This study aimed at investigating whether Angiotensin-(1-7) [Ang-(1-7)] could alleviate cardiac fibrosis by modulating the expression of Rac1 and Rad GTPase and evaluating the specific mechanism in rat cardiac fibroblasts (CFs) and in canine atrial rapid pacing models. Fresh CFs were isolated from ventricles of 1 ~ 3 day-old Sprague-Dawley rats, and were randomly divided into control group, LPS group, Ang-(1-7) group and LPS+Ang-(1-7) group. CCK8 was used to detect cell proliferation. qRT-PCR was to detect the mRNA expression of ACE2. Mongrel dogs were randomized into four groups: Sham-operated (Sham), AF-Control, AF-EGFP and AF-ACE2 groups. Immunoperoxidase staining was used to test Rac1 and Gem expression. Collagen fibers in heart were stained a red color and extent of cardiac fibrosis was evaluated. Histopathology was to observe actin,α-smooth. Enzyme-Linked Immunosorbent Assay was to detect Ang II and Ang-(1-7) expression in canine atrial tissue. And western blot analysis was to detect the protein expression of iNOS, COX2, α-SMA, Rac1, ACE, and AT1R in cells and tissues. Cellular experiments showed LPS promoted proliferation of fibroblasts, which could be inhibited by Ang-(1-7) (p < 0.05). Compared with control group, LPS-induced CFs showed a significant increase in protein expression of iNOS, COX2, and α-SMA, which promoted cell proliferation. However, the application of Ang-(1-7) reversed these effects and suppressed the ACE/AT1R signaling pathway in LPS-induced CFs. In animal experiments, ACE2 over-expression would up-regulate atrial Ang-(1-7) and AT2R and reduce atrial angiotensin II (Ang II) and AT1R expressions (p < 0.05). In AF-ACE2 group, Rac1 was down-regulated and Rad GTPase was up-regulated, in comparison with AF-Control and AF-EGFP groups, associated with reductions of CTGF, fibrosis-related factors α-SMA and N-cadherin. ACE2 was found to alleviate cardiac fibrosis through the Ang(1-7)/Mas signaling pathway in animal experiments.

Background: We previously demonstrated that re-expression of the embryonic transcription factor TBX18 is sufficient to reprogram ventricular myocytes (VMs) into sinoatrial node (SAN)-like induced pacemaker myocytes (iPMs). Here, we examined the timeline of this reprogramming and tested whether combined gene transfer of TBX18, SHOX2, and TBX3—transcription factors critical to SAN development—would lead to superior reprogramming. Methods: Neonatal rat VMs were isolated, cultured as monolayers, and transduced with adenoviral vectors expressing (1) GFP (control), (2) TBX18 alone, or (3) a combination of TBX18, SHOX2, and TBX3 (TST group). Spontaneous field potentials were recorded via multi-electrode arrays (MEA) on days 1, 3, 5, 7, 9, and 12. Single-cell RNA-seq (scRNA-seq) was performed on days 3, 6, and 14. Result: MEA data revealed that by d5, the TBX18 group exhibited significantly elevated beat rates compared to control (89.4±49.9 vs. 62.5±30.6 bpm, n =8, p <1e−50). Notably, TST group showed markedly faster pacing rates as early as d3 (181.6±89.5 bpm, p<1e-50), a difference that persisted throughout d12 (71.3±27.9 bpm, n=8, p<1e-50), indicating rapid onset and sustained efficacy of TST-mediated reprogramming. ScRNA-seq analysis revealed that reprogramming by TBX18 involved activation of signaling pathways such as Wnt and TGF-β, though significant changes in the expression of related molecules did not emerge until d7. In contrast, the TST group showed early enrichment (by d3) of differentially expressed genes (DEGs) involved in canonical Wnt signaling (GO:0060828, p =3.9e−4), cell-substrate adhesion (GO:0010810, p =2.1e−3), and inhibition of stem cell differentiation (GO:2000737, p =5.6e−3).Compared to VMs, the TST group exhibited early and robust expression of SAN-specific genes such as Hcn4 (log2FC=0.27, p =1.6e−22), Gjc1 (log2FC=0.27, p =1.4e−35), and Col1a1 (log2FC=1.25, p =4.1e−27), while downregulating ventricular-enriched genes including Gja1 (log2FC=1.48, p <1e−50), Ryr2 (log2FC=0.64, p <1e−50), and Slc8a1 (log2FC=0.18, p <1e−50). These findings were validated by qPCR and immunostaining. Conclusion: The results indicate that gene transfer of three transcription factors that figure prominently in the native SAN accelerates pacemaker cell reprogramming, accompanied by significantly higher spontaneous beat rates compared to TBX18 alone. This combinatorial approach enables faster onset and more sustained gene therapy for biological cardiac pacing.

