Imatinib­ and ponatinib­mediated cardiotoxicity in zebrafish embryos and H9c2 cardiomyoblasts.

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose­dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose­dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro, H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo, zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription­quantitative PCR (RT­qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose­dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose­dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose­dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI­induced cardiotoxicities.

DNAm scores for serum GDF15 and NT-proBNP levels associate with a range of traits affecting the body and brain.

BACKGROUND: Plasma growth differentiation factor 15 (GDF15) and N-terminal proB-type natriuretic peptide (NT-proBNP) are cardiovascular biomarkers that associate with a range of diseases. Epigenetic scores (EpiScores) for GDF15 and NT-proBNP may provide new routes for risk stratification. RESULTS: In the Generation Scotland cohort (N ≥ 16,963), GDF15 levels were associated with incident dementia, ischaemic stroke and type 2 diabetes, whereas NT-proBNP levels were associated with incident ischaemic heart disease, ischaemic stroke and type 2 diabetes (all PFDR < 0.05). Bayesian epigenome-wide association studies (EWAS) identified 12 and 4 DNA methylation (DNAm) CpG sites associated (Posterior Inclusion Probability [PIP] > 95%) with levels of GDF15 and NT-proBNP, respectively. EpiScores for GDF15 and NT-proBNP were trained in a subset of the population. The GDF15 EpiScore replicated protein associations with incident dementia, type 2 diabetes and ischaemic stroke in the Generation Scotland test set (hazard ratios (HR) range 1.36-1.41, PFDR < 0.05). The EpiScore for NT-proBNP replicated the protein association with type 2 diabetes, but failed to replicate an association with ischaemic stroke. EpiScores explained comparable variance in protein levels across both the Generation Scotland test set and the external LBC1936 test cohort (R2 range of 5.7-12.2%). In LBC1936, both EpiScores were associated with indicators of poorer brain health. Neither EpiScore was associated with incident dementia in the LBC1936 population. CONCLUSIONS: EpiScores for serum levels of GDF15 and Nt-proBNP associate with body and brain health traits. These EpiScores are provided as potential tools for disease risk stratification.

Serum microRNA-125b-5p expression in patients with dilated cardiomyopathy combined with heart failure and its effect on myocardial fibrosis.

OBJECTIVE: This paper was performed to decipher the serum microRNA (miR)-125b-5p expression in patients with dilated cardiomyopathy (DCM) combined with heart failure (HF) and its effect on myocardial fibrosis. METHODS: Serum miR-125b-5p expression, LVEDD, LVESD, LVEF, LVFS, and NT-proBNP levels were evaluated in clinical samples. A rat DCM model was established by continuous intraperitoneal injection of adriamycin and treated with miR-125b-5p agomir and its negative control. Cardiac function, serum TNF-α, hs-CRP, and NT-proBNP levels, pathological changes in myocardial tissues, cardiomyocyte apoptosis, and the expression levels of miR-125b-5p and fibrosis-related factors were detected in rats. RESULTS: In comparison to the control group, the case group had higher levels of LVEDD, LVESD, and NT-pro-BNP, and lower levels of LVEF, LVFS, and miR-125b-5p expression levels. Overexpression of miR-125b-5p effectively led to the improvement of cardiomyocyte hypertrophy and collagen arrangement disorder in DCM rats, the reduction of blue-stained collagen fibers in the interstitial myocardium, the reduction of the levels of TNF-α, hs-CRP, and NT-proBNP and the expression levels of TGF-1ß, Collagen I, and α-SMA, and the reduction of the number of apoptosis in cardiomyocytes. CONCLUSION: Overexpression of miR-125b-5p is effective in ameliorating myocardial fibrosis.

Diagnostic and prognostic significance of miR-320a-3p in patients with chronic heart failure.

