Lipid metabolism and signaling in cardiac lipotoxicity.

Lipid metabolism and signaling in cardiac lipotoxicity Kenneth D’Souza, Carine Nzirorera, Petra C. Kienesberger PII: DOI: Reference:

S1388-1981(16)30043-9 doi: 10.1016/j.bbalip.2016.02.016 BBAMCB 57911

To appear in:

BBA - Molecular and Cell Biology of Lipids

Received date: Revised date: Accepted date:

30 December 2015 19 February 2016 19 February 2016

Please cite this article as: Kenneth D’Souza, Carine Nzirorera, Petra C. Kienesberger, Lipid metabolism and signaling in cardiac lipotoxicity, BBA - Molecular and Cell Biology of Lipids (2016), doi: 10.1016/j.bbalip.2016.02.016

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ACCEPTED MANUSCRIPT Heart Lipid Metabolism

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BBA Molecular and Cell Biology of Lipids

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Lipid metabolism and signaling in

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cardiac lipotoxicity

Kenneth D’Souza1, Carine Nzirorera1, and Petra C. Kienesberger1,*

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Dalhousie Medicine New Brunswick, Department of Biochemistry and Molecular Biology, Dalhousie University, 100 Tucker Park Road, Saint John, New Brunswick, Canada

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Address correspondence to: Petra C. Kienesberger, Dalhousie Medicine New Brunswick,

Department of Biochemistry and Molecular Biology, Dalhousie University, 100 Tucker Park Road, Saint John E2L 4L5, New Brunswick, Canada. Tel: +1-506-636-6971; Fax: 506-6366001. E-mail: [email protected]

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ACCEPTED MANUSCRIPT BBA Molecular and Cell Biology of Lipids

Heart Lipid Metabolism

Abstract The heart balances uptake, metabolism and oxidation of fatty acids (FAs) to maintain ATP

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production, membrane biosynthesis and lipid signaling. Under conditions where FA uptake

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outpaces FA oxidation and FA sequestration as triacylglycerols in lipid droplets, toxic FA metabolites such as ceramides, diacylglycerols, long-chain acyl-CoAs, and acylcarnitines can

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accumulate in cardiomyocytes and cause cardiomyopathy. Moreover, studies using mutant mice

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have shown that dysregulation of enzymes involved in triacylglycerol, phospholipid, and sphingolipid metabolism in the heart can lead to the excess deposition of toxic lipid species that

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adversely affect cardiomyocyte function. This review summarizes our current understanding of lipid uptake, metabolism and signaling pathways that have been implicated in the development

Highlights

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ischemia-reperfusion.

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of lipotoxic cardiomyopathy under conditions including obesity, diabetes, aging, and myocardial

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Cardiomyocyte lipid overload can have toxic effects and cause cardiac dysfunction.

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Cardiac lipotoxicity is observed during pathophysiological conditions including obesity, insulin resistance, diabetes, aging, and myocardial ischemia-reperfusion.

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Studies using animal models have uncovered mechanisms of cardiac lipotoxicity.

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Proteins involved in FA uptake and oxidation as well as TAG, sphingolipid, and phospholipid metabolism have been implicated in cardiac lipotoxicity.

Keywords Heart; Cardiomyopathy; Lipids; Lipotoxicity; Obesity; Diabetes

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Abbreviations ACSL1, long-chain acyl-CoA synthetase 1

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ATGL, adipose triglyceride lipase

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CerS5, ceramide synthase 5 CPT, carnitine palmitoyltransferase

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DAG, diacylglycerol

ER, endoplasmic reticulum

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ERK, extracellular signal regulated kinase

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DGAT, diacylglycerol acyltransferase

FA, fatty acid

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FABPpm, plasma membrane isoform of fatty acid binding protein FoxO1, forkhead box protein O1

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HSL, hormone-sensitive lipase IκB, Inhibitor of NFκB IKK, IκB kinase

JNK, c-Jun N-terminal kinase

iPLA2, calcium-independent phospholipase A2 LPA, lysophosphatidic acid LPC, lysophosphatidylcholine LPL, lipoprotein lipase MAPK, mitogen activated protein kinases MLK3, mixed lineage kinase-3 MFBD, milk fat-based high fat diet NFκB, nuclear factor-κB 3



PGC1, peroxisome proliferator-activated receptor γ-coactivator 1 PIP2, phosphatidylinositol-4,5-bisphosphate

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PKC, protein kinase C

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PP2A, protein phosphatase 2A PPAR, peroxisome proliferator-activated receptor

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ROS, reactive oxygen species

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SAPK, stress activated protein kinase S1P, sphingosine-1-phosphate

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TAG, triacylglycerol TNFα, tumor necrosis factor α

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TLR4, toll-like receptor 4

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VLDLR, very low density lipoprotein receptor

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1. Introduction Lipotoxicity is defined as the process where excess accumulation of lipids and over-

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activation of lipid signaling pathways trigger cellular distress and dysfunction, which may

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manifest as insulin resistance, defective mitochondria, energy starvation, and endoplasmic reticulum stress and may ultimately lead to apoptotic cell death or lipoapoptosis (Figure 1) (1).

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When this process occurs in cardiomyocytes, the consequences can be cardiac dysfunction and

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heart failure, which is also referred to as lipotoxic cardiomyopathy (1,2). The archetypal conditions triggering a lipotoxic milieu within cardiomyocytes are metabolic disorders such as

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obesity, insulin resistance, and diabetes mellitus (3-6). In addition, cardiac lipotoxicity has also been observed during other patho/physiological conditions, e.g. myocardial ischemia-

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reperfusion/infarction and aging (7-9). Increased myocardial lipid deposition develops when cardiomyocyte fatty acid (FA) uptake outpaces FA oxidation, leading to an increased availability

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of FAs for non-oxidative metabolic pathways and accumulation of cardiotoxic FA metabolites (3). In the case of obesity, insulin resistance, and diabetes the main culprit is excess FA delivery due to augmented dietary fat intake and adipose tissue lipolysis (10). In addition, myocardial insulin resistance and impaired glucose utilization may further increase myocardial FA uptake and accumulation of toxic lipid species (11). While insulin resistance is likely a key contributing factor to obesity and diabetes-induced lipotoxic cardiomyopathy by causing a shift in cardiac substrate utilization from glucose to FAs and concomitant FA overload, it is also a consequence of cardiac lipotoxicity since toxic FA metabolites can impair myocardial insulin signaling (12,13). Due to its limited ability to synthesize FAs, the myocardium relies heavily on the uptake of FAs in the form of albumin-bound free FAs or lipoproteins - chylomicrons and very-low-

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density-lipoproteins - from the circulation to sustain cardiomyocyte lipid homeostasis and energy metabolism (14). In fact, uptake of FAs is a major determinant of FA oxidation and therefore

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mitochondrial ATP production (15-17). Although FAs are the principal energy substrate for the

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healthy adult heart, cardiomyocytes are omnivorous, deriving their ATP for contractile work from a variety of sources including glucose, lactate, and amino acids, allowing for metabolic

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flexibility to adjust substrate utilization to circulating substrate concentrations, work load,

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hormonal stimuli and oxygen supply (3,18,19). In contrast, diseased hearts often have decreased metabolic flexibility, which can contribute to cardiac dysfunction and lipotoxic cardiomyopathy

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(19-21).