Complete atrioventricular block (CAVB) is a largely intractable disease that leads to severe bradyarrhythmia. The only treatment is the implantation of a pacemaker device. Left untreated, CAVB patients experience QT prolongation and ventricular overload, increasing susceptibility to cardiac remodeling and heart failure, as well as potentially lethal tachyarrhythmias. Animal models of CAVB offer a direct avenue to investigate the disease and potential disease-modifying therapies. However, existing models have limitations due to phenotype instability and high attrition rate. We aimed to create an improved method for disease model creation and report beating rate and variability analysis of the model. We report a modified surgical model of CAVB in rats by adapting a clinical radiofrequency energy generator to ablate the atrioventricular node region of the rat heart. Compared to previous ablation methods that utilize sharp needle entry into the AV node (AVN) region, the modified method resulted in a significantly higher success rate with a lower attrition rate. The rat model of CAVB showed stable conduction block for at least four weeks after model creation, supporting their suitability as a preclinical model of severe bradyarrhythmia due to AV conduction block. Models showed severe beat-to-beat variability with decreased overall autonomic innervation. We present a rodent model of a complete and stable AV block, which enhances the rigor and reproducibility of the disease model and downstream applications.

Introduction: TBX18-mediated reprogramming converts ventricular myocytes (VMs) into pacemaker-like cells (iPMs), creating de novo pacing in vitro and in vivo. Here, we employ single-cell RNA sequencing (scRNA-seq) and ATAC-seq to elucidate the molecular mechanisms underlying this process. Methods: Neonatal rat ventricular myocytes were transduced with adenoviral TBX18 or GFP. ScRNA-seq and ATAC-seq were performed at days 3, 6, and 14 post-gene transfer. Functional assays, including field potential recordings and lineage tracing, assessed pacemaker function. Results: At d3, genes that encode chamber cardiomyocyte contractility and ion channels (e.g., Myh6, Tnni3, Scn5a ) were downregulated while dedifferentiation markers ( Alcam, Acta2 ) were upregulated in TBX18-NRVMs compared to control. By day 6, MEA data revealed the emergence of a pacemaker-like subpopulation exhibiting higher automaticity (>2 Hz spontaneous beats in 22.3% of TBX18-NRVMs vs. 2.6% in controls, p < 0.001). This stage was also characterized by epigenetic remodeling, evidenced by significantly increased chromatin accessibility at Hcn4 and Gjc1 , and extracellular matrix (ECM) reorganization, including upregulation of ECM-related genes ( Col1a1 , Col3a1 , Eln ). By day 14, 19.4% of TBX18-NRVMs exhibit Hcn4+ pacemaker-like characteristics, in contrast to only 0.2% in control GFP-NRVMs. TBX18-iPMs displayed upregulated nodal pacemaker gene expression ( Hcn4, Gjc1, Igfbp5 ), with reduced ventricular identity (low Kcnj2 and Gja1 ), and functional automaticity. Gene regulatory network analysis identified Klf4 and Junb as key transcriptional hubs in pacemaker reprogramming. Inhibition of TGF-β signaling or activation of Wnt signaling markedly impaired the induction of iPMs, as reflected by a reduced proportion of Hcn4 -positive cells and the loss of pacemaker-associated ion channel and ECM gene expression profiles. Lineage tracing using Hcn4 (+/eGFP) transgenic mice confirmed the persistence of TBX18-reprogrammed iPMs for over one year in vivo, supporting the long-term stability of the reprogramming process. Conclusions: TBX18 reprogramming follows a stepwise mechanism: (1) ventricular identity loss and dedifferentiation, (2) activation of pacemaker-specific gene programs. Cell-cell interaction predictions suggest TGF-β activation and Wnt inhibition enhance reprogramming efficiency. These findings provide mechanistic insights and identify potential molecular drivers for optimizing pacemaker cell generation.