AIM: The purpose of this study was to investigate the diagnostic and prognostic value of miR-320a-3p in chronic heart failure (CHF). METHODS: A total of 103 patients with CHF and 95 healthy controls were included in the study population. The expression level of serum miR-320a-3p was detected by qRT-PCR. The diagnostic effect of miR-320a-3p on CHF was evaluated by receiver operating characteristic curve. Kaplan-Meier curve and Cox regression were used to analyze the risk factors for 4-year prognosis of CHF patients. Bioinformatics analysis was used to analyze the possible target genes of miR-320a-3p and related signaling pathways. RESULTS: Serum miR-320a-3p expression was increased in CHF patients, and the levels of BNP and LVEF were positively and negatively correlated with miR-320a-3p, respectively. The AUC value of ROC curve was 0.866, indicating that miR-320a-3p had high diagnostic accuracy for CHF. Survival curve and Cox analysis showed that high expression of miR-320a-3p was associated with poor prognosis in CHF patients, and age and miR-320a-3p were independent risk factors for prognosis in CHF patients. GO and KEGG analysis showed that the downstream target genes of miR-320a-3p were involved in biological processes such as cell adhesion, stem cell differentiation and neural development, and were enriched in mTOR, TNF, AMPK and other signaling pathways. CONCLUSIONS: miR-320a-3p increased abnormally in CHF and was related to the severity of CHF. miR-320a-3p has the potential to be a diagnostic and prognostic marker for CHF.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and ßMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Cardiac biomarker responses following high-intensity interval and continuous exercise: the influence of ACE-I/D gene polymorphism and training status in men.

This study aimed to investigate the relationship between pre- and postexercise cardiac biomarker release according to athletic status (trained vs. untrained) and to establish whether the I/D polymorphism in the angiotensin-converting enzyme (ACE) gene had an influence on cardiac biomarkers release with specific regard on the influence of the training state. We determined cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in 29 trained and 27 untrained male soccer players before and after moderate-intensity continuous exercise (MICE) and high-intensity interval exercise (HIIE) running tests. Trained soccer players had higher pre (trained: 0.014 ± 0.007 ng/mL; untrained: 0.010 ± 0.005 ng/mL) and post HIIE (trained: 0.031 ± 0.008 ng/mL; untrained: 0.0179 ± 0.007) and MICE (trained: 0.030 ± 0.007 ng/mL; untrained: 0.018 ± 0.007) cTnI values than untrained subjects, but the change with exercise (ΔcTnI) was similar between groups. There was no significant difference in baseline and postexercise NT-proBNP between groups. NT-proBNP levels were elevated after both HIIE and MICE. Considering three ACE genotypes, the mean pre exercise cTnI values of the trained group (DD: 0.015 ± 0.008 ng/mL, ID: 0.015 ± 0.007 ng/mL, and II: 0.014 ± 0.008 ng/mL) and their untrained counterparts (DD: 0.010 ± 0.004 ng/mL, ID: 0.011 ± 0.004 ng/mL, and II: 0.010 ± 0.006 ng/mL) did not show any significant difference. To sum up, noticeable difference in baseline cTnI was observed, which was related to athletic status but not ACE genotypes. Neither athletic status nor ACE genotypes seemed to affect the changes in cardiac biomarkers in response to HIIE and MICE, indicating that the ACE gene does not play a significant role in the release of exercise-induced cardiac biomarkers indicative of cardiac damage in Iranian soccer players.NEW & NOTEWORTHY Our study investigated the impact of athletic status and angiotensin-converting enzyme (ACE) gene I/D polymorphism on cardiac biomarkers in soccer players. Trained players showed higher baseline cardiac troponin I (cTnI) levels, whereas postexercise ΔcTnI remained consistent across groups. N-terminal pro-brain natriuretic peptide increased after exercise in both groups, staying within normal limits. ACE genotypes did not significantly affect pre-exercise cTnI. Overall, athletic status influences baseline cTnI, but neither it nor ACE genotypes significantly impact exercise-induced cardiac biomarker responses in this population.

miR-1268a Regulates Fatty Acid Metabolism by Targeting CD36 in Angiotensin II-induced Heart Failure.

