Calreticulin reveals a critical Ca²⁺ checkpoint in cardiac myofibrillogenesis

Calreticulin (crt) is an ubiquitously expressed and multifunctional Ca²⁺-binding protein that regulates diverse vital cell functions, including Ca²⁺ 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 Ca²⁺ with EGTA or BAPTA, or by inhibiting Ca²⁺/calmodulin-dependent kinases (CaMKs). An imposed ionomycin-triggered cystolic-free Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_c increase. We conclude that the absence of calreticulin interferes with myofibril formation. Most importantly, calreticulin deficiency revealed the importance of a Ca²⁺-dependent checkpoint critical for early events during cardiac myofibrillogenesis.