Randomized controlled trial of ultra-protective vs. protective ventilation strategy in veno-arterial extracorporeal membrane oxygenation patients with refractory cardiogenic shock: a study protocol for the ultra-ECMO trial.

Background: A protective or ultra-protective tidal volume strategy is widely applied to patients with acute respiratory distress syndrome (ARDS). The use of very low tidal volume has the potential to further redece ventilation-induced lung injury (VILI) comparde with a "normal" lung protective management. Plus, cardiogenic pulmonary edema (CPE) caused by hydrostatic mechanisms in patients with cardiogenic shock has similar respiratory mechanics to those found in patients with ARDS. And no consensus exists on mechanical ventilation parameter settings in patients with VA-ECMO. The study aimed to investigate the impact of an ultra-protective tidal volume strategy on the 28-day ventilator-free day (VFD) number in VA-ECMO-supported patients with refractory cardiogenic shock, including cardiac arrest. Methods: The Ultra-ECMO trial is a randomized controlled, open-label, single-center prospective superiority trial. At the onset of ECMO initiation, we will divide patients randomly into an intervention group and a control group in a 1:1 ratio. The control group will adopt protective ventilation settings [initial tidal volume: 6 ml/kg of predicted body weight (PBW)] for ventilation, and the intervention group will adopt ultra-protective ventilation settings (initial tidal volume: 4 ml/kg of PBW) for ventilation. The procedure is expected to last 72 h, after which the ventilator settings will be at the intensivists' discretion. The primary outcome is the VFD number at 28 days after inclusion. The secondary outcomes will include respiratory mechanics; analgesic/sedation dosage; lung ultrasound score; interleukin-6, interleukin-8, and monocyte chemotactic protein-1 levels in broncho-alveolar lavage fluid at the moment of enrollment (T0), 24, 48, and 72 h (T1, T2, and T3, respectively) after ECMO initiation; total time (in days) required for ECMO weaning; length of stay in the intensive care unit; total cost of hospitalization; amounts of resuscitative fluids; and in-hospital mortality. Discussion: VA-ECMO-treated patients without ARDS possess abnormal lung function. CPE, thoracic compliance reduction, and poor pulmonary blood perfusion are frequently present, and these patients can more easily progress to ARDS. It seems that targeting the protective tidal volume can lower adverse outcome incidence rates, even in patients without ARDS. This trial seeks to answer the question of whether adopting an ultra-protective tidal volume strategy can lead to superior primary and secondary outcomes compared to adopting a protective tidal volume strategy in patients treated by VA-ECMO. The Ultra-ECMO trial will provide an innovative mechanical ventilation strategy for VA-ECMO-supported patients for improving treatment outcomes at biological and potentially clinical levels. Clinical Trial Registration: ChiCTR2200067118.

Impact of connecting methods of continuous renal replacement therapy device on patients underwent extracorporeal membrane oxygenation: A retrospectively observational study.

OBJECTIVE: The objective of this study was to compare the safety and efficiency of different extracorporeal membrane oxygenation (ECMO) and continuous renal replacement therapy (CRRT) connection methods. BACKGROUND: The number of patients receiving ECMO is increasing, and the fields of application are getting wider. However, patients receiving ECMO are prone to acute kidney injury and fluid overload requiring CRRT. There are few comparative studies of two different systems of connecting CRRT device and ECMO from safety and efficacy perspective. METHODS: This retrospective observational study included patients receiving ECMO in the extracorporeal life support centre of the First Affiliated Hospital of Nanjing Medical University from June, 2015, to December, 2020. Patients were divided into the parallel system group and integrated system group according to the connecting method between ECMO circuit and CRRT line. The outcomes were discharge survival rate, CRRT therapeutic dose completion rate, CRRT catheterisation time, CRRT initiating time, local bleeding at the CRRT catheter site, mean filter life, ECMO circuit thrombosis, ECMO air leakage, or blood leakage due to CRRT. RESULTS: Thirty patients in the parallel system group and 70 patients in the integrated system group were finally included. The discharge survival rate and CRRT therapeutic dose completion rate were not significantly different between the two groups. The parallel system group had significant longer CRRT initiating time (49.0 ± 12.1 min vs. 14.6 ± 2.1 min, P < 0.001) and shorter filter life (11.5 ± 3.2 h vs. 47.3 ± 14.0 h, P < 0.001) than the integrated system group. The occurrence rate of local bleeding was 93.3% in the parallel system group, and there is no bleeding case in the integrated system group. There was no case of ECMO circuit thrombosis from CRRT as well as ECMO air or blood leakage caused by CRRT in either group. ECMO therapy can be adapted by adjusting the position of the CRRT outlet in the integrated system. CONCLUSIONS: Connecting CRRT and ECMO as an integrated system might accelerate CRRT initiation, avoid local bleeding, and prolong filter life compared to the parallel system. The chance of developing CRRT-related ECMO circuit leak and thrombosis is manageable.

