BACH1 deficiency prevents neointima formation and maintains the differentiated phenotype of vascular smooth muscle cells by regulating chromatin accessibility.

The transcription factor BTB and CNC homology 1(BACH1) has been linked to coronary artery disease risk by human genome-wide association studies, but little is known about the role of BACH1 in vascular smooth muscle cell (VSMC) phenotype switching and neointima formation following vascular injury. Therefore, this study aims to explore the role of BACH1 in vascular remodeling and its underlying mechanisms. BACH1 was highly expressed in human atherosclerotic plaques and has high transcriptional factor activity in VSMCs of human atherosclerotic arteries. VSMC-specific loss of Bach1 in mice inhibited the transformation of VSMC from contractile to synthetic phenotype and VSMC proliferation and attenuated the neointimal hyperplasia induced by wire injury. Mechanistically, BACH1 suppressed chromatin accessibility at the promoters of VSMC marker genes via recruiting histone methyltransferase G9a and cofactor YAP and maintaining the H3K9me2 state, thereby repressing VSMC marker genes expression in human aortic smooth muscle cells (HASMCs). BACH1-induced repression of VSMC marker genes was abolished by the silencing of G9a or YAP. Thus, these findings demonstrate a crucial regulatory role of BACH1 in VSMC phenotypic transition and vascular homeostasis and shed light on potential future protective vascular disease intervention via manipulation of BACH1.

Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries.

BACKGROUND: Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway. METHODS: Type 2 diabetes models were established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein-coupled receptor-targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies. RESULTS: Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase-mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes Rip1, Rip3, and Mlkl. Under hyperglycemic conditions, high glucose induced CB2R internalization in a ß-arrestin 2-dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of Bach2 diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries. CONCLUSIONS: CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.

Cardiotoxic effects of common and emerging drugs: role of cannabinoid receptors.

Drug-induced cardiotoxicity has become one of the most common and detrimental health concerns, which causes significant loss to public health and drug resources. Cannabinoid receptors (CBRs) have recently achieved great attention for their vital roles in the regulation of heart health and disease, with mounting evidence linking CBRs with the pathogenesis and progression of drug-induced cardiotoxicity. This review aims to summarize fundamental characteristics of two well-documented CBRs (CB1R and CB2R) from aspects of molecular structure, signaling and their functions in cardiovascular physiology and pathophysiology. Moreover, we describe the roles of CB1R and CB2R in the occurrence of cardiotoxicity induced by common drugs such as antipsychotics, anti-cancer drugs, marijuana, and some emerging synthetic cannabinoids. We highlight the 'yin-yang' relationship between CB1R and CB2R in drug-induced cardiotoxicity and propose future perspectives for CBR-based translational medicine toward cardiotoxicity curation and clinical monitoring.

Coenzyme Q deficiency may predispose to sudden unexplained death via an increased risk of cardiac arrhythmia.

Cardiac arrhythmia is currently considered to be the direct cause of death in a majority of sudden unexplained death (SUD) cases, yet the genetic predisposition and corresponding endophenotypes contributing to SUD remain incompletely understood. In this study, we aimed to investigate the involvement of Coenzyme Q (CoQ) deficiency in SUD. First, we re-analyzed the exome sequencing data of 45 SUD and 151 sudden infant death syndrome (SIDS) cases from our previous studies, focusing on previously overlooked genetic variants in 44 human CoQ deficiency-related genes. A considerable proportion of the SUD (38%) and SIDS (37%) cases were found to harbor rare variants with likely functional effects. Subsequent burden testing, including all rare exonic and untranslated region variants identified in our case cohorts, further confirmed the existence of significant genetic burden. Based on the genetic findings, the influence of CoQ deficiency on electrophysiological and morphological properties was further examined in a mouse model. A significantly prolonged PR interval and an increased occurrence of atrioventricular block were observed in the 4-nitrobenzoate induced CoQ deficiency mouse group, suggesting that CoQ deficiency may predispose individuals to sudden death through an increased risk of cardiac arrhythmia. Overall, our findings suggest that CoQ deficiency-related genes should also be considered in the molecular autopsy of SUD.