To date, numerous rodent models have been generated and studied that were instrumental

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for the characterization of lipotoxic cardiomyopathy and using which lipid metabolic and signaling pathways were identified that mediate cardiomyocyte lipotoxicity (for a list of mouse

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models of cardiac lipotoxicity see Goldberg et al. (3)). In addition, it has also been revealed that the fruit fly can exhibit lipotoxic cardiomyopathy when fed a high fat diet, suggesting that the molecular mechanisms that lead to toxic lipid overload of the myocardium are evolutionarily conserved (22). It has become evident that chronically increased accumulation of certain FA metabolites in cardiomyocytes and the ensuing pathological sequelae are sufficient to precipitate cardiac dysfunction in these animal models. The FA metabolites linked to cardiac lipotoxicity include

ceramides,

diacylglycerols

(DAGs),

long-chain

acyl-CoAs,

acylcarnitines,

lysophospholipids, and triacylglycerols (TAG), although it is believed that the latter represents a marker of lipotoxicity rather than being directly cytotoxic (Figure 2) (4,23). Changes in FA uptake, storage, and/or oxidation in animal models of lipotoxic cardiomyopathy influence more than one lipid metabolism and signaling pathway, which renders it difficult to pinpoint a specific

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FA metabolite causative for cardiomyocyte dysfunction. It is unlikely to be the excess deposition of a single FA metabolite and over-activation of a single lipid signaling pathway, but rather a

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combination of these that triggers cardiac dysfunction in animal models of lipotoxic

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cardiomyopathy.

The etiology and functional relevance of cardiac lipotoxicity is less clear in the human

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heart when compared to animal models of lipotoxic cardiomyopathy. In this regard, the

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development of proton magnetic resonance spectroscopy for the noninvasive quantification of intramyocardial TAG in vivo was an important contribution towards the understanding of the

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relationship between increased cardiac lipid deposition and cardiac function in humans (24-27). Using this technique, it has been demonstrated that increased myocardial TAG deposition is not

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only associated with but precedes cardiac dysfunction in humans with type 2 diabetes mellitus (28) and correlates with body mass index (29,30). These findings indicate that there is a

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causative relationship between cardiac lipid accumulation and impaired myocardial contractility. Moreover, it has been suggested that cardiac steatosis and ensuing lipotoxic cardiomyopathy are widespread and clinically highly relevant given the global rise in the prevalence of obesity and metabolic complications (30).

It is currently poorly understood whether the cardiac accumulation of lipids other than TAGs, which are believed to contribute to cardiac lipotoxicity in animal models, are altered in the human heart during obesity, insulin resistance, diabetes, and other conditions associated with lipotoxic cardiomyopathy. Nevertheless, it is likely that increased TAG levels in the human heart are a marker for overall increased cardiac content of toxic lipid metabolites and the dysregulation of lipid metabolism and signaling pathways negatively impacting cardiac metabolism and function. The goal of this review is to provide an overview of metabolic pathways and proteins

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involved in the regulation of cardiomyocyte lipid levels and signaling that have been implicated in the development of lipotoxic cardiomyopathy.

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2. Cardiac fatty acid delivery and uptake

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The major sources of FAs for cardiomyocytes are albumin-bound free FAs and FAs released from lipoprotein TAGs either at the coronary lumen via lipoprotein lipase (LPL) or following

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receptor-mediated endocytosis. Uptake of these FAs occurs either via passive diffusion or

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protein carrier-mediated transport by fatty acid translocase (FAT)/CD36, the plasma membrane isoform of fatty acid binding protein (FABPpm), and fatty acid transport protein (FATP) 1/4/6

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(Figure 2)(18). Using positron emission tomography imaging, it has been demonstrated that increased postprandial FA uptake in hearts from overweight and obese subjects with impaired

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glucose tolerance is associated with early impairment of left ventricular function and increased myocardial oxidative metabolism (31). The latter findings indicate that chronically enhanced

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myocardial FA uptake in the setting of obesity precipitates lipotoxic cardiomyopathy in humans. A number of studies using rodent models have provided more mechanistic insight into how increased myocardial FA uptake can lead to excess accumulation of lipid metabolites and trigger lipotoxic cardiomyopathy (32-34). Cardiac FA transport was enhanced in obese-diabetic Zucker rats, which was paralleled by an increase in sarcolemmal FA transporters CD36 and FABPpm (35). Cardiomyocytes from obese-diabetic Zucker rats displayed increased CD36-mediated longchain FA uptake and TAG accumulation that preceded contractile dysfunction, suggesting that enhanced sarcolemmal expression of CD36 promotes diabetes-mediated cardiac lipotoxicity (36,37). Moreover, adult rat cardiomyocytes displayed increased sarcolemmal CD36 protein expression upon incubation with media mimicking obese/diabetic conditions (high insulin, high palmitate concentrations), which was associated with enhanced basal palmitate uptake, lipid

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accumulation, and decreased sarcomere shortening (38). Inhibition of CD36 in these cells protected from increased FA uptake, TAG deposition, and contractile dysfunction (38),

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demonstrating that CD36-mediated FA uptake plays a key role in cardiomyocyte lipotoxicity

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triggered by an obese/diabetic milieu.

CD36 has also been implicated in aging-induced lipotoxic cardiomyopathy (8). Aged

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mice displayed increased cardiac CD36 expression when compared to young mice, which was

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paralleled by increased cardiac lipid accumulation, decreased cardiac function, and hypertrophic remodeling (8). CD36 deficient mice were resistant to aging-induced lipotoxic cardiomyopathy

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(8). A role of CD36 in facilitating lipotoxic cardiomyopathy has also been shown in mice with cardiomyocyte-specific over-expression of peroxisome proliferator-activated receptor α

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(PPARα) (39), a lipostat which increases transcription of an array of genes involved in FA

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transport and utilization in the diabetic heart (40). Chronic activation of PPARα in hearts of these

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mice augmented FA uptake that exceeded FA oxidation resulting in increased myocardial lipid (TAG) deposition and systolic dysfunction, which was exacerbated by feeding a high fat diet rich in long-chain FAs or induction of diabetes (33,40). CD36 deficiency protected against cardiac steatosis and cardiomyopathy in PPARα over-expressing mice, suggesting that inhibition of CD36 may be an effective therapeutic intervention for diabetes-related lipotoxic cardiomyopathy (39). Indeed, because of its key role in cardiac FA uptake and lipotoxicity, CD36 is being explored as therapeutic target to ameliorate lipotoxic cardiomyopathy and insulin resistance via immunochemical inhibition of sarcolemmal CD36 and interference with the subcellular recycling of CD36 (41). Similar to the deletion of CD36, deficiency of LPL rescued myocardial TAG accumulation and contractile dysfunction in PPARα over-expressing mice (42). While these studies show that CD36 and LPL are required for lipotoxic cardiomyopathy in PPARα

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over-expressing hearts, the identity of the toxic FA metabolite(s) mediating these effects remains unclear.

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In addition to PPARα over-expression, forced expression of FATP1 specifically in

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cardiomyocytes resulted in increased FA uptake and lipotoxic cardiomyopathy that was associated with increased accumulation of free FAs but not TAGs, and diastolic dysfunction

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(32). Cardiac FA uptake was also upregulated indirectly by over-expressing long-chain acyl-CoA

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synthetase 1 (ACSL1), leading to increased deposition of TAGs, ceramides, and certain phospholipid species, lipoapoptosis and systolic dysfunction (2). Moreover, an important role in

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lipotoxicity following myocardial infarction has been ascribed to the very-low-density lipoprotein receptor (7). Specifically, decreasing FA uptake via very-low-density lipoprotein

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receptor deficiency protected from ischemia-induced TAG accumulation, endoplasmic reticulum stress, and apoptosis and improved survival in mice subjected to myocardial infarction (7).

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Taken together, these findings show that multiple proteins involved in cardiomyocyte FA uptake are implicated in the development of cardiac lipotoxicity and cardiomyopathy during conditions including obesity/diabetes, aging, and myocardial infarction.

