AMP-activated protein kinase (AMPK) is a heterotrimeric protein that senses cellular energy status and maintains energy homeostasis by switching off biosynthetic pathways and increasing catabolism. The subcellular localization of AMPK has been shown to affect its activation and function. The γ2 subunit has both nuclear localization sequence and nuclear export sequence suggesting that it can shuttle between the two compartments. By overexpressing GFP-tagged γ subunits in COS7 cells followed by glucose deprivation or AMPK activation (A769662), we demonstrated that AMPK containing γ2 but not γ1 or γ3 subunit translocates into nucleus. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylates and inactivates Pol I-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes and lowering preribosomal RNA (pre-rRNA) level. Down-regulation of rRNA synthesis attenuated expression of ER stress markers (spliced X-box binding protein-1 and C/EBP homologous protein) and ER stress-induced cell death. Deleting γ2-AMPK using CRISPR-Cas9 system led to increases in pre-rRNA level, ER stress markers and cell death during glucose deprivation. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac specific deletion of γ2-AMPK (cKO). Although the total AMPK activity was unaltered in cKO hearts due to upregulation of γ1-AMPK the lack of γ2-AMPK sensitizes the heart to myocardial ischemia-reperfusion (I/R, 30min ischemia followed by 24hr reperfusion) injury as evidenced by larger infarct size (Infarct size/area at risk: 34.7±5.7% vs. 50.6±8.9%, for control and cKO respectively, P<0.05). The cKO failed to suppress pre-rRNA level during I/R and showed higher levels of ER stress markers. Conversely, cardiac-specific overexpression (OE) of γ2-AMPK decreased ER stress markers and pre-rRNA level upon I/R injury and the infarct size was reduced (Infarct size/area at risk: 26.8±6.5% for control vs. 15.8±3.7% for OE, P<0.05), suggesting that γ2-AMPK protects against I/R injury and ER stress in the heart. Taken together, our study reveals isoform-specific functions of γ2-AMPK in modulating protein synthesis, cell survival and cardioprotection.