Deciphering the Role of Lipid Droplets in Cardiovascular Disease: A Report from the 2017 National Heart, Lung, and Blood Institute Workshop

Abstract

Lipid droplets (LDs) are distinct and dynamic organelles that affect the health of cells and organs. Much progress has been made in understanding how these structures are formed, how they interact with other cellular organelles, how they are used for storage of triacylglycerol (TAG) in adipose tissue, and how they regulate lipolysis. Our understanding of the biology of LDs in the heart and vascular tissue is relatively primitive compared to LDs in adipose tissue and liver. The National Heart, Lung, and Blood Institute (NHLBI) convened a working group to discuss how LDs affect cardiovascular diseases (CVD). The goal of the working group was to examine the current state of knowledge on the cell biology of LDs, including current methods to study them in cells and organs and reflect on how LDs influence the development and progression of CVDs. This review summarizes the working group discussion and recommendations on research areas ripe for future investigation that will likely improve our understanding of atherosclerosis and heart function.

Introduction

Lipid droplets (LDs) are dynamic organelles that store neutral lipids for later use during energy deficit. Imbalance of LD function has been implicated in various human diseases. Overabundant and/or enlarged LDs are the hallmarks of obesity, type 2 diabetes, liver steatosis, atherosclerosis, cardiac steatosis, and cardiomyopathy¹. Conversely, failure to develop or maintain adipose tissue in lipodystrophies, a group of heterogeneous disorders characterized by varying degrees of body fat loss or redistribution of fat, leads to insulin resistance and other metabolic complications. Several genetic causes of the familial forms of lipodystrophy are linked to LD biology and a number of relatively rare forms of cardiomyopathy are characterized by excess LDs.

Although major advances have been made in the past 20 years to characterize LDs and their relationship to adipose and liver biology, less investigation has focused on LDs and cardiovascular disease (CVD). We now understand that LDs exhibit a dual nature in cardiovascular health. In some situations, the storage of lipid within droplets is a marker for lipid overload and reduced cardiac function. Dysfunction in storage and turnover of endogenous triglyceride within the LD of the cardiomyocyte is related to impaired transcriptional regulation of metabolic gene expression in failing hearts and reduced mitochondrial generation of energy to support cell function, the energy demands of the contractile apparatus^2–6. In other circumstances, LDs may prevent lipid toxicity by sequestering toxic lipid species such as cholesterol, ceramides, and sn-1,2-diacylglycerols (DAGs)⁷. Another example is the accumulation of cholesterol esters in the LDs of macrophages (foam cells) in arteries, an important step in atherosclerosis development. Within the atherosclerotic plaque, LDs can serve as a safe reservoir to store cholesterol esters until they can be removed from macrophages. However, little is known regarding the protein compositions of LDs in foam cells and how formation and mobilization of LDs are regulated in the context of atherosclerosis.

To evaluate the state of the field in LD research, identify the gap areas in cardiovascular research, and foster collaborations among researchers in the scientific community, the National Heart, Lung, and Blood Institute (NHLBI) convened a working group in May 2017, to discuss the emerging importance of studying the role of LDs in CVD. The working group comprised 12 experts from diverse backgrounds in diabetes, liver diseases, cardiovascular research, proteomics, nuclear magnetic resonance, and cell and systems biology. A list of workshop participants can be found in the appendix. In this report, we provide a summary of the workshop presentations, discussion, and recommendations for future research on the role of LDs in CVD with three objectives: (1) to summarize the current information available on the roles of LDs in heart function and vascular disease, (2) to discuss the state-of-the-art technologies in the field that may be suitable for further development, and (3) to identify gaps in knowledge and opportunities for high impact research in the future.

Current State of Knowledge

What are the function, composition, and structure of LDs?

LDs are cytosolic organelles composed of a neutral lipid core (which may contain triacylglycerols (TAGs), cholesteryl esters (CEs), retinyl esters, ether lipids and other neutral lipids) surrounded by a phospholipid monolayer with many surface proteins. Traditionally, LDs were considered inert organelles whose sole purpose was to store lipids. This view has been overturned in the past decade, with better understanding of the biogenesis, composition, modification, and regulation of LDs. LDs (which have also been called “intracellular lipoproteins”) are now viewed as dynamic organelles with diverse properties and functions across cell types, playing a crucial role in lipid metabolism and energy homeostasis, metabolic gene expression, intracellular lipid and membrane trafficking, viral infection, and inflammatory responses.