Average mean arterial pressure in the first 6 hours of extracorporeal cardiopulmonary resuscitation in the prediction of the prognosis of neurological outcome: a single-center retrospective study.

OBJECTIVE: To study the correlation between the mean arterial pressure (MAP) level in the first 6 hours of extracorporeal cardiopulmonary resuscitation (ECPR) and patients' neurological outcomes. METHODS: Sex, age, basic comorbidities, the time from the first cardiac arrest to the start of CPR, the time from the first cardiac arrest to extracorporeal membrane oxygenation (ECMO), standardized ECMO flow, and the pH value at the beginning of ECMO and after 6 hours were recorded. MAP was recorded every 2 hours during the first 6 hours, and the average was calculated. The lactic acid clearance rate of the first 6 hours was calculated. Evaluated the neurological prognosis of patients at discharge. Then the patients were divided into groups according to their average MAP, and the above variables were compared in groups. RESULTS: Enrolled 63 adult ECPR patients. There were no statistically significant differences in sex, age, basic comorbidities, the time from the first cardiac arrest to the start of conventional CPR, the time from the first cardiac arrest to the start of ECMO, standardized ECMO flow, 6-hour lactic acid clearance rate, pH value at the sixth hour of operation between two groups. The pH value at the start of ECMO, survival rate, and good prognosis rate in low average MAP group were significantly lower. Low average MAP was associated with poor neurological outcomes (relative risk (RR) 1.50, 95% CI 1.17, 1.92). The RR of good neurological outcome for patients with average MAP ⩾65 mmHg was 5.91 (95% CI 1.45, 24.06), and the RR for average MAP ⩾100 mmHg was 1.18 (95% CI 0.19, 7.52). CONCLUSION: For ECPR patients, average MAP <65 mmHg in the first 6 hours of ECPR indicates a poor neurological prognosis. However, whether higher average MAP levels can improve the neurological prognosis of ECPR patient remains to be further studied.

Effect of arterial oxygen partial pressure inflection point on Venoarterial extracorporeal membrane oxygenation for emergency cardiac support.

BACKGROUND: Temporary circulatory support is a bridge between acute circulatory failure and definitive treatment or recovery. Currently, venoarterial extracorporeal membrane oxygenation (VA-ECMO) is considered to be one of the effective circulatory support methods, although cardiac function monitoring during the treatment still needs further investigation. Inflection point of arterial oxygen partial pressure (IPPaO2) may occur at an early stage in part of patients with a good prognosis after VA-ECMO treatment, and the relationship between time of IPPaO2 (tIPPaO2) and recovery of cardiac function or prognosis remains unclear. METHODS: To investigate this relationship, we retrospectively analyzed the clinical data of 71 patients with different conditions after treatment with VA-ECMO in the emergency center of Jiangsu Province Hospital between May 2015 and July 2020. Spearman's correlation analysis was used for the correlation between tIPPaO2 and quantitative data, and ROC curve for the predictive effect of tIPPaO2 on the 28-day mortality. RESULTS: Thirty-five patients were admitted because of refractory cardiogenic shock (26 of 35 survived) and the remaining 36 patients due to cardiac arrest (13 of 36 survived). The overall survival rate was 54.9% (39 of 71 survived). Acute physiology and chronic health evaluation II, ECMO time, tIPPaO2, continuous renal replacement therapy time, mechanical ventilation time, and bleeding complications in the survival group were lower than those in the non-survival group, with length of stay, intensive care unit stay, and platelet levels were being higher. The tIPPaO2 was negatively correlated with ejection fraction, and the shorter tIPPaO2 resulted in a higher 28-day survival probability, higher predictive value for acute myocardial infarction and fulminant myocarditis. CONCLUSIONS: Therefore, tIPPaO2 could be a reliable qualitative indicator of cardiac function in patients treated with VA-ECMO, which can reveal appropriate timing for adjusting VA-ECMO flow or weaning. TRIAL REGISTRATION: ChiCTR1900026105 .