Multiple RNAs have been involved in the progress of heart failure. However, the role of miR-1268a in heart failure is still unclear. The differentially expressed miRNAs in heart failure was analyzed based on GEO dataset GSE104150. AC16 cells were treated with Angiotensin II (Ang II) to explore the role of miR-1268a in heart failure. The web tool miRWalk was used to analyze the targets of miR-1268a. miR-1268a was up-regulated in Ang II-treated AC16 cells. Ang II treatment markedly inhibited cell proliferation, ATP production, fatty acid (FA) uptake and enhanced levels of HF markers BNP and ST2, and oxidative stress of AC16 cells. Notably, inhibition of miR-1268a eliminated the inhibiting effect of Ang II on cell proliferation, ATP production, FA uptake and decreased levels of BNP an ST2, and oxidative stress on AC16 cells. Furthermore, CD36 was a target of miR-1268a and the CD36 level was decreased by miR-1268a mimics but increased by miR-1268a inhibitor in AC16 cells. miR-1268a regulates FA metabolism and oxidative stress in myocardial cells by targeting CD36 in heart failure.

Relationship between lipid profile and B­type natriuretic peptide T­381C (rs198389) gene polymorphism in patients with stable coronary artery disease.

The research explored the link between Brain Natriuretic Peptides (BNP) gene promoter T-381C polymorphism, serum BNP, and lipid profiles in Kurdish people from Iraq with stable coronary artery disease (CAD). The study was conducted on 62 individuals with CAD and 31 without CAD (control group). DNA was extracted from each individual's sample using the Sanger sequencing method to study the BNP gene's polymorphism. The identified alleles were TT, TC, and CC. The frequency of the TT genotype decreased significantly among the patient group compared to the control group, while the CC genotype's frequency was higher (p<0.05). However, there was no significant increase in BNP levels in TC and CC genotypes compared to the TT genotype. Lipid profile values were not significantly different among the genotypes. The study utilized a cut-off value for BNP activity for predicting CAD and found that individuals with a BNP activity value less than the cut-off had significantly greater changes in lipid profile and renal function (p<0.05). Stepwise multivariate regression analysis showed that cholesterol was not the only primary determinant of BNP rate in subjects with stable CAD; oxidized low-density lipoprotein (Ox-LDL), a history of heart attacks, and oxidative stress malondialdehyde (MDA) had a significant effect. Homozygous C allele carriers at position 381 of the BNP precursors gene promoter were more likely to exhibit atherosclerosis lesions. We found that BNP rs198389 was not correlated with lipid profile and kidney disease.

Corin and Left Atrial Cardiomyopathy, Hypertension, Arrhythmia, and Fibrosis.

Two siblings presented with cardiomyopathy, hypertension, arrhythmia, and fibrosis of the left atrium. Each had a homozygous null variant in CORIN, the gene encoding atrial natriuretic peptide (ANP)-converting enzyme. A plasma sample obtained from one of the siblings had no detectable levels of corin or N-terminal pro-ANP but had elevated levels of B-type natriuretic peptide (BNP) and one of the two protein markers of fibrosis that we tested. These and other findings support the hypothesis that BNP cannot fully compensate for a lack of activation of the ANP pathway and that corin is critical to normal ANP activity, left atrial function, and cardiovascular homeostasis.

The underlying mechanism of transcription factor IRF1, PRDM1, and ZNF263 involved in the regulation of NPPB rs3753581 on pulse pressure hypertension.

