Ethanol (EtOH)-induced cardiotoxicity (EIC) can significantly damage cardiomyocytes and compromise cardiac function, finally leading to cardiomyopathy and heart failure [1]. Recently, numerous theories have been postulated for EIC, including disrupted oxidative stress, apoptotic cell death, decreased mitochondria number, deranged lipid metabolism, accelerated protein catabolism and dysregulated autophagic flux [[2], [3], [4]].

Lipid droplet (LD), a kind of singular phospholipid-containing cytosolic organelle, not only plays a critical role in modulating lipid metabolism, but also has the potential to establish contact sites with mitochondria, thereby mediating essential cellular functions [5]. These LD-Mito contacts are able to promote fatty acid (FA) flux from LD to mitochondria, then facilitate β-oxidation and prevent lipotoxicity by restricting free FA in cytosol [6]. In response to fasting or exercise, LD-Mito contacts in muscle or heart are expanded to ensure energy supply by enhancing FA flux from LD to mitochondria and its subsequent oxidation [[7], [8], [9], [10]]. In high-fat diet mice, increased LD-Mito contacts by overexpressing Mitofusin-2 (MFN2) significantly promote FA β-oxidation and improve cardiac function [11]. Besides, the LD-Mito contacts were also reported to augment mitochondria respiratory capacity independent of FA flux in cardiomyocytes [12] and promote LD biogenesis in brown adipocytes [13]. Despite intense involvements of LD-Mito contacts in lipid metabolism, whether it has a role in EtOH-induced dysregulated lipid metabolism within cardiomyocyte remains elusive.

PLIN5, a perilipin family protein located on the LD surface, is prevalent in tissues with highly oxidative activity, such as the heart. PLIN5 is crucial for LD dynamics by regulating LD formation, motion and lipolysis [14]. It is later proposed to link LD to mitochondria via its C-terminal domain [15]. Moreover, recent studies demonstrated that PLIN5 exerts protective effect in the ischemic heart and alleviates myocardial ischemia/reperfusion injury [16,17]. Lacking PLIN5 exacerbates pressure overload-induced cardiac hypertrophy and heart failure, yet prevents type 1 diabetes-induced cardiac malfunction in mice [18,19]. While numerous research has highlighted the close relationship between PLIN5 and cardiovascular diseases, the role of PLIN5 and PLIN5-mediated LD-Mito contacts in EIC remains unknown.

In the current study, we established an in vitro EIC model to evaluate the role of LD-Mito contacts in EIC. Our observations revealed that a reduction in LD-Mito contacts levels caused by PLIN5 downregulation subsequently resulted in a compromised FA flux from LD to mitochondria in EtOH-treated cardiomyocytes. Overexpression of full-length PLIN5 abolished the reduction in LD-Mito contacts and restored FA flux, yet did not ameliorate cardiomyocyte apoptosis or inflammatory responses. Intriguingly, overexpression of the truncated form of PLIN5 (PLIN5Δ) which was unable to tether the LD to mitochondria ameliorated apoptosis and inflammation. To eliminate the impact of multifunctional nature of PLIN5 and more directly evaluate the specific role of LD-Mito contacts in EIC, we constructed a LD-Mito Linker expressing cell line using GPAT4 sequences and mitochondria-targeting sequence of FIS1. Overexpression of synthetic LD-Mito Linker restored the LD-Mito contacts and FA flux as well as aggravated apoptosis, inflammatory response, oxidative stress, and Mitochondrial membrane potential depolarization in EtOH-treated cardiomyocytes. Taken together, we propose a mechanistic framework in which the reduction of LD-Mito contacts and consequently decreased FA flux may act as an adaptive response to stress, conferring protective effects against EIC.