Post-mortem genetic analysis of sudden unexplained death in a young cohort: a whole-exome sequencing study.

Sudden unexplained death (SUD) constitutes a considerable portion of unexpected sudden death in the young. Molecular autopsy has proved to be an efficient diagnostic tool in the multidisciplinary management of SUD. Yet, many cases remain undiagnosed using the widely adopted targeted genetic screening strategies. Here, we investigated the genetic substrates of a young SUD cohort (18-40 years old) from China using whole-exome sequencing (WES), with the primary aim to identify novel SUD susceptibility genes. Within 255 previously acknowledged SUD-associated genes, 21 variants with likely functional effects (pathogenic/likely pathogenic) were identified in 51.9% of the SUD cases. More importantly, a set of 33 candidate genes associated with myopathy were identified to be novel susceptibility genes for SUD. Comparative analysis of the cumulative PHRED-scaled CADD score and polygenetic burden score showed that the amount and deleteriousness of variants in the 255 SUD-associated genes and the 33 candidate genes identified by this study were significantly higher compared with 289 randomly selected genes. A significantly higher genetic burden of rare variants (MAF < 0.1%) in the 33 candidate genes also highlighted putative roles of these genes in SUD. After incorporating these novel genes, the genetic testing yields of the current SUD cohort elevated from 51.9 to 66.7%. Our study expands understanding of the genetic variants underlying SUD and presents insights that improve the utility of genetic screenings.

Endoplasmic reticulum stress-related secretory proteins as biomarkers of early myocardial ischemia-induced sudden cardiac deaths.

Early myocardial ischemia-induced sudden cardiac deaths (EMI-SCD) remain a great diagnostic challenge for forensic pathologists due to no gross or non-specific histological pathology. The goal of this study was to assess whether three secretory proteins, related with cellular endoplasmic reticulum stress, can be applied in forensic diagnosis of EMI-SCD. These markers included LMAN2, CAPN-1, and VCP and were compared with two clinically used markers (CK-MB and cTnI). A total of 21 EMI-SCD cases with a mean age of 53.0 (± 10.5) years and a mean ischemia interval of < 2.77 (± 2.56) hours were collected. Another 23 cases (mean 44.6 ± 15.0 year old) that died from non-cardiac causes served as control. Enzyme-linked immunosorbent assay (ELISA) was performed to detect target proteins' serum concentrations in the EMI-SCD and control groups. We found that LMAN2, CAPN-1, and VCP were all significantly increased in the EMI-SCD group as compared with control serum, with the fold changes ranging from 1.48 (p = 0.0022, LMAN2), 1.33 (p = 0.041, CAPN-1), to 1.26 (p = 0.021, VCP), respectively. The concentrations of these proteins remained highly stable within 6 h and were not affected by death time, postmortem interval (< 4 h), age, and month at death. Receiver operating characteristic (ROC) curves showed that the areas under the curve (AUC) were 0.8178 (LMAN2), 0.6988 (CAPN-1), and 0.7267 (VCP), all of which were higher than CK-MB (AUC 0.5590) and cTn-I (AUC 0.5911). The diagnostic specificity (all above 60%) was obviously higher than CK-MB (43.48%) and cTnI (34.78%). In conclusion, LMAN-2, CAPN-1, and VCP could be stable serological biomarkers for diagnosis of EMI-SCD cases.

Serum SELENBP1 and VCL Are Effective Biomarkers for Clinical and Forensic Diagnosis of Coronary Artery Spasm.

