Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.
Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca(2+)-binding protein that regulates diverse vital cell functions, including Ca(2+) storage in the ER and protein folding. Calreticulin deficiency in mice is lethal in utero due to defects in heart development and function. Herein, we used crt(-/-) embryonic stem (ES) cells differentiated in vitro into cardiac cells to investigate the molecular mechanisms underlying heart failure of knockout embryos. After 8 d of differentiation, beating areas were prominent in ES-derived wild-type (wt) embryoid bodies (EBs), but not in ES-derived crt(-/-) EBs, despite normal expression levels of cardiac transcription factors. Crt(-/-) EBs exhibited a severe decrease in expression and a lack of phosphorylation of ventricular myosin light chain 2 (MLC2v), resulting in an impaired organization of myofibrils. Crt(-/-) phenotype could be recreated in wt cells by chelating extracellular or cytoplasmic Ca(2+) with EGTA or BAPTA, or by inhibiting Ca(2+)/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca(2+) concentration ([Ca(2+)](c)) elevation restored the expression, phosphorylation, and insertion of MLC2v into sarcomeric structures and in turn the myofibrillogenesis. The transcription factor myocyte enhancer factor C2 failed to accumulate into nuclei of crt(-/-) cardiac cells in the absence of ionomycin-triggered [Ca(2+)](c) increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca(2+)-dependent checkpoint critical for early events during cardiac myofibrillogenesis.
Li
Jian
J
Biology of Aging Laboratory, Department of Geriatrics, Geneva University Hospitals, Geneva 1225, Switzerland.
Pucéat
Michel
M
Perez-Terzic
Carmen
C
Mery
Annabelle
A
Nakamura
Kimitoshi
K
Michalak
Marek
M
Krause
Karl-Heinz
KH
Jaconi
Marisa E
ME
D000818:Animals; D052160:Benzothiazoles; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037282:Calreticulin; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D003959:Diamines; D015180:Electrophoresis, Gel, Two-Dimensional; D005456:Fluorescent Dyes / Q000494:pharmacology; D006321:Heart / Q000196:embryology / Q000502:physiology*; D007150:Immunohistochemistry; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D024510:Muscle Development / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D009930:Organic Chemicals; D010641:Phenotype; D010766:Phosphorylation; D011804:Quinolines; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012261:Ribonucleoproteins / Q000378:metabolism*; D013997:Time Factors
D052160:Benzothiazoles; D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002614:Chelating Agents; D003959:Diamines; D005456:Fluorescent Dyes; D007476:Ionophores; D009930:Organic Chemicals; D011804:Quinolines; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C098022:SYBR Green I; D015759:Ionomycin; D002118:Calcium