To investigate the correlation between NPPB gene variants and pulse pressure hypertension and the underlying regulatory mechanisms and try to confirm that NPPB may be a potential molecular target of gene therapy for pulse pressure hypertension. A total of 898 participants were recruited from the First Affiliated Hospital of Fujian Medical University and the plasmids with differential expression of NPPB were constructed. Genotype distribution of NPPB(rs3753581, rs198388, and rs198389)was analyzed and the expression of N-terminal pro-B-type natriuretic peptide(NT-proBNP) and renin-angiotensin -aldosterone system(RAAS) related indicators were identified in the groups studied. According to a genotype analysis, there was a significant difference in the genotype distribution of NPPB rs3753581 among the groups (P = 0.034). In logistic regression analysis, NPPB rs3753581 TT was associated with a 1.8-fold greater risk of pulse pressure hypertension than NPPB rs3753581 GG (odds ratio = 1.801; 95% confidence interval: 1.070-3.032; P = 0.027). The expression of NT-proBNP and RAAS related indicators in clinical and laboratory samples showed striking differences. The activity of firefly and Renilla luciferase in pGL-3-NPPB-luc (-1299G) was higher than pGL-3-NPPBmut-luc(-1299 T)(P < 0.05). The binding of NPPB gene promoter rs3753581 (-1299G) with transcription factors IRF1, PRDM1, and ZNF263 was predicted and validated by the bioinformatics software TESS and chromatin immunoprecipitation(P < 0.05). NPPB rs3753581 was correlated with genetic susceptibility to pulse pressure hypertension and the transcription factors IRF1, PRDM1, and ZNF263 may be involved in the regulation of NPPB rs3753581 promoter (-1299G) on the expression of NT-proBNP/RAAS.

BNP protects against diabetic cardiomyopathy by promoting Opa1-mediated mitochondrial fusion via activating the PKG-STAT3 pathway.

Brain natriuretic peptide (BNP) belongs to the family of natriuretic peptides, which are responsible for a wide range of actions. Diabetic cardiomyopathy (DCM) is often associated with increased BNP levels. This present research intends to explore the role of BNP in the development of DCM and the underlying mechanisms. Diabetes was induced in mice using streptozotocin (STZ). Primary neonatal cardiomyocytes were treated with high glucose. It was found that the levels of plasma BNP started to increase at 8 weeks after diabetes, which preceded the development of DCM. Addition of exogenous BNP promoted Opa1-mediated mitochondrial fusion, inhibited mitochondrial oxidative stress, preserved mitochondrial respiratory capacity and prevented the development of DCM, while knockdown of endogenous BNP exacerbated mitochondrial dysfunction and accelerated DCM. Opa1 knockdown attenuated the aforementioned protective action of BNP both in vivo and in vitro. BNP-induced mitochondrial fusion requires the activation of STAT3, which facilitated Opa1 transcription by binding to its promoter regions. PKG, a crucial signaling biomolecule in the BNP signaling pathway, interacted with STAT3 and induced its activation. Knockdown of NPRA (the receptor of BNP) or PKG blunted the promoting effect of BNP on STAT3 phosphorylation and Opa1-mediated mitochondrial fusion. The results of this study demonstrate for the first time that there is a rise in BNP during the early stages of DCM as a compensatory protection mechanism. BNP is a novel mitochondrial fusion activator in protecting against hyperglycemia-induced mitochondrial oxidative injury and DCM through the activation of NPRA-PKG-STAT3-Opa1 signaling pathway.

Inhibition of Fap Promotes Cardiac Repair by Stabilizing BNP.

BACKGROUND: Myocardial infarction (MI) elicits cardiac fibroblast activation and extracellular matrix (ECM) deposition to maintain the structural integrity of the heart. Recent studies demonstrate that Fap (fibroblast activation protein)-a prolyl-specific serine protease-is an important marker of activated cardiac fibroblasts after MI. METHODS: Left ventricle and plasma samples from patients and healthy donors were used to analyze the expression level of FAP and its prognostic value. Echocardiography and histological analysis of heart sections were used to analyze cardiac functions, scar formation, ECM deposition and angiogenesis after MI. RNA-Sequencing, biochemical analysis, cardiac fibroblasts (CFs) and endothelial cells co-culture were used to reveal the molecular and cellular mechanisms by which Fap regulates angiogenesis. RESULTS: We found that Fap is upregulated in patient cardiac fibroblasts after cardiac injuries, while plasma Fap is downregulated and functions as a prognostic marker for cardiac repair. Genetic or pharmacological inhibition of Fap in mice significantly improved cardiac function after MI. Histological and transcriptomic analyses showed that Fap inhibition leads to increased angiogenesis in the peri-infarct zone, which promotes ECM deposition and alignment by cardiac fibroblasts and prevents their overactivation, thereby limiting scar expansion. Mechanistically, we found that BNP (brain natriuretic peptide) is a novel substrate of Fap that mediates postischemic angiogenesis. Fap degrades BNP to inhibit vascular endothelial cell migration and tube formation. Pharmacological inhibition of Fap in Nppb (encoding pre-proBNP) or Npr1 (encoding the BNP receptor)-deficient mice showed no cardioprotective effects, suggesting that BNP is a physiological substrate of Fap. CONCLUSIONS: This study identifies Fap as a negative regulator of cardiac repair and a potential drug target to treat MI. Inhibition of Fap stabilizes BNP to promote angiogenesis and cardiac repair.