Coronary artery spasm (CAS) plays an important role in the pathogenesis of many ischemic heart entities; however, there are no established diagnostic biomarkers for CAS in clinical and forensic settings. This present study aimed to identify such serum biomarkers by establishing a rabbit CAS provocation model and integrating quantitative serum proteomics, parallel reaction monitoring/mass spectrometry-based targeted proteomics, and partial least-squares discriminant analysis (PLS-DA). Our results suggested that SELENBP1 and VCL were potential candidate biomarkers for CAS. In independent clinical samples, SELENBP1 and VCL were validated to be significantly lower in serum but not blood cells from CAS patients, with the reasons for this possibly due to the decreased secretion from cardiomyocytes. The areas under the curve of the receiver operating characteristics (ROC) analysis were 0.9384 for SELENBP1 and 0.9180 for VCL when diagnosing CAS. The CAS risk decreased by 32.3% and 53.6% for every 10 unit increases in the serum SELENBP1 and VCL, respectively. In forensic samples, serum SELENBP1 alone diagnosed CAS-induced deaths at a sensitivity of 100.0% and specificity of 72.73%, and its combination with VCL yielded a diagnostic specificity of 100.0%, which was superior to the traditional biomarkers of cTnI and CK-MB. Therefore, serum SELENBP1 and VCL could be effective biomarkers for both the clinical and forensic diagnosis of CAS.

Second-generation antipsychotics induce cardiotoxicity by disrupting spliceosome signaling: Implications from proteomic and transcriptomic analyses.

Second-generation antipsychotics (SGAs) are first-line drugs that are prescribed for mental disorders in clinic. Severe cardiotoxicity has been widely reported and thus limits their clinical application. This study aimed to identify the common mechanism underlying SGAs-induced cardiotoxicity using dual-omics analyses. Balb/C mice were intraperitoneally injected with two representative SGAs, olanzapine (2.5 mg/kg) and clozapine (25 mg/kg), at clinically comparable doses for 0, 7, 14 and 21 days. Our results showed that both SGAs induced cardiomyocyte degeneration, inflammation infiltration, and cardiac fibrosis, all of which worsened with time. Proteomic analysis revelaed that 22 differentially expressed (DE) proteins overlapped in olanzapine and clozapine-treated hearts. These proteins were significantly enriched in muscle contraction, amino acid metabolism and spliceosomal assembly by GO term analysis and spliceosome signaling was among the top enriched pathways by KEGG analysis. Among the 22 DE proteins, three spliceosome signal proteins were validated in a dynamic detection, and their expression significantly correlated with the extent of SGAs-induced cardiac fibrosis. Following the spliceosome signaling dysregulation, RNA sequencing revealed that alternative splicing events in the mouse hearts were markedly enhanced by SGAs treatments, and the production of vast transcript variants resulted in dysregulation of multiple pathways that are critical for cardiomyocytes adaptation and cardiac remodeling. Pladienolide B, a specific inhibitor of mRNA splicing, successfully corrected SGAs-induced alternative splicing and significantly attenuated the secretion of pro-inflammatory factors and cell deaths induced by SGAs exposure. Our study concluded that the spliceosome signaling was a common pathway driving SGAs cardiotoxicity. Pharmacological inhibition of the spliceosome signaling represents a novel therapeutic strategy against SGAs cardiotoxicity.

CB1R Promotes Chronic Alcohol-Induced Neuronal Necroptosis in Mice Prefrontal Cortex.