Exercise-induced peptide TAG-23 protects cardiomyocytes from reperfusion injury through regulating PKG-cCbl interaction.

Recent studies have revealed that proper exercise can reduce the risk of chronic disease and is beneficial to the body. Peptides have been shown to play an important role in various pathological processes, including cardiovascular diseases. However, little is known about the role of exercise-induced peptides in cardiovascular disease. We aimed to explore the function and mechanism of TAG-23 peptide in reperfusion injury and oxidative stress. Treatment with TAG-23 peptide significantly improved cell viability, the mitochondrial membrane potential, and ROS levels and reduced LDH release, the apoptosis rate and caspase 3 activation in vitro. In vivo, TAG-23 ameliorated MI and heart failure induced by I/R or DOX treatment. Pull-down assays showed that TAG-23 can bind to PKG . The TAG-23-PKG complex inhibited PKG degradation through the UPS. We also identified cCbl as the E3 ligase of PKG and found that the interaction between these proteins was impaired by TAG-23 treatment. In addition, we provided evidence that TAG-23 mediated Lys48-linked polyubiquitination and subsequent proteasomal degradation. Our results reveal that a novel exercise-induced peptide, TAG-23, can inhibit PKG degradation by serving as a competitive binding peptide to attenuate the formation of the PKG-cCbl complex. Treatment with TAG-23 may be a new therapeutic approach for reperfusion injury.

Can Cardiopulmonary Rehabilitation Facilitate Weaning of Extracorporeal Membrane Oxygenation (CaRe-ECMO)? Study Protocol for a Prospective Multidisciplinary Randomized Controlled Trial.

Background: Mortality of patients suffering from critical illness has been dramatically improved with advanced technological development of extracorporeal membrane oxygenation (ECMO) therapy. However, the majority of ECMO-supported patients failed to wean from ECMO therapy. As one of several options, cardiopulmonary rehabilitation serves as effective intervention in the improvement of cardiovascular and respiratory function in various major critical illness. Nonetheless, its role in facilitating ECMO weaning has not yet been explored. The purpose of this study is to investigate the effectiveness of cardiopulmonary rehabilitation on rate of ready for ECMO weaning in ECMO-supported patients (CaRe-ECMO). Methods: The CaRe-ECMO trial is a randomized controlled, parallel group, clinical trial. This trial will be performed in a minimum number of 366 ECMO-supported eligible patients. Patients will be randomly assigned to either: (1) the CaRe-ECMO group, which will be treated with usual care including pharmacotherapy, non-pharmacotherapy, and specific nursing for ECMO therapy and the CaRe-ECMO program; or (2) the control group, which will receive usual care only. The CaRe-ECMO program consists of protocolized positioning, passive range of motion (PROM) training, neuromuscular electrical stimulation (NMES), surface electrical phrenic nerve stimulation (SEPNS), and pulmonary rehabilitation. The primary outcome of the CaRe-ECMO trial is the rate of ready for ECMO weaning at CaRe-ECMO day 7 (refers to 7 days after the CaRe-ECMO program initiation). Secondary outcomes include rate of ECMO and mechanical ventilation weaning, total length in day of ready for ECMO weaning, ECMO weaning and mechanical ventilation, all-cause mortality, rate of major post-ECMO complications, ECMO unit length of stay (LOS) and hospital LOS, total cost for hospitalization, cerebral performance category (CPC), activities of daily living (ADL), and health-related quality of life (HRQoL). Discussion: The CaRe-ECMO is designed to answer the question "whether cardiopulmonary rehabilitation can facilitate weaning of ECMO (CaRe-ECMO)." Should the implementation of the CaRe-ECMO program result in superior primary and secondary outcomes as compared to the controls, specifically the add-on effects of cardiopulmonary rehabilitation to the routine ECMO practice for facilitating successful weaning, the CaRe-ECMO trial will offer an innovative treatment option for ECMO-supported patients and meaningfully impact on the standard care in ECMO therapy. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT05035797.