Transcriptional cofactor dyxin mediates hypertrophic response in the heart during angiotensin II-induced hypertension.

Dyxin is a LIM-domain containing transcriptional regulator protein shown to play a role in a hypertrophic response in the heart. Here, the effect of adenoviral dyxin overexpression was studied on cardiac function and gene expression in the normal heart and in angiotensin II (Ang II)-induced hypertension in rats. The adenovirus-mediated intramyocardial gene transfer of dyxin (1.5x109 infectious units/animal) was performed into the left ventricle (LV) of Sprague-Dawley rats with and without the Ang II (33 µg/kg/h) infusion, administered via osmotic minipumps for 1 and 2 weeks. Echocardiography was used to assess the structural and functional changes. Dyxin expression and localization in the heart was analyzed with quantitative RT-PCR and immunohistochemistry, respectively. In the normal rat heart, the adenoviral overexpression of dyxin did not alter LV function in normal hearts as assessed by echocardiography. Dyxin was found to be localized in the cardiomyocytes as shown by the immunohistochemical staining. In Ang II-induced hypertrophy, echocardiographic data revealed a significant increase in the posterior wall diameter both in systole (21%, P<0.05) and diastole (21%, P<0.01) as well as in the diameter of the interventricular septum in systole (19%, P<0.05) in the dyxin-injected group compared with the LacZ-injected animals after two weeks of Ang II infusion. Interestingly, a significant decrease in the levels of both atrial natriuretic peptide (ANP) mRNA (55%, P<0.01) and B-type natriuretic peptide (BNP) mRNA (68%, P<0.05) was observed in the dyxin-injected group compared with the LacZ control group after one week of Ang II infusion. These results indicate that dyxin overexpression was deteriorative against pressure overload by inducing structural changes in the LV in rats. Interestingly, simultaneous adenoviral overexpression of dyxin suppressed the Ang II-induced changes of ANP and BNP genes suggesting that dyxin might have a role as a regulator of the cardiac hypertrophic gene program.

Substantial impact of 3-iodothyronamine (T1AM) on the regulations of fluorescent thermoprobe-measured cellular temperature and natriuretic peptide expression in cardiomyocytes.

There is growing interest in 3-iodothyronamine (T1AM), an active thyroid hormone metabolite, that induces negative inotropic and chronotropic actions in the heart and exerts systemic hypothermic action. We explored the direct impact of T1AM on cardiomyocytes with a focus on the regulation of the intracellular temperature and natriuretic peptide (NP) expression. A thermoprobe was successfully introduced into neonatal rat cardiomyocytes, and the temperature-dependent changes in the fluorescence intensity ratio were measured using a fluorescence microscope. After one-hour incubation with T1AM, the degree of change in the fluorescence intensity ratio was significantly lower in T1AM-treated cardiomyocytes than in equivalent solvent-treated controls (P < 0.01), indicating the direct hypothermic action of T1AM on cardiomyocytes. Furthermore, T1AM treatment upregulated B-type NP (BNP) gene expression comparable to treatment with endothelin-1 or phenylephrine. Of note, ERK phosphorylation was markedly increased after T1AM treatment, and inhibition of ERK phosphorylation by an MEK inhibitor completely cancelled both T1AM-induced decrease in thermoprobe-measured temperature and the increase in BNP expression. In summary, T1AM decreases fluorescent thermoprobe-measured temperatures (estimated intracellular temperatures) and increases BNP expression in cardiomyocytes by activating the MEK/ERK pathway. The present findings provide new insight into the direct myocardial cellular actions of T1AM in patients with severe heart failure.

