A single-cell genomic strategy for alternative transcript start sites identification.

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

Highly Similar Tetramerization Domains from the p53 Protein of Different Mammalian Species Possess Varying Biophysical, Functional and Structural Properties.

The p53 protein is a transcriptional regulatory factor and many of its functions require that it forms a tetrameric structure. Although the tetramerization domain of mammalian p53 proteins (p53TD) share significant sequence similarities, it was recently shown that the tree shrew p53TD is considerably more thermostable than the human p53TD. To determine whether other mammalian species display differences in this domain, we used biophysical, functional, and structural studies to compare the properties of the p53TDs from six mammalian model organisms (human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum). The results indicate that the p53TD from the opossum and tree shrew are significantly more stable than the human p53TD, and there is a correlation between the thermostability of the p53TDs and their ability to activate transcription. Structural analysis of the tree shrew and opossum p53TDs indicated that amino acid substitutions within two distinct regions of their p53TDs can dramatically alter hydrophobic packing of the tetramer, and in particular substitutions at positions corresponding to F341 and Q354 of the human p53TD. Together, the results suggest that subtle changes in the sequence of the p53TD can dramatically alter the stability, and potentially lead to important changes in the functional activity, of the p53 protein.

Biomineralization through a Symmetry-Controlled Oligomeric Peptide.

Biomineralization peptides are versatile tools for generating nanostructures since they can make specific interactions with various inorganic metals, which can lead to the formation of intricate nanostructures. Previously, we examined the influence that multivalency has on inorganic structures formed by p53 tetramer-based biomineralization peptides and noted a connection between the geometry of the peptide and its ability to regulate nanostructure formation. To investigate the role of multivalency in nanostructure formation by biomineralization peptides more thoroughly, silver biomineralization peptides were engineered by linking them to additional self-assembling molecules based on coiled-coil peptides and multistranded DNA oligomers. Under mild reducing conditions at room temperature, these engineered biomineralization peptides self-assembled and formed silver nanostructures. The trimeric forms of the biomineralization peptides were the most efficient in forming a hexagonal disk nanostructure, with both the coiled-coil peptide and DNA-based multimeric forms. Together, the results suggest that the spatial arrangement of biomineralization peptides plays a more important role in regulating nanostructure formation than their valency.

Bilayer Hydrogel Composed of Elastin-Mimetic Polypeptides as a Bio-Actuator with Bidirectional and Reversible Bending Behaviors.

Biologically derived hydrogels have attracted attention as promising polymers for use in biomedical applications because of their high biocompatibility, biodegradability, and low toxicity. Elastin-mimetic polypeptides (EMPs), which contain a repeated amino acid sequence derived from the hydrophobic domain of tropoelastin, exhibit reversible phase transition behavior, and thus, represent an interesting starting point for the development of biologically derived hydrogels. In this study, we succeeded in developing functional EMP-conjugated hydrogels that displayed temperature-responsive swelling/shrinking properties. The EMP-conjugated hydrogels were prepared through the polymerization of acrylated EMP with acrylamide. The EMP hydrogel swelled and shrank in response to temperature changes, and the swelling/shrinking capacity of the EMP hydrogels could be controlled by altering either the amount of EMP or the salt concentration in the buffer. The EMP hydrogels were able to select a uniform component of EMPs with a desired and specific repeat number of the EMP sequence, which could control the swelling/shrinking property of the EMP hydrogel. Moreover, we developed a smart hydrogel actuator based on EMP crosslinked hydrogels and non-crosslinked hydrogels that exhibited bidirectional curvature behavior in response to changes in temperature. These thermally responsive EMP hydrogels have potential use as bio-actuators for a number of biomedical applications.

Development of a fluorescence reporter system to quantify transcriptional activity of endogenous p53 in living cells.

The tumor suppressor p53 (also known as TP53) plays a central role in cellular stress responses by regulating transcription of multiple target genes. The temporal dynamics of p53 are thought to be important for its function; these encode input information and are decoded to induce distinct cellular phenotypes. However, it remains unclear to what extent the temporal dynamics of p53 reflect the activity of p53-induced gene expression. In this study, we report a multiplexed reporter system that allows us to visualize the transcriptional activity of p53 at the single-cell level. Our reporter system features simple and sensitive observation of the transcriptional activity of endogenous p53 to the response elements of various target genes. Using this system, we show that the transcriptional activation of p53 exhibits strong cell-to-cell heterogeneity. The transcriptional activation of p53 after etoposide treatment is highly dependent on the cell cycle but this is not seen after UV exposure. Finally, we show that our reporter system allows simultaneous visualization of the transcriptional activity of p53 and cell cycle. Our reporter system can thus be a useful tool for studying biological processes involving the p53 signaling pathway.