AIMS: Alcohol abuse induces multiple neuropathology and causes global burden to human health. Prefrontal cortex (PFC) is one of the most susceptible regions to alcohol-induced neuropathology. However, precise mechanisms underlying these effects on PFC remain to be elucidated. Herein, we investigated whether RIP1/RIP3/MLKL-mediated necroptosis was involved in the alcohol-induced PFC injury, and explored the effect that cannabinoid receptors (CBRs) exerted on the neurotoxicity of alcohol. METHODS: In this study, dynamic development of neuronal necroptosis in the PFC region was monitored after 95% (v/v) alcohol vapor administration for 15 and 30 days, respectively. Selective CBRs agonists or inverse agonists were pretreated according to the experimental design. All the PFC tissues were isolated and further examined by biochemical and histopathological analyses. RESULTS: It was found that chronic alcohol exposure increased the protein level of MLKL and also the phosphorylated levels of RIP1, RIP3 and MLKL in a time-dependent manner, all of which indicated the activation of necroptosis signaling. Particularly, compared to astrocytes, neurons from the PFC showed more prototypical necrotic morphology in response to alcohol insults. In parallel, an increased protein level of CB1R was also found after 15 and 30 days alcohol exposure. Administration of specific inverse agonists of CB1R (AM251 and AM281), but not its agonists or CB2R modulators, significantly alleviated the RIP1/RIP3/MLKL-mediated neuronal necroptosis. CONCLUSION: We reported the involvement of RIP1/RIP3/MLKL-mediated necroptosis in alcohol-induced PFC neurotoxicity, and identified CB1R as a critical regulator of neuronal necroptosis that enhanced our understanding of alcohol-induced neuropathology in the PFC.

Cannabinoid Receptors in Myocardial Injury: A Brother Born to Rival.

Cannabinoid receptors typically include type 1 (CB1) and type 2 (CB2), and they have attracted extensive attention in the central nervous system (CNS) and immune system. Due to more in-depth studies in recent years, it has been found that the typical CB1 and CB2 receptors confer functional importance far beyond the CNS and immune system. In particular, many works have reported the critical involvement of the CB1 and CB2 receptors in myocardial injuries. Both pharmacological and genetic approaches have been used for studying CB1 and CB2 functions in these studies, revealing that the brother receptors have many basic differences and sometimes antagonistic functions in a variety of myocardial injuries, despite some sequence or location identity they share. Herein, we introduce the general differences of CB1 and CB2 cannabinoid receptors, and summarize the functional rivalries between the two brother receptors in the setting of myocardial injuries. We point out the importance of individual receptor-based modulation, instead of dual receptor modulators, when treating myocardial injuries.

Single-cell RNA analysis on ACE2 expression provides insights into SARS-CoV-2 potential entry into the bloodstream and heart injury.

Coronavirus disease-2019 (COVID-19) is a global pandemic with high infectivity and pathogenicity, accounting for tens of thousands of deaths worldwide. Recent studies have found that the pathogen of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), shares the same cell receptor angiotensin converting enzyme II (ACE2) as SARS-CoV. The pathological investigation of COVID-19 deaths showed that the lungs had characteristics of pulmonary fibrosis. However, how SARS-CoV-2 spreads from the lungs to other organs has not yet been determined. Here, we performed an unbiased evaluation of cell-type-specific expression of ACE2 in healthy and fibrotic lungs, as well as in normal and failed adult human hearts, using published single-cell RNA-seq data. We found that ACE2 expression in fibrotic lungs mainly locates in arterial vascular cells, which might provide a route for bloodstream spreading of SARS-CoV-2. Failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, and SARS-CoV-2 might attack cardiomyocytes through the bloodstream in patients with heart failure. Moreover, ACE2 was highly expressed in cells infected by respiratory syncytial virus or Middle East respiratory syndrome coronavirus and in mice treated by lipopolysaccharide. Our findings indicate that patients with pulmonary fibrosis, heart failure, and virus infection have a higher risk and are more susceptible to SARS-CoV-2 infection. The SARS-CoV-2 might attack other organs by getting into the bloodstream. This study provides new insights into SARS-CoV-2 blood entry and heart injury and might propose a therapeutic strategy to prevent patients from developing severe complications.

Pharmacological activation of CB2 receptor protects against ethanol-induced myocardial injury related to RIP1/RIP3/MLKL-mediated necroptosis.