3. TAG synthesis and mobilization It has been reported that a significant proportion, if not the majority, of FAs that enter the cardiomyocyte are shuttled through the intracellular TAG pool for temporary storage prior to oxidation (43). While TAGs per se are generally believed to be non-toxic, sequestration and release of FAs via intracellular TAG metabolism is likely a major determinant of toxic FA and FA metabolite accumulation (Figure 2). The concept that TAG accumulation can protect from FA-induced lipotoxicity by sequestering FAs that could otherwise stimulate adverse signaling

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events was first established by Listenberger et al. (44) using non-cardiac cells. Specifically, while incubation of cells with excess palmitate, a saturated FA which poorly incorporates into

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TAGs, led to lipotoxic cell death, co-incubation with the unsaturated FA oleate mitigated

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palmitate-induced lipotoxicity by diverting palmitate away from apoptosis-stimulating pathways into the TAG pool (44). Furthermore, incubation of cells with oleate induced lipotoxicity when

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TAG synthesis was impaired (44). These studies show that sequestration of FAs in TAGs can

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rescue lipotoxic effects of saturated FAs. A recent study by Bosma et al. (45) highlights that this concept also applies to cardiomyocytes. In line with this notion, cardiac-specific over-expression

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of diacylglycerol acyltransferase (DGAT) 1, which catalyzes the final step in TAG synthesis, increased cardiac TAG content ~2-fold and decreased the accumulation of diacylglycerols

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(DAGs), ceramides, and free FAs without affecting heart function at baseline (46). The cardiac phenotype of DGAT1 over-expressing mice is reminiscent of changes in cardiac lipid

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accumulation that occur following exercise (46). Furthermore, forced DGAT1 expression ameliorated lipotoxic cardiomyopathy in mice with cardiomyocyte-specific over-expression of either ACSL1 or PPARγ, which was associated with decreased cardiac DAG and ceramide levels, but unchanged TAG accumulation (46,47). However, a study using another cardiacspecific DGAT1 over-expression model reported that DGAT1-induced cardiac steatosis was associated with cardiac dysfunction, as was evidenced primarily by impaired diastolic function (48). Diastolic dysfunction was accompanied by increased cardiac fibrosis and decreased mitochondrial biogenesis (48). Despite increased myocardial TAG levels, cardiac content of free FAs and DAGs was unchanged in this model (48). The reasons for the divergent effects of DGAT1 over-expression on myocardial lipid levels, most notably free FAs and DAGs, and cardiac function are presently unclear, but may involve differences in background strain or

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transgene construct design. Cardiomyocyte-specific DGAT1 knockout mice generated by Lui et al. (49) developed heart failure and died prematurely (49). Hearts from these mice exhibited

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severely increased cardiac DAG and ceramide levels (49), suggesting that decreased

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sequestration of toxic FA metabolites in TAGs due to DGAT1 deficiency in the myocardium produces a lipotoxic milieu that contributes to heart failure. Interestingly, this was associated

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with increased PPARα and CD36 expression leading to enhanced myocardial FA uptake (49),

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suggesting that cardiomyocyte-specific DGAT1 deficiency not only reduces the ability to sequester toxic lipid metabolites in TAGs but also leads to increased FA uptake which further

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exacerbates lipotoxic cardiomyopathy. Surprisingly, global DGAT1 knockout mice did not exhibit cardiac lipotoxicity or dysfunction (50). Expression of PPAR and genes involved in FA

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utilization, including CD36, was reduced in the heart from DGAT1 knockout mice, which was accompanied by decreased myocardial FA oxidation and increased glucose uptake (50). These

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findings suggest that global DGAT1 deficiency leads to a shift in substrate utilization away from FAs to glucose, thus preventing lipotoxicity. The presence or absence of DGAT1 in the intestine may contribute to the differences in cardiac phenotypes observed between global versus cardiomyocyte-specific DGAT1 knockout mice (49,51). Intestinal DGAT1 stimulates dietary fat secretion out of enterocytes and therefore influences the availability of dietary fats in tissues including the heart (51). As such, mice expressing DGAT1 in the intestine only negated the protection from diet-induced hepatic steatosis and obesity in global DGAT1 knockout mice (51). Moreover, when cardiomyocyte-specific DGAT1 knockout mice were crossed with intestinal DGAT1 knockout mice, heart function and survival improved, which was associated with a reduction in the accumulation of DAGs and ceramides in the myocardium of double-knockout mice (49). DGAT1 expression in the heart is markedly decreased in humans with heart failure,

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which is associated with increased cardiac accumulation of DAGs and ceramides - reminiscent of cardiac-specific DGAT1 knockout mice - and reduced levels of TAGs and FAs (52). Unlike

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the cardiomyocyte-specific DGAT1 knockout mouse, expression of PGC1α, CD36, and other

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proteins involved in lipid metabolism was decreased in the failing human heart (52). It is possible that the degree of DGAT1 inhibition (knockout in mice versus partial deficiency in

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humans), the duration of DGAT1 inhibition, developmental effects of DGAT1 deficiency in

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knockout mice, and/or species-dependant mechanisms contribute to this discrepancy. Compared to DGAT1, relatively little information is currently available about the role of

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glycerol-3-phosphate acyltransferases (53) and lipins/phosphatidate phosphatases (54), which are key enzymes in glycerolipid biosynthesis upstream of DGAT, in modulating myocardial lipid

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levels and lipotoxic cardiomyopathy. Lipin1 was identified as the major lipin isoform in the heart

energy

metabolism

and

lipotoxicity.

Lipin1

expression

and

phosphatidate

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cardiac

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and was found to be regulated by PGC1α/estrogen related receptor (55), important modulators of

phosphohydrolase activity were downregulated in the failing heart from TG9 mice (55). Interestingly, lipin mRNA expression and phosphatidate phosphohydrolase 1 activity was also reduced in Zucker diabetic fatty rats and type 2 diabetic patients (56). Insulin therapy led to increased lipin expression and phosphatidate phosphohydrolase 1 activity (56). Likewise, phosphatidate phosphohydrolase 1 and 2 activity was decreased in the heart from obese-insulin resistant JCR:LA corpulent rats (57). Taken together, these studies suggest that lipins may be involved in lipotoxic cardiomyopathy induced by insulin resistance-diabetes. The first and rate-limiting step in TAG hydrolysis in the heart is mediated by adipose triglyceride lipase (ATGL/PNPLA2) (58,59). Loss of ATGL function in mice either via deletion of ATGL or its co-activating protein CGI-58 led to massive cardiac TAG accumulation and

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precipitated heart failure (58-60). Cardiac steatosis was accompanied by fibrotic and hypertrophic remodeling and a depression of mitochondrial FA oxidation in ATGL knockout

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mice (58,59). The latter was due to a reduction in PPARα and PPARγ-coactivator (PGC) 1 activation in hearts from constitutive whole body and muscle-specific ATGL deficient mice (58),

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although a drastic decrease in PPARα signaling was not obvious in inducible cardiomyocyte-

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specific ATGL deficient mice (59). It is likely that, irrespective of changes in lipid signaling,

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mitochondrial function and metabolism that compromise cardiac contractility, the excessive deposition of TAG-filled lipid droplets per se in ATGL deficient hearts is mechanically

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obstructing and impairs proper contractile functioning of cardiomyocytes. Mutations in the ATGL/PNPLA2 gene have also been linked to heart failure in humans, constituting a new clinical

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entity termed “triglyceride deposit cardiomyovasculopathy” (61,62). TAG content in hearts from humans with defective ATGL is also massively (~upwards of 30-times) increased (62), similar to