[Value of cardiac troponin T in predicting the prognosis of patients with cardiogenic shock receiving veno-arterial extracorporeal membrane oxygenation treatment: a consecutive 5-year retrospective study].

OBJECTIVE: To explore the changing trend of cardiac troponin T (cTnT) in patients with cardiogenic shock (CS) receiving veno-arterial extracorporeal membrane oxygenation (V-A ECMO) and its predictive value. METHODS: A retrospective study was conducted. The data of patients with CS receiving V-A ECMO admitted to the First Affiliated Hospital of Nanjing Medical University from March 2015 to May 2020 were enrolled. The baseline data, ECMO related parameters, serum cTnT levels at 1, 2, 3 days after ECMO and intensive care unit (ICU) prognosis were recorded. The parameters with clinical significance and significant difference in univariate analysis were analyzed by binary multivariate Logistic regression analysis. Meanwhile, receiver operating characteristic (ROC) curve was drawn, area under ROC curve (AUC) was analyzed, and the threshold, sensitivity and specificity of serum cTnT level and its reduction rate for predicting clinical outcome were evaluated. RESULTS: A total of 72 patients were enrolled, of which 42 survived and 30 died at ICU discharge, and the ICU mortality was 41.7%. Univariate analysis results: compared with the survival group, the patients in the death group had higher acute physiology and chronic health evaluation II (APACHE II) score [32 (30, 34) vs. 29 (25, 30)], and the incidence of cardiac arrest before ECMO (70.0% vs. 31.0%), the ratios of invasive mechanical ventilation and continuous renal replacement therapy during ECMO were higher (96.7% vs. 66.7%, 83.3% vs. 42.9%), and the differences were statistically significant (all P < 0.05). Serum cTnT levels (ng/L) at 2 days and 3 days after ECMO in the death group were significantly higher than those in the survival group [2 days: 6 373.5 (898.3, 15 251.5) vs. 1 760.5 (933.0, 4 257.8), 3 day: 6 202.0 (758.9, 16 554.3) vs. 1 678.0 (623.3, 3 407.8), both P < 0.05], and the decrease rates of cTnT within 2 days and 3 days after ECMO were significantly lower than those in the survival group [2 days: 17.3% (-44.2%, 34.7%) vs. 36.8% (18.1%, 60.6%), 3 days: 32.4% (-30.0%, 55.5%) vs. 53.2% (38.3%, 72.3%), both P < 0.05]. Binary multivariate Logistic regression analysis showed that cardiac arrest before ECMO [odds ratio (OR) = 4.564, 95% confidence interval (95%CI) was 1.212-17.193, P = 0.025] and the decrease rate of cTnT level within 2 days after ECMO (OR = 1.617, 95%CI was 1.144-4.847, P = 0.026) were independent prognostic risk factors for the ICU death of CS patients receiving V-A ECMO. ROC curve analysis showed that the decline rate of cTnT within 2 days after ECMO transfer had a certain predictive value for the ICU death of CS patients receiving V-A ECMO. The AUC was 0.704 (95%CI was 0.584-0.824). The optimal diagnostic threshold was 40.0%, the sensitivity was 86.7%, the specificity was 52.4%, the positive predictive value was 66.9%, and the negative predictive value was 89.1%. CONCLUSIONS: The early decline rate of cTnT in CS patients who received V-A ECMO treatment in death group was lower than that of survival patients. The cTnT decline rate 2 days after ECMO was an independent risk factor for the death of such patients.