Optogenetic termination of atrial tachyarrhythmias by brief pulsed light stimulation.

AIMS: The most efficient way to acutely restore sinus rhythm from atrial fibrillation (AF) is electrical cardioversion, which is painful without adequate sedation. Recent studies in various experimental models have indicated that optogenetic termination of AF using light-gated ion channels may provide a myocardium-specific and potentially painless alternative future therapy. However, its underlying mechanism(s) remain(s) incompletely understood. As brief pulsed light stimulation, even without global illumination, can achieve optogenetic AF termination, besides direct conduction block also modulation of action potential (AP) properties may be involved in the termination mechanism. We studied the relationship between optogenetic AP duration (APD) and effective refractory period (ERP) prolongation by brief pulsed light stimulation and termination of atrial tachyarrhythmia (AT). METHODS AND RESULTS: Hearts from transgenic mice expressing the H134R variant of channelrhodopsin-2 in atrial myocytes were explanted and perfused retrogradely. AT induced by electrical stimulation was terminated by brief pulsed blue light stimulation (470 nm, 10 ms, 16 mW/mm2) with 68% efficacy. The termination rate was dependent on pulse duration and light intensity. Optogenetically imposed APD and ERP changes were systematically examined and optically monitored. Brief pulsed light stimulation (10 ms, 6 mW/mm2) consistently prolonged APD and ERP when light was applied at different phases of the cardiac action potential. Optical tracing showed light-induced APD prolongation during the termination of AT. CONCLUSION: Our results directly demonstrate that cationic channelrhodopsin activation by brief pulsed light stimulation prolongs the atrial refractory period suggesting that this is one of the key mechanisms of optogenetic termination of AT.

Empagliflozin suppresses mitochondrial reactive oxygen species generation and mitigates the inducibility of atrial fibrillation in diabetic rats.

Introduction: Recent studies have demonstrated that sodium-glucose co-transporter-2 inhibitors (SGLT2-i) reduce the risk of atrial fibrillation (AF) in patients with diabetes mellitus (DM), in which oxidative stress due to increased reactive oxygen species (ROS) contributes to the pathogenesis of AF. We aimed to further investigate this, and examine whether the SGLT2-i empagliflozin suppresses mitochondrial-ROS generation and mitigates fibrosis. Methods: A high-fat diet and low-dose streptozotocin treatment were used to induce type-2 DM (T2DM) in Sprague-Dawley rats. The rats were randomly divided into three groups: control, DM, and DM treated with empagliflozin (30 mg/kg/day) for 8 weeks. The mitochondrial respiratory capacity and ROS generation in the atrial myocardium were measured using a high-resolution respirometer. Oxidative stress markers and protein expression related to mitochondrial biogenesis and dynamics as well as the mitochondrial morphology were examined in the atrial tissue. Additionally, mitochondrial function was examined in H9c2 cardiomyoblasts. Atrial tachyarrhythmia (ATA) inducibility, interatrial conduction time (IACT), and fibrosis were also measured. Results: Inducibility of ATA, fibrosis, and IACT were increased in rats with DM when compared to controls, all of which were restored by empagliflozin treatment. In addition, the rats with DM had increased mitochondrial-ROS with an impaired complex I-linked oxidative phosphorylation capacity. Importantly, empagliflozin seemed to ameliorate these impairments in mitochondrial function. Furthermore, empagliflozin reversed the decrease in phosphorylated AMPK expression and altered protein levels related to mitochondrial biogenesis and dynamics, and increased mitochondrial content. Empagliflozin also improved mitochondrial function in H9c2 cells cultured with high glucose medium. Discussion: These data suggest that empagliflozin has a cardioprotective effect, at least in part, by reducing mitochondrial ROS generation through AMPK signaling pathways in the atrium of diabetic rats. This suggests that empagliflozin might suppress the development of AF in T2DM.

