In this study using postmortem myocardial specimens from septic patients and non-septic controls, we found that eight miRNAs were up-regulated and two down-regulated in septic myocardial specimens compared to non-septic control specimens. The miRNAs regulating genes were related to cell adhesion, cardiovascular growth and development, cell viability, and inflammation and immune response. These findings may be associated with the development of septic cardiomyopathy.
To the best of our knowledge, the present study is the first to investigate miRNA expression using FFPE cardiac tissue from patients with sepsis. A previous study investigated myocardial specimens obtained from patients who died due to sepsis but focused on messenger RNA (mRNA) expression in tissue samples that were snap frozen or placed on dry ice [19]. In that study, the mRNA expression pattern in the hearts of septic patients showed significant reductions in the levels of mRNAs associated with proteins responsible for cardiac energy production and contractility. In our study, the predicted miRNA target genes were associated with cardiovascular functions, cell adhesion, cell viability, and inflammation and immune response.
Compared to controls, septic heart tissue samples had the highest up-regulated fold change in miR-146b-5p, which has been shown to be up-regulated in fibroblasts, endothelial cells, and macrophages under hypoxic conditions during cardiac injury and repair in the infarcted myocardium of mice and the serum of myocardial ischemia patients. [20]. Its predicted target genes regulate cell adhesion, apoptosis, and inflammation and immune response. MiR-146-5p up-regulation has been found to be cardioprotective and local inhibition of the miRNA significantly restores cardiac remodeling and function in both mouse and porcine infarct models [20]. In contrast to a mouse sepsis model of cecal ligation and puncture, a previous study reported that miR-146b protected against sepsis-induced myocardial injury by suppressing expression of IL-1β and myocardium apoptosis [21]. The largest increase in the mean counts in sepsis occurred with miR-12136, the specific function of which in sepsis or septic cardiomyopathy is not known, but the miRNA is involved in the process of mRNA translation [22].
We also found that miR-155-5p was up-regulated in septic patients’ myocardial tissue samples compared to controls. The biological function regulated by the predicted target genes of miR-155-5p include cardiovascular growth and development, as well as inflammation and immune response. Previous data indicate that miR-155-5p exerts a significant influence on multiple pathways associated with sepsis and renal damage, and a direct relationship exists between the activation of miR-155-5p and increased expression of pro-inflammatory IL-6 cytokine and chemoattractant IL-8 cytokine [23, 24]. Earlier studies showed that up-regulation of miR-155 has cardioprotective properties [25]. In rat hearts, miR-155 levels were increased after intraperitoneal lipopolysaccharide injection. Inhibition of miR-155 significantly down-regulated the apoptosis of cardiomyocytes, whereas overexpression of miR-155 significantly up-regulated the apoptosis of cardiomyocytes and in vivo ejection fraction and significantly increased fractional shortening and heart weight [26]. MiR-155 has been shown to be highly up-regulated in endotoxemic mice, as well as in human endothelial cells, increasing endothelial leakage in a tight junction protein-dependent manner [27]. Septic endothelial dysfunction and capillary leakage cause tissue edema, and myocardial edema has been suggested to play a role in the pathophysiology of septic cardiomyopathy [4]. The upregulation of miR-155 in the present study may support these previous findings. In a Chinese study with elderly severe septic shock patients, miR-155 and miR-143 were found to be potential useful biomarkers with relation to serum TNF-α, IL-6, CK-MB, and cTnI levels [28].
We also identified increased levels of miR-21-3p in septic hearts. In a previous study, miR-21-3p was shown to be up-regulated in mouse hearts with LPS-induced cardiac dysfunction, and pharmacological inhibition of miR-21-3p led to preservation of cardiac function (preserved ejection fraction and fractional shortening) and improved survival [29]. The same study also found that miR-21-3p levels were significantly increased in the plasma of sepsis patients with cardiac dysfunction compared to patients without septic cardiac dysfunction, but the mechanistic link between miR-21-3p and septic cardiac dysfunction in humans has not been shown. Overexpression of miR-21-3p has also been shown to exacerbate myocardial inflammation, whereas downregulation suppresses cardiomyocyte apoptosis in LPS-treated rats [30].
