
  • MAP Kinase Kinase Kinase 5
  • Map3k5 protein, mouse
  • Casp3 protein, mouse
  • Casp9 protein, mouse
  • Caspase 3
  • Caspase 9
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Atp2a2 protein, mouse
  • molecular chaperone GRP78