Chronic alcohol abuse can lead to myocardial contractile dysfunction and myocardial remodeling, accompanied by disturbances in mitochondrial homeostasis, altered autophagic function and dysregulation of protein metabolism, and the main manifestations of ACM are cardiac hypertrophy, fibrosis, cardiomyocyte death and heart failure [1]. The metabolic process of ethanol in rats was similar to humans and the heart is the main organ being attacked by alcohol [34]. To date, the pathogenesis of ACM is thought to be related to various factors such as apoptosis, autophagy and ROS production, but many of these pathogenic mechanisms have not been fully theorized [1]. In this study, we have carried out the research of mechanisms focusing on hypertrophy, autophagy and apoptosis to improve the molecular healthcare and therapeutic method against ACM. We found that alcohol treatment could induce hypertrophy in H9C2 cells and rat myocardial tissue, and it also upregulated the expression levels of hypertrophy, autophagy and apoptotic marker proteins both in vitro and in vivo. Consistent with the report by Peng et al., our results also suggested that alcohol could induce hypertrophic changes both in vitro and in vivo [35].
As a transmembrane protein, LOX-1 has been reported to take part in the pathogenic process of many cardiovascular diseases. Its expression was maintained at a relatively low level in normal physiological states. However, under pathological conditions, some external chemicals, inflammatory mediators and cytokines could up-regulate LOX-1 expression and accelerate the occurrence of cardiovascular diseases, so it could be used as a diagnostic marker or even a therapeutic target [36]. In fact, there were few reports on the effect of LOX-1 expression on cardiac hypertrophy and autophagy in ACM. Our data presented that overexpression of LOX-1 enhanced the harmful effects of alcohol by increasing surface area and autophagy of rat cardiomyocytes, whereas LOX-1 knockdown showed the opposite effect. Besides, the knockdown of LOX-1 in vivo also diminished the upregulated expression levels of hypertrophy, autophagy and apoptotic markers caused by alcohol in the myocardial tissues of rats. Our results were consistent with the promoting role of LOX-1 in rats ventricular hypertrophy proposed by Zhu et al [37]. Recently, Lin’s research also pointed out that LOX-1 was a key molecule of cardiomyocyte hypertrophic responses [38]. Besides these, the knockdown of LOX-1 was also reported to relieve apoptosis and autophagic response as well as restored the morphology of cardiomyocytes in vitro [39]. Therefore, targeting LOX-1 should be a novel therapeutic method against ACM.
In our last research, we demonstrated that the knockdown of LOX-1 could mitigate the fibrotic changes in the heart by blocking P38MAPK signaling [4]. Here, we further presented that the knockdown of LOX-1 could also lighten the hypertrophic changes in the heart as well as reduce the autophagy and apoptosis of cardiomyocytes. The mechanism of myocardial hypertrophy is complex and involves multiple signaling pathways. It has also been shown that the P38MAPK signaling pathway is significantly activated in hypertrophied cardiomyocytes. On the one hand, it was found that retardation of P38MAPK signaling showed alleviative influences on the hypertrophy and autophagy of cardiomyocytes, which was related to MEF2C [24, 40]. Specifically, the P38MAPK signaling pathway was activated, leading to increased expression level of P38MAPK protein as well as increased levels of phosphorylation, which stimulated increased expression levels of downstream MEF2C protein, resulting the conversion of contractile α-MHC into embryonic β-MHC in cardiomyocytes, and then lead to cardiomyocyte hypertrophy [22, 40, 41].