Genetic Variants in AC092159.2 and Risk of Gestational Diabetes Mellitus in a Chinese Population.

Long-noncoding RNA AC092159.2, located ∼247 bp upstream of the TMEM18 gene, may play integral roles in metabolic processes, including adipocyte differentiation and glycometabolism. AC092159.2 may be an important regulator of TMEM18, which is an important susceptibility gene related to obesity and type 2 diabetes. We designed a case-control study (including 964 gestational diabetes mellitus [GDM] cases and 1021 controls) to assess the associations of 14 single-nucleotide polymorphisms (SNPs) in AC092159.2 with the GDM risk. Logistic regression analyses showed that rs11127496 A > G, rs12714417 C > T, and rs1320334 A > G conferred a decreased GDM risk in the recessive and additive model, whereas rs11691220 T > C conferred an increased GDM risk in the dominant and additive model. Also, with increasing number of protective alleles of the four SNPs, the risk of GDM was significantly decreased in a dose-dependent manner (ptrend = 0.007). Further function annotation indicated that these four SNPs may fall on the function elements of human pancreatic islets. The genotype-phenotype associations suggested that these SNPs may contribute to GDM by affecting TMEM18 expression. AC092159.2 SNPs (rs11127496 A > G, rs11691220 T > C, rs12714417 C > T, and rs1320334 A > G) may be susceptibility makers for risk of GDM in Chinese females.

[Predictive values of different critical scoring systems for survival rate after discharge in critically ill patients supported by extracorporeal membrane oxygenation].

OBJECTIVE: To determine the predictive values of different critical scoring systems for survival rate after discharge in critically ill patients supported by extracorporeal membrane oxygenation (ECMO). METHODS: The clinical data of 34 critically ill patients supported by ECMO admitted to Department of Emergency of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Provincial People's Hospital) from July 2015 to September 2017 were retrospectively analyzed. The general information and the worst values of vital signs and related pathophysiological indicators within 12 hours before ECMO treatment of patients were collected, and sequential organ failure assessment (SOFA), multiple organs dysfunction score (MODS), simplified acute physiology score II (SAPS II), and acute physiology and chronic health evaluation IV (APACHE IV) scores were calculated. The patients were divided into survival group and non-survival group according to 28-day survival after hospital discharge. General clinical characteristics and aforementioned scores were compared between the two groups. Scoring systems for predicting prognosis were assessed by using the receiver operating characteristic (ROC) curve. The Kaplan-Meier method was used to depict the surviving curve. RESULTS: Thirty-four patients were finally enrolled, 13 of whom were dead at the follow-up period of 28 days after hospital discharge, and 21 survived. Duration of ECMO support in non-survival group was significantly shorter than that in survival group (hours: 101.4±7.8 vs. 134.4±12.6), SOFA, SAPS II, and APACHE IV scores were significantly higher than those of survival group (SOFA score: 10.6±3.6 vs. 8.8±3.3, SAPS II score: 38.7±14.3 vs. 31.8±12.5, APACHE IV score: 46.5±15.5 vs. 38.1±11.3, all P < 0.05). There was no significant difference in gender, age, body mass index (BMI), vital signs or related pathophysiological indicators within 12 hours before ECMO treatment, or MODS score between the two groups. ROC curve analysis showed that the area under ROC curve (AUC) of SAPS II score for predicting 28-day survival rate was the highest, which was significantly higher than that of SOFA, MODS, and APACHE IV score (0.880 vs. 0.694, 0.654, 0.682, all P < 0.05). When the best cut-off value of SAPS II score was 43, the sensitivity was 81.2%, and the specificity was 77.9%. Kaplan-Meier survival analysis showed that 28-day survival rate after hospital discharge in patients with SAPS II score < 43 (n = 18) was significantly higher than that in patients with SAPS II score ≥ 43 (n = 16; χ2 = 2.444, P = 0.018). CONCLUSIONS: Four critical scoring systems of SOFA, MODS, SAPS II and APACHE IV have been proved to have good prognostic ability to predict 28-day survival after hospital discharge in critically ill patients supported by ECMO. Among them, SAPS II score system has more accurate prediction value.

