br Results br Discussion Cardiac dysfunction common

Results
Discussion Cardiac dysfunction, common complication of severe sepsis, is one cause of death in intensive care units. Accumulated evidence revealed the regulatory effect of autophagy on sepsis-induced cardiac dysfunction [15], [22], although the mechanisms in young and aging are not elucidated. Our study demonstrated a notable increase in autophagy level in young mice hearts challenged with LPS. Also, we detected a significant attenuation of p-AMPK STF 083010 level in the aged mice with septic cardiac dysfunction, suggesting a closed relevance of AMPK in sepsis-induced cardiac dysfunction. Given the demonstration that both phosphorylated AMPK and phosphorylated mTOR contributed to the stimulation of cardiomyocytes contractility, we wondered to know whether the crosstalk between AMPK and mTOR exists. We found that pretreatment of A769662, an activator of AMPK, restored the cardiomyocytes contractility partly due to the blunted phosphorylation of S6 which is the downstream protein of mTOR. Our study further demonstrated preserved autophagy against LPS in aged mice following A769662 treatment. It is highly needed to clarify the functional roles of autophagy in sepsis-cardiac dysfunction in young and aged mice. Interestingly, our data show that pharmacological induction of AMPK that enhanced the autophagy improves contractile responses in aged murine cardiomyocytes challenged with LPS. A new published study showed that myocardial aging is a T-cell-mediated phenomenon that heart-directed immune responses may spontaneously arise in the elderly [23]. Improvement of LPS-induced mitochondrial dysfunction by fasudil was attributed to inhibition of ROCK-dependent Drp1 phosphorylation and activation of autophagic processes [24]. Overall our data indicate the relationship between AMPK and autophagy response to LPS treatment in heart dysfunction. Autophagy which has been demonstrated to be essential for cellular homeostasis, is the catabolic process for delivering cytosolic cargo to the lysosome for degradation. The role of autophagy in cardiovascular injuries of different experimental models is still a controversial topic. Excessive autophagy can induce cell death, the process of which is called autophagic cell death. A study had indicated that inhibition of autophagy protects the heart from pathological cardiac dysfunction in animal models of ischemia-reperfusion and hypertrophy [25]. Alleviation of autophagy might have therapeutic benefit in treating several cardiac diseases [26]. A study newly demonstrates that TFEB mediated autophagy is crucial for protection against LPS induced myocardial injury particularly in aging senescent heart [15]. In our results, the Atg5, p62 and LC3 were elevated in response to LPS treatment in mice hearts in this study. This may indicate that LPS can stimulate severe autophagy, which is reported in previous studies [27]. A study showed that the a maladaptive role for autophagy in hearts subjected to LPS challenge [28]. Our data suggested that LPS challenge-induced myocardial autophagy is an adaptive response as evidenced by the favorable response from A769662 against LPS-induced cardiac injury which is expected to stimulate AMPK. More importantly, our results indicated that aging accentuated LPS-induced myocardial dysfunction and survival possibly through abating myocardial autophagy induction in response to LPS challenge demonstrating by cardiomyocytes contractility and intracellular Ca2+ homeostasis. Although induction of AMPK has been demonstrated to prevent cardiac injury after LPS treatment in aged mice, the interaction between cardiac dysfunction and myocardial autophagy during sepsis still remains to be clarified, especially whether there is a “bridge” between them. In order to elucidate the upstream and downstream signals that regulate autophagy in our study, we examined several key molecules which may be involved in. The data here showed that PP2A, PP2Cα, mTOR and S6 were key factors in this process. It is known that The PP2 heterotrimeric protein phosphatase is ubiquitously expressed. Its serine/threonine phosphatase activity has broad substrate specificity and diverse cellular functions. Moreover, the mTOR contributes to cell survival in cardiomyocytes and regulates cell proliferation, apoptosis, cell migration and metabolism. In our study, PP2A and PP2Cαdecreased the phosphorylation of AMPK accompanied by inhancing autophagy. Further experiments showed that inhibition of AMPK could increase the p-mTOR and p-S6 proteins expression. The expression of phosphorylation of mTOR and S6 decreased in the presence of the AMPK activator A769662. These results favor the formation the AMPK/mTOR/S6 pathway.