In addition, Zhou et al presented that the expression of Beclin-1 in myocardial tissue of rats with long-term heavy alcohol consumption was significantly increased, resulting in the aggregation of autophagic vesicles and impaired autophagic flux, along with apoptosis of cardiomyocytes and impaired cardiac function [42]. Cardiomyocyte-specific autophagy activation could exacerbate hypertrophic remodeling and systolic dysfunction [25, 43]. It was found that multiple ATG proteins such as ATG5 could catalyze the formation of phosphatidylethanolamine-conjugated LC3 (LC3-II) and promote the autophagosome formation process [44]. Besides, lysosomal-associated membrane proteins (LAMPs) were a group of highly glycosylated transmembrane proteins located on the Lysosome membrane, and it was essential for successful completion of phagosome maturation [45]. In addition, lysosomal-associated membrane protein 1 (LAMP1) was commonly used as a lysosomal marker [45, 46]. What’s more, basal autophagy uses adaptor proteins, such as p62/SQSTM, that identify and deliver misfolded or aggregated proteins and damaged organelles to the autophagosome for degradation, and it was inversely proportional to the degradation of the autophagolysosome [44, 47]. We also agreed with Zhao et al’s research conclusions which proposed that alcohol stimulated myocardial autophagy and strengthened the expressions of autophagy and apoptosis-associated proteins in cardiomyocytes [29]. On the other hand, the activated P38MAPK pathway was found to be closely related to cardiomyocyte apoptosis [48]. Apoptosis could lead to the reduction of the number of cardiomyocytes and the change of myocardial structure. Apoptosis was regulated by intracellular signaling and was followed by nucleoplasmic condensation, nucleolus cleavage, DNA degradation, and ultimately the formation of apoptotic vesicles. With heavy alcohol consumption, mast cells and macrophages could release TNF-α, which activates caspase-8, followed by caspase-3 and caspase-7 in cardiomyocytes, leading to protein degradation. In the mitochondria, alcohol-induced oxidative stress could lead to DNA damage and induce apoptosis, a process regulated by Bax and Bcl. During apoptosis, Bax was responsible for the formation of small pores in the mitochondrial membrane through which cytochrome C flowed out and recruited the apoptotic protein kinase factor APAF-1, leading to the formation of apoptotic vesicles that activated caspase-9, caspase-7, and caspase-3, causing apoptosis. [49]. And P38MAPK signaling pathway played an important role in this process [49]. Therefore, targeting upstream activators of the P38MAPK pathway was an alternative and effective therapeutic choice for the treatment of ACM.
The present study investigated the effect of LOX-1 on ACM cardiac hypertrophy in both in vitro and in vivo models. The results showed that LOX-1 could significantly aggravate alcohol-induced cardiomyocyte hypertrophy, autophagy and apoptosis, but LOX-1 knockdown could protect H9C2 cardiomyocytes from alcohol-induced injury by blocking the P38MAPK signaling pathway in vitro. Furthermore, the protective roles of LOX-1 knockdown were verified in ACM rat models in vivo. We have investigated the molecular biological mechanism of ACM pathogenesis and made some further exploration of the potential and specific role of LOX-1 as an important intervention target in ACM treatment, thus providing experimental data reference and pointing out the future research direction for the prevention and treatment of ACM.
After alcohol treatment, the up-regulated LOX-1 promoted P38MAPK expression, then P38MAPK induced β-MHC expression through MEF2C to promote alcoholic hypertrophy. In addition, P38MAPK could activate autophagy through phosphorylation of ULK1, and then promote alcoholic hypertrophy and apoptosis, causing cardiac dysfunction (Fig. 5). However, LOX-1 expression was regulated by a variety of pro-inflammatory cytokines, oxidative stress response and mechanical stimulation [4–7]. It was reported that alcohol could cause inflammation and oxidative stress [50, 51]. Therefore, alcohol may induce LOX-1 expression through oxidative stress and the production of pro-inflammatory cytokines but this has not been specifically verified, which was a limitation of our study. Next, studies have shown that in addition to P38MAPK, LOX-1 and alcoholic cardiomyopathy may be related to other mechanisms, such as ROS or inflammation [4, 8, 37, 52–58]. What’s more, P38MAPK was a group of kinases activated by inflammation and stress, which should also include oxidative stress. The interaction between LOX-1, P38MAPK, oxidative stress, and inflammation can be studied in the future.
Alcohol increases the expression and phosphorylation of P38MAPK by up-regulating the expression of LOX-1. Phosphorylated P38MAPK then enters the nucleus to promote β-MHC expression via MEF2C, causing cardiac hypertrophy and leading to alcoholic cardiomyopathy. In addition, activated P38MAPK can phosphorylate ULK1, and then activate Beclin1, which can increase the expression of ATG5, LC3 and LAMP1 to induce autophagy, promoting cardiac hypertrophy and cardiomyocyte apoptosis, and ultimately lead to alcoholic cardiomyopathy.