Adipocytes are uniquely specialized to store large amounts of lipid in the form of LDs. Nevertheless, other tissues also store lipids, particularly in times of calorie excess. Exceeding the normal LD storage capacity of adipocytes, as can occur in obesity and lipodystrophy, results in ectopic lipid accumulation in other tissues (including skeletal muscle, liver, pancreatic β cells) which contributes to insulin resistance, fatty liver, and CVD. Liver, heart, and kidneys also harbor increased LDs during fasting/starvation when levels of non-esterified fatty acids (NEFAs) in the circulation rise.

Several proteins and enzymes are associated with LDs, and these proteins synthesize TAGs and phospholipids, and liberate stored lipids. Probably the best studied LD structural proteins are the five members of the perilipin (PLIN) family. Perilipins regulate the actions of lipases. Under basal conditions, PLINs limit LD hydrolysis and fatty acid release, and under stimulated conditions (such as catecholamine treatment) PLIN1 and PLIN5 promote LD hydrolysis to provide fatty acids for processes such as oxidation.

How is LD size regulated?

The regulation of LD size and abundance is critical for metabolic homeostasis. Conditions associated with increased LD size and number (obesity) or inability to form or store LDs (lipodystrophy) are associated with increased risk for dyslipidemia, type 2 diabetes, atherosclerosis, coronary artery disease, and cardiomyopathy. The enzymes that catalyze TAG synthesis are a determinant of LD formation and have been explored as therapeutic targets to reduce TAG storage⁸. However, complete inhibition of LD synthesis leads to severe disease, as mutations in TAG biosynthetic enzymes AGPAT2 (encoding 1-acylglycerol 3-phosphate o-acyltransferase 2) or Lpin1 (encoding lipin 1, phosphatidic acid phosphatase) cause lipodystrophy associated with insulin resistance, elevated circulating free fatty acid levels, fatty liver, and increased CVD risk^9–12. LD size also is dependent on droplet coat proteins. Heterozygous frameshift mutations in the gene for PLIN1 have been identified in 3 families with autosomal dominant partial lipodystrophy. The histological phenotype of the subcutaneous adipose tissue in these cases included smaller adipocyte size, macrophage infiltration and fibrosis¹³.

In addition to synthesis of the neutral lipid core, the expansion of LDs requires increased phospholipid surface, particularly phosphatidylcholine (PC). During LD expansion, the rate-limiting enzyme in PC synthesis, CCTα (CTP: phosphocholine cytidylyltransferase α), senses the phospholipid deficiency on growing LDs and is activated at their surface to maintain the balance between LD core and shell¹⁴. Several additional proteins are required to convert nascent to mature LDs, most notably seipin, which appears to act at contact sites between nascent LDs and the endoplasmic reticulum (ER) to promote LD maturation¹⁵. Generalized lipodystrophy results from deficiency in seipin, as well as from deficiencies in additional proteins with incompletely understood roles in LD biology (e.g., caveolin 1, polymerase I and transcript release factor)^{9, 16}.

An opposing force to LD TAG synthesis is the mobilization of fatty acids. Fatty acids are important sources of energy and function as signaling mediators and biosynthetic substrates. Because excessive fatty acid mobilization can result in the accumulation of toxic lipid mediators¹⁷, the core intracellular lipolysis machinery is under tight control^{18, 19}. In the heart, this core lipolysis machinery consists of adipose triglyceride lipase (ATGL), the rate-limiting enzyme of triglyceride hydrolysis; alpha-beta hydrolase domain-containing 5 (ABHD5, also known as CGI-58), an essential coactivator of ATGL; and hormone sensitive lipase, which mediates hydrolysis of diglycerides generated by ATGL. Mutations of these core proteins are involved in a range of important diseases that include cardiovascular and metabolic disease, as well as cancer^20–24. Intracellular lipolysis is regulated by extracellular signals, such as catecholamines and insulin, in addition to intracellular signal transduction and metabolite levels that together serve to precisely match mobilization of fatty acids with oxidative demand. As such, lipolysis is a highly dynamic process that is spatially compartmentalized and involves rapid (on the order of seconds) trafficking and interactions within the core lipolysis machinery on the surface of LDs²⁵.