Chronic ethanol abuse can lead to harmful consequences for the heart, resulting in systolic dysfunction, variability in the heart rate, arrhythmia, and cardiac remodelling. However, the precise molecular mechanism responsible for ethanol-induced cardiomyopathy is poorly understood. In this regard, the present study aimed to describe the RIP1/RIP3/MLKL-mediated necroptotic cell death that may be involved in ethanol-induced cardiomyopathy and characterize CBR-mediated effects on the signalling pathway and myocardial injury. We performed an ethanol vapour administration experiment to analyse the effects of ethanol on cardiac structure and function in male C57BL/6J mice. Ethanol induced a significant decline in the cardiac structure and function, as evidenced by a decline in ejection fraction and fractional shortening, and an increase in serum Creatine Kinase levels, myocardial collagen content, and inflammatory reaction. Furthermore, ethanol also upregulated the expression levels of necroptosis-related markers such as p-RIP1, p-RIP3, and p-MLKL in the myocardium. Nec-1 treatment exerted significant cardioprotective effects by salvaging the heart tissue, improving the cardiac function, and mitigating inflammation and necroptosis. In addition, ethanol abuse caused an imbalance in the endocannabinoid system and regulated two cannabinoid receptors (CB1R and CB2R) in the myocardium. Treatment with selective CB2R agonists, JWH-133 or AM1241, markedly improved the cardiac dysfunction and reduced the ethanol-induced necroptosis in the myocardium. Altogether, our data provide evidence that ethanol abuse-induced cardiotoxicity can possibly be attributed to the RIP1/RIP3/MLKL-mediated necroptosis. Moreover, pharmacological activation of CB2R may represent a new cardioprotective strategy against ethanol-induced cardiotoxicity.

Aberrant endoplasmic reticulum stress mediates coronary artery spasm through regulating MLCK/MLC2 pathway.

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.

Surrogate analyte-based quantification of main endocannabinoids in whole blood using liquid chromatography-tandem mass spectrometry.

Endocannabinoids (eCBs) are endogenous ligands of the endocannabinoid system that are known to regulate several physiological and behavioral processes. Previous studies have developed methods for the detection of main eCBs including arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), mostly in serum or plasma. Whole blood is a superior biomaterial for eCBs analysis owing to the nature of the shortened isolation procedure and decreased risk of 2-AG isomerization during preparation. In this study, a surrogate analyte-based liquid chromatography-tandem mass spectrometry assay was developed for the measurement of AEA, 2-AG and its isomer 1-arachidonoylglycerol (1-AG) using a maximum of 100 µL whole blood. Chromatographic separation was achieved using a reverse-phase column and a gradient elution. Detection was performed in selected reaction monitoring mode with an electrospray ionization source. The limits of detection of three eCBs were 0.05-0.1 ng/mL. Good linearity was observed over the concentration range. Intra- and inter-assay accuracy and precision were ≤10.9 and ≤8.7% at four quality control levels. The response factor and parallelism experiment illustrated that the surrogate analytes were suitable for accurate quantification of the main eCBs in whole blood. This surrogate analyte approach was successfully applied to authentic blood samples obtained from alcohol negative drivers and those under the influence of alcohol.

Causes of Sudden Unexpected Death in Schizophrenia Patients: A Forensic Autopsy Population Study.