Tissue elasticity is essential to a broad spectrum of cell biology and organ function including the heart. Routine cell culture models on rigid polystyrene dishes are limited in studying the impact of tissue elasticity in distinct regions of the myocardium such as the cardiac conduction system. Gelatin, a derivative of collagen, is a simple and tunable platform for modeling tissue elasticity. We sought to study the effects of increasing tissue stiffness on cardiac pacemaker cell function by using transcription factor-reprogrammed pacemaker cells cultured on gelatin hydrogels with specific elasticity. Our data indicate that automaticity of the pacemaker cells, measured in rhythmic contractions and oscillating intracellular Ca2+ transients, was enhanced when cultured on a stiffer matrix of 14 kPa. This was accompanied by increased expression of cardiac pacemaker ion channel, Hcn4, and a reciprocal decrease in Cx43 expression compared with control conditions. Propagation of Ca2+ transients was slower in the pacemaker cell monolayers compared with control, which recapitulates a hallmark feature in the native pacemaker tissue. Ca2+ transient propagation of pacemaker cell monolayer was slower on stiffer than on softer hydrogel, and this was dependent on enhanced proliferation of cardiac fibroblasts rather than differences in gap junctional coupling. Culturing the pacemaker cells on rigid plastic plates led to irregular or loss of synchronous contractions as well as unusually long Ca2+ transient durations. Taken together, our data demonstrate that automaticity of pacemaker cells is augmented by stiffer extracellular matrix substrates within the elasticity range of the healthy myocardium. This simple approach presents a physiological in vitro model to study mechanoelectric feedback of cardiomyocytes including the conduction system cells. NEW & NOTEWORTHY The major achievement of this work is development of a robust and straightforward approach to model cardiac conduction system cells with a range of cardiac tissue elasticity with a goal to understand the impact of tissue stiffness on cardiac pacing. Our data provide a framework for further investigation of the heart rhythm in health and disease in the context of fibrosis.

Introduction: Tissue elasticity is essential to proper cell biology and organ function including the heart. Cell culture models on rigid polystyrene dishes are limited in studying the impact of tissue elasticity on specific cardiac cell types such as the cardiac conduction system cells. Hypothesis: We hypothesized that gelatin hydrogel at varying stiffness can uncover tissue elasticity-dependent changes to cardiac pacemaker cell physiology. Methods: Neonatal rat ventricular myocytes (NRVMs) were cultured as monolayers on polystyrene culture plates or on 1, 6, and 14 kPa gelatin hydrogels. All culture surface was coated with fibronectin. NRVMs were transduced with Adv-TBX18 or Adv-GFP at day 0 (d0). Results: The rates of rhythmic contractions were faster in TBX18-NRVMs than in GFP-NRVMs on the gelatin hydrogels from d1 to d7 (n≥4, p <0.05). TBX18-NRVMs cultured on the 14 kPa gelatin hydrogels showed faster contractions than those on 1 kPa or on plastic plates at d3 and d7 (d3: 54±12, 9±5, 0±0 bpm, d7: 31±10, 4±7, 24±10 bpm, n≥4, p <0.05). TBX18-NRVMs exhibited faster Ca 2+ transients on 14 kPa than on 1 and 6 kPa gelatin hydrogel or on plastic at d8 (33±2 vs. 9±2, 19±8, 11±5 transients/min, respectively, n=3, p<0.05). Hcn4 protein expression was higher in TBX18-NRVMs cultured on 14 kPa than on 1 kPa gelatin hydrogels or plastic plates (n=4/group, p <0.05). Cx43 protein expression was lower in TBX18-NRVMs compared to control at d8 regardless of the basement elasticity (n=4/group, p <0.05). However, Ca 2+ transient propagation velocity of TBX18-NRVMs was slower on 14 kPa than on 1 kPa hydrogels at d9 (5±1 vs. 8±1 cm/s, n≥3, p <0.05). Vimentin+ fibroblasts were more numerous in TBX18-NRVMs on 14 kPa than on 1 or 6 kPa hydrogels at d6 (40±2 vs. 28±4 or 27±2%, n=4, p <0.05), suggesting that the slower propagation velocity in TBX18-NRVMs on 14 kPa was due to increase in fibroblast content. Conclusions: The data demonstrate that automaticity of pacemaker cells is augmented by stiffer ECM substrates within the elasticity range of the healthy myocardium. This approach presents an easy-to-deploy in vitro model to study effects of tissue elasticity on cardiac conduction system cell physiology.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers. Intramyocardial injection of synthetic mRNA coding for the embryonic T-box transcription factor 18 gene generates rate-adaptive cardiac pacing and limits innate and inflammatory immune responses, as shown in rodents and pigs.