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mice with ATGL deficiency. In contrast to ATGL knockout mice, patients with PNPLA2 mutations had increased expression of PPARα, PPARγ, and CD36 in the heart, suggesting that enhanced lipid uptake and TAG synthesis further augment myocardial TAG deposition and contribute to heart failure in these individuals (62). It is presently unclear whether levels of lipids other than TAGs, which are typically associated with lipotoxicity, are changed in ATGL deficient mice and patients with mutated PNPLA2. These studies suggest that although TAGs can sequester toxic FAs and lipid intermediates in lipid droplets, massive TAG accumulation in the heart beyond a certain threshold may also lead to insufficient mechanical contraction and contribute to heart failure. While lack of ATGL function leads to heart failure in mice and humans, mice with cardiac-specific over-expression of ATGL were protected from type 1 diabetes-induced

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augmentation of cardiac TAG content and lipotoxic cardiomyopathy (63). Constitutive ATGL over-expression not only prevented an increase in myocardial TAG deposition in streptozotocin-

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diabetic mice, but also prevented diabetes-induced increases in palmitoyl-CoA and ceramide

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accumulation and reduced PPARα and PPARα target gene expression (63). Interestingly, in human cardiomyocyte-derived AC16 cells, adenoviral overexpression of ATGL reduced

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intracellular TAG storage and increased free FA levels, which was associated with stimulation of

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ER stress (45); these findings suggest that acute ATGL activation can trigger lipotoxicity in cardiomyocytes due to the inability of cells to sequester FAs in lipid droplets (45) while chronic

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ATGL activation in vivo protects from lipotoxicity likely due to counter-regulatory downregulation of PPARα and overall lipid utilization (63-65). Similar to chronic ATGL

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overexpression in type 1 diabetic mice, cardiac-specific over-expression of hormone-sensitive lipase (HSL), which hydrolyzes predominantly DAGs and, to a lesser extent, TAGs, prevented

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cardiac steatosis, fibrosis, and cardiomyopathy due to streptozotocin-induced diabetes (66). Moreover, it has been demonstrated using a high fat diet-fed Drosophila obesity model that cardiac-specific over-expression of the ATGL ortholog, Brummer lipase, prevents obesityinduced cardiac TAG accumulation and cardiac dysfunction (22). A similar effect was also achieved upon inhibition of lipogenesis and stimulation of lipolysis via impairment of the insulin-target of rapamycin pathway in hearts from obese Drosophila (22). These findings suggest that regulation of cardiac TAG catabolism via ATGL and HSL modulates cardiac lipotoxicity under conditions of obesity and diabetes, although the precise mechanisms for this effect remain to be fully uncovered. Recent studies also showed that cardiomyocyte-specific over-expression of perilipin 5, a lipid droplet coating protein that connects lipid droplets with mitochondria and limits

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intracellular TAG catabolism, resulted in cardiac steatosis (67,68), reduced PPARα activation (67,68), mitochondrial dysfunction (68), left ventricular hypertrophy (67,68), and mild

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impairment of cardiac function (68). The absence of more severe cardiac TAG accumulation and

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dysfunction, similar to that observed following ATGL ablation, in mice with perilipin 5 overexpression has been ascribed to increased protein kinase A-mediated perilipin 5

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phosphorylation, allowing for some lipolysis to occur (69). Deficiency of perilipin 5 led to the

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depletion of cardiac TAGs due to excess lipolysis, increased cardiac FA oxidation, and formation of reactive oxygen species (ROS) that contributed to age-related cardiomyopathy, suggesting that

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perilipin 5 function needs to be finely balanced to maintain cardiac TAG homeostasis and protect from lipotoxicity (70).

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Apart from the aforementioned TAG metabolic pathways that are centered on the formation and degradation of cytosolic lipid droplets, the heart is also able to synthesize and

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secrete TAGs in the form of lipoproteins (14,71,72). This TAG secretion pathway is upregulated in the heart in diet-induced obesity via increased microsomal triglyceride transfer protein expression (71), which has been suggested to limit cardiac TAG accumulation. Moreover, activation of cardiomyocyte lipoprotein secretion via over-expression of apolipoprotein B abolished cardiac TAG accumulation in high fat diet-fed and streptozotocin-diabetic mice and protected against obesity/diabetes-induced cardiac dysfunction (71). Similarly, apolipoprotein B overexpression protected against lipotoxic cardiomyopathy induced by cardiomyocte-specific over-expression of glycosylphosphatidylinositol-anchored human lipoprotein lipase (LpLGPI) (73). Taken together, these studies suggest that the metabolism of TAGs in lipid droplets and TAG secretion via lipoproteins play a major role in the pathogenesis of lipotoxic cardiomyopathy.

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4. Mitochondrial FA metabolism

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To enable the transport of long-chain acyl-CoAs through the mitochondrial double-membrane,

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they are converted to acylcarnitines by carnitine palmitoyltransferase (CPT) 1 at the outer mitochondrial membrane (Figure 2). After passing the inner mitochondrial membrane,

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acylcarnitines are converted back to acyl-CoAs by CPT2 and subjected to β-oxidation for ATP

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production. CPT1β is the predominant CPT1 isoform in the heart (74). Although mice with heterozygous CPT1β deficiency exhibited unchanged myocardial FA oxidation and no overt

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cardiac phenotype at baseline, decreased FA oxidation and increased TAG and ceramide accumulation were evidenced following transverse aortic constriction-induced pressure overload

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(74). Additionally, cardiac hypertrophy, cardiomyocyte apoptosis, and cardiac dysfunction were

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exacerbated in these mice, suggesting that partial CPT1β deficiency causes myocardial

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lipotoxicity following hemodynamic stress (74). Mice with homozygous cardiac- and skeletal muscle-specific CPT1β deficiency died prematurely from heart failure even in the absence of hemodynamic stress (75). Hearts from these mice also exhibited massive hypertrophy and increased neutral lipid accumulation as was assessed by Oil Red O staining. Although this increase in lipid accumulation is indicative of lipotoxicity, levels of specific toxic lipids have not been assessed (75). While targeted CPT1β deletion in mice appears to cause lipotoxic cardiomyopathy (75), inhibition of CPT1 and CPT2 using the antianginal drug, perhexiline, led to improved left ventricular ejection fraction, resting and peak stress myocardial function, oxygen consumption and improved skeletal muscle energetics in patients with chronic heart failure (76). The proposed mechanism for this effect is that by inhibiting FA utilization, perhexiline induces a metabolic shift toward utilization of glucose and lactate for ATP

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production (76). Perhexiline treatment may thereby improve insulin sensitivity and oxygenefficiency in the failing heart (76). The discrepancy between the detrimental effect of CPT1β

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deletion in mice and the beneficial effect of pharmacological targeting of CPT1/2 using

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perhexiline in humans with heart failure could be due to differing degrees and duration of CPT1 inhibition, species-specific effects of CPT1 inhibition, non-specific effects of perhexiline and/or

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pre-existing cardiac morbidity (63). Notably, while short-term (8 days) inhibition of CPT1 using

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etomoxir did not result in excess cardiac lipid accumulation in rats, 10-day administration of etomoxir precipitated cardiomyopathy in mice (75,77,78).

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Cardiomyocyte-specific ablation of PPARδ, a transcriptional regulator of FA oxidation, resulted in decreased basal myocardial FA oxidation, which was accompanied by increased

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myocardial TAG deposition, cardiac hypertrophy and heart failure, suggestive of lipotoxic cardiomyopathy (79). Moreover, mice with deficiency of long-chain acyl-CoA dehydrogenase,

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which initiates FA β-oxidation, had increased myocardial TAG content at baseline and, more so, following a 24-h fast, and exhibited elevated cardiac ceramide levels and systolic dysfunction in the fasted state (80). Conversely, cardiomyocyte-specific over-expression of pyruvate dehydrogenase kinase 4, an inhibitor of pyruvate dehydrogenase, increased myocardial FA oxidation and activated cardiac expression of PGC1α, a regulator of mitochondrial biogenesis and function, and prevented increased TAG accumulation following short-term (4 weeks) high fat diet feeding (81). Taken together, these studies suggest that insufficient FA oxidation can contribute to lipotoxic cardiomyopathy by producing an environment wherein uptake of FAs outstrips their degradation and oxidative metabolism, leading to cardiomyocyte lipid accumulation.

