The LKB1fl/fl mice have 5- to 10-fold lower expression and activity of LKB1 in various tissues, including skeletal muscle mass, heart, testis, lung, liver, and kidney (26). Echocardiographic analysis Mice were anesthetized by intraperitoneal injection of 0.3 mg/g body wt of Avertin (tribromoethanol). muscle Erythromycin Cyclocarbonate mass LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPK1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced excess weight and enlarged atria. These findings show that LKB1 plays a crucial role in regulating AMPK2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an option upstream kinase can activate AMPK1 in cardiac muscle mass. (14, 15, 21, 29) indicated that enzymes homologous to the mammalian LKB1 tumor suppressor kinase and calmodulin-dependent protein kinase kinase (CAMKK) would mediate the activation of the yeast homolog of AMPK. This prompted studies in the mammalian system that resulted in the finding that LKB1 phosphorylated AMPK at Thr172 in vitro and that, in LKB1-deficient cell lines, AMPK could not be activated by a variety of agonists and stresses (12, 27, 33). More recently, muscle mass contraction and other agonists were unable to activate AMPK2 in mouse skeletal muscle mass lacking the expression of LKB1 (26), and AMPK phosphorylation at Thr172 was markedly diminished in mouse liver deficient in LKB1 (28). Although these studies support the SIR2L4 notion that LKB1 is usually a regulator of AMPK, the finding that AMPK possessed significant basal activity and phosphorylation at Thr172 in LKB1-deficient cells (12, 27) suggested that there were option regulators. Recent studies revealed that CAMKK isoforms are likely to also phosphorylate AMPK at Thr172, based on the finding that the CAMKK inhibitor, STO-609, as well as short interfering (si)RNA-mediated knockdown of CAMKK isoforms, inhibited the basal AMPK activity in LKB1-deficient cell lines, as well as the activation of AMPK that is observed in response to brokers that elevate cellular Ca2+ levels (13, 16, 32). CAMKK isoforms are highly expressed in Erythromycin Cyclocarbonate neuronal tissue, and K+-induced depolarization of rat cerebrocortical slices, which increases Ca2+ without affecting ATP levels, was observed to activate AMPK in a manner that was inhibited by STO-609. This study suggested that CAMKK rather than LKB1 controls AMPK in Ca2+-regulated pathways, at least in neuronal tissues. Although expression of CAMKK isoforms was detected in tissues, including testis, spleen, and heart at low levels (3), whether CAMKKs function to activate AMPK in these tissues is unknown. AMPK plays a key role in regulating lipid and glucose metabolism in cardiac muscle mass, where it is activated when oxygen and/or blood supply is compromised during hypoxic and/or ischemic conditions. Activation of AMPK in cardiac muscle mass stimulates fatty acid oxidation (18), glucose uptake (23), and glycolysis (20), to generate ATP and thereby safeguard cardiac tissues during and following ischemic or hypoxic stress. Mice that have reduced AMPK activity in cardiac muscle mass caused by the overexpression of a dominant-negative form of AMPK are more susceptible to cardiac damage during ischemia and reperfusion experiments (24). Although LKB1 appears to be a major regulator of AMPK2 in skeletal muscle mass (26), the identity of the upstream kinase(s) that regulates AMPK in the cardiac muscle mass is less certain. Previous studies have suggested that at least two individual activities that phosphorylate and activate AMPK could be resolved from heart extracts, and the activity of one of these was reportedly stimulated by ischemia (2, 4). Even though identity of this ischemia-stimulated enzyme is usually unknown, immunoprecipitation studies indicated that it was not LKB1 (2). In this study, we employed mice that were deficient in cardiac and skeletal muscle mass LKB1, to define the role that cardiac muscle mass LKB1 plays in regulating the activity of AMPK isoforms, as well as cellular energy levels, in the heart under normoxic, no-flow ischemic, and anoxic conditions. EXPERIMENTAL PROCEDURES Materials Protease inhibitor cocktail tablets were obtained from Roche (no. 1697498, Erythromycin Cyclocarbonate Lewes, Sussex, UK), protein G-Sepharose, and [-32P]ATP were purchased from Amersham Biosciences (Little Chalfont, UK), precast SDS-polyacrylamide Bis-Tris gels were from Invitrogen, phosphocellulose P81 paper was from Whatman. Medium.