In the heart, these interactions are coordinated by PLIN5, which functions as a dynamic scaffold on the surface of LDs²⁶ to regulate interactions between ATGL and CGI-58. PLIN5 also has been proposed to play a central role in creating a “metabolic synapse” between LDs and mitochondria, where fatty acids are oxidized to generate ATP for contraction²⁷. PLIN5 in the nucleus has been shown to function as an activator of the PGC-1α transcriptional program to promote mitochondrial biogenesis and oxidative metabolism²⁸. Nuclear lipid droplets have been observed in cultured hepatocytes²⁹. It is unknown whether the nuclear PLIN5 involved in transcription also coats nuclear LDs. PLIN5 enriches in the nuclear fraction of the fasting mouse heart, but whether PLIN5 regulates gene expression in the heart requires future investigation. Importantly, the proteins of the core lipolysis machinery can each be targeted by synthetic activators and inhibitors^{27, 30, 31}, providing an opportunity for development of new therapeutic approaches for the treatment of CVD.

What is the role of LDs in the heart?

To support the high energy demands of persistent contraction against a pressure load, the heart oxidizes more fat than any other organ. As in some other organs and tissues (with obvious exclusions such as the brain and glycolytic skeletal muscle), long chain fatty acids (LCFAs) are the primary fuel for oxidative ATP generation for the cardiomyocyte. However, LCFAs also contribute as ligands for nuclear hormone receptors and as substrates for the formation of physiologically active acyl-intermediates and synthesis of membrane lipids. Upon uptake into the heart, fatty acids are CoA-esterified via the actions of the acyl CoA synthetase (ASCL) and fatty acid transport proteins (FATPs). The fatty acyl CoAs are esterified onto a glycerol backbone and incorporated into LDs as TAG for subsequent hydrolysis by lipases to supply FA for mitochondrial oxidation and also as ligands for PPARα^{6, 32}.Incorporation of fatty acids into the neutral lipid pool within LDs and their controlled release from LDs protects the heart from excess concentrations of acyl intermediates, which can disrupt signaling pathways and lead to tissue dysfunction or injury, known as lipotoxicity.

Perilipins regulate the actions of lipases to either limit the hydrolysis of TAG under basal conditions or to promote such action under stimulated conditions, such as catecholamine treatment. In the heart, most attention has focused on PLIN5, which is preferentially expressed in tissues with high oxidative capacity. Studies in humans have suggested that reduced PLIN5 expression correlates with impaired cardiac function following myocardial infarction³³ and that failing hearts have reduced PLIN5 RNA expression compared with donor hearts³⁴. In mice, constitutive, whole-body gene knockout of Plin5 results in the absence of myocardial lipid droplets, as well as in age-related cardiomyopathy that is prevented by anti-oxidant therapy³⁵ and in increased infarct size and cardiac dysfunction following ischemia-reperfusion³⁶. Conversely, heart-specific overexpression of PLIN5 has been associated with cardiac steatosis and cardiac hypertrophy^{37, 38}. Thus, having the right amount of PLIN5 in the heart at the right time likely is important for maintenance of normal heart structure and/or function. Future work in genetically engineered mouse models is needed to address the mechanisms and downstream pathways that underlie the effects of PLIN5 dose on cardiac lipid metabolism, histology, and function.

In hyperlipidemic states, LDs accumulate in the heart (i.e., cardiac steatosis) at least in part due to delivery of surplus metabolic substrates to the heart, which expresses the biochemical enzymes and possesses the cellular machinery for LD formation. Accumulation of lipids in myocardial LDs is associated with heart failure in obesity and diabetes mellitus, diseases characterized by hyperlipidemia³⁹. Studies using localized ¹H magnetic resonance spectroscopy to detect cardiac steatosis in patients with impaired glucose tolerance or type 2 diabetes indicate that an increase in the lipid content of the myocardium can precede the development of heart failure⁴⁰. In experimental rodent models, studies combining cardiac tagging magnetic resonance imaging and proton magnetic resonance spectroscopy demonstrate that even a short-term high fat diet expands the pool of LDs and induces early impairment of contractility⁴¹. The hearts of chronically diabetic mice are characterized by very high TAG content and turnover rates, associated with chronically high PPARα expression and activation^{2, 3}. Elevated TAG turnover results from PPARα activation that is known to induce target gene expression for LCFAs uptake, β-oxidation enzymes, and both TAG synthesis and lipolysis. Under such conditions, the endogenous TAG pool of the diabetic heart contributes to the already elevated mitochondrial beta-oxidation of LCFAs within the mitochondria, oxidative stress, and formation of ceramides⁴².