Serum cystatin C levels are associated with coronary artery disease and its severity.

OBJECTIVES: Serum cystatin C has been established as a predictor of cardiovascular events. The aim of this study was to evaluate the role of cystatin C in determining the presence and the severity of patients with coronary artery disease (CAD). DESIGN AND METHODS: A total of 936 subjects without overt renal disease were included in this cross-sectional study. Among them were 714 patients with CAD and 222 without based on coronary angiography. Subjects were further divided into four groups according to cystatin C quartile. Serum cystatin C was measured using particle-enhanced immunoassay method. The study analyzed the relationship of cystatin C levels with the presence and severity of CAD, including the number of stenotic vessels involved and Gensini score. RESULTS: Serum cystatin C levels were significantly higher in patients with CAD than those without (P<0.001), and significantly increased as the involvement of coronary vessels increased (P<0.001). The prevalence of CAD and its severity assessed by Gensini score were also significantly greater in the highest quartile of cystatin C (P<0.001). Moreover, cystatin C levels were independently correlated with the presence of CAD in a multivariate logistic regression model (P=0.023) and were positively correlated with Gensini score by linear regression analysis (standardized ß=0.083, P=0.010). CONCLUSIONS: Elevated serum cystatin C levels were significantly associated with the presence and severity of CAD in patients with normal renal function. It is suggested that cystatin C might play a role in CAD diagnosis and serve as a marker of CAD severity.

Effects of miR-19b overexpression on proliferation, differentiation, apoptosis and Wnt/ß-catenin signaling pathway in P19 cell model of cardiac differentiation in vitro.

MicroRNA (miR)-19b is part of the miR-17-92 cluster associated with cardiac development. Here, we investigated the effects of overexpressing miR-19b on proliferation, differentiation, apoptosis, and regulation of the Wnt/ß-catenin signaling pathway in the multipotent murine P19 cell line that can be induced to undergo cardiogenesis. P19 cells were transfected with the miR-19b plasmid or empty vector, and miR-19b overexpression was verified by Quantitative Real-Time PCR (qPCR). The miR-19b or vector control stable cell lines were selected using Blasticidin S HCl, and their proliferation, cell cycle, and apoptosis levels were analyzed using the Cell Counting Kit-8 and flow cytometry. P19 cell differentiation markers, apoptosis-related genes (bax, bcl-2), and Wnt/ß-catenin signaling pathway-related genes were detected by qPCR, the corresponding proteins by Western blot. Expression of the Wnt pathway and differentiation marker proteins was also verified by immunofluorescence. Morphological changes associated with apoptosis were observed by electron microscopy and Hoechst staining. On the basis of these results, we demonstrated that miR-19b overexpression promoted proliferation and differentiation but inhibited apoptosis in P19 cells; Wnt and ß-catenin expressions were decreased, while that of GSK3ß was increased with miR-19b overexpression. Overexpression of miR-19b inhibited activation of the Wnt/ß-catenin signaling pathway in P19 cells, which may regulate cardiomyocyte differentiation. Our findings may bring new insights into the mechanisms underlying cardiac diseases and suggest that miR-19b is a potential new therapeutic target for cardiovascular diseases.

Effect of organic matter on the performance of granular anammox process.

The presence of organic matter (OM) is considered to affect anammox process adversely, while practically wastewaters containing ammonia are not free from OM. In this study, the performance of anammox granules in presence of OM was evaluated under different COD to N ratios. Low OM concentration did not affect ammonia and nitrite removal significantly but improved the total nitrogen removal via denitrifiers. High OM could suppress anammox activity, resulting in a lower ammonia removal. PCR tests revealed that there was a reduction in the number of anammox bacteria and denitrifiers quantity increased when 400mg COD/L influent was applied. A COD to N threshold ratio for anammox inhibition, defined when ammonia removal dropped to 80%, was 3.1, higher than that of flocculent sludge. This study revealed that the coexistence of denitrification and anammox was an effective strategy to treat wastewaters containing high levels of nitrogen and OM.