Background: Atrial fibrillation (AF) is affected by both environmental and genetic factors. Previous genetic association studies, especially genome-wide association studies, revealed a large group of AF-associated genes. However, little is known about the functions and interactions of these genes. Moreover, established genetic variants of AF contribute modestly to AF variance, implying that numerous additional AF-associated genetic variations need to be identified. Hence, a systematic network and pathway analysis is needed.Methods: We retrieved all AF-associated genes from genetic association studies in various databases and performed integrative analyses including pathway enrichment analysis, pathway crosstalk analysis, network analysis, and microarray meta-analysis.Results: We collected 254 AF-associated genes from genetic association studies in various databases. Pathway enrichment analysis revealed the top biological pathways that were enriched in the AF-associated genes related to cardiac electromechanical activity. Pathway crosstalk analysis showed that numerous neuro-endocrine-immune pathways connected AF with various diseases including cancers, inflammatory diseases, and cardiovascular diseases. Furthermore, an AF-specific subnetwork was constructed with the prize-collecting Steiner forest algorithm based on the AF-associated genes, and 24 novel genes that were potentially associated with AF were inferred by the subnetwork. In the microarray meta-analysis, six of the 24 novel genes (APLP1, CREB1, CREBBP, PRMT1, IRAK1, and PLXND1) were expressed differentially in patients with AF and sinus rhythm.Conclusions: AF is not only an isolated disease with abnormal electrophysiological activity but might also share a common genetic basis and biological process with tumors and inflammatory diseases as well as cardiovascular diseases. Moreover, the six novel genes inferred from network analysis might help detect the missing AF risk loci.

Background: Pacemaker implantation is standard treatment for patients with complete atrioventricular (AV) block. However, contraindications for device implantations create a need for alternative non-hardware cardiac pacing solutions. We developed a porcine model of complete heart block with junctional bradycardia to test a novel transcription factor-based gene therapy consisting of Tbx18, Shox2 and Tbx3 (TST). Somatic reprogramming of ventricular cardiomyocytes by TST induces pacemaker-like activity and provides biological pacing in vitro . Methods: Eight domestic swine (Yorkshire/Landrace) received catheter ablation of the AV node/His Bundle resulting in complete AV block, confirmed by dissociation of P and R waves on ECG. An implanted device pacemaker (VVI mode) maintained cardiac output during occurrences of junctional bradycardia below 50 bpm. Telemetric 3-lead ECG was recorded continuously. Subjects received TST gene therapy or control gene (GFP) via catheter-based intramuscular injection into the high right ventricular septum. Pacemaker usage and ECG were continuously monitored for 8 weeks. Results: Following AV node ablation, junctional escape rhythm decelerated into junctional bradycardia. After 3 weeks of AV block, 49.9 ± 13.1% of heartbeats were initiated by the device pacemaker. Upon reaching pacemaker dependence of 95% in an average of 27.9 ± 5.5 days, subjects received TST gene therapy or control injection. In subjects receiving TST therapy, daily pacemaker reliance decreased to 50% dependency in an average of 14.7 ± 2.3 days, while in GFP controls, daily pacemaker dependency at 50 bpm remained unchanged at >98% throughout follow-up. In TST-treated subjects, reductions in pacemaker dependence were associated with increases in ventricular rates exceeding 70 bpm. Efficacy of gene therapy waned after about one month; an improvement of >10% pacemaker dependency over two consecutive days was still observed on average 29.0 ± 4.1 days after TST injection. Conclusion: In a porcine model of complete AV block, ventricular junctional escape rhythm decelerated into bradycardia in less than one month. Subsequent gene therapy by TST injection into the high ventricular septum alleviated pacemaker dependency and enhanced heart rate for 29.0 ± 4.1 days. Somatic reprogramming of cardiomyocytes by TST gene delivery may provide a biological pacemaker alternative to hardware device implantation for therapeutic cardiac pacing.

