A genomics perspective of personalized prevention and management of obesity.

This review discusses the landscape of personalized prevention and management of obesity from a nutrigenetics perspective. Focusing on macronutrient tailoring, we discuss the impact of genetic variation on responses to carbohydrate, lipid, protein, and fiber consumption. Our bioinformatic analysis of genomic variants guiding macronutrient intake revealed enrichment of pathways associated with circadian rhythm, melatonin metabolism, cholesterol and lipoprotein remodeling and PPAR signaling as potential targets of macronutrients for the management of obesity in relevant genetic backgrounds. Notably, our data-based in silico predictions suggest the potential of repurposing the SYK inhibitor fostamatinib for obesity treatment in relevant genetic profiles. In addition to dietary considerations, we address genetic variations guiding lifestyle changes in weight management, including exercise and chrononutrition. Finally, we emphasize the need for a refined understanding and expanded research into the complex genetic landscape underlying obesity and its management.

A junctional cAMP compartment regulates rapid Ca2+ signaling in atrial myocytes.

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.

CYP1A2 polymorphisms modify the association of habitual coffee consumption with appetite, macronutrient intake, and body mass index: results from an observational cohort and a cross-over randomized study.

BACKGROUND/OBJECTIVES: Evidence regarding the influence of coffee on appetite and weight control is equivocal and the influence of covariates, such as genetic variation in caffeine metabolism, remains unknown. Herein, we addressed the novel hypothesis that genetic variation in CYP1A2, a gene responsible for more than 95% of caffeine metabolism, differentially impacts the association of coffee consumption with appetite and BMI among individuals with different genetic predispositions to obesity. SUBJECTS/METHODS: A cross-over randomized intervention study involving 18 volunteers assessed the effects of coffee consumption on dietary intake, appetite, and levels of the appetite-controlling hormones asprosin and leptin. Data on habitual coffee intake, BMI, and perceived appetite were obtained from an observational cohort of 284 volunteers using validated questionnaires. Participants were stratified according to a validated genetic risk score (GRS) for obesity and to the -163C > A (rs762551) polymorphism of CYP1A2 as rapid (AA), intermediate (AC), or slow (CC) caffeine metabolizers. RESULTS: Coffee consumption led to lower energy and dietary fat intake and circulating asprosin levels (P for interaction of rs762551 genotype*coffee consumption=0.056, 0.039, and 0.043, respectively) as compared to slow/intermediate metabolizers. High coffee consumption was more prevalent in rapid compared to slow metabolizers (P = 0.008 after adjustment for age, sex, and BMI) and was associated with lower appetite perception and lower BMI only in rapid metabolizers (P for interaction of rs762551 genotype*coffee consumption = 0.002 and 0.048, respectively). This differential association of rs762551 genotype and coffee consumption with BMI was more evident in individuals at higher genetic risk of obesity (mean adjusted difference in BMI = -5.82 kg/m2 for rapid versus slow/intermediate metabolizers who consumed more than 14 cups of coffee per week). CONCLUSIONS: CYP1A2 rs762551 polymorphism modifies the association of habitual coffee consumption with BMI, in part by influencing appetite, energy intake and circulating levels of the orexigenic hormone asprosin. This association is more evident in subjects with high genetic predisposition to obesity. ClinicalTrials.gov: registered Clinical Trial NCT04514588.

Genome-wide transcriptomics identifies an early preclinical signature of prion infection.

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

Structure-function analysis of naturally occurring apolipoprotein A-I L144R, A164S and L178P mutants provides insight on their role on HDL levels and cardiovascular risk.

Naturally occurring point mutations in apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), may affect plasma HDL-cholesterol levels and cardiovascular risk. Here, we evaluated the effect of human apoA-I mutations L144R (associated with low HDL-cholesterol), L178P (associated with low HDL-cholesterol and increased cardiovascular risk) and A164S (associated with increased cardiovascular risk and mortality without low HDL-cholesterol) on the structural integrity and functions of lipid-free and lipoprotein-associated apoA-I in an effort to explain the phenotypes of subjects carrying these mutations. All three mutants, in lipid-free form, presented structural and thermodynamic aberrations, with apoA-I[L178P] presenting the greatest thermodynamic destabilization. Additionally, apoA-I[L178P] displayed reduced ABCA1-mediated cholesterol efflux capacity. When in reconstituted HDL (rHDL), apoA-I[L144R] and apoA-I[L178P] were more thermodynamically destabilized compared to wild-type apoA-I, both displayed reduced SR-BI-mediated cholesterol efflux capacity and apoA-I[L144R] showed severe LCAT activation defect. ApoA-I[A164S] was thermodynamically unaffected when in rHDL, but exhibited a series of functional defects. Specifically, it had reduced ABCG1-mediated cholesterol and 7-ketocholesterol efflux capacity, failed to reduce ROS formation in endothelial cells and had reduced capacity to induce endothelial cell migration. Mechanistically, the latter was due to decreased capacity of rHDL-apoA-I[A164S] to activate Akt kinase possibly by interacting with endothelial LOX-1 receptor. The impaired capacity of rHDL-apoA-I[A164S] to preserve endothelial function may be related to the increased cardiovascular risk for this mutation. Overall, our structure-function analysis of L144R, A164S and L178P apoA-I mutants provides insights on how HDL-cholesterol levels and/or atheroprotective properties of apoA-I/HDL are impaired in carriers of these mutations.