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Apart from lipotoxic effects associated with impaired mitochondrial FA import and βoxidation, it has also been proposed that cardiac lipotoxicity can be induced by conditions where

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mitochondrial FA β-oxidation exceeds tricarboxylic acid cycle capacity and subsequent oxidative

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phosphorylation, leading to the accumulation of acylcarnitines (82,83). For instance, drastically increased myocardial acylcarnitine accumulation manifests during myocardial ischemia, wherein

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lack of oxygen supply and ensuing accrual of reducing equivalents prevent complete FA

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oxidation (82,83). While the increased conversion of mitochondrial FA β-oxidation intermediates may be initially protective, in that it prevents excess deposition of potentially cytotoxic acyl-

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CoAs and other lipid metabolites (84), acylcarnitines themselves have been shown to disrupt sarcolemmal integrity and electrophysiologic functions, possibly leading to myocardial

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arrhythmias (82,83), which suggests that acylcarnitines are potent cardiolipotoxins. It is important to note that during myocardial ischemia, accumulation of acylcarnitines reflects

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impaired FA oxidation; specifically, long-chain acylcarnitines are increased while short-chain acylcarnitines are reduced in the ischemic heart (85). On the contrary, obesity/diabetes results in an increase in the accumulation of both short- and long-chain acylcarnitines caused by enhanced myocardial FA delivery and mitochondrial FA import (83,86,87). Su et al. (83) observed a several-fold increase in the concentrations of acylcarnitines, particularly long-chain acylcarnitines and 3-hydroxy acylcarnitines, in the streptozotocin-diabetic rat heart, which could be reversed by insulin treatment (83). 3-Hydroxy acylcarnitine accumulation in the diabetic heart is suggestive of incomplete FA β-oxidation due to substrate overload and demonstrates that acylCoA and acylcarnitine pools can be reversibly converted (83). These findings also suggest that 3hydroxy acyl-CoA dehydrogenase is rate-limiting for myocardial β-oxidation (83). Interestingly, calcium-independent phospholipase A2 (iPLA2) activity was increased in the ischemic and

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diabetic heart and, by liberating FAs from phospholipids, was found to contribute substantially to the pool of FAs converted to myocardial acylcarnitines (83). Consistent with the notion that

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acylcarnitine accumulation is cardiotoxic, improvement of cardiac function in mice with cardiac-

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specific over-expression of PPARγ, which develop lipotoxic cardiomyopathy, by PPARα deletion was associated with decreased cardiac acylcarnitine levels (88). Surprisingly, these

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PPARα/PPARγ double-mutant mice exhibited increased FA oxidation and lipid droplet size,

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while cardiac TAG, DAG, and ceramide levels were unchanged when compared to the PPARγ over-expressors (88). The reasons for the paradoxical increase in FA oxidation and decrease in

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acylcarnitine concentrations upon superimposition of PPARα deficiency on the PPARγ transgenic background are puzzling. It is possible, however, that the double mutants have

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improved mitochondrial FA oxidation capacity, which limits incomplete FA oxidation events and acylcarnitine accumulation and thus ameliorates lipotoxic cardiomyopathy induced by

AC CE P

PPARγ over-expression.

Another transcription factor which regulates mitochondrial FA metabolism in the heart and which has more recently been linked to lipotoxic cardiomyopathy is forkhead box protein O1 (FoxO1) (89). FoxO1 activity is increased in the hearts from mice with diabetes induced either genetically or by high fat diet feeding (89). Cardiomyocyte-specific FoxO1 ablation prevented high fat diet-induced cardiomyopathy and cardiac insulin resistance, which was associated with reduced mitochondrial FA oxidation and increased glucose utilization (89). Deletion of FoxO1 also protected from myocardial lipid accumulation in mice with high fat diet-induced obesity, as was assessed by TAG measurements and Oil Red O staining of cardiac sections (89). In addition, overexpression of constitutively active FoxO1 in neonatal cardiomyocytes increased inhibitory serine phosphorylation of insulin receptor substrate 1, which impairs insulin signal transduction

20



(89). It is conceivable that FoxO1 contributes to obesity/diabetes-induced cardiac lipotoxicity by not only increasing myocardial insulin resistance and FA oxidation but overall FA uptake and

PT

utilization, leading to the accumulation of toxic lipid species.

RI

By producing ATP via oxidative phosphorylation, mitochondria are the principal source of ROS in cardiomyocytes (90). Leakage of electrons from complexes I and III in the electron

SC

transport chain leads to the continuous production of superoxide, which can react with nitric

NU

oxide to form the highly reactive peroxynitrite (90). Peroxynitrite and ROS then target phospholipids in the mitochondrial membrane to generate lipid peroxides (90). Cardiolipin, an

MA

abundant phospholipid in the mitochondrial membrane critically involved in mitochondrial function, is a primary target of lipid peroxidation since a large proportion of FAs contained in

TE

D

cardiolipin are unsaturated linoleic acid (90,91). Lipid peroxides that evade the antioxidant response can subsequently be converted to reactive aldehydes, which have the potential to

AC CE P

modulate membrane properties and impair the function of targeted proteins (90). Outcomes of lipid peroxidation product accumulation include calcium overload, increased mitochondrial uncoupling, and DNA mutagenesis, which can precipitate cardiac dysfunction. Increased accumulation of lipid peroxidation products has been observed in diabetic cardiomyopathy, myocardial infarction, and heart failure, suggesting that lipotoxicity induced by lipid peroxidation products may contribute to cardiomyopathies associated with increased oxidative stress (90). Further research is warranted to provide definitive evidence for a mechanistic link between myocardial lipid peroxidation product accumulation and lipotoxic cardiomyopathy.

5. Cardiomyocyte phospholipid metabolism and signaling

21



Phospholipids have vital biological roles as structural components of membranes, modulating the function of membrane-associated proteins, and signaling messengers. It is not surprising then

PT

that phospholipid and energy metabolism are interlinked and that alterations in phospholipid

RI

homeostasis can cause myocardial lipotoxicity (92-96). For example, the widely used antianginal and cardioprotective compound trimetazidine not only partially blocks FA oxidation by

SC

inhibiting 3-ketoacyl-coenzyme A thiolase, the final enzyme in β-oxidation, but also stimulates

NU

phospholipid synthesis and turnover (94). In doing so, trimetazidine may alter membrane properties and indirectly lower FA availability for mitochondrial oxidation by promoting

MA

sequestration of FAs in phospholipids (94). Interestingly, in the streptozotocin-diabetic rat heart, trimetazidine treatment protected from adverse remodeling of mitochondrial membrane FA

TE

D

composition and partially prevented a diabetes-induced decline in creatine kinase functional activity, suggesting that trimetazidine ameliorates diabetes-related mitochondrial dysfunction

AC CE P

and membrane lipotoxicity via sustained phospholipid homeostasis (94). A particularly bioactive phospholipid, lysophosphatidylcholine (LPC), has been identified as potently toxic upon its accumulation in the heart (93). LPCs are formed by the phospholipase A-mediated release of one of the two FAs contained in phospholipids either in the sn-1 or sn-2 position (97). LPCs have pro-inflammatory and pro-arrhythmogenic properties and myocardial accumulation of LPCs is associated with mitochondrial dysfunction (93). Increased LPC accumulation was evidenced during atherosclerosis and myocardial ischemia and hypoxia (93,97,98). By modulating cardiac ion channel function, LPC accumulation has been shown to contribute to cardiac arrhythmia and sudden cardiac death (93). Interestingly, mice lacking PGC1β, an important regulator of mitochondrial biogenesis and energy metabolism, exhibited increased cardiac concentrations of LPCs, presenting an example where mitochondrial

22



dysfunction is linked to alterations in (lyso)phospholipid metabolism (93). Lysophospholipid accumulation in PGC1β deficient hearts was associated with ion channel remodeling and

PT

deranged calcium handling, causing ventricular arrhythmia (93).