To determine whether lipid overload alone can cause heart dysfunction, several laboratories created genetically modified mice with enhanced cardiomyocyte lipid uptake or reduced cardiac fatty acid metabolism^43–45. In these models of cardiac lipotoxicity, imbalance between lipid import and metabolism leads to lipid accumulation, cardiomyopathy, and in some cases, sudden death. Thus, although metabolic channeling of excess fatty acids to intracellular TAG stores may serve an initial cytoprotective role by sequestering excess fatty acids away from mitochondria, ER, lysosomes, and other organelles^{4, 46, 47}, the capacity for physiological storage of lipids in the heart is limited. In the setting of prolonged lipid excess, storage capacity is overwhelmed and/or TAG hydrolysis exceeds synthesis. Lipotoxicity ensues, initially manifesting as organelle dysfunction, and ultimately leading to cell death and tissue damage^48–50.

In contrast to these examples of lipid-induced toxicity, greater numbers or increased size of LDs, occurring during prolonged fasting or via overexpression of DAG acyltransferase (DGAT)-1, do not cause toxicity⁵¹, likely because fatty acids esterified as TAGs and stored in LDs are protected from enzymatic conversion to potentially toxic metabolites. In some animal models, enhanced sequestration of fatty acids in LDs improves cardiac function in the setting of lipid overload, whereas decreased LD capacity promotes oxidative stress and functional decline. In other models, such as mice with germline loss-of-function of ATGL, marked cardiac steatosis causes heart failure and premature death⁵². Experiments in whole-body, constitutive ATGL knockout mice suggested that lipolytic release from LDs of lipid ligand(s) for PPARα is required for activation of target genes necessary for maintenance of normal mitochondrial substrate oxidation and respiration⁵³. Thus, it appears that cardiac lipid storage and hydrolysis may be physiologic or pathologic. This parallels lipid storage in skeletal muscle, in which excess LD accumulation is associated with insulin resistance in obese and sedentary individuals, but may also be associated with improved muscle function and greater insulin sensitivity in other cases (the latter being known as the “athlete’s paradox”).

These genetic models have served as platforms for discovery of the pathophysiology of lipid overload in the heart, providing novel insights into the effects of different diet compositions as well as the crosstalk between sex hormones and lipid metabolism^{2, 54}. In addition, a signature of metabolic and signaling pathways mediated by lipid overload in vivo has emerged. In the lipid-overloaded heart, palmitate serves as substrate for de novo synthesis of ceramides, which have been shown to be cardiotoxic⁵⁵. As has been demonstrated in cell culture models of lipotoxicity, lipid overload incites oxidative and ER stress in the heart^{2, 53, 56}. Genetic screens have also uncovered novel roles for several non-coding RNAs in the regulation of lipotoxicity^{57, 58}, although the contributions of these RNAs to lipotoxicity in vivo remain to be determined. Despite recent advances, the endogenous mechanisms that determine the transition between adaptive lipid storage and lipotoxicity are not well understood. This represents an important area for investigation with the potential to identify new therapeutic targets.

How do LDs interact with membrane and intracellular signaling pathways?

Nutrient sensing plays an important role in how cells remodel LD metabolism to adapt to energy transitions^{59, 60}. At the plasma membrane, the fatty acid receptor CD36 transduces intracellular signals that function to regulate nutrient metabolism. In cardiomyocytes, CD36 mediates both fatty acid activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) and inhibition of the insulin receptor. It also influences cytosolic calcium transients by regulating inositol trisphosphate (IP3) generation and store-operated calcium entry^{61, 62}, a pathway that can be activated by fatty acids. CD36 signaling controls mobilization of LDs, cellular production of eicosanoids and the secretion of neurotransmitters (serotonin) and enteropeptides (cholestvstokinin, secretin) that influence cellular fat processing⁶³. In the heart, CD36 might help link membrane signal transduction to LD metabolism by coordinating several events that could influence LD trafficking and/or composition. Membrane signaling events could include initiation of calcium transients, activation of phospholipase for phospholipid remodeling, and phosphorylation of proteins linked to AMPK activation and intracellular TAG breakdown^{64, 65}. Interestingly, the LD proteome includes a substantial number of proteins that function in cell signaling, membrane traffic, and interaction between organelles^{66, 67}. More focus on the mechanisms that coordinate plasma membrane signaling with LD trafficking, composition, and turnover is needed. In this context, a better understanding of the role of calcium dynamics in coordinating metabolic and functional adaptation of the heart is important.