Background: COVID-19 induces a pro-coagulant state with thrombotic events. This meta-analysis explores the efficacy and safety of antiplatelet-based therapy in COVID-19 patients through randomized controlled trials (RCTs). Materials and methods: A systematic literature search until March 10, 2023, identified 7 RCTs involving 23,415 inpatients. Of these, 11,891 received antiplatelet-based treatment, and tistical analysis was performed using Review Manager 5.4. Results: The included trials involved patients with a mean age ranging from 54.3 to 62.0 years and a prevalence of hypertension ranging from 10.9 to 65.0% and coronary artery disease ranging from 3.2 to 32.7%. The pooled analysis showed no significant difference in overall mortality between groups (RR 1.0, 95% CI 0.99 - 1.01, p = 0.76). However, antiplatelet therapy significantly reduced major thrombotic events (RR 0.86, 95% CI 0.75 - 0.99, p = 0.04). Conversely, it increased major bleeding risks (RR 1.62, 95% CI 1.24 - 2.12, p = 0.0005). There was no significant difference in the incidence of invasive mechanical ventilation and retherapy does not confer mortality benefit in COVID-19 patients but lowers major thromrisks. Ongoing large RCTs will provide more therapy.

Introduction: Somatic gene transfer of an embryonic transcription factor, TBX18, could directly reprogram ventricular myocytes to induced pacemaker myocytes. We and others have previously reported that direct myocardial gene delivery of TBX18 created ventricular pacing in heart block animals in vivo. However, the de novo biological pacing was short-lived, diminishing its translational value. Hypothesis: Our preliminary RNAseq and mass spec data indicate pronounced Tgfβ signaling in TBX18-transduced cardiomyocytes. Tgfβ signaling induces fibrosis in multiple organs including the heart. We hypothesized that TBX18 triggers Tgfβ signaling and results in fibrosis, leading to loss of durable ventricular pacing. Methods: Adenoviral vectors expressing TBX18 or GFP was delivered to neonatal rat ventricular myocytes (NRVMs) in vitro or to the left ventricular apex of adult rats with complete atrioventricular block (CAVB). Results: In vitro experiments showed increased population of vimentin+ fibroblasts and alpha smooth muscle actin+ myofibroblasts in TBX18-NRVMs compared to GFP-NRVMs at 2 weeks after gene transfer. Single cell transcriptome data indicated that TBX18 triggered substantial changes in extracellular matrix-related gene expression and Tgfβ signaling. Quantitative RT-PCR and ELISA validated scRNA-seq data, indicating fibroblast activation. TBX18-NRVMs showed faster automaticity than that control did, but their automaticity began wavering after day 7. Treatment of TBX18-NRVMs with a Tgfβ signaling inhibitor, A83-01, mitigated fibroblast activation by TBX18 and preserved their automaticity. Direct delivery of TBX18 into ventricular myocardium resulted in focal fibrosis as well as expansion of vimentin+ fibroblasts at day 7. TBX18 gene transfer initiated ventricular pacing in a rat model of CAVB, but ventricular pacing diminished after one week. In contrast, treating TBX18-injected animals with A83-01 enabled sustained biological pacing beyond three weeks of after gene delivery, accompanied by reduced fibrosis in situ. Conclusions: Inhibition of Tgfβ signaling is sufficient to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

