Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and antioxidants in a cell. In the heart, oxidative stress may deteriorate calcium handling, cause arrhythmia, and enhance maladaptive cardiac remodeling by the induction of hypertrophic and apoptotic signaling pathways. Consequently, dysregulated ROS production and oxidative stress have been implicated in numerous cardiac diseases, including heart failure, cardiac ischemia–reperfusion injury, cardiac hypertrophy, and diabetic cardiomyopathy. Lipid droplets (LDs) are conserved intracellular organelles that enable the safe and stable storage of neutral lipids within the cytosol. LDs are coated with proteins, perilipins (Plins) being one of the most abundant. In this review, we will discuss the interplay between oxidative stress and Plins. Indeed, LDs and Plins are increasingly being recognized for playing a critical role beyond energy metabolism and lipid handling. Numerous reports suggest that an essential purpose of LD biogenesis is to alleviate cellular stress, such as oxidative stress. Given the yet unmet suitability of ROS as targets for the intervention of cardiovascular disease, the endogenous antioxidant capacity of Plins may be beneficial.

Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg ^–/– mice). In 9- to 10-week-old hUgcg ^–/– mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg ^–/– mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg ^–/– mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors. Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function. Cardiac glycosphingolipids are required to maintain β-adrenergic signaling and contractile capacity in cardiomyocytes and to preserve normal heart function.

Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9^−/− mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9^−/− mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9^−/− hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9^−/− mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9^−/− mice. Circulating lipid levels were unchanged in CM-Pcsk9^−/− mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9^−/− mice had an increased number of mitochondria–endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production. Model of the effects of Pcsk9 deletion in the heart. We propose that cardiac PCSK9 maintains the normal composition of cholesterol and phospholipids in the mitochondrial membrane by modulating their transport through mitochondria-associated ER membrane contacts (MAMs). Reduced cardiac Pcsk9 expression alters the lipid composition of mitochondrial membranes and cristae morphology, leading to impaired assembly and activity of mitochondrial respiratory complexes (especially ATP synthase), and thereby decreased OXPHOS and ATP production, and increased ROS production. These impairments indicate metabolic inflexibility characterized by reduced mitochondrial oxidative metabolism and insufficient compensatory energy production by glycolytic pathways, making the heart less efficient in generating energy to support demand. PCSK9 cardiac deficiency leads to progressive DCM, impaired heart function/structure, and compromised energy production. TCA, tricarboxylic acid cycle

The adult heart develops hypertrophy to reduce ventricular wall stress and maintain cardiac function in response to an increased workload. Although pathological hypertrophy generally progresses to heart failure, physiological hypertrophy may be cardioprotective. Cardiac-specific overexpression of the lipid-droplet protein perilipin 5 (Plin5) promotes cardiac hypertrophy, but it is unclear whether this response is beneficial. We analyzed RNA-sequencing data from human left ventricle and showed that cardiacPLIN5expression correlates with up-regulation of cardiac contraction–related processes. To investigate how elevated cardiac Plin5 levels affect cardiac contractility, we generated mice with cardiac-specific overexpression ofPlin5(MHC-Plin5mice). These mice displayed increased left ventricular mass and cardiomyocyte size but preserved heart function. Quantitative proteomics identified sarcoplasmic/endoplasmic reticulum Ca²⁺ATPase 2 (SERCA2) as a Plin5-interacting protein. In situ proximity ligation assay further confirmed the Plin5/SERCA2 interaction. Live imaging showed increases in intracellular Ca²⁺release during contraction, Ca²⁺removal during relaxation, and SERCA2 function in MHC-Plin5versus WT cardiomyocytes. These results identify a role of Plin5 in improving cardiac contractility through enhanced Ca²⁺signaling.

BACKGROUND: Cholesterol is critical for mitochondrial membrane structure and function. Given the emergence of mitochondria as a key factor in the pathogenesis of heart failure, mitochondrial cholesterol homeostasis may be crucial for maintaining mitochondrial properties and thus cardiac function. We previously showed that CM- Pcsk9 ^–/– mice (mice with cardiomyocyte-specific deletion of the gene encoding PCSK9 [proprotein convertase subtilisin-kexin type 9]) have impaired cardiomyocyte mitochondrial bioenergetics and heart function, paralleled by cardiomyocyte mitochondrial cholesterol accumulation and an increased number of mitochondria–endoplasmic reticulum contacts. However, the mechanisms linking PCSK9 to mitochondrial cholesterol homeostasis remain unclear. We hypothesized that PCSK9 acts on proteins involved in mitochondrial cholesterol trafficking in the heart to maintain cardiac mitochondrial function. METHODS: By performing RNA sequencing and immunoblot on hearts from CM- Pcsk9 ^–/– and CM- Pcsk9 ^+/+ (without cardiomyocyte-specific deletion of Pcsk9 ) mice, we showed that TSPO (translocator protein) was increased by Pcsk9 deficiency. To investigate the relationship between TSPO levels and heart function in humans, we compared the transcriptome of human left ventricles with high versus low TSPO levels. We used H9c2 (a rat cardiomyoblast cell line) cardiomyocytes to explore the mechanism linking PCSK9/TSPO to mitochondrial cholesterol content and function. The impact of reduced TSPO levels on cardiac function and mitochondrial oxidation in CM- Pcsk9 ^–/– mice was tested using adeno-associated virus serotype 9 short hairpin TSPO. RESULTS: Both gene and protein levels of TSPO, a mitochondrial protein involved in cholesterol transport, were increased in CM- Pcsk9 ^–/– mouse hearts. Transcriptome analysis showed that high TSPO expression in human left ventricles was associated with impaired mitochondrial and cardiac function. We showed that PCSK9 induced TSPO degradation through a proteasomal mechanism that occurs in cardiomyocytes but not hepatocytes and contributes to maintaining normal mitochondrial cholesterol composition and function. At the molecular level, endoplasmic reticulum–resident PCSK9 interacted with GRP78 (glucose regulatory protein 78) , reducing GRP78-TSPO interactions and leading to TSPO misfolding and degradation by the ubiquitin-proteasome pathway. Importantly, gene therapy–induced downregulation of TSPO in CM- Pcsk9 ^{− / −} mice prevented mitochondrial cholesterol accumulation and improved cardiac function. CONCLUSIONS: These findings indicate that PCSK9 regulates mitochondrial cholesterol levels by modulating the TSPO degradation in the heart. Modulation of mitochondrial cholesterol by targeting TSPO may be a promising therapeutic approach for heart failure.

Aberrant activation of fibroblasts is a pivotal component of cardiac fibrosis predisposing to heart failure. However, the molecular regulation of the functional state of cardiac fibroblasts in fibrosis resolution remains largely unexplored, and therefore, effective antifibrosis therapies are still lacking. By translating mouse transcriptomics to humans, we unlocked common molecular denominators connecting the fibroblast phenotypic state and fibrogenic signaling pathways in cardiac fibrosis. Through the construction of a fibroblast-specific transcriptional gene regulatory network, we found ITGAL and DUSP9 as key druggable targets for human myocardial fibrosis. A computational drug repurposing approach predicted 367 antifibrotic candidate compounds for heart disease. In primary cardiac fibroblasts derived from patients with heart failure, we provided experimental validation of the top 2–ranked repositioned drug candidates and their combination. These innovative approaches facilitate the identification of potential targets and drug candidates for cardiac fibrosis, providing actionable opportunities for clinical translation.