Ectopic lipid deposition also plays a major role in the pathogenesis of hepatic insulin resistance and type 2 diabetes^{12, 68}. Nonalcoholic fatty liver disease (NAFLD) affects 1 in 3 Americans, is a major predisposing factor for nonalcoholic steatohepatitis (NASH) and hepatocellular cancer, and is an independent risk factor for CVD. Understanding the cellular and molecular mechanisms by which ectopic lipid promotes insulin resistance in liver, and identifying the key lipid mediators in this process, is therefore of great interest. In a model of high fat feeding NAFLD, sn-1,2-DAGs have been shown to trigger hepatic insulin resistance through translocation/activation of protein kinase C (PKC)-epsilon, resulting in phosphorylation of insulin receptor Thr¹¹⁶⁰ (mouse Thr¹¹⁵⁰) and inhibition of insulin receptor kinase activity⁶⁹. However, it is important to note that alterations in the cellular compartmentation of sn-1,2-DAG play an important role in lipid-induced hepatic insulin resistance as reflected by the increase in hepatic DAG content in LDs in CGI-58 antisense oligonucleotide treated rats despite the lack of PKCε translocation/activation and hepatic insulin resistance⁷⁰. This hypothesis can also explain the lack of association of hepatic steatosis and hepatic insulin resistance in other conditions of increased lipid storage in LDs (e.g. Apo B deficiency, MTP deficiency, etc.). In addition, alterations in hepatic acetyl-CoA, an allosteric activator of pyruvate carboxylase, have been shown to mediate insulin suppression of hepatic gluconeogenesis and promote increased rates of hepatic gluconeogenesis in animal models of poorly controlled type 1 and 2 diabetes⁷¹. Other studies have implicated ceramides as intracellular lipids that may also block insulin-signaling pathways⁷². Thus, the regulation of cytosolic signaling lipids affects insulin actions and hepatic gluconeogenesis independent of hepatocellular insulin signaling.

What is known about the structure and function of cholesterol-enriched LDs within atherosclerotic plaques?

Because free cholesterol cannot accumulate safely in cells, as its content increases, progressively more is esterified to cholesteryl ester (CE) and stored. Thus, macrophages in atherosclerotic plaques frequently become foamy in appearance because of the accumulation of CE that eventually concentrates in LDs. CE formation is mediated by the ACAT (acyl-CoA cholesterol acyltransferase) enzymes⁷³. Classic studies by Brown and Goldstein showed that like TAG-rich LDs in other tissues, there is a dynamic turnover of CE in macrophage foam cells⁷⁴. While there are candidate neutral CE hydrolases, controversy persists over which enzyme hydrolyzes CE in different species⁷⁵. Macrophage formation of LDs appears to be cytoprotective. Deletion of ACAT1 from macrophages in LDL-receptor deficient mice prone to develop atherosclerosis results in increased macrophage lipotoxicity and atherogenic load⁷. Conversely, limiting cholesterol accumulation, for instance by reducing the absorption of dietary cholesterol in ACAT2 deficient mice, protects from atherogenesis⁷⁶. Additionally, oxidized CE have been found to accumulate in human atherosclerotic plaques⁷⁷. Over 50 unique lipid products derived from cholesteryl-linoleate alone, accounting for 20 to 90% of the lipid mass in plaques, have been characterized⁷⁷. Recent reports suggest that some oxidized CE can exert untoward biological activity⁷⁸. TAGs are also found in atherosclerotic plaques from humans and animals, and are very likely to comprise part of the LDs in macrophages as admixtures in a liquid crystalline, isotropic state⁷⁹. In cell culture, the efflux of cholesterol differs from pure CE-LDs vs. CE/TAG-LDs^{79, 80}, with higher rates in the latter. LDs are thought to be not only storage containers in macrophage foam cells, but also direct and indirect contributors to the pathology of progressing plaques^{81, 82}.