ABSTRACTImplantable cardiac pacemaker devices are generally effective for patients with symptomatic bradyarrhythmia. However, device-dependent cardiac pacing is far from ideal and often inadequate, particularly for pediatric patients who need to go through invasive revision of the indwelling hardware. Biological pacemakers have been proposed as device-free alternatives to the current treatment, but sustained, unwavering biological pacing beyond days after the biologic delivery has not been demonstrated. We have previously demonstrated that re-expression of an embryonic transcription factor, TBX18, could reprogram ventricular cardiomyocytes into induced pacemaker myocytes (iPMs). Here, we report that exogenous expression of TBX18 per se leads to severe fibrosis in situ, impairing the iPMs’ ability to pace together. Acute fibrosis is accompanied with proliferation and activation of cardiac fibroblasts via Tgfβ-Smad2/3 pathway. Small molecule inhibition of Tgfβ signaling mitigated the interstitial remodeling, independent from TBX18-induced iPM reprogramming at the single-cell level. Direct and focal gene transfer of TBX18 into the left ventricular myocardium created ventricular pacing in a rat model of chronic atrioventricular block, but such activity began to wane in a week. In contrast, a combination therapy consisting of TBX18 gene transfer and Tgfβ inhibition enabled sustained biological pacing beyond the four-week study period. Our data demonstrate that inhibition of Tgfβ signaling suffices to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

Background: We have previously demonstrated reprogramming of ventricular myocytes into induced pacemaker cells (iPMs) using an embryonic transcription factor TBX18. We have also discovered that Tgfβ pathway is significantly upregulated in TBX18-iPMs, and triggers fibrosis upon gene transfer in vitro and in vivo. Here, we sought to investigate the impact of Tgfβ signaling on iPMs’ automaticity. Methods: Neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno-TBX18 or control Adeno-GFP. Single cell RNAseq was performed at days 3, 6, and 14. Spontaneous field potentials from NRVM monolayers were recorded with microelectrode array. Results: TBX18 gene transfer strongly repressed working cardiomyocyte gene expression, e.g., Gja1, Scn5a, Pln throughout the 14-day period. TBX18-NRVMs progressed toward pacemaker cells, evidenced by increase in nodal pacemaker-related ion channel gene expression. TBX18 also triggered cytoskeletal remodeling, and activated nonmyocyte gene expression throughout the 2-week window. This was illustrated by ECM remodeling gene expression such as Acta2, Col1a1, and Postn. Treatment with A83-01, a small molecule inhibitor of Tgfβ receptors, mitigated the increase in nonmyocyte gene expression. TBX18-NRVMs exhibited spontaneously oscillating field potentials, reaching a peak pacing frequency of 196±36 bpm during week 1, which is comparable to the sinus rhythm of newborn rats. However, the pacing frequency gradually wavered after week 1. In contrast, A83-01-treated TBX18-iPMs showed peak pacing frequency beyond week 1, with the majority of their total beat counts near their sinus rhythm (63.3%) while only 14.9% of total beats were near the sinus rhythm in TBX18-iPMs without Tgfβ inhibition (P<0.001). Interestingly, A83-01 did not impact PM-related ion channel gene expression (such as Hcn4, Gjc1) nor the percentage of TBX18-iPMs (5.1% vs. 5.4%, with or without A83-01, p=0.561), suggesting that improved automaticity may be due to its impact on nonmyocytes. Conclusion: TBX18-iPMs exhibit increased Tgfβ signaling and nonmyocyte gene expression, which negatively impacts their automaticity. Inhibition of Tgfβ signaling modulates nonmyocyte gene expression and enhances automaticity of TBX18-iPMs.

BACKGROUND:Previous observational studies and meta-analyses have suggested that statins could be beneficial in reducing the risk of adverse clinical outcomes. This study is the first to conduct a meta-analysis of recently published randomized controlled clinical trials investigating the potential therapeutic benefits of statins for COVID-19.MATERIALS AND METHODS:A thorough search was conducted using databases such as PubMed and Embase until May 2023 to identify randomized controlled clinical trials investigating the use of statins in patients with COVID-19. Review Manager 5.4 was used to analyze the selected studies.RESULTS:Seven randomized controlled trials comprising a total of 2,370 patients were included in this study. Of these, 1,295 patients received statin therapy, while 1,075 received placebo or other drugs. All included studies were conducted on inpatients with an average age of 45 - 61 years, and the proportion of patients with diabetes and coronary heart disease was less than 30%. One study only included severely ill patients. Our results showed that statin treatment did not significantly reduce hospitalized patient mortality (11.5 vs. 13.4%, p = 0.94), the proportion of patients transferred to intensive care due to disease changes (14.2 vs. 11.2%, p = 0.41), or the proportion of patients requiring mechanical ventilation (5.3 vs. 7.9%, p = 0.71) compared to controls. However, the use of statins was associated with a slight increase in hospital stay.CONCLUSION:A critical appraisal of published randomized controlled trials on statin therapy in COVID-19 did not show any significant effect on mortality, the risk of transfer to intensive care, or mechanical ventilation.

