Threonine-17 phosphorylation of phospholamban: a key determinant of frequency-dependent increase of cardiac contractility

Multiple studies have shown that phospholamban (PLN) plays a key role in regulation of frequency-dependent increase of cardiac contraction, a hallmark of the contractile reserve in myocardium. However, the mechanisms underlying this relationship remain elusive. Phosphorylation of PLN occurs on residues: serine-16 (Ser(16)) and threonine-17 (Thr(17)) in vivo. In isolated wild-type cardiomyocytes, we found that increases of stimulation frequency from 0.5 to 5 Hz were associated with increased Thr(17) phosphorylation of PLN and cardiac contractility. To further delineate the role of PLN phosphorylation in the frequency-dependent increases of cardiac function, three transgenic mouse models, expressing wild-type, Ser16Ala (S16A), or Thr17Ala (T17A) mutant PLN in the null background were generated. Transgenic lines expressing similar levels of wild-type or mutant PLN were selected and isolated cardiomyocytes were paced from 0.5 to 5 Hz. Upon increases in pacing frequency, the fractional shortening (FS) and rates of contraction (+dL/dt) and relaxation (-dL/dt) increased in wild-type and S16A mutant PLN cardiomyocytes. In contrast, in myocytes expressing the T17A mutant PLN, there were no increases in FS and +/-dL/dt upon increasing the frequency of stimulation. The time to 50% peak shortening (TTP(50)) and to 50% relaxation (TTR(50)) were also abbreviated to a much higher extent (two-fold) in wild-type and S16A mutant compared to T17A mutant PLN cardiomyocytes. These results indicate that Thr(17) phosphorylation of PLN is the major contributor to frequency-dependent increases of contractile and relaxation parameters in mouse cardiomyocytes, although some increases in these parameters occur even in the absence of PLN phosphorylation. Thus, the positive force-frequency relationship in cardiomyocytes is mechanistically and mainly related to PLN phosphorylation.     

Structural plasticity of 4-α-helical bundles exemplified by the puzzle-like molecular assembly of the Rop protein