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis.

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a's Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability.

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-ß, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERß-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERß. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful.

Phosphorylation of serine96 of histidine-rich calcium-binding protein by the Fam20C kinase functions to prevent cardiac arrhythmia.

Precise Ca cycling through the sarcoplasmic reticulum (SR), a Ca storage organelle, is critical for proper cardiac muscle function. This cycling initially involves SR release of Ca via the ryanodine receptor, which is regulated by its interacting proteins junctin and triadin. The sarco/endoplasmic reticulum Ca ATPase (SERCA) pump then refills SR Ca stores. Histidine-rich Ca-binding protein (HRC) resides in the lumen of the SR, where it contributes to the regulation of Ca cycling by protecting stressed or failing hearts. The common Ser96Ala human genetic variant of HRC strongly correlates with life-threatening ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy. However, the underlying molecular pathways of this disease remain undefined. Here, we demonstrate that family with sequence similarity 20C (Fam20C), a recently characterized protein kinase in the secretory pathway, phosphorylates HRC on Ser96. HRC Ser96 phosphorylation was confirmed in cells and human hearts. Furthermore, a Ser96Asp HRC variant, which mimics constitutive phosphorylation of Ser96, diminished delayed aftercontractions in HRC null cardiac myocytes. This HRC phosphomimetic variant was also able to rescue the aftercontractions elicited by the Ser96Ala variant, demonstrating that phosphorylation of Ser96 is critical for the cardioprotective function of HRC. Phosphorylation of HRC on Ser96 regulated the interactions of HRC with both triadin and SERCA2a, suggesting a unique mechanism for regulation of SR Ca homeostasis. This demonstration of the role of Fam20C-dependent phosphorylation in heart disease will open new avenues for potential therapeutic approaches against arrhythmias.

The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics.

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.

The future of apolipoprotein E mimetic peptides in the prevention of cardiovascular disease.

PURPOSE OF REVIEW: This review aims to discuss the recent developments in the area of apolipoprotein E (apoE) mimetics and their therapeutic potential for treating cardiovascular disease, the leading cause of mortality worldwide. RECENT FINDINGS: Ongoing research efforts target the development of novel therapies that would not only reduce circulating levels of atherogenic lipoproteins, but could also increase high density lipoprotein cholesterol (HDL-C) levels and/or improve HDL function. Among them, synthetic peptides that mimic the structure of natural human apoE, a component of triglyceride-rich lipoproteins and HDL, have been designed and proven to be functionally similar to apoE. In specific, apoE mimetic peptides mediate hepatic clearance of circulating atherogenic lipoproteins, dramatically reduce plasma cholesterol, and lead to attenuation of atherosclerosis development in vivo. These peptides also exhibit pleiotropic antiatherogenic properties, such as macrophage cholesterol efflux capacity, as well as anti-inflammatory and antioxidative functions. SUMMARY: ApoE mimetics are undergoing preclinical and clinical evaluation with promising results to date that render them attractive candidates in cardiovascular disease prevention and treatment.

Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation.

Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies.

HAX-1 regulates SERCA2a oxidation and degradation.

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.

Prion infections and anti-PrP antibodies trigger converging neurotoxic pathways.

Prions induce lethal neurodegeneration and consist of PrPSc, an aggregated conformer of the cellular prion protein PrPC. Antibody-derived ligands to the globular domain of PrPC (collectively termed GDL) are also neurotoxic. Here we show that GDL and prion infections activate the same pathways. Firstly, both GDL and prion infection of cerebellar organotypic cultured slices (COCS) induced the production of reactive oxygen species (ROS). Accordingly, ROS scavenging, which counteracts GDL toxicity in vitro and in vivo, prolonged the lifespan of prion-infected mice and protected prion-infected COCS from neurodegeneration. Instead, neither glutamate receptor antagonists nor inhibitors of endoplasmic reticulum calcium channels abolished neurotoxicity in either model. Secondly, antibodies against the flexible tail (FT) of PrPC reduced neurotoxicity in both GDL-exposed and prion-infected COCS, suggesting that the FT executes toxicity in both paradigms. Thirdly, the PERK pathway of the unfolded protein response was activated in both models. Finally, 80% of transcriptionally downregulated genes overlapped between prion-infected and GDL-treated COCS. We conclude that GDL mimic the interaction of PrPSc with PrPC, thereby triggering the downstream events characteristic of prion infection.

Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias.

A hallmark of human and experimental heart failure is deficient sarcoplasmic reticulum (SR) Ca-uptake reflecting impaired contractile function. This is at least partially attributed to dephosphorylation of phospholamban by increased protein phosphatase 1 (PP1) activity. Indeed inhibition of PP1 by transgenic overexpression or gene-transfer of constitutively active inhibitor-1 improved Ca-cycling, preserved function and decreased fibrosis in small and large animal models of heart failure, suggesting that inhibitor-1 may represent a potential therapeutic target. We recently identified a novel human polymorphism (G109E) in the inhibitor-1 gene with a frequency of 7% in either normal or heart failure patients. Transgenic mice, harboring cardiac-specific expression of G109E inhibitor-1, exhibited decreases in contractility, Ca-kinetics and SR Ca-load. These depressive effects were relieved by isoproterenol stimulation. Furthermore, stress conditions (2Hz +/- Iso) induced increases in Ca-sparks, Ca-waves (60% of G109E versus 20% in wild types) and after-contractions (76% of G109E versus 23% of wild types) in mutant cardiomyocytes. Similar findings were obtained by acute expression of the G109E variant in adult cardiomyocytes in the absence or presence of endogenous inhibitor-1. The underlying mechanisms included reduced binding of mutant inhibitor-1 to PP1, increased PP1 activity, and dephosphorylation of phospholamban at Ser16 and Thr17. However, phosphorylation of the ryanodine receptor at Ser2808 was not altered while phosphorylation at Ser2814 was increased, consistent with increased activation of Ca/calmodulin-dependent protein kinase II (CaMKII), promoting aberrant SR Ca-release. Parallel in vivo studies revealed that mutant mice developed ventricular ectopy and complex ventricular arrhythmias (including bigeminy, trigeminy and ventricular tachycardia), when challenged with isoproterenol. Inhibition of CaMKII activity by KN-93 prevented the increased propensity to arrhythmias. These findings suggest that the human G109E inhibitor-1 variant impairs SR Ca-cycling and promotes arrhythmogenesis under stress conditions, which may present an additional insult in the compromised function of heart failure carriers.

Forced swim test induces divergent global transcriptomic alterations in the hippocampus of high versus low novelty-seeker rats.

BACKGROUND: Many neuropsychiatric disorders, including stress-related mood disorders, are complex multi-parametric syndromes. Susceptibility to stress and depression is individually different. The best animal model of individual differences that can be used to study the neurobiology of affect regards spontaneous reactions to novelty. Experimentally, when naive rats are exposed to the stress of a novel environment, they display a highly variable exploratory activity and are classified as high or low responders (HR or LR, respectively). Importantly, HR and LR rats do not seem to exhibit a substantial differentiation in relation to their 'depressive-like' status in the forced swim test (FST), a widely used animal model of 'behavioral despair'. In the present study, we investigated whether FST exposure would be accompanied by phenotype-dependent differences in hippocampal gene expression in HR and LR rats. RESULTS: HR and LR rats present a distinct behavioral pattern in the pre-test session but develop comparable depressive-like status in the second FST session. At 24 h following the second FST session, HR and LR rats (stressed and unstressed controls) were sacrificed and hippocampal samples were independently analyzed on whole rat genome Illumina arrays. Functional analysis into pathways and networks was performed using Ingenuity Pathway Analysis (IPA) software. Notably, hippocampal gene expression signatures between HR and LR rats were markedly divergent, despite their comparable depressive-like status in the FST. These molecular differences are reflected in both the extent of transcriptional remodeling (number of significantly changed genes) and the types of molecular pathways affected following FST exposure. A markedly higher number of genes (i.e., 2.28-fold) were statistically significantly changed following FST in LR rats, as compared to their HR counterparts. Notably, genes associated with neurogenesis and synaptic plasticity were induced in the hippocampus of LR rats in response to FST, whereas in HR rats, FST induced pathways directly or indirectly associated with induction of apoptotic mechanisms. CONCLUSIONS: The markedly divergent gene expression signatures exposed herein support the notion that the hippocampus of HR and LR rats undergoes distinct transcriptional remodeling in response to the same stress regimen, thus yielding a different FST-related 'endophenotype', despite the seemingly similar depressive-like phenotype.

Reprogramming of the microRNA transcriptome mediates resistance to rapamycin.

The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation that is often deregulated in cancer. Inhibitors of mTOR, including rapamycin and its analogues, are being evaluated as antitumor agents. For their promise to be fulfilled, it is of paramount importance to identify the mechanisms of resistance and develop novel therapies to overcome it. Given the emerging role of microRNAs (miRNAs) in tumorigenesis, we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Long-term rapamycin treatment showed extensive reprogramming of miRNA expression, characterized by up-regulation of miR-17-92 and related clusters and down-regulation of tumor suppressor miRNAs. Inhibition of members of the miR-17-92 clusters or delivery of tumor suppressor miRNAs restored sensitivity to rapamycin. This study identifies miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. It also identifies potential markers to assess the efficacy of treatment and provides novel therapeutic targets to treat rapamycin-resistant tumors.

A novel role for phospholamban in the thalamic reticular nucleus.

The thalamic reticular nucleus (TRN) is a brain region that influences vital neurobehavioral processes, including executive functioning and the generation of sleep rhythms. TRN dysfunction underlies hyperactivity, attention deficits, and sleep disturbances observed across various neurodevelopmental disorders. A specialized sarco-endoplasmic reticulum calcium (Ca2+) ATPase 2 (SERCA2)-dependent Ca2+ signaling network operates in the dendrites of TRN neurons to regulate their bursting activity. Phospholamban (PLN) is a prominent regulator of SERCA2 with an established role in myocardial Ca2+-cycling. Our findings suggest that the role of PLN extends beyond the cardiovascular system to impact brain function. Specifically, we found PLN to be expressed in TRN neurons of the adult mouse brain, and utilized global constitutive and innovative conditional genetic knockout mouse models in concert with electroencephalography (EEG)-based somnography and the 5-choice serial reaction time task (5-CSRTT) to investigate the role of PLN in sleep and executive functioning, two complex behaviors that map onto thalamic reticular circuits. The results of the present study indicate that perturbed PLN function in the TRN results in aberrant TRN-dependent phenotypes in mice (i.e., hyperactivity, impulsivity and sleep deficits) and support a novel role for PLN as a critical regulator of SERCA2 in the TRN neurocircuitry.

Small heat shock protein 20 interacts with protein phosphatase-1 and enhances sarcoplasmic reticulum calcium cycling.

BACKGROUND: Heat shock proteins (Hsp) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling, and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. The objective of this study was to determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in calcium (Ca) cycling. METHODS AND RESULTS: Hsp20 overexpression in intact animals resulted in significant enhancement of cardiac function, coupled with augmented Ca cycling and sarcoplasmic reticulum Ca load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing nonphosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20 overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1-PLN axis. This hypothesis was supported by cell fractionation, coimmunoprecipitation, and coimmunolocalization studies, which revealed an association between Hsp20, PP1, and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73 to 160 in Hsp20 and AA 163 to 330 in PP1. CONCLUSIONS: Hsp20 is a novel regulator of sarcoplasmic reticulum Ca cycling by targeting the PP1-PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.

Regulation of adverse remodelling by osteopontin in a genetic heart failure model.

AIMS: Desmin, the muscle-specific intermediate filament protein, is a major target in dilated cardiomyopathy and heart failure in humans and mice. The hallmarks of desmin-deficient (des(-/-)) mice pathology include pronounced myocardial degeneration, extended fibrosis, and osteopontin (OPN) overexpression. We sought to identify the molecular and cellular events regulating adverse cardiac remodelling in des(-/-) mice and their potential link to OPN. METHODS AND RESULTS: In situ hybridization, histology, and immunostaining demonstrated that inflammatory cells and not cardiomyocytes were the source of OPN. RNA profile comparison revealed that activation of inflammatory pathways, sustained by innate immunity mechanisms, predominated among all changes occurring in degenerating des(-/-) myocardium. The expression of the most highly up-regulated genes (OPN: 226×, galectin-3: 26×, osteoactivin/Gpnmb/DC-HIL: 160× and metalloprotease-12: 98×) was associated with heart infiltrating macrophages. To evaluate the role of OPN, we generated des(-/-)OPN(-/-) mice and compared their cardiac function and remodelling indices with those of des(-/-). Osteopontin promoted cardiac dysfunction in this model since des(-/-)OPN(-/-) mice showed 53% improvement of left ventricular function, paralleled to an up to 44% reduction in fibrosis. The diminished fibrotic response in the absence of OPN could be partly mediated by a dramatic reduction in myocardial galectin-3 levels, associated with an impaired galectin-3 secretion by OPN-deficient infiltrating macrophages. CONCLUSION: Cardiomyocyte death due to desmin deficiency leads to inflammation and subsequent overexpression of a series of remodelling modulators. Among them, OPN seems to be a major regulator of des(-/-) adverse myocardial remodelling and it functions at least by potentiating galectin-3 up-regulation and secretion.