Significances of viable synergistic autophagy-associated cathepsin B and cathepsin D (CTSB/CTSD) as potential biomarkers for sudden cardiac death

Sudden cardiac death (SCD) is an unexpected death caused by the sudden cessation of cardiac activity. It is the most significant cause of natural death in the world, accounting for 170,000 to 450,000 adult deaths in western countries, including the USA, and 544,000 adult deaths in China annually [1, 2]. The pathogenesis of SCD is extremely complex, coronary heart disease (CHD) is certainly the most common disease contributing to SCD [3]. Despite discovery of risk factors, innovative diagnostic modalities, therapeutic interventions, and new drug targets development, have led to mortality rate reduction, but the incidence of SCD as a proportion of overall cardiovascular death has remained relatively constant [4]. Therefore, identifying accurate molecular markers is very important for SCD diagnosis and treatment.

Autophagy is an essential process of the catabolic mechanism, which promotes cell survival by eliminating damaged or defective organelles and releasing energy substrates via the degradation of cellular constituents. However, an uncontrolled and excessive autophagic activation can trigger cell death via the depletion of essential organelles and molecules [5]. Because autophagy can eliminate misfolded proteins and damaged organelles [6], and supply substrates for ATP regeneration during ischemia and starvation [7, 8], these functions can maintain cardiac structure and function. So, autophagy is considered as essential for the maintenance of cardiovascular homeostasis and function [9]. Coronary obstruction and microcirculation disorder lead to myocardial ischemia and hypoxia in the pathogenesis of CHD, autophagy was activated, it could be emove damaged organelles, and inhibit inflammation, leading to anti-inflammatory effects and stabilization of atherosclerotic plaques. The process is considered an adaptive response with cardioprotective effects [10‐12]. On the contrary, studies have shown that if autophagy becomes dysfunctional, which stimulates hyperactivation of inflammasomes to promote atherogenesis [13].

Cathepsins are the primary lysosomal proteases, they are naturally expressed in the lysosomes of various cells and tissues. Cathepsins contribute to arterial plaque formation and underlie clinical events by extracellular matrix digestion, thereby rendering plaques prone to rupture. In addition, lysosomes also mediate a variety of homeostatic processes such as nutrient breakdown and removal of damaged organelles [14, 15]. Cathepsins are subdivided into 3 subfamilies based on the active-site amino acids: Serine cathepsin (CTSA/G), aspartic cathepsin (CTSD/E), cysteine cathepsin (CTSB/C/F/H/K/L/O/S/V/W/Z) [6]. CTSB and CTSD were extracted as the autophagy-related genes from Human Autophagy Database [16] (HADb, http://​www.​autophagy.​lu/​index.​html). Studies conducted in vitro or in vivo models have revealed that over-expression of CTSB/D can contribute to the formation of arterial plaques and increase the risk of coronary artery disease. However, the relationship between CTSB/D and coronary heart disease in autophagy regulation remains unclear. It is necessary to have a comprehensive and in-depth understanding of the role of autophagy of the above two genes in the process of SCD.

We studied CTSB/D expression in data from coronary arteries of SCD in private protein datasets. Moreover, we analyzed genomic alterations and functional networks related to autophagy-related proteins CTSB/D to determine their expression patterns, the potential functions, and the correlation between autophagy and risk factors and autophagy pathways. Thus, our results could potentially reveal new autophagy-related targets and strategies for SCD diagnosis and treatment.

SCD is a significant public health issue, which accounts for half of all cardiovascular-related deaths worldwide [2, 23, 24]. In autopsies performed on adult male who are victims of sudden cardiac death, we found that these deceased had atherosclerotic plaque led to various degrees of coronary artery stenoses, which resulted in the dysfunction of cardiomyocytes and cardiac functions.

To investigate the role of autophagy in SCD, we analyzed the datasets which performed protein quantification of the anterior descending branch of the left coronary artery in six cadavers (SCD = 3, control = 3) using label-free proteomics. we identified 148 DEPs, and we screened out 6 members of the cathepsins family (CTSZ, CTSC, CTSF, CTSD, CTSB, CTSA) as hub proteins through Functional enrichment, PPI module analysis. The relative expression level was significantly higher in the CHD group compared with the normal group, and their functional enrichment analysis were involved in inflammation, immune response, and lysosome pathway, the same results were still obtained with Metascape, GSEA and Hierarchical clustering. We focused on autophagy-associated cathepsins in cathepsins family which were obtained by comparing the human autophagy database so as to analyze the expression levels of expressed, regulatory networks, and potential mechanism of CTSB/CTSD in SCD.

Cathepsins are the major lysosomal hydrolases [25]. Their activities potentially affect crucial atherogenic cascades, such as the inflammation, lipid metabolism, and autophagy [26]. Previous clinical studies suggested that CTSB and CTSD were known as plays a crucial role in arterial stiffening and atherosclerotic vascular disease [23, 27]. The serum levels all associated with increased risk and poorer outcome of coronary events severity [28‐31]. Above conclusion was also confirmed via the quantification of proteomics and western blotting in our study. In addition, we also found that the expression of CTSB/CTSB in SCD were positively correlated with the Gensini score (which is a well-recognized scoring system that evaluate the severity of CHD [32, 33]). Yet, a few studies have a contrasting conclusion, they reported that decreased myocardial and serum CTSD levels were relatively lower in SCD with cardiac hypertrophy [34]. The different conclusion may be related to a number of factors (e.g., the basal diseases, source of samples, sample collection time et al.). Despite the opposite conclusion, to be sure, CTSB/CTSD played an important role in the development of SCD and involved the severity of atherosclerosis.