The dimeric Repressor of Primer (Rop) protein, a widely used model system for the study of coiled-coil 4-α-helical bundles, is characterized by a remarkable structural plasticity. Loop region mutations lead to a wide range of topologies, folding states, and altered physicochemical properties. A protein-folding study of Rop and several loop variants has identified specific residues and sequences that are linked to the observed structural plasticity. Apart from the native state, native-like and molten-globule states have been identified; these states are sensitive to reducing agents due to the formation of nonnative disulfide bridges. Pro residues in the loop are critical for the establishment of new topologies and molten globule states; their effects, however, can be in part compensated by Gly residues. The extreme plasticity in the assembly of 4-α-helical bundles reflects the capacity of the Rop sequence to combine a specific set of hydrophobic residues into strikingly different hydrophobic cores. These cores include highly hydrated ones that are consistent with the formation of interchain, nonnative disulfide bridges and the establishment of molten globules. Potential applications of this structural plasticity are among others in the engineering of bio-inspired materials.Recurrent motifs of tertiary structure are convenient model systems for studying protein folding and potentially also for the design of bio-inspired materials. For protein design purposes, structural plasticity is an important, although poorly understood, parameter to be considered, as it is among the main reasons that the re-engineering of proteins toward novel materials is not yet satisfactorily manageable (1, 2).The present study focuses on the structural plasticity associated with the 4-α-helical bundle (4HB) motif. 4HBs consist of four amphipathic α-helices packed in a parallel or antiparallel fashion (3, 4). Their folding is largely determined by a repeating pattern of hydrophobic and hydrophilic residues, organized on the basis of seven-residue repeats (heptads) (5). Being the simplest tertiary motif, 4HBs have been subject to numerous protein-folding studies; attempts have been made to exploit them as building blocks for bio-inspired materials (6).A paradigm of a highly regular 4HB is the RNA-binding ColE1 Repressor of Primer (Rop) protein (7–9), also referred to as RNA-one-modulator (ROM). Each monomer is an α-helical hairpin consisting of two antiparallel α-helices connected by a short loop. The sequence of Rop displays a heptad repeats pattern that is interrupted only in the loop region.Structural simplicity makes Rop an attractive model system for the study of the folding of 4HBs. The loop region and the hydrophobic core have thereby attracted particular attention, as these regions are linked with the remarkable ability of Rop mutants to adopt altered topologies and properties (10–15). Striking examples of loop variants include mutant Loopless Rop (LLR), in which an uninterrupted pattern of heptad repeats is established through a five-residue deletion in the loop. In this “loopless” mutant, the α-helical hairpin of the monomer is converted into a single helix (15, 16). The complete LLR molecule is a tetramer that is completely reorganized relative to the dimeric wild-type (WT) Rop, thereby becoming a hyper-thermostable protein (16). On the other hand, establishment of an uninterrupted heptad periodicity through a two-residue insertion in the loop produces minimal changes relative to WT in terms of structure and properties (12). Thus, these two mutants with uninterrupted patterns of heptads reveal that there is a considerable structural plasticity inherent to the Rop sequence, but the relationship between heptad periodicity and the structural/physicochemical properties is complex.Extreme structural plasticity producing completely altered 4HB topologies is also associated with point mutations in the loop region. Replacement of loop residue Ala31 by Pro (17) results in a complete reorganization of the entire protein, which is converted from the canonical left-handed, all-antiparallel form into a right-handed mixed-parallel and antiparallel 4-α-helical bundle, displaying a “bisecting U” topology that is to a large extent determined by the local conformation at residue 31 (18). Mutant A31P displays two variations of the bisecting U topology; these differ in the relative juxtaposition of the α-helices (19). These conformations crystallize in different space groups (orthorhombic and monoclinic); both space groups have been occasionally observed in the same crystallization drop, indicating the coexistence of the two forms in solution. Molecular dynamics simulations for A31P have demonstrated a potential for the interconversion between the two conformations (14).Hydrophobic core mutants occasionally also display structural plasticity producing a new (“syn”) topology (20) that results from the “anti”-topology of WT through a 180° flip of one monomer around the dyad axis normal to the long axis of WT. The competition between the anti and syn topologies and the mixture of the two structures have been studied in detail for some Rop mutants (21, 22).Apart from structural plasticity, a closely related issue associated with the folding of Rop is the role of Cys residues (Cys-38 and Cys-52). Both residues are buried in the hydrophobic core and are not involved in the formation of disulfide bridges in any of the known structures of WT and its mutants. Surprisingly, however, a Cys-free variant (CYSfree) that conserves the structure, stability, and in vivo activity of WT exhibits dramatically faster unfolding kinetics (23).The present study focuses on the role of the loop region and Cys residues in the structural plasticity of Rop. To explore the conversion of the WT anti-topology into the bisecting U topology of A31P, the three double mutants D30P/A31G (PG), D30G/A31P (GP), and D30P/A31P (PP) have been constructed for loop positions 30 and 31. These mutations combine the effects of the most constrained amino acid (Pro) and of the least constrained one (Gly). In addition, the potential role of Cys residues in Rop folding is explored by following the effects of reducing agents.     

