Estrogen receptors interact with the alpha catalytic subunit of AMP-activated protein kinase Running title: Estrogen receptors interact with AMPK

SYNOPSIS Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signaling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity, and cancer. AMPK is targeted by 17β-estradiol (E2), the main circulating estrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an estrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit binding domain. Finally, both ERs interacted with the upstream LKB1 kinase complex, which is required for E2-dependent activation of AMPK. We conclude that estradiol activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. Summary statement: We identified a novel interaction between the classical estrogen receptors (ERα and ERβ) and the catalytic subunit of AMPK in several cell types. In addition we demonstrate that estradiol activates AMPK through ERα and requires the upstream kinase complex LKB1

Length-dependent activation in three striated muscle types of the rat

The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca2+ is termed length-dependent activation. Length-dependent activation is a property of all striated muscles, yet the relative extent of length-dependent activation between skeletal muscle and cardiac muscle is unclear. Although length-dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length-dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length-dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca2+-force relationship was determined at sarcomere lengths (SL) of 1.95 μm, 2.10 μm and 2.25 μm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca2+ than CM. Length-dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca2+-force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X-ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL-LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length-dependent activation differs among the three types of striated muscle and (2) these differences in the length-dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations

Improved metabolism and redox state with a novel preservation solution: Implications for donor lungs after cardiac death (DCD)

textabstractLungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n=3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant