Neighborhood Social and Physical Environments and Sedentary Behaviors: Importance of Early Childhood in Shaping Health Behaviors.

This study aims to investigate the persistence of sedentary behaviors from early childhood to adolescence and the longitudinal association between neighborhood social and physical environments and childhood sedentary behaviors. Using the Future of Families & Child Wellbeing Study (N = 2716), we conducted a cross-lagged path analysis to examine the longitudinal association between neighborhood social and physical environments (neighborhood socioeconomic status, observed physical disorder, and crime rates) and sedentary behaviors at subsequent measurement points, after controlling for demographic factors and family socioeconomic status. We observed the continuity of sedentary behaviors in early childhood, extending throughout adolescence. Neighborhood socioeconomic status was significantly associated with sedentary behaviors at subsequent measurement points from early childhood to adolescence. Observation-based physical disorder and crime rates were associated with engagement in greater sedentary behaviors in adolescence but not in early and middle childhood. We also observed a partial mediation effect of neighborhood physical disorder and crime rates in the association between neighborhood socioeconomic status and sedentary behaviors in adolescence. Our results demonstrated the importance of early childhood in shaping healthy behaviors and highlighted the contribution of unfavorable environments to sedentary behaviors. These findings call for the need to support disadvantaged neighborhoods, particularly by addressing safety concerns and physical disorder, for promoting physical activity and reducing sedentary behaviors among adolescents.

Bioinformatics and integrated pharmacology network to identify the therapeutic targets and potential molecular mechanism of alpha-lipoic acid on primary ovarian insufficiency.

Women experiencing primary ovarian insufficiency (POI) are more likely to experience infertility, and its incidence is increasing worldwide annually. Recently, the role of alpha-lipoic acid (ALA) in the treatment of POI has been reported. However, details of the potential pharmacological targets and related molecular pathways of ALA remain unclear and need to be elucidated. Thus, this study aims to elucidate the potential therapeutic target and related molecular mechanism of ALA on POI. First, the potential targets of POI and ALA-related targets were downloaded from online public databases. Subsequently, the overlapped target genes between POI and ALA were acquired, and gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG) analysis, protein-protein interaction (PPI) networks were performed and constructed. Finally, molecular docking was performed to verify protein-to-protein effect. A total of 152 potential therapeutic targets were identified. The biological processes of the intersecting targets were mainly involved in the cellular response to peptides, response to xenobiotic stimuli, and response to peptide hormones. The highly enriched pathways were the cAMP, PI3K/AKT, estrogen, progesterone mediated oocyte maturation, and apoptosis signaling pathways. The top 10 hub targets for ALA in the treatment of POI were STAT3, STAT1, CASP3, MTOR, PTGS2, CASP8, HSP90AA1, PIK3CA, MAPK1, and ESR1. The binding between ALA and all top hub targets were verified using the molecular docking analysis. In summary, using the systematic integrated pharmacology network and bioinformatics analysis, this study illustrated that ALA participates in the treatment of POI via multiple targets and multiple pathways mechanisms.

Identification and Characterization of p300-Mediated Lysine Residues in Cardiac SERCA2a.

Impaired calcium uptake resulting from reduced expression and activity of the cardiac sarco-endoplasmic reticulum Ca2+ ATPase (SERCA2a) is a hallmark of heart failure (HF). Recently, new mechanisms of SERCA2a regulation, including post-translational modifications (PTMs), have emerged. Our latest analysis of SERCA2a PTMs has identified lysine acetylation as another PTM which might play a significant role in regulating SERCA2a activity. SERCA2a is acetylated, and that acetylation is more prominent in failing human hearts. In this study, we confirmed that p300 interacts with and acetylates SERCA2a in cardiac tissues. Several lysine residues in SERCA2a modulated by p300 were identified using in vitro acetylation assay. Analysis of in vitro acetylated SERCA2a revealed several lysine residues in SERCA2a susceptible to acetylation by p300. Among them, SERCA2a Lys514 (K514) was confirmed to be essential for SERCA2a activity and stability using an acetylated mimicking mutant. Finally, the reintroduction of an acetyl-mimicking mutant of SERCA2a (K514Q) into SERCA2 knockout cardiomyocytes resulted in deteriorated cardiomyocyte function. Taken together, our data demonstrated that p300-mediated acetylation of SERCA2a is a critical PTM that decreases the pump's function and contributes to cardiac impairment in HF. SERCA2a acetylation can be targeted for therapeutic aims for the treatment of HF.

Intraocular Pressure-Lowering and Retina-Protective Effects of Exosome-Rich Conditioned Media from Human Amniotic Membrane Stem Cells in a Rat Model of Glaucoma.

Glaucoma is one of the most devastating eye diseases, since the disease can develop into blindness and no effective therapeutics are available. Although the exact mechanisms and causes of glaucoma are unknown, increased intraocular pressure (IOP) has been demonstrated to be an important risk factor. Exosomes are lipid nanoparticles secreted from functional cells, including stem cells, and have been found to contain diverse functional molecules that control body function, inhibit inflammation, protect and regenerate cells, and restore damaged tissues. In the present study, exosome-rich conditioned media (ERCMs) were attained via hypoxic culture (2% O2) of human amniotic membrane mesenchymal stem cells (AMMSCs) and amniotic membrane epithelial stem cells (AMESCs) containing 50 times more exosome particles than normoxic culture (20% O2) medium (NCM). The exosome particles in ERCM were confirmed to be 77 nm in mean size and contain much greater amounts of growth factors (GFs) and neurotrophic factors (NFs) than those in NCM. The glaucoma-therapeutic effects of ERCMs were assessed in retinal cells and a hypertonic (1.8 M) saline-induced high-IOP animal model. CM-DiI-labeled AMMSC exosomes were found to readily penetrate the normal and H2O2-damaged retinal ganglion cells (RGCs), and AMMSC-ERCM not only facilitated retinal pigment epithelial cell (RPEC) proliferation but also protected against H2O2- and hypoxia-induced RPEC insults. The IOP of rats challenged with 1.8 M saline increased twice the normal IOP (12-17 mmHg) in a week. However, intravitreal injection of AMMSC-ERCM or AMESC-ERCM (3.9-4.5 × 108 exosomes in 10 µL/eye) markedly recovered the IOP to normal level in 2 weeks, similar to the effect achieved with platelet-derived growth factor-AB (PDGF-AB, 1.5 µg), a reference material. In addition, AMMSC-ERCM, AMESC-ERCM, and PDGF-AB significantly reversed the shrinkage of retinal layers, preserved RGCs, and prevented neural injury in the glaucoma eyes. It was confirmed that stem cell ERCMs containing large numbers of functional molecules such as GFs and NFs improved glaucoma by protecting retinal cells against oxidative and hypoxic injuries in vitro and by recovering IOP and retinal degeneration in vivo. Therefore, it is suggested that stem cell ERCMs could be a promising candidate for the therapy of glaucoma.

Blue Perovskite Nanocrystal Light-Emitting Diodes: Overcoming RuddlesdenPopper Fault-Induced Nonradiative Recombination via Post-Halide Exchange.

Metal halide perovskites (MHPs) have gained traction as emitters owing to their excellent optical properties, such as facile bandgap tuning, defect tolerance, and high color purity. Nevertheless, blue-emitting MHP light-emitting diodes (LEDs) show only marginal progress in device efficiency compared with green and red LEDs. Herein, the origin of the drop in efficiency of blue-emitting perovskite nanocrystals (PNCs) by mixing halides and the genesis of Ruddlesden-Popper faults (RPFs) in CsPbBrX Cl3-X nanocrystals is investigated. Using scanning transmission electron microscopy and density functional theory calculations, the authors have found that RPFs induce possible nonradiative recombination pathways owing to the high chloride vacancy concentration nearby. The authors further confirm that the blue-emitting PNCs do not show RPFs post-halide exchange in the CsPbBr3 nanocrystals. By introducing the post-halide exchange treatment, high-efficiency pure blue-emitting (464 nm) PNC-based LEDs with an external quantum efficiency of 2.1% and excellent spectral stability with a full-width at half-maximum of 14 nm are obtained.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro.

OBJECTIVES: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. DESIGN: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. MATERIALS: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. SETTINGS: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. METHODS: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. RESULTS: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 µm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 µm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). LIMITATIONS: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. CONCLUSIONS: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

MicroRNAs in Dystrophinopathy.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which represent the range of dystrophinopathies, account for nearly 80% of muscle dystrophy. DMD and BMD result from the loss of a functional dystrophin protein, and the leading cause of death in these patients is cardiac remodeling and heart failure. The pathogenesis and progression of the more severe form of DMD have been extensively studied and are controlled by many determinants, including microRNAs (miRNAs). The regulatory role of miRNAs in muscle function and the differential miRNA expression in muscular dystrophy indicate the clinical significance of miRNAs. This review discusses the relevant microRNAs as potential biomarkers and therapeutic targets for DMD and DMD cardiomyopathy as examples of dystrophinopathies.

Ubiquitin and Ubiquitin-like Proteins in Cancer, Neurodegenerative Disorders, and Heart Diseases.

Post-translational modification (PTM) is an essential mechanism for enhancing the functional diversity of proteins and adjusting their signaling networks. The reversible conjugation of ubiquitin (Ub) and ubiquitin-like proteins (Ubls) to cellular proteins is among the most prevalent PTM, which modulates various cellular and physiological processes by altering the activity, stability, localization, trafficking, or interaction networks of its target molecules. The Ub/Ubl modification is tightly regulated as a multi-step enzymatic process by enzymes specific to this family. There is growing evidence that the dysregulation of Ub/Ubl modifications is associated with various diseases, providing new targets for drug development. In this review, we summarize the recent progress in understanding the roles and therapeutic targets of the Ub and Ubl systems in the onset and progression of human diseases, including cancer, neurodegenerative disorders, and heart diseases.

Use of Telehealth Amid the COVID-19 Pandemic: Experiences of Mental Health Providers Serving Rural Youth and Elderly in Pennsylvania.

Stay-at-home orders and public health safety concerns precipitated by the COVID-19 pandemic facilitated rapid changes in policy and reimbursement regulations and resulted in the sudden uptake of telehealth in mental health practices across the United States. This study explored how mental health service providers experienced the use of telehealth in serving their rural clients who are youth and older adults. A purposive sampling strategy was employed to identify and recruit mental health service providers and insurance agency representatives for this study. By means of online focus groups, this statewide study explored the experiences of 147 mental health and public insurance providers using telehealth to serve rural youth and elderly in Pennsylvania amid the pandemic in 2020. NVivo 12 qualitative analysis software was used in data analysis. The findings suggest that telehealth is perceived as both: silver linings during the pandemic (service continuation during the pandemic, improved parental involvement and responsiveness, easing the transportation challenges, and decrease in no-show rates) and some roadblocks to success (Not for every youth!, Technology challenges among the older adults, and "Dead zones" without internet and cellphone reception). Policy and practice recommendations are suggested including incentives for proactive telehealth uptake, telehealth parity laws and reimbursement policies, and incentivizing innovative use of technology for specific populations and therapeutic modalities. Continuous policy support and organizational efforts to provide customized telemental health are called for to remediate rural disparities in access to mental health services beyond the pandemic period.

Role of SIRT1 in Modulating Acetylation of the Sarco-Endoplasmic Reticulum Ca2+-ATPase in Heart Failure.

RATIONALE: SERCA2a, sarco-endoplasmic reticulum Ca2+-ATPase, is a critical determinant of cardiac function. Reduced level and activity of SERCA2a are major features of heart failure. Accordingly, intensive efforts have been made to develop efficient modalities for SERCA2a activation. We showed that the activity of SERCA2a is enhanced by post-translational modification with SUMO1 (small ubiquitin-like modifier 1). However, the roles of other post-translational modifications on SERCA2a are still unknown. OBJECTIVE: In this study, we aim to assess the role of lysine acetylation on SERCA2a function and determine whether inhibition of lysine acetylation can improve cardiac function in the setting of heart failure. METHODS AND RESULTS: The acetylation of SERCA2a was significantly increased in failing hearts of humans, mice, and pigs, which is associated with the reduced level of SIRT1 (sirtuin 1), a class III histone deacetylase. Downregulation of SIRT1 increased the SERCA2a acetylation, which in turn led to SERCA2a dysfunction and cardiac defects at baseline. In contrast, pharmacological activation of SIRT1 reduced the SERCA2a acetylation, which was accompanied by recovery of SERCA2a function and cardiac defects in failing hearts. Lysine 492 (K492) was of critical importance for the regulation of SERCA2a activity via acetylation. Acetylation at K492 significantly reduced the SERCA2a activity, presumably through interfering with the binding of ATP to SERCA2a. In failing hearts, acetylation at K492 appeared to be mediated by p300 (histone acetyltransferase p300), a histone acetyltransferase. CONCLUSIONS: These results indicate that acetylation/deacetylation at K492, which is regulated by SIRT1 and p300, is critical for the regulation of SERCA2a activity in hearts. Pharmacological activation of SIRT1 can restore SERCA2a activity through deacetylation at K492. These findings might provide a novel strategy for the treatment of heart failure.

Semantic Cardiac Segmentation in Chest CT Images Using K-Means Clustering and the Mathematical Morphology Method.

Whole cardiac segmentation in chest CT images is important to identify functional abnormalities that occur in cardiovascular diseases, such as coronary artery disease (CAD) detection. However, manual efforts are time-consuming and labor intensive. Additionally, labeling the ground truth for cardiac segmentation requires the extensive manual annotation of images by the radiologist. Due to the difficulty in obtaining the annotated data and the required expertise as an annotator, an unsupervised approach is proposed. In this paper, we introduce a semantic whole-heart segmentation combining K-Means clustering as a threshold criterion of the mean-thresholding method and mathematical morphology method as a threshold shifting enhancer. The experiment was conducted on 500 subjects in two cases: (1) 56 slices per volume containing full heart scans, and (2) 30 slices per volume containing about half of the top of heart scans before the liver appears. In both cases, the results showed an average silhouette score of the K-Means method of 0.4130. Additionally, the experiment on 56 slices per volume achieved an overall accuracy (OA) and mean intersection over union (mIoU) of 34.90% and 41.26%, respectively, while the performance for the first 30 slices per volume achieved an OA and mIoU of 55.10% and 71.46%, respectively.

Deep-Learning-Based Coronary Artery Calcium Detection from CT Image.

One of the most common methods for diagnosing coronary artery disease is the use of the coronary artery calcium score CT. However, the current diagnostic method using the coronary artery calcium score CT requires a considerable time, because the radiologist must manually check the CT images one-by-one, and check the exact range. In this paper, three CNN models are applied for 1200 normal cardiovascular CT images, and 1200 CT images in which calcium is present in the cardiovascular system. We conduct the experimental test by classifying the CT image data into the original coronary artery calcium score CT images containing the entire rib cage, the cardiac segmented images that cut out only the heart region, and cardiac cropped images that are created by using the cardiac images that are segmented into nine sub-parts and enlarged. As a result of the experimental test to determine the presence of calcium in a given CT image using Inception Resnet v2, VGG, and Resnet 50 models, the highest accuracy of 98.52% was obtained when cardiac cropped image data was applied using the Resnet 50 model. Therefore, in this paper, it is expected that through further research, both the simple presence of calcium and the automation of the calcium analysis score for each coronary artery calcium score CT will become possible.

Apigenin Ameliorates Scopolamine-Induced Cognitive Dysfunction and Neuronal Damage in Mice.

We investigated the protective effect and mechanisms of apigenin against cognitive impairments in a scopolamine-injected mouse model. Our results showed that intraperitoneal (i.p.) injection of scopolamine leads to learning and memory dysfunction, whereas the administration of apigenin (synthetic compound, 100 and 200 mg/kg/day) improved cognitive ability, which was confirmed by behavioral tests such as the T-maze test, novel objective recognition test, and Morris water maze test in mice. In addition, scopolamine-induced lipid peroxidation in the brain was attenuated by administration of apigenin. To further evaluate the protective mechanisms of apigenin on cognitive and memory function, Western blot analysis was carried out. Administration of apigenin decreased the B-cell lymphoma 2-associated X/B-cell lymphoma 2 (Bax/Bcl-2) ratio and suppressed caspase-3 and poly ADP ribose polymerase cleavage. Furthermore, apigenin down-regulated the ß-site amyloid precursor protein-cleaving enzyme, along with presenilin 1 (PS1) and PS2 protein levels. Apigenin-administered mice showed lower protein levels of a receptor for advanced glycation end-products, whereas insulin-degrading enzyme, brain-derived neurotrophic factor (BDNF), and tropomyosin receptor kinase B (TrkB) expression were promoted by treatment with apigenin. Therefore, this study demonstrated that apigenin is an active substance that can improve cognitive and memory functions by regulating apoptosis, amyloidogenesis, and BDNF/TrkB signaling pathways.

miR-146a Suppresses SUMO1 Expression and Induces Cardiac Dysfunction in Maladaptive Hypertrophy.

RATIONALE: Abnormal SUMOylation has emerged as a characteristic of heart failure (HF) pathology. Previously, we found reduced SUMO1 (small ubiquitin-like modifier 1) expression and SERCA2a (sarcoplasmic reticulum Ca2+-ATPase) SUMOylation in human and animal HF models. SUMO1 gene delivery or small molecule activation of SUMOylation restored SERCA2a SUMOylation and cardiac function in HF models. Despite the critical role of SUMO1 in HF, the regulatory mechanisms underlying SUMO1 expression are largely unknown. OBJECTIVE: To examine miR-146a-mediated SUMO1 regulation and its consequent effects on cardiac morphology and function. METHODS AND RESULTS: In this study, miR-146a was identified as a SUMO1-targeting microRNA in the heart. A strong correlation was observed between miR-146a and SUMO1 expression in failing mouse and human hearts. miR-146a was manipulated in cardiomyocytes through AAV9 (adeno-associated virus serotype 9)-mediated gene delivery, and cardiac morphology and function were analyzed by echocardiography and hemodynamics. Overexpression of miR-146a reduced SUMO1 expression, SERCA2a SUMOylation, and cardiac contractility in vitro and in vivo. The effects of miR-146a inhibition on HF pathophysiology were examined by transducing a tough decoy of miR-146a into mice subjected to transverse aortic constriction. miR-146a inhibition improved cardiac contractile function and normalized SUMO1 expression. The regulatory mechanisms of miR-146a upregulation were elucidated by examining the major miR-146a-producing cell types and transfer mechanisms. Notably, transdifferentiation of fibroblasts triggered miR-146a overexpression and secretion through extracellular vesicles, and the extracellular vesicle-associated miR-146a transfer was identified as the causative mechanism of miR-146a upregulation in failing cardiomyocytes. Finally, extracellular vesicles isolated from failing hearts were shown to contain high levels of miR-146a and exerted negative effects on the SUMO1/SERCA2a signaling axis and hence cardiomyocyte contractility. CONCLUSIONS: Taken together, our results show that miR-146a is a novel regulator of the SUMOylation machinery in the heart, which can be targeted for therapeutic intervention.

Inhibition of miR-25 improves cardiac contractility in the failing heart.

Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.

miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure.

MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.

Cardiac Stim1 Silencing Impairs Adaptive Hypertrophy and Promotes Heart Failure Through Inactivation of mTORC2/Akt Signaling.

BACKGROUND: Stromal interaction molecule 1 (STIM1) is a dynamic calcium signal transducer implicated in hypertrophic growth of cardiomyocytes. STIM1 is thought to act as an initiator of cardiac hypertrophic response at the level of the sarcolemma, but the pathways underpinning this effect have not been examined. METHODS AND RESULTS: To determine the mechanistic role of STIM1 in cardiac hypertrophy and during the transition to heart failure, we manipulated STIM1 expression in mice cardiomyocytes by using in vivo gene delivery of specific short hairpin RNAs. In 3 different models, we found that Stim1 silencing prevents the development of pressure overload-induced hypertrophy but also reverses preestablished cardiac hypertrophy. Reduction in STIM1 expression promoted a rapid transition to heart failure. We further showed that Stim1 silencing resulted in enhanced activity of the antihypertrophic and proapoptotic GSK-3ß molecule. Pharmacological inhibition of glycogen synthase kinase-3 was sufficient to reverse the cardiac phenotype observed after Stim1 silencing. At the level of ventricular myocytes, Stim1 silencing or inhibition abrogated the capacity for phosphorylation of Akt(S473), a hydrophobic motif of Akt that is directly phosphorylated by mTOR complex 2. We found that Stim1 silencing directly impaired mTOR complex 2 kinase activity, which was supported by a direct interaction between STIM1 and Rictor, a specific component of mTOR complex 2. CONCLUSIONS: These data support a model whereby STIM1 is critical to deactivate a key negative regulator of cardiac hypertrophy. In cardiomyocytes, STIM1 acts by tuning Akt kinase activity through activation of mTOR complex 2, which further results in repression of GSK-3ß activity.

SUMO1-dependent modulation of SERCA2a in heart failure.

The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca(2+) re-uptake during excitation-contraction coupling. Impaired Ca(2+) uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca(2+) decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure.

Cigarette Smoking Condensate Disrupts Endoplasmic Reticulum-Golgi Network Homeostasis Through GOLPH3 Expression in Normal Lung Epithelial Cells.

INTRODUCTION: Cigarette smoke (CS) is associated with a broad range of diseases including lung cancer. Many researchers have suggested that cigarette smoke condensate (CSC) may be more toxic compared to cigarette smoke extract (CSE) because CSC contains the lipid-soluble faction of smoke while CSE contains the hydrophilic or gas phase. The aim of this research is to investigate the effects of CSC on the disruption of endoplasmic reticulum (ER)-Golgi homeostasis in normal lung epithelial cells. METHODS: CS was generated according to the ISO 3308 method. To ascertain the mechanistic effects of CSC on lung toxicity, normal lung epithelial cells of the cell line 16HBE14o- were treated with CSC (0.1mg/mL) for 48 hours. The toxic effects of CSC on ER-Golgi homeostasis and GOLPH3 expression were observed through diverse molecular tools including transmission electron microscope analysis. RESULTS: Our results demonstrated that CSC treatment increased reactive oxygen species generation in lung cells and led to the alteration of ER-Golgi homeostasis in conjunction with increased autophagy. In particular, GOLPH3, known as an oncogene and a marker protein for the trans-Golgi network, was upregulated in CSC-treated cells. GOLPH3 protein overexpression was also confirmed in the lungs of human lung cancer patients as well as NNK-treated mice. CONCLUSION: Our study revealed that CSC caused lung damage through the disruption of ER-Golgi homeostasis and autophagy induction. The expression level of the trans-Golgi marker protein GOLPH3 could serve as a reliable bio-indicator for CS-related lung cancer. IMPLICATIONS: CS is a harmful factor in the development of many diseases including cancer. In this research, we demonstrated that CSC treatment led to malfunction of the ER-Golgi network, with the disrupted ER and Golgi causing GOLPH3 overexpression and abnormal autophagy accumulation. In addition, although the value of GOLPH3 as a predictor remains to be fully elucidated, our data suggest that GOLPH3 levels may be a novel prognostic biomarker of tobacco related lung disease.

Selective regulation of cyclic nucleotide phosphodiesterase PDE3A isoforms.

Inhibitors of cyclic nucleotide phosphodiesterase (PDE) PDE3A have inotropic actions in human myocardium, but their long-term use increases mortality in patients with heart failure. Two isoforms in cardiac myocytes, PDE3A1 and PDE3A2, have identical amino acid sequences except for a unique N-terminal extension in PDE3A1. We expressed FLAG-tagged PDE3A1 and PDE3A2 in HEK293 cells and examined their regulation by PKA- and PKC-mediated phosphorylation. PDE3A1, which is localized to intracellular membranes, and PDE3A2, which is cytosolic, were phosphorylated at different sites within their common sequence. Exposure to isoproterenol led to phosphorylation of PDE3A1 at the 14-3-3-binding site S312, whereas exposure to PMA led to phosphorylation of PDE3A2 at an alternative 14-3-3-binding site, S428. PDE3A2 activity was stimulated by phosphorylation at S428, whereas PDE3A1 activity was not affected by phosphorylation at either site. Phosphorylation of PDE3A1 by PKA and of PDE3A2 by PKC led to shifts in elution on gel-filtration chromatography consistent with increased interactions with other proteins, and 2D electrophoresis of coimmunoprecipitated proteins revealed that the two isoforms have distinct protein interactomes. A similar pattern of differential phosphorylation of endogenous PDE3A1 and PDE3A2 at S312 and S428 is observed in human myocardium. The selective phosphorylation of PDE3A1 and PDE3A2 at alternative sites through different signaling pathways, along with the different functional consequences of phosphorylation for each isoform, suggest they are likely to have distinct roles in cyclic nucleotide-mediated signaling in human myocardium, and raise the possibility that isoform-selective inhibition may allow inotropic responses without an increase in mortality.