Polypyrimidine tract binding proteins (PTB) regulate the expression of apoptotic genes and susceptibility to caspase-dependent apoptosis in differentiating cardiomyocytes

Abstract

Cardiac morphologic abnormalities in mice deficient for key regulators of the caspase-dependent signaling underscored its role in heart development. However, the mechanisms regulating apoptotic gene expression in the developing heart are unknown. As polypyrimidine tract binding proteins (PTB) determine gene isoform expression during myoblast differentiation and contribute to Apaf-1 translation in cell lines, we investigated whether PTB regulate apoptotic gene expression in differentiating cardiomyocytes. Our results show that PTB are expressed in the embryonic heart and are silenced during development, coinciding with a reduction in the expression of apoptotic genes. Overexpression of PTB in postnatal cardiomyocytes, which express low levels of PTB and apoptotic genes, induced an increase in the amount of pro-apoptotic proteins without affecting abundance of their respective transcripts. Translation of the reporter gene Firefly Luciferase preceded by the 5 0 -untranslated region of Apaf-1 or Caspase-3 was enhanced by PTB in cardiomyocytes. PTB silencing in fibroblasts induced a decrease of apoptotic protein levels. PTB overexpression in cardiomyocytes induced caspase activity and caspase-dependent DNA fragmentation during ischemia, which is otherwise caspase-independent in differentiated cardiomyocytes. Our results show that PTB contribute to apoptotic gene expression and modulate the susceptibility to caspase activation in differentiating rat cardiomyocytes.

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

“…The loss of CLK1 activity may disrupt a feedback loop because its kinase activity is critical for the phosphorylation of multiple SR proteins that regulate splicing (63,70,71). The inclusion of CASP3 exon 2 with EWS-FLI1 reduction places an internal ribosome entry site into the 5′-UTR of the mRNA (72). This 5′-UTR may enhance the translation of caspase-3, thus increasing its protein levels and in part explaining the increased apoptosis seen with YK-4-279 treatment (21).…”

RNA-Binding Proteins in Heart Development

“…While Kalsotra et al ( 2008 ) reported that their levels do not change from PN1 to adult stages, Zhang et al ( 2009 ) and Ye et al ( 2013 ) demonstrated its downregulation in rat and mouse. A correlation between the PTB developmental downregulation and the developmental downregulation of apoptotic genes was demonstrated in mouse (Zhang et al 2009 ). Consistently, over-expression of PTB in adult CM induces the expression of pro-apoptotic proteins without affecting their transcript levels suggesting that PTB regulates translation of apoptotic genes.…”

“…When PTB is re-expressed in CM, caspase activity is enhanced and ischemia triggers caspase-dependent DNA fragmentation. By contrast, in the absence of PTB differentiated CM show caspaseindependent DNA fragmentation (Zhang et al 2009 ). These results suggest that PTB proteins play important roles in CM maturation similarly to what has been shown in C2C12 myoblast and neuron differentiation (Boutz et al 2007a , b ;Makeyev et al 2007 ).…”

“…Many predicted RBFOX1/2 targets encode neuromuscular proteins, which consistently were reported to be disrupted in muscular dystrophy and neurological, sensory, and cardiac diseases ( McKusick 1998 ;Zhang et al 2009 ). Zhang et al found that those targets are more likely to be disease-associated genes than what would be expected by chance (Zhang et al 2009 ). This evidence supports the idea that RBFOX proteins are important regulators of heart functions.…”

Translation of Myocyte Enhancer Factor-2 is induced by hypertrophic stimuli in cardiomyocytes through a Calcineurin-dependent pathway

“…Ptb nucleotide sequence GCACAGTC CTGAAGATCAT was chosen for PTB-specific expression silencing using free RNAi design interfaces and has been validated previously [28]. Primers for small hairpin RNA interference (shRNA) were subcloned into the pLVTHM plasmid as described before [28]. pEIGW or pLVTHM constructs were transfected into HEK293T packing cell line and tittered as described previously [28].…”