ZAKß Alleviates Oxidized Low-density Lipoprotein (ox-LDL)-Induced Apoptosis and B-type Natriuretic Peptide (BNP) Upregulation in Cardiomyoblast.

Oxidized low-density lipoprotein (ox-LDL) is a type of modified cholesterol that promotes apoptosis and inflammation and advances the progression of heart failure. Leucine-zipper and sterile-α motif kinase (ZAK) is a kinase of the MAP3K family which is highly expressed in the heart and encodes two variants, ZAKα and ZAKß. Our previous study serendipitously found opposite effects of ZAKα and ZAKß in which ZAKß antagonizes ZAKα-induced apoptosis and hypertrophy of the heart. This study aims to test the hypothesis of whether ZAKα and ZAKß are involved in the damaging effects of ox-LDL in the cardiomyoblast. Cardiomyoblast cells H9c2 were treated with different concentrations of ox-LDL. Cell viability and apoptosis were measured by MTT and TUNEL assay, respectively. Western blot was used to detect apoptosis, hypertrophy, and pro-survival signaling proteins. Plasmid transfection, pharmacological inhibition with D2825, and siRNA transfection were utilized to upregulate or downregulate ZAKß, respectively. Ox-LDL concentration-dependently reduces the viability and expression of several pro-survival proteins, such as phospho-PI3K, phospho-Akt, and Bcl-xL. Furthermore, ox-LDL increases cleaved caspase-3, cleaved caspase-9 as indicators of apoptosis and increases B-type natriuretic peptide (BNP) as an indicator of hypertrophy. Overexpression of ZAKß by plasmid transfection attenuates apoptosis and prevents upregulation of BNP. Importantly, these effects were abolished by inhibiting ZAKß either by D2825 or siZAKß application. Our results suggest that ZAKß upregulation in response to ox-LDL treatment confers protective effects on cardiomyoblast.

Circ-TLR4 promotes cardiac hypertrophy through recruiting FUS to stabilize TLR4 mRNA.

BACKGROUND: Cardiac hypertrophy is an adaptive and compensatory mechanism preserving cardiac output during detrimental stimuli. Circular RNAs (circRNAs) have been illustrated to exert important implications in the pathogenesis of multiple cardiovascular diseases (CVD) including demonstrated cardiac hypertrophy. Toll-like receptor 4 (TLR4) has been previously reported to be a crucial regulator in inflammatory response and cardiac hypertrophy. However, the role of circular isoforms derived from TLR4 in cardiac hypertrophy remains unclear. METHODS: Expression of circ-TLR4 and TLR4 in cardiomyocytes was detected by RT-qPCR. The indicators of cardiac hypertrophy responses, including cell surface area, atrial natriuretic factor (ANF), B-type natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC) were measured by immunofluorescence staining and western blot. RIP assay was used to validate the interaction between circ-TLR4 and TLR4. RESULTS: Circ-TLR4 and TLR4 was up-regulated in cellular models of cardiac hypertrophy. Circ-TLR4 knockdown attenuated angiotensin II (Ang II)-induced hypertrophy responses in cardiomyocytes. Moreover, circ-TLR4 positively regulated TLR4 expression through recruiting FUS to stabilize TLR4 mRNA. Furthermore, TLR4 overexpression rescued the cardiac responses mediated by circ-TLR4 silencing. CONCLUSION: Circ-TLR4 promotes cardiac hypertrophy through recruiting FUS to stabilize TLR4 mRNA.

ASA Status, NPPA/NPPB Haplotype and Coronary Artery Disease Have an Impact on BNP/NT-proBNP Plasma Levels.