RI

Cardiac ischemia activates calcium-independent phospholipase A2 (iPLA2), which metabolizes phospholipids to produce FAs and lysophospholipids (99). When hearts from

SC

transgenic mice with cardiomyocyte-specific iPLA2β over-expression were subjected to

NU

ischemia, a drastic increase in free FAs released into the venous eluent and elevated LPC concentrations in ischemic zones were observed, which was accompanied by ventricular

MA

tachyarrhythmias (99). These findings suggest that excessive hydrolysis of membrane phospholipids by iPLA2β can trigger malignant ventricular tachyarrhythmias during acute

TE

D

cardiac ischemia (99). These changes were not observed in wild type hearts, which has been attributed to the fact that the murine myocardium generally contains extremely low amounts of

AC CE P

iPLA2 activity when compared to the human heart (99). Besides iPLA2β, iPLA2γ has also been linked to myocardial lipotoxicity and dysfunction (100). Mice with cardiomyocyte-specific iPLA2γ over-expression exhibited not only a drastic decrease in myocardial phospholipid content but also a pronounced increase in TAG levels and changes in TAG molecular species composition following brief caloric restriction, as well as fasting-induced hemodynamic dysfunction (100). Moreover, iPLA2γ over-expressing hearts had markedly increased lysophosphospholipid and acylcarnitine content (100), suggesting that over-active iPLA2γ and ensuing phospholipid breakdown in the myocardium leads to cardiac lipotoxicity. Arachidonic acid is sequestered in phospholipids and can be liberated upon activation of iPLA2s in the heart (95). In fact, the release of arachidonic acid by iPLA2s constitutes the ratelimiting step in the generation of eicosanoids, which is mediated by the subsequent arachidonic

23



acid oxidation via lipooxygenases, cyclooxygenases, and cytochrome P450 (95). Cardiac eicosanoid signaling plays important physiological and pathological roles by activating protein

PT

kinases and ion channels and is implicated in myocardial infarction, arrhythmogenesis, and

RI

hypertrophic remodeling (101). Therefore, it is likely that iPLA2 activation can cause cardiac lipotoxicity not only by producing excess lysophospholipids, but also by promoting adverse

NU

SC

eicosanoid accumulation and signaling.

6. Cardiac sphingolipid metabolism

MA

Ceramides are the most widely studied sphingolipids involved in lipotoxic cardiomyopathy. Cardiomyocytes can either synthesize ceramides or acquire these lipids from circulating

TE

D

lipoproteins. De novo synthesis of sphingolipids, including ceramides, is initiated by serine palmitoyltransferase (SPT) (Figure 2)(102). Alternatively, ceramides can also be generated via

AC CE P

sphingomyelinase or ceramide synthases (102), which can be modulated by Sirt3-mediated deacetylation (103). Cardiac ceramide levels were increased in mice with cardiomyocyte-specific over-expression of a glycosylphosphatidylinositol-anchored human lipoprotein lipase (LpLGPI), which develop dilated lipotoxic cardiomyopathy secondary to excess FA uptake (104). Consistent with the notion that increased ceramide accumulation is cytotoxic, incubation of a human cardiomyocyte cell line with C6-ceramide resulted in upregulation of the heart failure/hypertrophy markers, atrial natriuretic peptide and brain natriuretic peptide (104). A role of ceramide accumulation in lipotoxic cardiomyopathy in vivo was shown by inhibiting the ratelimiting enzyme of ceramide biosynthesis, serine palmitoyltransferase, either pharmacologically using myriocin or genetically using heterozygous deletion of a serine palmitoyltransferase subunit, which ameliorated systolic heart dysfunction in LpLGPI mice (104). This improvement

24



of in vivo heart function was also associated with reduced mortality rates, decreased expression of atrial natriuretic peptide and brain natriuretic peptide, and a shift in cardiac energy metabolism

PT

from FA oxidation towards increased glucose oxidation (104). While this study suggests that

RI

increased ceramide accumulation contributes to cardiomyopathy, it remains unclear whether other sphingolipids, which would be lowered with general inhibition of sphingolipid synthesis,

SC

also play a role in lipotoxic cardiomyopathy as observed in LpLGPI mice.

NU

A recent study implicated increased cardiac C14-ceramide concentrations in cardiac hypertrophy, dysfunction, and autophagy induced by a milk fat-based high fat diet (MFBD) rich

MA

in myristate (C14:0 FA) (105). Inhibition of de novo ceramide synthesis using myriocin restored cardiac function and prevented cardiac hypertrophy in MFBD-fed mice (105). Interestingly,

TE

D

myristate-induced cardiomyocyte autophagy and hypertrophy was dependent on ceramide synthase 5 (CerS5), which produces ceramide by N-acylation of sphingosine (ceramide salvage

AC CE P

pathway) (105). Consumption of a MFBD also resulted in increased de novo synthesis of noncanonical myristate-derived d16 sphingolipids, which were shown to promote cell death and cleavage of poly(ADP-ribose) polymerase (PARP) in cardiomyocytes (106). Taken together, these studies pinpointed specific sphingolipid species and metabolic pathways in promoting lipotoxic cardiomyopathy.

Besides inhibition of ceramide synthase, increasing ceramide catabolism via ceramidase may lower cardiomyocyte ceramide levels. A study by Holland et al. (107) found that adiponectin, an adipokine which promotes cell survival, insulin signaling, and inhibits inflammation, activates ceramide catabolism via ceramidase action and increases the subsequent formation

of

sphingosine-1-phosphate

(S1P),

a

potent

anti-apoptotic

signaling

sphingolipid/lysophospholipid (107). Mice with inducible cardiomyocyte-specific apoptosis via

25



caspase-8 activation exhibited improved survival upon inhibition of ceramide synthesis by myriocin treatment or increased adiponectin signaling (107). Conversely, adiponectin deficiency

PT

exacerbated cardiomyocyte apoptosis (107). Adiponectin over-expression was associated with an

RI

increase in circulating S1P and S1P administration was sufficient to completely prevent mortality in mice with induced cardiomyocyte apoptosis (107). These findings suggest that a reduction in

SC

ceramide concentrations and increased formation of S1P, which is subsequently secreted to

NU

activate S1P receptor signaling in an autocrine/paracrine manner, are key mechanisms by which adiponectin protects against cardiomyocyte apoptosis (107). Adiponectin levels decrease with

MA

obesity in rodents and humans (108,109), suggesting that increased ceramide and decreased S1P formation due to diminished adiponectin signaling and ceramidase activation contributes to

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D

obesity-induced lipotoxic cardiomyopathy.

A recent study suggested that lowering certain sphingolipid species in the normal heart

AC CE P

can precipitate cardiac dysfunction (110). Mice with cardiomyocyte-specific deficiency of the serine palmitoyltransferase subunit 2 (Sptlc2) exhibited reduced cardiac concentrations of C18:0 and very long-chain ceramides, dihydroceramides, as well as the sphingoid bases, sphingosine and sphinganine (110). These alterations in sphingolipid content were paralleled by increases in certain fatty acyl-CoA species, elevated cardiolipin content, and altered phospholipid FA composition (110). Interestingly, Sptlc2 deficiency led to increased cardiac ER stress and apoptosis, ventricular wall thinning, cardiac fibrosis, left ventricular dilatation and systolic heart dysfunction (110). These findings demonstrate that reduced cardiac sphingolipid synthesis via Sptlc2 not only decreases certain sphingolipid species but also leads to significant changes in concentrations and composition of other cardiac lipids and dilated cardiomyopathy (110). This

26



study also highlights that more research is required to elucidate the link between metabolism of ceramides/sphingolipids and other lipids that may have toxic effects.