BACKGROUND: Schizophrenia is a detrimental psychiatric disorder, with an increased mortality from natural and nonnatural causes. METHODS: This study was a retrospective review of autopsy cases of all the individuals with history of schizophrenia investigated by the Office of the Chief Medical Examiner, State of Maryland, for a 5-year period from 2008 to 2012. RESULT: A total of 391 schizophrenia patients were autopsied at the Office of the Chief Medical Examiner because they died suddenly and unexpectedly. Their age ranged from 15 to 100 years with the mean age of 49.5 years. Of the 391 deaths, 191 (48.8%) were white, 185 (47.3%) were African American, and 15 (3.9%) were either Hispanic or Asian. The male and female ratio was 1.5:1. The majority of deaths (64.2%) were caused by natural diseases, 12.0% deaths were accidents, 11.5% deaths were suicides, and 9.7% deaths were homicides. The manner of death remained undetermined in 38 cases (9.7%). Of the 251 natural deaths, 198 cases (78.9%) were owing to cardiovascular diseases. Cause of death was listed as cardiac arrhythmia in 11 cases. This diagnosis of cardiac arrhythmia was made by exclusion based on death scene investigation, review of medical history, complete autopsy, and toxicological tests. Drug intoxication was the second most common cause of death. CONCLUSIONS: The study shows high fatality caused by cardiovascular diseases and drug intoxication among schizophrenia patients, which calls attention of the medical community to closely monitor the high risk factors of sudden death among schizophrenia patients.

Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/ß-catenin signaling.

Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/ß-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/ß-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/ß-catenin signaling. XAV-939, an inhibitor of Wnt/ß-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/ß-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/ß-catenin signaling might be promising in relation to RS-induced myocardial injury.

Evaluating the potential of housekeeping genes, rRNAs, snRNAs, microRNAs and circRNAs as reference genes for the estimation of PMI.

The precise estimation of postmortem interval (PMI) is a critical step in death investigation of forensic cases. Detecting the degradation of RNA in tissues by real time quantitative polymerase chain reaction (RT-qPCR) technology provides a new theoretical basis for estimation of PMI. However, most commonly used reference genes degrade over time, while previous studies seldom consider this when selecting suitable reference genes for the estimation of PMI. Studies have shown microRNAs (miRNAs) are very stable and circular RNAs (circRNAs) have recently emerged as a novel class of RNAs with high stability. We aimed to evaluate the stability of the two kinds of RNAs and normal reference genes using geNorm and NormFinder algorithms to identify tissue-specific reference genes for PMI estimation. The content of candidate RNAs from mouse heart, liver and skeletal muscle tissues were dynamically examined in 8 consecutive days after death. Among the 11 candidate genes (ß-actin, Gapdh, Rps18, 5S, 18S, U6, miR-133a, miR-122, circ-AFF1, LC-Ogdh and LC-LRP6), the following genes showed prioritized stability: miR-122, miR-133a and 18S in heart tissues; LC-Ogdh, circ-AFF1 and miR-122 in liver tissues; and miR-133a, circ-AFF1 and LC-LRP6 in skeletal muscle tissues. Our results suggested that miRNAs and circRNAs were more stable as reference genes than other kinds of RNAs regarding PMI estimation. The appropriate internal control genes were not completely the same across tissue types.

Synergistic effects of c-Jun and SP1 in the promotion of TGFß1-mediated diabetic nephropathy progression.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Transforming growth factor beta 1 (TGFß1) is a well-distinguished mediator of progressive renal fibrosis in DN. However, the molecular mechanisms contributing to enhanced TGFß1 expression in the progression of DN are not fully understood. Herein, we reported that c-Jun and specificity protein 1 (SP1) were critical upstream regulators of TGFß1 expression in DN. The increase in c-Jun and SP1 expressions was positively correlated with TGFß1 in both high glucose-treated human renal mesangial cells (HRMCs) and diabetic kidneys. Furthermore, c-Jun dose-dependently promoted SP1-mediated TGFß1 transcription and vice versa. The synergistic effects of c-Jun and SP1 were attributed to their auto-regulation and cross-activation. Moreover, enhanced phosphorylation levels of c-Jun and SP1 were accompanied with increased TGFß1 expression in diabetic kidneys. Accordingly, dephosphorylation of c-Jun and SP1 by the specific c-Jun N-terminal kinase (JNK) inhibitor SP600125 prevented the increase in TGFß1 expression. These results suggested that c-Jun and SP1 synergistically activated profibrotic TGFß1 expression in the development of DN by auto-regulation, cross-activation and phospho-modification.

Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm.

BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2.