Empagliflozin attenuates arrhythmogenesis in diabetic cardiomyopathy by normalizing intracellular Ca2+ handling in ventricular cardiomyocytes.

Diabetic cardiomyopathy has been reported to increase the risk of fatal ventricular arrhythmia. The beneficial effects of the selective sodium-glucose cotransporter-2 inhibitor have not been fully examined in the context of antiarrhythmic therapy, especially its direct cardioprotective effects despite the negligible SGLT2 expression in cardiomyocytes. We aimed to examine the antiarrhythmic effects of empagliflozin (EMPA) treatment on diabetic cardiomyocytes, with a special focus on Ca2+ handling. We conducted echocardiography and hemodynamic studies and studied electrophysiology, Ca2+ handling, and protein expression in C57BLKS/J-leprdb/db mice (db/db mice) and their nondiabetic lean heterozygous Leprdb/+ littermates (db/+ mice). Preserved systolic function with diastolic dysfunction was observed in 16-wk-old db/db mice. During arrhythmia induction, db/db mice had significantly increased premature ventricular complexes (PVCs) than controls, which was attenuated by EMPA. In protein expression analyses, calmodulin-dependent protein kinase II (CaMKII) Thr287 autophosphorylation and CaMKII-dependent RyR2 phosphorylation (S2814) were significantly increased in diabetic hearts, which were inhibited by EMPA. In addition, global O-GlcNAcylation significantly decreased with EMPA treatment. Furthermore, EMPA significantly inhibited ventricular cardiomyocyte glucose uptake. Diabetic cardiomyocytes exhibited increased spontaneous Ca2+ events and decreased sarcoplasmic reticulum (SR) Ca2+ content, along with impaired Ca2+ transient, all of which normalized with EMPA treatment. Notably, most EMPA-induced improvements in Ca2+ handling were abolished by the addition of an O-GlcNAcase (OGA) inhibitor. In conclusion, EMPA attenuated ventricular arrhythmia inducibility by normalizing the intracellular Ca2+ handling, and we speculated that this effect was, at least partly, due to the inhibition of O-GlcNAcylation via the suppression of glucose uptake into cardiomyocytes.NEW & NOTEWORTHY SGLT2is are known to improve cardiovascular outcomes regardless of the presence of diabetes and decrease traditional cardiovascular risk factors. We demonstrated, for the first time, that EMPA inhibited PVCs by normalizing Ca2+ handling in diabetic mice. Our data suggest that the effects of SGLT2is on calcium handling may occur because of suppression of O-GlcNAcylation through inhibition of glucose uptake and not because of NHE inhibition, as previously suggested.

Fragmented QRS on 12-lead electrocardiogram predicts long-term prognosis in patients with cardiac sarcoidosis.

Fragmented QRS (fQRS) on a 12-lead electrocardiogram is a known marker of fatal arrhythmias or cardiac adverse events in ischemic and non-ischemic cardiomyopathy patients. Nonetheless, the association between fQRS and clinical outcomes in patients with cardiac sarcoidosis (CS) remains unclear. Herein, we investigated whether fQRS is associated with long-term clinical outcomes in CS patients. A total of 78 patients who received immunosuppressive therapy (IST) for clinically diagnosed CS were retrospectively examined. Patients were classified into two groups according to the presence (n = 19) or absence (n = 59) of fQRS on electrocardiogram before IST. The primary outcome was the composite event of all-cause death, ventricular tachyarrhythmias (VTs), and hospitalization for heart failure. Results of late gadolinium enhancement on cardiac magnetic resonance imaging were also analyzed. During a median follow-up period of 3.7 years (interquartile range: 1.6-6.2 years), the primary outcome occurred more frequently in patients with fQRS than in those without (47% vs. 13%, log-rank p = 0.002). Multivariable Cox regression analyses showed that fQRS was an independent determinant of the primary outcome. The incidence of VTs, within 12 months of IST initiation, was comparable between the two groups; however, late-onset VTs, defined as those occurring ≥ 12 months after IST initiation, occurred more frequently in the fQRS group (21% vs. 2%, log-rank p = 0.002). The scar zone and scar border zone were greater in patients with fQRS than in those without it. In conclusion, our analysis suggests that fQRS is an independent predictor of adverse events, particularly late-onset VTs, in patients with CS.

Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA.

The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low.

Lipid Droplet Formation Is Regulated by Ser/Thr Phosphatase PPM1D via Dephosphorylation of Perilipin 1.

Hypertrophy and hyperplasia of white adipocytes induce obesity, leading to diseases such as type 2 diabetes and hypertension, and even cancer. Hypertrophy of white adipocytes is attributed to the excessive storage of the energy form of triglycerides in lipid droplets (LDs). LDs are fat storage organelles that maintain whole-body energy homeostasis. It is important to understand the mechanism of LD formation for the development of obesity therapy; however, the regulatory mechanisms of LD size and formation are not fully understood. In this study, we demonstrated that the PPM family phosphatase PPM1D regulates LD formation. PPM1D specific inhibitor, SL-176 significantly decreased LD formation via two different pathways: dependent of and independent of adipocyte-differentiation processes. In the mature white adipocytes after differentiation, LD formation was found to be controlled by PPM1D via dephosphorylation of Ser511 of perilipin 1. We found that inhibition of PPM1D in mature white adipocytes significantly reduced the size of the LDs via dephosphorylation of Ser511 of perilipin 1 but did not change the lipolysis sensitivity and the total amount of lipid in cells. Collectively, the results of this study provide evidence that PPM1D plays an important role in LD formation in mature adipocytes.

Pharmacological nNOS inhibition modified small-conductance Ca2+-activated K+ channel without altering Ca2+ dynamics.

Atrial fibrillation (AF) is associated with electrical remodeling processes that promote a substrate for the maintenance of AF. Although the small-conductance Ca2+-activated K+ (SK) channel is a key factor in atrial electrical remodeling, the mechanism of its activation remains unclear. Regional nitric oxide (NO) production by neuronal nitric oxide synthase (nNOS) is involved in atrial electrical remodeling. In this study, atrial tachyarrhythmia (ATA) induction and optical mapping were performed on perfused rat hearts. nNOS is pharmacologically inhibited by S-methylthiocitrulline (SMTC). The influence of the SK channel was examined using a specific channel inhibitor, apamin (APA). Parameters such as action potential duration (APD), conduction velocity, and calcium transient (CaT) were evaluated using voltage and calcium optical mapping. The dominant frequency was examined in the analysis of AF dynamics. SMTC (100 nM) increased the inducibility of ATA and apamin (100 nM) mitigated nNOS inhibition-induced arrhythmogenicity. SMTC caused abbreviations and enhanced the spatial dispersion of APD, which was reversed by apamin. By contrast, conduction velocity and other parameters associated with CaT were not affected by SMTC or apamin administration. Apamin reduced the frequency of SMTC-induced ATA. In summary, nNOS inhibition abbreviates APD by modifying the SK channels. A specific SK channel blocker, apamin, mitigated APD abbreviation without alteration of CaT, implying an underlying mechanism of posttranslational modification of SK channels.NEW & NOTEWORTHY We demonstrated that pharmacological nNOS inhibition increased the atrial arrhythmia inducibility and a specific small-conductance Ca2+-activated K+ channel blocker, apamin, reversed the enhanced atrial arrhythmia inducibility. Apamin mitigated APD abbreviation without alteration of Ca2+ transient, implying an underlying mechanism of posttranslational modification of SK channels.

Stimulation of the mitochondrial calcium uniporter mitigates chronic heart failure-associated ventricular arrhythmia in mice.