In our series, the expression of vascular smooth muscle regulator (miR-4787-5p) was up-regulated compared to controls. MiR-4787-5p regulates vascular smooth muscle cell apoptosis, and its overexpression has been shown to have diagnostic value in patients with acute aortic dissection [31].
Our study demonstrated that miR-320c, miR-320d, miR-3960, and miR-4488 were up-regulated in heart tissue specimens. Their predicted target genes regulate genes related to cell adhesion. However, studies of these miRNAs are sparse. In an experimental study, miR-4488 was found to inhibit the accumulation of inflammatory proteins in venous endothelial cells (vECs) [32].
MiR-451a had the largest difference in mean counts and highest fold decrease in septic heart specimens compared to controls. A rat model has shown that miR-451a expression decreases in ischemia reperfusion injury, and up-regulation of miR-451a in myocardial tissue reduces the area of myocardial infarction, attenuates myocardial injury, and reduces myocardial cell apoptosis [33]. Human studies have shown that miR-451a expression is elevated in the plasma of patients with acute myocardial infarction compared with unstable coronary disease and healthy control groups [34], ánd that miR-451 has a regulatory role in ischemic heart injury [35]. The decreased miR-451a count in septic hearts compared with controls in the present study may support the assumption that myocardial dysfunction in sepsis is not caused by ischemia. MiR-144-5p was also down-regulated in sepsis and has been shown to be associated with the macrophage response to vascular inflammation [36].
Clinical significance
As miRNAs have tissue-specific expression and can be secreted into blood, they could offer a biomarker tool for detecting myocardial dysfunction or possibly help guide treatment and monitor the treatment response in sepsis. Potential clinical use in diagnostics or prognostication of miRNA blood levels in the ICU setting would require accurate, rapid, and low-cost assays with multimarker panels of numerous miRNAs because of the heterogenous nature of the septic response. It is logical to think that the use of miRNAs as biomarkers will become routine following technological development [37]. In addition to biomarkers, the first animal studies imply that miRNAs may offer possible therapeutic interventions through restoration of the dysregulated immune system [38].
Limitations
We were not able to differentiate distributive shock and septic cardiomyopathy. It would have required systematic echocardiography studies or other methods to record cardiac function, such as routine use of a pulmonary artery catheter. However, these two forms of cardiovascular dysfunction often coexist. Furthermore, the collection of myocardial tissue samples was not standardized, as the autopsies were performed for clinical diagnostic purposes.
The selected control subjects were heterogeneous, deceased due to traffic accident, self-inflicted injury, or sudden cardiac death. They were matched for age, gender, and history of coronary artery disease, and there was no evidence of infection. Due to the small sample size and wide 95% CIs for differences in the mean miRNA counts, we chose a 2-fold increase as significant. As far as we know, there is no scientifically established clinically meaningful difference in each miRNA. MiRNA profiling studies have demonstrated that even subtle alterations in miRNA expression, such as a 1.5-fold difference, may exert a notable influence on cellular biology [39]. Finally, due to the small sample size, the reported associations cannot reliably be drawn as causal. The results must be interpreted as observational and hypothesis-generating. Despite its limitations, our study brings new insight into the cardiac response in sepsis at the organ level. Several miRNAs seem to be integrated in the pathophysiology of the septic myocardial response, either promoting or inhibiting cardiac damage. The up-regulated miRNAs were related to endothelial adhesion, inflammation, and apoptosis, and the down-regulated miRNAs to cardiovascular functions, cell viability, and immune response. Further studies are needed to explore whether soluble miRNAs may serve as biomarkers or prognostic tools for septic myocardial dysfunction.