Vascular smooth muscle cells (VSMCs) can also become foam cells in atherosclerotic plaques. Recent research has shown that these foam cells can become macrophage-like in their phenotype^{83, 84} and can comprise in excess of 30% of the cells identified as macrophages with standard immunostaining in human and murine plaques⁸⁵. In vitro, VSMC transdifferentiation to a macrophage-like state can be accomplished by loading the cells with cholesterol, which accumulates in CE-LDs^{83, 84}. It is unknown whether it is the free cholesterol pool or the CE-LD that is integral in the transdifferentiation of VSMCs to macrophage, and the regulatory process involved is undefined.

What do we know about the cellular metabolism of LDs?

LDs make dynamic contacts with nearly every other organelle in the cell⁸⁶. These contacts are thought to mediate transfer of lipids (including phospholipids, sterols, and fatty acids) between the different cellular compartments^{87, 88}. Contact sites between lipid droplets and other organelles may allow for metabolic channeling, since various steps in lipid anabolic and catabolic pathways may occur in different organelles, including the ER, Golgi, plasma membrane, lysosomes, mitochondria, and peroxisomes. Recently, progress has been made in identifying some of the proteins that mediate LD-organelle membrane contacts^{87, 88}. For example, FATP1 and DGAT2 at the ER-LD interface are involved in LD expansion⁸⁹, while PLIN5 has been implicated in forming LD-mitochondria contacts and in channeling fatty acids from LDs to mitochondria in liver and skeletal muscle⁹⁰. However, for most LD-organelle contacts, the proteins and/or lipids involved have not been identified. In addition, in many cases it is unclear what types of lipids are transferred and what the directionality of transfer is at these contact sites. To answer these questions, tools for visualizing lipid trafficking in live cells will be necessary.

Studies to assess TAG storage as well as the formation and dissolution of LDs in the heart require sophisticated nuclear magnetic resonance (NMR) methods and stable isotope kinetics. As discussed in an earlier section, turnover of the TAG pool in cardiomyocytes is intimately linked to expression of peroxisome proliferator-activated receptor (PPAR)α target genes Stable isotope-based measurements of TAG turnover within cardiomyocytes of the intact, functioning heart have been performed on rat hearts subjected to chronic pressure overload^{5, 6}. Measurements from pathologically hypertrophied hearts revealed reduced TAG content and turnover, associated with reduced expression of PPARα target genes. A consequence of the reduction in TAG dynamics is the accumulation of a lipotoxic acyl-derivative, the 16-carbon species of ceramide, which is consistent with measurements made in sampled human failing hearts^{6, 91}. However, dietary LCFAs can modulate heart TAG content, with monounsaturated oleate (18:1) supporting higher TAG dynamics than saturated palmitate (16:0)⁶. Oleate-dependent changes in TAG turnover were associated with either attenuated losses or normalized expression levels of metabolic enzymes induced by PPARα activity.

Conclusions

Summary and Future Directions

Current research is mostly focused on the basic biology of LDs and the elucidation of the roles of LDs in energy storage. Although much emphasis has been placed on understanding how LDs function in adipocytes and hepatocytes, LDs are known to be present in almost every cell type, where they may interact (in a cell-type specific manner) with various proteins to exert broader functions beyond lipid storage. Indeed, it is appreciated that the composition and turnover of lipids in LDs is an active component of metabolic signaling within both cardiomyocytes and endothelial cells, and mediates the cellular response to pathological stress. The NHLBI working group concluded that a better understanding of key regulatory pathways and functions of LDs in different cell types may yield mechanistic insights into the pathological processes that lead to ectopic lipid storage, insulin resistance, inflammation, and associated CVD. To guide research in exploring the roles of LDs in CVD, the working group members identified gap areas and recommended research opportunities to investigate the roles of LDs in the pathogenesis of obesity– and metabolic syndrome–related CVD. Some of these gap areas are described below and in Table 1; a complete list of research opportunities that were identified by the working group can be found online (https://www.nhlbi.nih.gov/events/2017/role-lipid-droplets-obesity-and-metabolic-syndrome-related-cardiovascular-diseases).

Table 1.

