AMPK Modulates Contractile Function of Cardiomyocytes
The present study will focus on the AMP-activated protein kinase (AMPK), a molecular stress response pathway that is activated by an increase in the intracellular concentration of AMP (Young 2008). AMPK is expressed throughout the brain, including areas that are involved in the control of food intake and neuroendocrine function(Lim, Kola et al. 2010). AMPK is also involved in the pathogenesis of Alzheimer's disease (AD) and Huntington's disease (HD) (Ramamurthy and Ronnett 2012). In this thesis I am using the heart as a model for understanding AMPK, which will hopefully provide insights that can be applied to the brain. Current knowledge regarding the impact of AMPK activation or its deficiency in both humans and animals promotes the notion that AMPK activation possesses a beneficial effect in the ischemic brain and in heart function. However, the specific role of AMPK alpha1 or AMPK alpha2 in mediating cardiomyocytes contractile function remains elusive. We are also interested in urocortin2 (Ucn2), endogenous corticotrophin releasing factor (CRF)-like peptides that exert beneficial cardiovascular effects (Turnley and Bartlett 1999). However, the underlying cellular mechanism behind this is not completely understood. Studies performed in Chapter 2 were designed to investigate how AMPK activation modulates the contractility of isolated cardiomyocytes. We found that AMPK activator, A-769662 induced maximal velocity of shortening (-dL/dt) and relengthening (+d L /d t ), peak height and peak shortening (PS) amplitude in both WT and AMPK alpha2 KO cardiomyocytes, but did not affect time-to-90% peak shortening (TPS90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPK alpha2 KO hearts. However, the rise of intracellular Ca 2+ levels as well as resting intracellular Ca 2+ levels have no significant difference between WT, AMPK alpha2 KO and AMPK KD groups with and without the presence of A-769662. It is also known that, WT, AMPK alpha2 KO and AMPK KD group displayed a robust phosphorylation of AMPK and downstream acetylCoA carboxylase (ACC). Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at the Ser 150 site which is related to the contractility of cardiomyocytes. Furthermore, immunoprecipitation analysis revealed that the AMPK alpha1 of cardiomyocytes was phosphorylated by A-769662. Studies performed in Chapter 3 demonstrate the potential role of Ucn2 in regulating myocardial function and elucidates the underlying signaling pathways and mechanisms. Results elicited that Ucn2 alone significantly increased maximal velocity of relenthening (+d L /d t ), peak height, peak shortening (PS) amplitude, maximal velocity of shortening (-d L /d t ), intracellular Ca 2+ levels and intracellular ATP levels without changing the resting sarcomere length in WT cardiomyocytes. However, these positive effects were abolished by anti-sauvagine-30 (a-SVG-30), a specific antagonist of corticotrophin releasing factor (CRF) CRF2 receptors. Results also showed that cardiomyocytes responded to Ucn2 via multiple signaling pathways including AMPK signaling and PKA signaling.