Abstract
FasL only induces cardiomyocyte (CM) apoptosis when transcription is inhibited. To identify transcription-dependent cardioprotective pathways, we performed microarray analyses on CM that were untreated or stimulated with FasL, in the absence or presence of low-dose Actinomycin D (ActD, to inhibit transcription). 23 transcripts were identified as upregulated by FasL and inhibited by ActD, including Serum- and Glucocorticoid-responsive Kinase-1 (SGK1), a serine-threonine kinase that is expressed in the heart and previously reported to regulate sodium channels. Studies of rats after aortic constriction demonstrated that both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CM stimulated in vitro with IGF-I or phenylephrine (PE). To examine the functional effects of SGK1 in CM, we generated recombinant Adenoviral vectors (Ad) carrying HA-tagged, constitutively-active (CA, S422D) and kinase-dead (KD, K127M) mutants of SGK1. In the absence of stimulation, Ad.SGK1.CA increased SGK1 activity 2.45|[plusmn]|0.21-fold (p<0.05), while Ad.SGK1.KD reduced it (0.44|[plusmn]|0.02-fold, p<0.05) compared to control-Ad infected CMs. Stimulation of control-Ad infected CMs with IGF-I induced a comparable, 2.3-fold increase in SGK1 activity. Treatment of Ad.SGK1.CA-infected CM with IGF-I further increased SGK1 activity while Ad. SGK1.KD inhibited it (2.26|[plusmn]|0.07- and 0.40|[plusmn]|0.02-fold, respectively, p<0.05 vs IGF-I-treated control cells). Thus, Ad.SGK1.CA increases SGK1 activity in CMs but remains responsive to further stimulation, and Ad.SGK1.KD acts as a dominant negative to inhibit endogenous SGK1 activity. Neither Ad.SGK1.CA nor Ad.SGK1.KD had significant effects on CM apoptosis in unstimulated CMs. In contrast, Ad.SGK1.CA substantially protected CMs against both serum deprivation (SD)- and hypoxia-induced apoptosis, while inhibition of SGK1 significantly increased the number of apoptotic nuclei compared with control virus-infected CM in both models. These changes in nuclear morphology correlated well with DNA laddering, the biochemical hallmark of apoptosis, and cleavage (activation) of caspase-3, as well as overall cell viability, as indicated by the MTT assay. Of note, the protective effect of IGF-I was significantly but incompletely reversed by expression of SGK1.KD (p<0.05). Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of Tuberin, p70s6kinase, and GSK3|[beta]| in CM which may contribute to its effects. We conclude that SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis. Thus, SGK1 may represent a novel therapeutic target for conditions such as heart failure or ischemic injury characterized by CM death.
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