Molybdenum plays an important role in marine biological activity, especially in nitrogen cycling as a cofactor for N2 fixation and nitrate reductase. However, the dissolved Mo (dMo) behavior and its interaction with N cycling in the coastal waters is still unclear. In this study, the dMo concentrations and parameters related to Mo distribution and N cycling in surface and bottom seawaters of the Bohai (BS) and Yellow Seas (YS) were examined. The results showed that dMo concentrations ranged from 36.4 nmol L-1 to 125.0 nmol L-1, most of which deviated significantly from the conservative line, indicating nonconservative behavior of Mo relative to salinity. The highest dMo concentrations occurring in 36°N section of north of the South YS (SYS), were close to conservative value (105 nmol L-1). Significant depletion up to 40-50 nmol L-1 of dMo mainly appeared in the BS, NYS and south of the SYS, suggesting the possible removal of dMo by biological utilization and particle adsorption. Particularly, the increasing dMo concentrations away the Yellow River estuary indicated that freshwater dilution was one of reasons for dMo distributions in the BS. The similar spatial distribution of dMo and dissolved Mn concentrations suggested the possible scavenging by MnOx phases for Mo removal. The negative correlation between dMo and chlorophyll-a (Chl-a) concentrations in surface seawaters suggested that biological uptake was involved in dMo removal. The depleted dMo in most of sites corresponded with the higher nitrite concentrations, implying the possible involvement of nitrate reduction process. Although the highest N2 fixation rates and relative abundances of cyanobacteria appeared in 36°N section, corresponding with the conservative dMo, suggesting that Mo may play a minor role in N2 fixation process there. The ten-folds of relative abundance of bacteria with nitrate reduction function than that with N2 fixation function suggested that dMo seems to play more important role in nitration reduction than nitrogen fixation in the BS and YS.

Background: We have demonstrated that TBX18 suffices to reprogram postnatal ventricular cardiomyocytes (CMs) to induced pacemaker cells. Cell-cell interactions and cell-extracellular matrix (ECM) are important regulators of microtissue organization and assembly. Here, we sought to gain a finer understanding of self-organization and assembly of pacemaker microtissue with myocytes and non-myocytes driven by TBX18. Methods: 2D monolayer of neonatal rat ventricular myocytes (NRVMs) were transduced with Adeno- GFP and Adeno-TBX18 in vitro model. AAV9-TBX18 was delivered via tail vein injection of Hcn4 (+/eGFP) transgenic mice to track the induced pacemaker myocytes. Result: TBX18-NRVMs showed more compact nodal-like aggregates with a higher density of CMs and surrounded by nonmyocytes. The number of alpha smooth muscle actin (αSMA) + myofibroblasts producing specific ECM and facilitating cell motility was increased by 190% in TBX18-NRVMs as well. Systemic delivery of AAV9-TBX18 via tail vein of mice created aggregation of de novo Hcn4+ induced pacemaker myocytes (iPMs) surrounded by αSMA+ myofibroblasts in vivo. Then, we treated pre-plated neonatal rat ventricular cells (NRVCs, including CMs and nonmyocytes) to explore the potential role of cell-cell communication and cell-ECM interaction in the formation of nodal-like aggregates through scRNA-seq. The data indicated that TBX18+ CM (28.6% % of all CMs) and TBX18-negative CMs (71.4% of all CMs) presented highly consistent transcriptomic profiles at day 3. No significant difference in Hcn4 transcript levels and the ratio of iPMs (defined as Hcn4+, Gja1 low , Nkx2.5 low , Tnni3 high , Actn2 high CM) was observed between them, as well as other pacemaker genes, suggesting cross-communication between them. Despite of TBX18+ fibroblasts (FBs) accounting for only 5% of all FBs, a higher proportion of activated myofibroblasts (39.3% vs. 25.4%) and lower proportion of quiescent FBs were observed in TBX18-NRVCs compared to control. Enrichment analysis revealed that TBX18-CMs lost ventricular specific electrical coupling and sarcomere organization (down-regulated Gja1, Cdh2, Irx3 ), but reconstructed hemidesmosome assembly and cell-ECM interaction enriched in sinoatrial node myocytes (Upregulated Lamc1, Itgb1, Plec, Des, Actn1, and Acta2 ) and activated Tgfβ signaling. Furthermore, treatment with gap junction inhibitor palmitoleic acid as well as Tgfβ receptor inhibitor A83-01 significantly suppressed the activation of quiescent fibroblasts to αSMA+ myofibroblasts and the formation of nodal-like aggregation of TBX18-NRVMs. Conclusion: TBX18 drives self-organization and nodal-like assembly of pacemaker microtissue with myocytes and non-myocytes through cell-cell communication of gap junction and Tgfβ signal.