PT

Although there is strong evidence from animal models that ceramide accumulation

RI

contributes to cardiomyocyte lipotoxicity and apoptosis in obesity and diabetes, it remains unclear whether similar relationships exist in the human heart (111). For example, although

SC

markers of apoptosis were increased in the atrial appendage of obese subjects with and without a

NU

history of diabetes, total ceramide and sphingoid base levels were unchanged, despite altered mRNA expression of several enzymes involved in sphingolipid metabolism (111). Additional

MA

studies are necessary to better understand the role of ceramides and other sphingolipids and their

TE

D

metabolizing enzymes in cardiac lipotoxicity in humans.

AC CE P

7. Extracellular lipid signaling

While studies on cardiac lipotoxicity generally examine the intracellular accumulation of lipids and ensuing activation of signaling pathways from within the cardiomyocyte, it should also be noted that it is plausible that signaling induced by extracellular lipids contributes to cardiac injury in conditions

such as obesity/diabetes and myocardial

ischemia-reperfusion.

Cardiomyocytes express sarcolemmal receptors through which circulating lipids, e.g. lysophosphatidic acid (LPA), S1P, and free FAs, induce downstream signaling. Signaling cascades initiated by these receptors can have profound effects on cell survival, structure, function, and metabolism (112-118). Toll-like receptor 4 (TLR4) is best known for binding to lipopolysaccharide from Gramnegative bacteria and thereby activating the innate immune system. It has been suggested that

27



FAs can also bind to TLR4 indirectly through Fetuin A and trigger an inflammatory response via TLR4 signaling, which contributes to FA-induced insulin resistance (119-121). Alternatively,

PT

FAs can be incorporated into lipid raft domains, to enhance TLR4 recruitment and dimerization (122). It has been proposed that lipid-induced insulin resistance mediated by TLR4 is dependent

RI

on ceramide synthesis through activation of IKKβ-NFκB signaling (117). Interestingly, TLR4

SC

expression was upregulated in cardiomyocytes from type 1 diabetic NOD mice (118). TLR4

NU

deficiency in these mice resulted in reduced myocardial TAG accumulation, which was accompanied by decreased LPL protein expression and ameliorated cardiac dysfunction (118).

MA

These findings suggest that elevated circulating FAs could mediate diabetes-induced lipotoxic cardiomyopathy in part through TLR4 signaling. However, additional studies are required to

TE

D

examine the role of FA-induced TLR4 activation in lipotoxic cardiomyopathy. Most of the LPA in blood is produced by autotaxin, a secreted lysophospholipase D

AC CE P

which catabolizes lysophospholipids such as LPC and lysophosphatidylserine (123). LPA signals through G protein-coupled receptors at the plasmamembrane, activating a variety of downstream signaling pathways, including PI3Kinase-Akt, adenylate cyclase-cAMP, Ras-p42/p44MAPK, Rho-Rho kinase, and phospholipase C signaling (123). Although our understanding of how signaling through extracellular LPA influences cardiomyocytes is still in its infancy, emerging evidence suggests that LPA receptor activation plays an important pathophysiologic role (113,123-125). Specifically, LPA signaling has been implicated in the development of cardiomyocyte hypertrophy in part through LPA1 and LPA3 receptors (126). LPA1/3 protein levels were increased significantly in rat hearts that were subjected to myocardial infarction (126). Interestingly, LPA1 and LPA3 may play opposite roles in mediating cardiomyocyte hypertrophy. Knockdown of LPA1 enhanced LPA-induced hypertrophy, whereas knockdown of

28



LPA3 ameliorated LPA-induced hypertrophy in neonatal rat cardiomyocytes (127). The mechanisms underlying LPA mediated hypertrophy are incompletely understood, however, it has

PT

been suggested that LPA can enhance the expression of miR-23a, a known inducer of cardiac

RI

hypertrophy (127-129). A study by Pulinilkunnil et al. (125) also suggested that LPA signaling can enhance FA uptake in cardiomyocytes via stimulation of LPL trafficking. By triggering F-

SC

actin polymerisation through RhoA and Rho kinase, LPA promoted the transport of LPL-

NU

containing vesicles from the Golgi apparatus to the cardiomyocyte surface, which was followed by translocation of LPL to the coronary lumen (125).

MA

Recent studies showed that circulating levels of autotaxin, which critically influences extracellular LPA concentrations, correlates with measures of insulin resistance in humans

TE

D

(130,131). Moreover, in animal models, autotaxin-mediated LPA signaling has been implicated in the development of obesity and insulin resistance (132,133). Therefore, it is tempting to

AC CE P

speculate that the autotaxin-LPA signaling axis contributes to cardiac lipotoxicity – via upregulation of LPL-mediated FA delivery – and hypertrophic remodeling under conditions of obesity-induced insulin resistance. In addition, LPA signaling has been shown to impair cardiomyocyte contractility, suggesting that increased circulating LPA, as observed under conditions such as myocardial ischemia and obesity-insulin resistance, could contribute to cardiac dysfunction (124). Future studies are required to examine the detailed contributions of cardiomyocyte signaling induced by extracellular LPA and autotaxin to cardiac lipotoxicity. While autotaxin is a relatively novel adipokine and its role in cardiac lipotoxicity remains unexplored, several other adipokines including adiponectin (see also section 6.) and leptin have been suggested to play an important role in lipotoxic cardiomyopathy by modulating cardiac energy metabolism (6,134). For example, leptin increases cardiac FA oxidation and decreases

29



lipid accumulation (135). Moreover, overexpression of leptin protected against lipotoxic cardiomyopathy in acyl CoA synthase transgenic mice, suggesting that hyperleptinemia as

PT

observed during obesity may mitigate obesity-induced cardiac lipotoxicity (136), although the

RI

development of leptin resistance during obesity (137) may counter the cardioprotective effects of

SC

leptin.

NU

8. Lipotoxic signaling induced by DAGs and ceramides

DAGs and ceramides are arguably the most intensely studied toxic lipid species to date.

MA

DAGs are potent lipid second messengers that can activate classical (α, βI, βII, and γ) and novel (δ, ε, ε, and ζ) isoforms of PKC, both of which have been implicated in the development of

D

myocardial disease including cardiac hypertrophy, fibrosis, inflammation, and diabetic

TE

cardiomyopathy (138-142) (Figure 3). PKCβ2 expression is increased in the myocardium of

AC CE P

diabetic patients and animals (139,141,143,144). Forced expression of PKCβ in the myocardium leads to a cardiomyopathy characterized by hypertrophy, necrosis and an expansion of the interstitial extracellular matrix (139,145,146), suggesting that PKCβ over-activation by DAGs leads to cardiac dysfunction and remodeling. PKCβ may activate pro-inflammatory and prohypertrophic cytokines such as tumor necrosis factor α (TNFα) through NFκB in cardiomyocytes under hyperglycemic conditions, thereby decreasing Akt phosphorylation and insulin signaling (141,147-149). PKCs can activate NFκB by directly phosphorylating its inhibitor IκB or through the generation of reactive oxygen species, which can secondarily activate IκB kinases (IKKβ) (150,151). PKCζ, a novel PKC isoform, can directly inhibit insulin signaling through phosphorylation of insulin receptor substrate 1 (IRS1) on Ser307 and Ser1101 in skeletal and possibly cardiac muscle (152). PKCε, another novel PKC isoform, is activated in the rat heart in

30



response to streptozotozin-induced diabetes (153). PKCε is known to promote DAG-induced insulin resistance through inhibition of IRS2 and insulin receptor tyrosine kinase in the liver

PT

(154).