It is increasingly appreciated that autophagy can be both protective and deleterious in atherosclerotic. Under the ischemic, hypoxia and hyperlipidemia stress, in order to deal with the cytotoxic effects of excessive inflammation, autophagy is activated to protect cardiomyocytes against ischemic or hypoxia injury and inhibition of inflammation, so as to contributes to enhances atherosclerotic plaque stability and attenuate cardiac injury. As an indicator of autophagic activity, CTSB and CTSD were involved in the regulation of cell death and survival in the development of atherosclerosis [35]. Si Ming Mand et al. believed that under homeostatic conditions CTSB cleaves the calcium channel MCOLN1/TRPML1 in the lysosomes, maintaining suppression of TFEB and reducing expression of lysosomal and autophagy-related proteins [36]. As another indicator of autophagic activity. Cardiac autophagy activity was increased after myocardial infarction, if up-regulation of CTSD was prevented during myocardial infarction exacerbates poor cardiac remodeling and dysfunction in mice [31]. Some studies had also recognized that after the silencing of cathepsin D, apoptosis and necrosis significantly increased, while stress-induced autophagy was abrogated [37]. From the above viewpoints, upregulation of CTSB/CTSD by atherosclerosis promotes autophagic flux and protects against cardiac remodeling and heart failure. Based on the above, CTSB/CTSD can be considered as a potential biomarker.

Besides its protective activities, more and more evidence showed that dysfunction of autophagy is an essential contributor to the development of advanced atherosclerotic lesions [38],it perhaps play a detrimental role in plaque formation, excessive or uncontrolled levels of autophagy are able to induce autophagy-dependent cell death and promoted atherosclerotic plaque instability [4]. Our results prefer to this conclusion. In our analysis, overexpression of CTSB and CTSD in SCD, we considered that CTSB/CTSD was involved in mechanisms of early atherogenesis and activated an autophagic response, but from the advanced atherosclerosis stage to SCD, the cholesterol crystals in the continuous formation of plaque destroy the lysosomal membrane, disrupt the autophagy process [39],and autophagy promotes atherosclerosis through of excessive inflammasome activation [13],the other is persistent hypoxic or ischemia, autophagy fails to deal with the excessive amount of oxidative stress in the plaque, finally lead to cell death, i.e., apoptosis. However, the two different perspectives indicating the controversial effect of autophagy in atherosclerosis need more thorough research.

In order to further explore regulatory mechanisms of CTSB/CTSD in autophagy, we performed a correlation analysis of CTSD/CTSB with other autophagy-related genes. The results obtained in this study showed that the expression of CTSB was negatively correlated with ATG4D, but positively correlated with FOXO3, the expression of CSTD, was negatively correlated with BNIP3, but positively correlated with TP53.However, the results of our analysis do not fit the trend of BNIP3 and ATG4D in autophagy in the literature [40‐43],The specific reasons and mechanism merit further exploration.

Although changes in CTSD/CTSB affect the occurrence of CHD, the main causes and underlying mechanisms are unclear. In this study, to better understand the mechanism of CTSD/CTSB in sudden coronary heart disease death, we also analyzed the TF-mRNA-miRNA relationship to obtain the co-regulatory network. SP1-CTSD-(has-miR-24-3p) and SP1-CTSB-(has-miR-128-3p) from the TF-mRNA-miRNA network were identified to play crucial roles in CHD. SP1 is a zinc finger transcription factor that regulates target gene transcription by binding to their promoter contain GC boxes, including regulate the expression of cathepsin [44]. It is also associated with several cellular processes such as chromatin remodeling, cell growth, responses to DNA damage, apoptosis, and cell differentiation. Experimental studies have revealed an essential role for microRNAs in regulating molecular and cellular processes related to the development of atherosclerosis. has-mir-24-3p is associated with plaque progression and plaque instability [45], while has-mir-128-3p is a key regulator of VSMC, affecting proliferation, migration, differentiation, and contraction of VSMC [46]. Thus, in the present study, we gain further insight into the mechanisms of target genes in the disease from the regulatory.

In our present study, we used a proteomic dataset of the left anterior descending branch of the human coronary artery from sudden cardiac death due to coronary heart disease, and screening the DEGs to focus on the cathepsin family members. The expression and function of the gene family members in CHD were comprehensively analyzed, and the CTSD/CTSB associated with autophagy were selected for mechanism study. Moreover, we have to admit that our study had limitations, although the use of human specimens avoids the drawbacks of tissue specificity, and the experimental results were more convincing for the development of the disease, but the sample size was small and only the male gender was analyzed. It may have had some influence on the results, so in the next step, it requires us to take into account plausibility to design the dataset. we will perform the relation between Cathepsins and autophagy by in vitro study as well by using western blot, immunofluorescence, immunoprecipitation. In summary, our findings provide new insights into the pathogenesis of CTSB/CTSD in SCD.