Research Opportunities Recommended by the Working Group

Study biological functions and physiological roles of LDs in the cardiovascular system

Understand the capacity of cells and organs to store metabolic energy and how this capacity is regulated under physiological conditions.

Comprehensively assess the key players involved in LD biology across cell types using unbiased screening approaches

Understand the interaction of LDs with other intracellular organelles

Understand tissue-specific fluxes in energy under physiological conditions

Understand the impact of diet and life-style on LD biology

Understand sex, race/ethnic, and age-dependent differences in LD biology that influence cardiovascular health

Understand how individual variations in LD biology may contribute to differences in cardiovascular health among populations and resilience to CVD

Investigate pathophysiological roles of LDs

Understand what happens when a cell exceeds its capacity to store lipid in LDs

Understand how abnormalities in LD function contribute to atherosclerosis, hypertension, cardiomyopathy, heart failure, and obesity- or metabolic syndrome-related CVD

Understand the mechanisms linking LDs to inflammation and insulin resistance

Understand the role of LDs in regulating gene transcription and RNA epigenetics

Understand how genetic and environmental factors alter LDs homeostasis and lead to pathological conditions

Understand how individual variations in LD biology contribute to differences in onset and progression of CVD, as well as to different responses to medications

Understand changes in LD composition and dynamics in response to metabolic stress

Develop specialized methodologies, workforce, and shared resources

Develop and validate novel imaging techniques and probes that permit monitoring of spatial and temporal lipid trafficking in cells or tissues in situ in real-time with minimal perturbation to the physiological actions of the lipids

Establish resource centers to provide services to all investigators and develop research protocols for the characterization of lipid content within LDs and proteins associated with LDs using multidisciplinary approaches

Encourage collaboration and resource sharing among investigators with diverse expertise, including biochemistry and chemistry, structural biology, mass spectrometry, NMR, cardiovascular physiology, epidemiology, genetics, and bioinformatics.

What are the function, composition, and structure of LDs?

One fundamental gap in knowledge is the biological roles of LDs in the cardiovascular system. More research is needed to understand the similarity and differences in function, composition and structure of LDs in the cardiovascular system compared with LDs in adipose tissue and liver. Once we know the key components, regulators, and interactions of LDs in cardiovascular cells and tissues, we will have a better understanding of the physiological and pathological roles of LDs in the cardiovascular system. To address this unmet need, a combination of methods and tools will be needed to analyze LDs in cardiovascular tissues and relevant cell types, including structural information of the enzymes and key players, metabolic flux analysis (mass spectrometry), measurements of the dynamic composition of LDs using proteomics/lipidomics, and new imaging approaches. One critical challenge is the lack of reliable methodologies to identify and quantify intracellular lipids and proteins associated with LDs. Modern techniques in mass spectrometry can provide information about the concentration of abundant lipids and the minor lipid species that may be active in picomolar or nanomolar concentrations. Stable isotope dilution quantitation by mass spectrometry has unsurpassed precision and accuracy in lipid analysis. Lipidomic analysis based on tandem mass spectrometry has emerged as a powerful strategy to reveal changes in the hundreds of neutral lipids and phospholipid species found in LDs. An additional valuable technique is NanoSIMS (nanoscale secondary ion mass spectrometry) that combines imaging of cells or tissues with mass spectrometry, which has been used to study the movement of fatty acids across capillaries and the distribution of cholesterol in the plasma membrane^{92, 93}. It is important to validate the initial hits from discovery study to differentiate contaminants from true LD-associated proteins or lipids. Developing and sharing methodologies and best practices of isolating LDs and establishing a LD protein and lipid databases will be critical to the field.

How is LD size regulated? What do we know about the cellular metabolism of LDs?