BACKGROUND: An aberrant increase in the diastolic calcium concentration ([Ca2+]i) level is a hallmark of heart failure (HF) and the cause of delayed afterdepolarization and ventricular arrhythmia (VA). Although mitochondria play a role in regulating [Ca2+]i, whether they can compensate for the [Ca2+]i abnormality in ventricular myocytes is unknown. OBJECTIVE: The purpose of this study was to investigate whether enhanced Ca2+ uptake of mitochondria may compensate for an abnormal increase in the [Ca2+]i of ventricular myocytes in HF to effectively mitigate VA. METHODS: We used a HF mouse model in which myocardial infarction was induced by permanent left anterior descending coronary artery ligation. The mitochondrial Ca2+ uniporter was stimulated by kaempferol. Ca2+ dynamics and membrane potential were measured using an epifluorescence microscope, a confocal microscope, and the perforated patch-clamp technique. VA was induced in Langendorff-perfused hearts, and hemodynamic parameters were measured using a microtip transducer catheter. RESULTS: Protein expression of the mitochondrial Ca2+ uniporter, as assessed by its subunit expression, did not change between HF and sham mice. Treatment of cardiomyocytes with kaempferol, isolated from HF mice 28 days after coronary ligation, reduced the appearance of aberrant diastolic [Ca2+]i waves and sparks and spontaneous action potentials. Kaempferol effectively reduced VA occurring in Langendorff-perfused hearts. Intravenous administration of kaempferol did not markedly affect left ventricular hemodynamic parameters. CONCLUSION: The effects of kaempferol in HF of mice implied that mitochondria may have the potential to compensate for abnormal [Ca2+]i. Mechanisms involved in mitochondrial Ca2+ uptake may provide novel targets for treatment of HF-associated VA.

Predictors of cardiovascular mortality after an electrical storm in patients with structural heart disease.

BACKGROUND: Electrical storms (ESs) in patients with structural heart disease (SHD) have been reported to be associated with a poor prognosis. However, the detailed cause of death and influence of implantable cardioverter defibrillator (ICD) therapy in ES patients have not been fully investigated. Therefore, we sought to explore the detailed clinical course after an ES and the impact of the ICD therapy in patients with SHDs. METHODS: We retrospectively analyzed 31 consecutive patients with ESs who had undergone an ICD implantation. ESs were defined as three or more ventricular arrhythmias within 24 h. RESULTS: During a mean follow up of 4.5 years, 13 patients died. Among them, cardiovascular death (CVD) was observed in 11/13 (85%), and the leading cause of the CVD was end-stage heart failure. A New York Heart Association class ≥III at the time of the ES occurrence (HR 6.51 95% CI 1.94-25.1, p = 0.003) and any shock therapy (HR 5.94 95% CI 1.06-112.2, p = 0.04) were associated with CVD. CONCLUSION: In the current single center study, the major cause of death in ES patients with SHDs was end-stage heart failure. Any shock therapy was associated with CVD. Arrhythmia management to avoid ICD shocks might reduce the mortality in ES patients.

Intrinsic anti-tachycardia pacing terminated ventricular tachycardia resistant to traditional anti-tachycardia pacing.

Implantable cardioverter-defibrillators (ICDs) serve to reduce the risk of sudden death; however, ICD shocks worsen patient prognosis. Therefore, attempts have been made to terminate life-threatening arrhythmias without ICD shocks. A 71-year-old man with non-ischemic cardiomyopathy, who previously underwent cardiac resynchronization therapy-defibrillator (CRT-D) placement, was hospitalized for ventricular tachyarrhythmia (VT) that was refractory to traditional anti-tachycardia pacing (ATP). Endocardial and epicardial ablation failed to prevent VT recurrence. Since the CRT-D battery was exhausted, it was replaced with a Cobalt™ XT HF CRT-D (Medtronic, Minneapolis, MN, USA), and the intrinsic ATP (iATP) algorithm was employed. Although VT recurred frequently, recurrent VTs were terminated by the iATP, which created a conduction block in the circuit without VT acceleration or shock. This is the first reported case wherein an iATP algorithm was effective against VT resistant to traditional anti-tachycardia pacing. This novel ATP algorithm has the potential to terminate refractory VT without ICD shocks and provide a better prognosis.

Hybrid epicardial ventricular tachycardia ablation with lateral thoracotomy in a patient with a history of left ventricular reconstruction surgery.

Although a hybrid procedure involving surgical access may be feasible for epicardial catheter ablation in individuals with prior cardiac surgery, surgical approaches in thoracotomy are important in patients with advanced adhesions. We performed an epicardial ventricular tachycardia (VT) ablation in a patient with dilated phase hypertrophic cardiomyopathy after left ventricular reconstruction. We gained surgical epicardial access via lateral thoracotomy based on the anticipated VT circuit in the apical anteroseptal area, which was estimated using prior endocardial mapping. The remaining epicardial myocardium around the surgical incision was involved in the central isthmus, and the VT was eliminated by radiofrequency catheter ablation. .

Role of active site arginine residues in substrate recognition by PPM1A.

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.