PUMA is critical for neonatal cardiomyocyte apoptosis induced by endoplasmic reticulum stress.
OBJECTIVE
Puma (p53-upregulated modulator of apoptosis), a proapoptotic BH3-only member of the Bcl-2 protein family, has been implicated in the pathomechanism of several diseases, including cancer, AIDS, and ischemic brain disease. We have recently shown that Puma is required for cardiac cell death upon ischemia/reperfusion of mouse hearts. Since ischemia/reperfusion is also associated with endoplasmic reticulum (ER) stress, in the present study we investigated whether Puma contributes to the ER stress-dependent component of cardiomyocyte apoptosis.


METHODS
Primary cultures of rat and mouse neonatal cardiomyocytes were treated with 3 muM thapsigargin or 100 ng mL(-1) tunicamycin. Puma levels were suppressed by adenoviral delivery of shRNA or targeted deletion of the puma gene. Puma expression was detected by RT-PCR and Western blotting. Apoptosis was assessed by TUNEL assay, caspase-3 cleavage, and cytochrome c release.


RESULTS
We have shown that in rat neonatal cardiac myocytes, thapsigargin or tunicamycin treatment led to ER-stress, transcriptional upregulation of Puma, and apoptosis. Most importantly, cardiac myocytes acquired resistance to ER stress-induced apoptosis if Puma expression was downregulated by adenoviral delivery of shRNA or eliminated by targeted deletion in knockout mice.


CONCLUSIONS
Taken together, our data indicate that Puma is a critical component of ER stress-induced apoptosis in cardiac myocytes, and inhibition of Puma activity may be used to treat cardiac infarcts or prevent heart failure by blocking ER stress-induced apoptosis.
Nickson
Philip
P
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
Toth
Ambrus
A
Erhardt
Peter
P
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D053148:Caspase 3 / Q000032:analysis; D002460:Cell Line; D002478:Cells, Cultured; D045304:Cytochromes c / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D017353:Gene Deletion; D015870:Gene Expression; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2; D034622:RNA Interference; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D015415:Biomarkers; C488588:PUMA protein, mouse; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D014415:Tunicamycin; D019284:Thapsigargin; D045304:Cytochromes c; D053148:Caspase 3; D000252:Calcium-Transporting ATPases