Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory ␥2 subunit have been shown to cause an essentially cardiacrestricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart.Objective: The ␥ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of ␥2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results:A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of ␥2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca 2؉ Key Words: familial hypertrophic cardiomyopathy Ⅲ myocardial contractility Ⅲ phosphorylation A MP-activated protein kinase (AMPK) is a crucial component of a highly conserved serine/threonine protein kinase cascade central to the control of energy balance at the cellular and whole-body levels. 1,2 AMPK exists as a ␣␤␥ heterotrimer, with ␣ being the catalytic subunit, and the ␤ and ␥ subunits performing structural and regulatory functions. Isoforms of all subunits have been identified (␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3), each being encoded by a different gene (PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, and PRKAG3, respectively). The ␣ subunits consist of a typical serine/threonine protein kinase domain at the N-terminus (which also contains the critical phosphorylation site for AMPK activation, Thr172 3 ) and a C-terminal domain involved in the binding of the ␤ and ␥ subunits. 1,2 The ␤ subunits are myristoylated at their N-terminus, contain a conserved C-terminal domain that is involved in binding of the ␣ and ␥ subunits, and a carbohydrate binding domain. The carbohydrate binding domain may allow AMPK to sense the status of cellular energy reserves in the form of glycogen in addition to responding to AMP/ATP levels. 4 The ␥ subunits have a high degree of homology in their C-terminal Original received October 31, 2011; revision received March 14, 2012; accepted March 19, 2012. In February 2012 sequences, all containing 2 pairs of highly conserved cystathionine ␤-synthase domains, which have been shown to be directly involved in the binding of adenine nucleotides. [5][6][7] In contrast, their N-terminal regions are highly variable, with ␥2 and ␥3 possessing different long N-terminal extensions compared with the shorter ␥1 isoform (Figure 1). The ␥2 and ␥3 N-terminal sequences appear to be unique in that they do not share sequence identity with each other nor with any known protein. ...

Transthyretin (TTR) binds Aβ peptide, preventing its deposition and toxicity. TTR is decreased in Alzheimer’s disease (AD) patients. Additionally, AD transgenic mice with only one copy of the TTR gene show increased brain and plasma Aβ levels when compared to AD mice with both copies of the gene, suggesting TTR involvement in brain Aβ efflux and/or peripheral clearance. Here we showed that TTR promotes Aβ internalization and efflux in a human cerebral microvascular endothelial cell line, hCMEC/D3. TTR also stimulated brain-to-blood but not blood-to-brain Aβ permeability in hCMEC/D3, suggesting that TTR interacts directly with Aβ at the blood-brain-barrier. We also observed that TTR crosses the monolayer of cells only in the brain-to-blood direction, as confirmed by in vivo studies, suggesting that TTR can transport Aβ from, but not into the brain. Furthermore, TTR increased Aβ internalization by SAHep cells and by primary hepatocytes from TTR+/+ mice when compared to TTR−/− animals. We propose that TTR-mediated Aβ clearance is through LRP1, as lower receptor expression was found in brains and livers of TTR−/− mice and in cells incubated without TTR. Our results suggest that TTR acts as a carrier of Aβ at the blood-brain-barrier and liver, using LRP1.

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-β (Aβ) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aβ interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aβ interaction, iododiflunisal (IDIF), in Aβ deposition and other AD features, using AβPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aβ levels were reduced in plasma suggesting TTR promoted Aβ clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

Abstract-Familial hypertrophic cardiomyopathy (HCM) is caused by mutations in 9 sarcomeric protein genes. The most commonly affected is ␤-myosin heavy chain (MYH7), where missense mutations cluster in the head and neck regions and directly affect motor function. Comparable mutations have not been described in the light meromyosin (LMM) region of the myosin rod, nor would these be expected to directly affect motor function. We studied 82 probands with HCM in whom no mutations had been found in MYH7 exons encoding the head and neck regions of myosin nor in the other frequently implicated disease genes. Primers were designed to amplify exons 24 to 40 of MYH7. These amplimers were subjected to temperature modulated heteroduplex analysis by denaturing high-performance liquid chromatography. An Ala1379Thr missense mutation in exon 30 segregated with disease in three families and was not present in 200 normal chromosomes. The mutation occurred on two haplotypes, indicating that it was not a polymorphism linked with another disease-causing mutation. The position of this residue within the LMM region of myosin suggests that it may be important for thick filament assembly or for accessory protein binding. A further missense mutation in exon 37, Ser1776Gly, segregated with disease in a single family and was absent from 400 population-matched control chromosomes. Because the Ser1776 residue occupies a core position in the myosin rod at which the substitution of glycine is extremely energetically unfavorable, it is likely to disrupt the coiled-coil structure. We conclude that mutation of the LMM can cause HCM and that such mutations may act through novel mechanisms of disease pathogenesis involving myosin filament assembly or interaction with thick filament binding proteins.