Knockdown of plakophilin 2 downregulates MIR-184 through CpG hypermethylation and suppression of the E2F1 pathway and leads to enhanced adipogenesis in vitro

Rationale: PKP2, encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. Objective: To characterize miRNA expression profile in PKP2-deficient cells. Methods and results: Control and PKP2-knockdown HL-1 (HL-1(Pkp2-shRNA)) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. Conclusions: PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells

Plasma homocyst(e)ine concentration, but not MTHFR genotype, is associated with variation in carotid plaque area

Background and Purpose - Elevated plasma homocyst(e)ine [H(e)] concentration is associated with premature atherosclerosis. A common cause of elevated plasma H(e) concentration is a thermolabile mutation (677T) in the gene encoding methylenetetrahydrofolate reductase (MTHFR). We sought to determine whether plasma H(e) concentration or MTHFR genotype would be more strongly associated with carotid plaque area (CPA), a potential intermediate phenotype of atherosclerosis. Methods - In 307 subjects who were ascertained through a premature atherosclerosis clinic, we measured CPA with 2- dimensional ultrasound and also determined traditional atherosclerosis risk factors, in addition to plasma H(e) concentration and MTHFR genotypes. Results - We found that the frequency of the MTHFR 677T allele was 0.363 in this sample. Mean plasma H(e) concentration was significantly higher in 677T/T homozygotes than in 677T/C heterozygotes and 677C/C homozygotes (17.1±13.7 versus 13.5±6.1 versus 12.6±5.9 μmol/L, respectively, P\u3c0.001). Analysis of variance showed that CPA was significantly associated with age, sex, smoking, diabetes, hypertension, and hyperlipidemia (each P\u3c0.05). When plasma H(e) concentration was included in the model, it was significantly associated with CPA (P\u3c0.05). However, when the MTHFR genotype was included in the model, it was not associated with CPA (P=0.50). Furthermore, there was a significant correlation of CPA with plasma H(e) (r=0.23, P\u3c0.0001). However, the mean CPA did not differ between subjects according to genotype. Conclusions - Thus, plasma H(e), but not MTHFR genotype, is significantly associated with carotid atherosclerosis, suggesting that the biochemical test may be sufficient to identify patients who may be at increased risk of atherosclerosis through this mechanism

Does the presence of an environmental committee strengthen the impact of board gender diversity on corporate environmental disclosure? Evidence from Sub-Saharan Africa

open access articleThis study examines the relationship between board gender diversity, the presence of environmental committees and corporate environmental disclosure (CED). Using 1130 firm-year observations of 113 firms listed across five Sub-Saharan Africa (SSA) stock markets from 2010 to 2019, we find that the extent of CED in SSA is low compared with developed countries. However, panel quantile regression analysis reveals that the presence of women directors is positively associated with CED, and the relationship is contingent on the presence of an environmental committee. The study makes three principal contributions: it adds to the limited literature on the relationship between board gender diversity and CED, where virtually all previous studies have been conducted in developed countries; it is the first to examine the direct relationship between environmental committees and CED in the developing world; and, most importantly, it is the first study to examine the possible moderating influence of environmental committees

BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice

Mutation in the LMNA gene, encoding lamin A/C, causes a diverse group of diseases called laminopathies. Cardiac involvement is the major cause of death and manifests as dilated cardiomyopathy, heart failure, arrhythmias, and sudden death. There is no specific therapy for LMNA-associated cardiomyopathy. We report that deletion of Lmna in cardiomyocytes in mice leads to severe cardiac dysfunction, conduction defect, ventricular arrhythmias, fibrosis, apoptosis, and premature death within 4 weeks. The phenotype is similar to LMNA-associated cardiomyopathy in humans. RNA sequencing, performed before the onset of cardiac dysfunction, led to identification of 2338 differentially expressed genes (DEGs) in Lmna-deleted cardiomyocytes. DEGs predicted activation of bromodomain-containing protein 4 (BRD4), a regulator of chromatin-associated proteins and transcription factors, which was confirmed by complementary approaches, including chromatin immunoprecipitation sequencing. Daily injection of JQ1, a specific BET bromodomain inhibitor, partially reversed the DEGs, including those encoding secretome; improved cardiac function; abrogated cardiac arrhythmias, fibrosis, and apoptosis; and prolonged the median survival time 2-fold in the myocyte-specific Lmna-deleted mice. The findings highlight the important role of LMNA in cardiomyocytes and identify BET bromodomain inhibition as a potential therapeutic target in LMNA-associated cardiomyopathy, for which there is no specific effective therapy

Histone Acetylation-Mediated Regulation of the Hippo Pathway

The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer. © 2013 Basu et al

RNA sequencing-based transcriptome profiling of cardiac tissue Implicados novela putative disease mechanisms in FLNC-associated arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) encompasses a group of inherited cardiomyopathies including arrhythmogenic right ventricular cardiomyopathy (ARVC) whose molecular disease mechanism is associated with dysregulation of the canonical WNT signalling pathway. Recent evidence indicates that ARVC and ACM caused by pathogenic variants in the FLNC gene encoding filamin C, a major cardiac structural protein, may have different molecular mechanisms of pathogenesis. We sought to identify dysregulated biological pathways in FLNC-associated ACM. RNA was extracted from seven paraffin-embedded left ventricular tissue samples from deceased ACM patients carrying FLNC variants and sequenced. Transcript levels of 623 genes were upregulated and 486 genes were reduced in ACM in comparison to control samples. The cell adhesion pathway and ILK signalling were among the prominent dysregulated pathways in ACM. Consistent with these findings, transcript levels of cell adhesion genes JAM2, NEO1, VCAM1 and PTPRC were upregulated in ACM samples. Moreover, several actin-associated genes, including FLNC, VCL, PARVB and MYL7, were suppressed, suggesting dysregulation of the actin cytoskeleton. Analysis of the transcriptome for biological pathways predicted activation of inflammation and apoptosis and suppression of oxidative phosphorylation and MTORC1 signalling in ACM. Our data suggests dysregulated cell adhesion and ILK signalling as novel putative pathogenic mechanisms of ACM caused by FLNC variants which are distinct from the postulated disease mechanism of classic ARVC caused by desmosomal gene mutations. This knowledge could help in the design of future gene therapy strategies which would target specific components of these pathways and potentially lead to novel treatments for ACM