Plasma concentrations of natriuretic peptides (NP) contribute to risk stratification and management of patients undergoing non-cardiac surgery. However, genetically determined variability in the levels of these biomarkers has been described previously. In the perioperative setting, genetic contribution to NP plasma level variability has not yet been determined. A cohort of 427 patients presenting for non-cardiac surgery was genotyped for single-nucleotide polymorphisms (SNPs) from the NPPA/NPPB locus. Haplotype population frequencies were estimated and adjusted haplotype trait associations for brain natriuretic peptide (BNP) and amino-terminal pro natriuretic peptide (NT-proBNP) were calculated. Five SNPs were included in the analysis. Compared to the reference haplotype TATAT (rs198358, rs5068, rs632793, rs198389, rs6676300), haplotype CACGC, with an estimated frequency of 4%, showed elevated BNP and NT-proBNP plasma concentrations by 44% and 94%, respectively. Haplotype CGCGC, with an estimated frequency of 9%, lowered NT-proBNP concentrations by 28%. ASA classification status III and IV, as well as coronary artery disease, were the strongest predictors of increased NP plasma levels. Inclusion of genetic information might improve perioperative risk stratification of patients based on adjusted thresholds of NP plasma levels.

Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

FGF21 (Fibroblast Growth Factor 21) Defines a Potential Cardiohepatic Signaling Circuit in End-Stage Heart Failure.

BACKGROUND: Extrinsic control of cardiomyocyte metabolism is poorly understood in heart failure (HF). FGF21 (Fibroblast growth factor 21), a hormonal regulator of metabolism produced mainly in the liver and adipose tissue, is a prime candidate for such signaling. METHODS: To investigate this further, we examined blood and tissue obtained from human subjects with end-stage HF with reduced ejection fraction at the time of left ventricular assist device implantation and correlated serum FGF21 levels with cardiac gene expression, immunohistochemistry, and clinical parameters. RESULTS: Circulating FGF21 levels were substantially elevated in HF with reduced ejection fraction, compared with healthy subjects (HF with reduced ejection fraction: 834.4 [95% CI, 628.4-1040.3] pg/mL, n=40; controls: 146.0 [86.3-205.7] pg/mL, n=20, P=1.9×10-5). There was clear FGF21 staining in diseased cardiomyocytes, and circulating FGF21 levels negatively correlated with the expression of cardiac genes involved in ketone metabolism, consistent with cardiac FGF21 signaling. FGF21 gene expression was very low in failing and nonfailing hearts, suggesting extracardiac production of the circulating hormone. Circulating FGF21 levels were correlated with BNP (B-type natriuretic peptide) and total bilirubin, markers of chronic cardiac and hepatic congestion. CONCLUSIONS: Circulating FGF21 levels are elevated in HF with reduced ejection fraction and appear to bind to the heart. The liver is likely the main extracardiac source. This supports a model of hepatic FGF21 communication to diseased cardiomyocytes, defining a potential cardiohepatic signaling circuit in human HF.

Impact of natriuretic peptide polymorphisms on diastolic and metabolic function in a populational cohort: insights from the STANISLAS cohort.

AIMS: Elevated brain natriuretic peptide (BNP) and the N-terminal fragment of its pro-hormone (NT-proBNP) have become established biomarkers for heart failure and are associated with cardiovascular morbidity and mortality. Investigating sources of inter-individual heterogeneity, particularly genetic factors, could help better identify patients at risk of future cardiovascular disease. The aim of this study was to estimate the heritability of circulating NT-proBNP levels, to perform a genome-wide association study (GWAS) and gene-candidate analysis focused on NPPB-NPPA genes on these levels, and to examine their association with cardiovascular or metabolic outcomes. METHODS AND RESULTS: A total of 1555 individuals from the STANISLAS study were included. The heritability of circulating NT-proBNP levels was estimated at 15%, with seven single nucleotide polymorphisms (SNPs) reaching the significant threshold in the GWAS. All above SNPs were located on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. NPPA gene expression was also associated with NT-proBNP levels. Moreover, six other SNPs from NPPA-NPPB genes were associated with diastolic function (lateral e' on echocardiography) and metabolic features (glycated haemoglobin). CONCLUSIONS: The heritability of natriuretic peptides appears relatively low (15%) and mainly based on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. Natriuretic peptide polymorphisms are associated with natriuretic peptide levels and diastolic function. These results suggest that natriuretic peptide polymorphisms may have an impact in the early stages of cardiovascular and metabolic disease.