BACKGROUND:As reported, CHADS2 scoring system moderately predicts the atrial fibrillation (AF) recurrence, a common event after cryoballoon ablation. We aimed to improve the diagnostic accuracy of the CHADS2 score by adding several routine auxiliary detection indicators into the scoring system and constructing a CHADS2 score-based nomogram to predict AF recurrence in patients with paroxysmal AF undergoing cryoballoon ablation.METHODS:Eighty-four patients with paroxysmal AF undergoing cryoballoon ablation were enrolled. Baseline characteristics were collected. The multivariable Cox proportional hazards model was used to identify the significantly related predictors of recurrence and to construct the nomogram whose performance was evaluated by the discrimination and calibration tests.RESULTS:Thirty-five patients developed AF recurrence after a mean follow up of 19.0 ± 15.77 months. In the Cox multivariate model, CHADS2 (>2) (hazard ratio [HR]: 2.38; 95% confidence interval [CI]: 1.14-4.98, p = .021) and albumin-to-globulin ratio (AGR) (HR: 2.49; 95% CI: 1.26-4.92, p < .008) were independent risk factors associated with AF recurrence. In addition to CHADS2 , AGR and red blood cell distribution width were used to construct the nomogram. As a result, the discrimination of the concordance index for the predictive model of AF recurrence was increased from 0.56 (95% CI: 0.494-0.632) to 0.712 (95% CI: 0.631-0.811). The 24-month one well matched the ideal 45° line among the calibration plots for 6, 12, and 24 months' recurrence-free survival.CONCLUSION:This novel easy-to-use CHADS2 score-based nomogram may be used to predict AF recurrence for patient of paroxysmal AF undergoing cryoballoon ablation. Further external validation is still needed.

Attributed to poor signal uniformity and external interference, ultrasensitive surface-enhanced Raman spectroscopy (SERS) still faces difficulties in the reliable and quantitative detection of trace molecules. Here, a facile Ag/Si/sodium carboxy methyl cellulose (NaCMC) film with internal standard (IS) was promoted for quantitative determination of thiram. The effects of preparation conditions on SERS activity of the film were systematically investigated and then a flexible SERS substrate with high sensitivity and uniformity was fabricated. The enhancement factor was calculated to be 1.12 × 106 and SERS mapping was recorded with a relative standard deviation value of 19.8% by utilizing 4-mercaptobenzoic acid (4-MBA) as target molecule. Additionally, the dominant contribution of the IS from encapsulated Si nanoparticles (NPs) was confirmed in the quantitative assay of 4-MBA and thiram, facilitating attractive fitting coefficients (R2) as 0.991 and 0.998. Besides that, the proposed flexible film was conducted to scrub trace thiram from the surfaces of apple, orange, and cucumber, resulting in recoveries of 89%, 94%, and 91%. A smart and facile quantitative SERS substrate was developed here for monitoring trace biochemical molecules, verifying its potential utilizations in monitoring pesticide residues.