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BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

Aims: Circulating biomarkers can provide important information for the diagnosis and prognosis of dilated cardiomyopathy (DCM). We explored novel biomarkers for the diagnosis and prognosis of DCM to improve clinical decision‐making. Methods and results: A total of 238 DCM patients and 65 control were consecutively enrolled at Zhongshan Hospital between January 2017 and January 2019. In the screening set, four DCM patients and four controls underwent measurements of serum proteomic analysis. Seventy‐six differentially expressed circulating proteins were screened by data‐independent acquisition proteomics, and three of these proteins (S100A4, S100A8/A9, and S100A12) were validated by multiple‐reaction monitoring‐mass spectrometry. In the validation set, subsequently, a total of 234 DCM patients and 61 control subjects were evaluated by enzyme‐linked immunosorbent assay. Circulating S100A4, S100A8/A9, and S100A12 were significantly increased in DCM patients (P < 0.001). These three proteins were significant positively correlated with other parameters, such as Lg (NT‐proBNP), IL‐1β, TGF‐β, CRP, left ventricular end‐diastolic diameter, and left ventricular end‐systolic diameter, whereas they were negatively correlated with left ventricular ejection fraction, respectively (P < 0.05). The receiver operator characteristic curve showed the combination of S100A4, S100A8/A9, and S100A12 [area under curve (AUC) 0.88, 95% confidence interval (CI) 0.84–0.93] was better than single S100A4 (AUC 0.74, 95% CI 0.68–0.81), S100A8/A9 (AUC 0.82, 95% CI 0.77–0.88), or S100A12 (AUC 0.80, 95% CI 0.72–0.88) in the diagnosis of DCM (P < 0.01). After a median follow‐up period of 33.5 months, 110 patients (47.01%) experienced major adverse cardiac events (MACEs), including 46 who had cardiac deaths and 64 who had heart failure rehospitalizations. Kaplan–Meier analysis indicated that the DCM patients with ≥75th percentile level of S100A4 had a significantly higher incidence of MACEs than those with <75th percentile level of S100A4 (61.40% vs. 42.37%, P < 0.05). There were no significant differences of MACE rate among DCM patients with different concentrations of S100A8/A9 and S100A12 (P > 0.05). Cox proportional hazards regression analysis revealed that S100A4 [≥75th percentile vs. <75th percentile: hazard ratio (HR) 1.65; 95% CI 1.11–2.45] remained significant independent predictors for MACEs (P < 0.05); however, S100A8/A9 and S100A12 were not independent factors for predicting MACE (P ≥ 0.05). Conclusions: S100A4, S100A8/A9, and S100A12 may be additional diagnostic tools for human DCM recognition, and the combination of these three indicators helped to improve the accuracy of a single index to diagnose DCM. Additionally, S100A4 was identified as a significant predictor of prognosis in patients with DCM. [ABSTRACT FROM AUTHOR]

To explore the role of autophagic flux in the increased susceptibility of the experimental diabetic heart to ischaemia‐reperfusion (I/R) injury, we established STZ‐induced diabetic mice and performed I/R. In vitro, neonatal mouse cardiomyocytes were subjected to high glucose and hypoxia/reoxygenation challenge to mimic diabetic I/R injury. We found that experimental diabetes aggravated I/R‐induced injury than compared with nondiabetic mice. Autophagic flux was impaired in I/R hearts, and the impairment was exacerbated in diabetic mice subjected to I/R with defective autophagosome formation and clearance. Calpains, calcium‐dependent thiol proteases, were upregulated and highly activated after I/R of diabetes, while calpain inhibition attenuated cardiac function and cell death and partially restored autophagic flux. The expression levels of Atg5 and LAMP2, two crucial autophagy‐related proteins, were significantly degraded in diabetic I/R hearts, alterations that were associated with calpain activation and could be reversed by calpain inhibition. Co‐overexpression of Atg5 and LAMP2 reduced myocardial injury and normalized autophagic flux. In conclusion, experimental diabetes exacerbates autophagic flux impairment of cardiomyocytes under I/R stress, resulting in worse I/R‐induced injury. Calpain activation and cleavage of Atg5 and LAMP2 at least partially account for the deterioration of autophagic flux impairment. [ABSTRACT FROM AUTHOR]