Maternal and cord blood hormones in relation to birth size

Birth size has been associated with adult life diseases, but the endocrine factors that are likely involved are not established. We evaluated the associations of maternal and cord blood hormones with birth size in normal pregnancies, and examined possible effect modification by maternal height, on the basis of prior suggestive evidence. In a prospective study of normal singleton pregnancies in Boston, USA and Shanghai, China, maternal hormone levels at the 27th gestational week were available for 225 pregnancies in Boston and 281 in Shanghai and cord blood measurements for 92 pregnancies in Boston and 110 in Shanghai. Pearson partial correlation coefficients of log-transformed hormone levels with birth weight and length were calculated. Overall, positive correlations with birth weight were found for maternal estriol (r = 0.19; p < 0.001) and progesterone (r = 0.15; p < 0.001) and these associations were more evident among taller mothers. There was an inverse association of cord blood progesterone (r = ?0.16; p < 0.03) with birth weight. In Boston, cord blood IGF-1 was positively associated with birth weight (r = 0.22; p < 0.04) and length (r = 0.25; p < 0.02), particularly among taller mothers (r = 0.43 and 0.38, respectively; p < 0.02), whereas among taller mothers in Shanghai the associations of IGF-2 with birth size appeared to be at least as strong as those of IGF-1. In conclusion, maternal estriol and progesterone, and cord blood IGF-1 were positively correlated with birth size. All correlations tended to be more pronounced among offspring of taller mothers. Among taller mothers in Shanghai, IGF-2 appeared to be at least as strongly associated with birth size as IGF-1.     

Sleep Deprivation: Effects on Weight Loss and Weight Loss Maintenance

This narrative review presents the findings from intervention studies on the effects of sleep deprivation on eating habits, metabolic rate, and the hormones regulating metabolism, and discusses their relevance to weight loss efforts. Disturbed sleeping patterns lead to increased energy intake, partly from excessive snacking, mainly on foods high in fat and carbohydrates. The studies focused mainly on the effects of sleep duration, but also of sleep quality, on dietary intake during weight loss trials, and on weight loss maintenance. It is important to explore sleep routines that could enhance the efforts of obese and overweight people to lose weight, maintain their weight loss, and improve their overall health.     

Fat-Secreted Ceramides Regulate Vascular Redox State and Influence Outcomes in Patients With Cardiovascular Disease

BackgroundObesity is associated with increased cardiovascular risk; however, the potential role of dysregulations in the adipose tissue (AT) metabolome is unknown.ObjectivesThe aim of this study was to explore the role of dysregulation in the AT metabolome on vascular redox signaling and cardiovascular outcomes.MethodsA screen was conducted for metabolites differentially secreted by thoracic AT (ThAT) and subcutaneous AT in obese patients with atherosclerosis (n = 48), and these metabolites were then linked with dysregulated vascular redox signaling in 633 patients undergoing coronary bypass surgery. The underlying mechanisms were explored in human aortic endothelial cells, and their clinical value was tested against hard clinical endpoints.ResultsBecause ThAT volume was associated significantly with arterial oxidative stress, there were significant differences in sphingolipid secretion between ThAT and subcutaneous AT, with C16:0-ceramide and derivatives being the most abundant species released within adipocyte-derived extracellular vesicles. High ThAT sphingolipid secretion was significantly associated with reduced endothelial nitric oxide bioavailability and increased superoxide generated in human vessels. Circulating C16:0-ceramide correlated positively with ThAT ceramides, dysregulated vascular redox signaling, and increased systemic inflammation in 633 patients with atherosclerosis. Exogenous C16:0-ceramide directly increased superoxide via tetrahydrobiopterin-mediated endothelial nitric oxide synthase uncoupling and dysregulated protein phosphatase 2 in human aortic endothelial cells. High plasma C16:0-ceramide and its glycosylated derivative were independently related with increased risk for cardiac mortality (adjusted hazard ratios: 1.394; 95% confidence interval: 1.030 to 1.886; p = 0.031 for C16:0-ceramide and 1.595; 95% confidence interval: 1.042 to 2.442; p = 0.032 for C16:0-glycosylceramide per 1 SD). In a randomized controlled clinical trial, 1-year treatment of obese patients with the glucagon-like peptide-1 analog liraglutide suppressed plasma C16:0-ceramide and C16:0-glycosylceramide changes compared with control subjects.ConclusionsThese results demonstrate for the first time in humans that AT-derived ceramides are modifiable regulators of vascular redox state in obesity, with a direct impact on cardiac mortality in advanced atherosclerosis. (The Interaction Between Appetite Hormones; NCT02094183)