Differential expression of microRNAs in cardiac myocytes compared to undifferentiated P19 cells.

microRNA (miRNA) expression is tightly controlled in a tissue-specific and developmental stage-specific manner; some are highly and specifically expressed in cardiovascular tissues. miRNA expression profiling, using miRNA microarrays facilitates studying the biological function of miRNAs. We investigated changes in miRNA expression profiles during differentiation of P19 cells into cardiac myocytes in order to elucidate the mechanisms of heart development. The morphology of P19 cells during differentiation was observed using an inverted microscope. Western blot analysis was performed to detect cardiac troponin I (cTnI) expression. Total RNA was extracted from P19 cells for microarray and real-time quantitative reverse transcription-polymerase chain reaction (real-time qRT-PCR) analyses to determine the miRNA expression profile. The miRNA microarray revealed differential expression of 49 miRNAs, of which 26 were down-regulated and 23 were up-regulated in differentiated cardiac myocytes, compared to normal P19 cells. This was confirmed by real-time qRT-PCR. We also utilized target prediction analysis to identify gene targets. Some miRNAs may have important roles in cardiac development and congenital heart defects (CHDs). Further analysis of miRNA function to confirm their target genes during cardiac development will determine the potential for novel miRNA-based therapeutic strategies.

LYRM1, a gene that promotes proliferation and inhibits apoptosis during heart development.

Congenital heart disease (CHD) is the most common type of birth defect, but its underlying molecular mechanisms remain unidentified. Previous studies determined that Homo sapiens LYR motif containing 1 (LYRM1) is a novel nucleoprotein expressed at the highest level in adipose tissue and in high levels in heart tissue. The LYRM1 gene may play an important role in the development of the human heart. This study was designed to identify the biological characteristics of the LYRM1 gene in heart development. On the basis of expression-specific differentiation markers identified with quantitative real-time RT-PCR and the morphology of LYRM1-overexpressing cells during differentiation, ectopic expression was not found to significantly affect differentiation of P19 cells into cardiomyocytes. MTT assays and cell cycle analysis showed that LYRM1 dramatically increases the proliferation of P19 cells. Furthermore, data from annexin V-FITC binding and caspase-3 activity revealed that LYRM1 can inhibit the apoptosis of P19 cells. Our data suggest that LYRM1 might have the potential to modulate cell growth, apoptosis, and heart development.

GATA-4 promotes the differentiation of P19 cells into cardiac myocytes.

The aim of this study was to investigate the effects of GATA-4 on the differentiation of P19 cells into cardiomyocytes and to examine the relationship between GATA-4 and cardiomyocytes. We constructed vectors to overexpress and silence GATA-4. These vectors, as well as empty ones were transfected into P19 cells. Subsequently, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis were performed. The morphology of P19 cells during differentiation was observed using an inverted microscope. Total RNA was extracted from P19 cells. We used real-time PCR to evaluate the expression levels of 6 genes: GATA-4, GATA-6, transthyretin (TTR), alpha-fetoprotein (AFP), Nkx2.5, and alpha-myosin heavy chain (alpha-MHC). The gene expression pattern of these 6 genes is graphically shown for each group. The GATA-4 mRNA level in cells overexpressing GATA-4 was notably higher than that in the controls, whereas the levels in the controls were notably higher than those in the GATA-4-silenced P19 cells. The cell lines overexpressing GATA-4 expressed higher levels of Nkx2.5 and alpha-MHC than the controls. However, the controls expressed higher levels of AFP, GATA-6 and TTR than the cells overexpressing GATA-4. The RNAi group expressed lower levels of TTR, Nkx2.5, and alpha-MHC than the controls, but there were no differences in the RNAi group and the controls with regard to the expression levels of AFP and GATA-6. The gene expression pattern in the cells overexpressing GATA-4 was biased toward the Nkx2.5 and alpha-MHC. On the other hand, the gene expression pattern in GATA-4-silenced cells and the controls was biased toward the TTR and AFP. The overexpression of GATA-4 enhances the differentiation of P19 cells into cardiac myocytes, whereas its down-regulation suppresses this trend.