The size of LDs changes in response to physiological stimuli. Understanding how the formation and catabolism of LDs are regulated will shed light on the mechanisms underlying the transition between adaptive lipid storage and lipotoxicity. One major hurdle is the lack of tools to study the dynamics of LDs in vivo in real time. Strategies that have been used to visualize lipid trafficking within cells, including naturally fluorescent lipids, fluorophore-conjugated lipids, and lipid-binding probes (such as proteins, toxins, and antibodies⁹⁴), have limitations. Naturally fluorescent lipids are not very bright or photostable, while labeling lipids with fluorophores tends to alter their physical properties and metabolism. In addition, for many classes of lipids, no specific probes are currently available. Therefore, the development of new probes that minimally disrupt lipid function, as well as further validation of existing tools, will greatly advance the field. The analysis of intracellular lipolysis will also benefit from development of chemical and genetic probes for manipulating and monitoring critical temporal and spatial domains. Importantly, recent work indicates that elements of the core lipolysis pathway are highly tractable targets for chemical/pharmacological manipulation^{27, 95, 96}. Because the core lipolysis machinery is highly conserved and exhibits co-evolution among regulatory elements, comparative analysis across species is likely to lead to new insights and understanding of this important pathway^97–99.

What is the role of LDs in the heart?

Greater understanding of the biology of LD formation, its relationship with cellular levels of toxic lipid intermediates, and its role in cardiac responses to pathological processes are needed. The questions that are incompletely answered are the following: What controls uptake of lipids into the heart and the targeting of lipids to oxidation versus storage? Do membrane-initiated signaling events influence LD phospholipid remodeling, LD trafficking or turnover? Are LDs a necessary intermediate for myocardial fatty acid oxidation? How do fatty acids traffic from LDs to mitochondria? Does increased fatty acid oxidation improve or worsen the response to ischemia and increased afterload, and do the effects differ in acute compared to chronic situations? The biological pathways leading to both pathological and physiological (protective) LD formation need to be defined. How do LDs in healthy and unhealthy states differ in composition? These LDs likely have different effects on hearts that are stressed with greater afterload and ischemic insults. Finally, how do these two types of LDs differ in pathological conditions such as diabetes, obesity, and underlying cardiac diseases?

How do LDs interact with membrane and intracellular signaling pathways?

LD are important hubs that physically interact with numerous organelles⁸⁷. In addition to LD formation, ER interactions have been implicated in ER stress and quality control during protein synthesis. Furthermore, the interface between LD and mitochondria appears to form a specialized synapse in cardiomyocytes that provides tight control of fuel mobilization and oxidation for ATP production. LD are also known to associate with peroxisomes, lysosomes and nuclei in various experimental systems, although the nature of these interactions and their functional significance in the cardiovascular system has not been addressed. A more intense focus on the signaling, metabolite trafficking, and protein-protein interaction between LD and various organelles is critical for uncovering the physiological role of LD and the impact of these processes on CVD. Such analyses will require development and implementation of high-resolution techniques that establish the relationships among organelles, as well as novel molecular probes that directly assess dynamic nutrient flux and protein interactions in living cells.

What is known about the structure and function of cholesterol-enriched LDs within atherosclerotic plaques?

While important features of cholesterol-enriched LDs have been discovered, there are opportunities to expand the molecular information about the required proteins/signaling cascades and their regulation. The major enzyme that esterifies cholesterol to CE in CE-LDs is well characterized (ACAT), but there is still controversy over the neutral CE hydrolase that mediates the reverse reaction. Moreover, not all lipids in foam cell LDs are likely to be CE. Atherosclerotic plaques also have a substantial amount of TAG. What is the relative contribution of each neutral lipid class to foam cell formation? What are the roles of other lipids and proteins, found within other LDs, in foam cell LD biology? Does the composition (lipids and proteins) of macrophage foam cell LDs influence the ability to efflux cholesterol or inflammatory activation? The current methods, using neutral lipid staining of tissue or lipid extracts of aortic homogenates will not adequately address these issues. Higher resolution imaging and analytical methods applied to specific cell types under conditions of plaque progression and regression are needed. Both macrophages and smooth muscle cells become foam cells but are the LDs in these cells different?

Non-standard Abbreviations and Acronyms

LD

lipid droplet

TAG

triacylglycerol

DAG

diacylglycerol

CVD

cardiovascular disease

CE

cholesteryl ester

PLIN

perilipin

NEFA

non-esterified fatty acids

PC

phosphatidylcholine

VSMC

vascular smooth muscle cells

ATGL

adipose triglyceride lipase

ABHD5

alpha-beta hydrolase domain-containing 5

ASCLs

acyl CoA synthetase

FATPs

fatty acid transport proteins

LCFAs

long chain fatty acids

DGAT-1

DAG acyltransferase

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

ACAT

acyl-CoA cholesterol acyltransferase.