RI

It has been suggested that the DAG acyl chain composition, DAG steroisomer type, and cellular localization play an important role in activating signaling proteins such as PKCs by

SC

DAG signaling (155). For example, DAGs generated through phospholipase C cleavage of

NU

phosphatidylinositol-4,5-bisphosphate (PIP2) differentially induce translocation and activation of several PKC isoforms when compared to DAGs generated by phospholipase D cleavage of

MA

phosphatidylcholine (156,157). It has been proposed that cellular DAGs exist in three distinct pools of stereoisomers (155). These consist of sn-1,2 DAG generated through de novo

TE

D

lipogenesis via DGAT at the endoplasmic reticulum, sn-1,2 and sn-2,3 DAG generated by ATGL on lipid droplets, and sn-1,2 DAG generated by phospholipase C enzymes via hydrolysis of PIP2

AC CE P

at plasma and ER membranes (155). PKC activation is believed to occur mainly by the latter DAG pool and it has been suggested that TAG/ATGL-derived DAG would require isomerization prior to activation of PKC enzymes (155). Ceramides function as key components of lipotoxic signaling pathways linking lipidinduced inflammation and inhibition of insulin signaling (102) (Figure 3). For example, TLR4 signaling induces de novo ceramide synthesis through IKKβ-NFκB signaling (117). Moreover, activation of TLR4 signaling is dependent on the production of ceramide by acid sphingomyelinase (158). Ceramide is critically required for TLR4-induced insulin resistance (117) and has been suggested to induce insulin resistance by targeting activation of Akt through two independent mechanisms (159). First, ceramide can activate protein phosphatase 2A (PP2A), which dephosphorylates Akt and second, ceramide can prevent Akt translocation to the plasma

31



membrane through activation of PKCδ (159,160). Indeed, treatment of AC16 human cardiomyocytes with C6 ceramide decreased phosphorylation of Akt and its downstream target

PT

glycogen synthase kinase 3β (GSK3β) in a concentration dependent manner, which is consistent

RI

with findings in skeletal muscle cells and adipocytes (104). In addition to inhibiting insulin signaling via reducing Akt phosphorylation, ceramide can also inhibit phosphorylation of IRS1

SC

(161). Moreover, ceramide appears to activate mixed lineage kinase-3 (MLK3), which may

NU

inhibit IRS through stress activated protein kinases (SAPK) including p38 mitogen activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) (162-166). Kinases in the MLK

MA

family regulate the stress response in cardiomyocytes, however, it remains to be determined whether ceramide also inhibits cardiomyocyte insulin signaling through these kinases (167).

TE

D

Apart from inhibiting insulin signaling, ceramides also signal through mitogen activated protein kinases (MAPKs), including extracellular signal regulated kinase (ERK), p38MAPK and

AC CE P

JNK to induce apoptosis (168). Specifically, ceramide triggers ERK dephosphorylation and stimulates phosphorylation of p38MAPK and JNK (168). MAPK signaling has been implicated in heart failure, cardiomyopathy, cardiac remodeling, and injury induced by hypertrophy and ischemia/reperfusion (169,170), suggesting that ceramide-MAPK signaling may play a key role in various forms of heart disease. Indeed, ceramide-induced apoptosis in rat cardiomyocytes could be attenuated by inhibiting JNK1 using antisense oligonucleotides (169). JNK can directly activate the mitochondrial apoptosis machinery in adult cardiac myocytes through the release of cytochrome c from mitochondria and activation of caspase 9 (171). It has also been proposed that ceramides induce apoptosis in neonatal rat cardiomyocytes through caspase 3 and 8-related proteases (172). Inhibition of ceramide synthesis using fumonisin B1 impaired palmitic acidinduced cytochrome c release, apoptosis and myofibril degeneration in adult rat cardiomyocytes

32



(173). Interestingly, incubation with sphinogomyelinase, which metabolizes sphingomyeline to ceramide, had no adverse effects on cardiomyocytes, suggesting that palmitate-induced

RI

PT

cardiomyocyte apoptosis is primarily mediated by de novo ceramide synthesis (173).

9. Concluding remarks

SC

Intense research and the generation of numerous animal models in the last fifteen years have

NU

been instrumental in improving our understanding of cardiac lipotoxicity, mostly in the context of obesity, insulin resistance, and diabetes. Certain lipids are believed to be particularly

MA

cardiotoxic when they accumulate in excess. These include ceramides, DAGs, long chain acylCoAs, acylcarnitines, and lysophospholipids. TAGs, on the other hand, are generally considered

D

to be non-toxic and likely serve as marker for increased lipid deposition in the heart.

TE

Nevertheless, enzymes involved in the synthesis and catabolism of TAGs do play a role in

AC CE P

cardiac lipotoxicity by regulating the sequestration of FAs in the TAG pool. A major limitation of most studies examining cardiac lipotoxicity in animal models is that only select lipids and lipid metabolism and signaling pathways were examined. More comprehensive lipidomics analysis will be necessary to uncover how accumulation and metabolism of toxic lipids in the myocardium are interconnected. Moreover, research examining cardiac lipotoxicity in the human heart is still in its infancy and future studies need to determine whether findings from animal models of lipotoxic cardiomyopathy translate to the human myocardium. Based on our understanding of the etiology of cardiac lipotoxicity from the study of animal models, it is apparent that limiting FA delivery and uptake produce the most consistent results in protecting from cardiac lipotoxicity not only during obesity/insulin resistance/diabetes, but also aging and myocardial ischemia/infarction (7,8,38,39,42). This is not entirely surprising since FA overload due to a mismatch between cardiomyocyte FA transport and FA metabolism 33



through oxidation or TAG synthesis is the primary culprit in lipotoxic cardiomyopathy. Therefore, targeting FA uptake, for example through CD36 inhibitors (41), may currently be the

PT

most promising avenue for the development of therapies for lipotoxic cardiomyopathy of

SC

RI

different etiologies.

NU

Acknowledgements

This work was supported by grants from the Natural Sciences and Engineering Research

Conflicts of Interest

TE

D

Research Foundation (NBHRF) to P.K.

MA

Council of Canada (NSERC), the Banting Research Foundation, and the New Brunswick Health

References 1. 2.

3. 4. 5.

6. 7.

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We have no conflict of interest to declare.

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8.


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26.

27.

28.

29. 30.

31.

32.

33.

34.

PT

RI

SC

NU

25.

MA

24.

D

23.

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Lipid peroxides Free fatty acids Acylcarnitines Ceramides

Lipotoxicity

Diacylglycerols

Long-chain acyl-CoAs

Apoptotic cell death Cardiac dysfunction Cardiac remodeling Arrhythmia Heart failure

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Insulin resistance Mitochondrial damage ATP starvation ER stress

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Obesity Insulin resistance Diabetes Aging Myocardial ischemia/ reperfusion etc.

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Lysophospholipids

Figure 1. Causes and consequences of cardiac lipotoxicity. Stressors including metabolic

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diseases, aging, and myocardial ischemia-reperfusion can lead to increased accumulation of lipids and lipid metabolites in the heart that have toxic effects on cardiomyocytes. Cardiac

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lipotoxicity can trigger cell stress and impair myocardial function.

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PIP2 PLC

Lipoprotein

Lipid droplet PL

DAG DAG

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Lyso-PL, eicosanoids

HSL

TAG

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FA/acylATGL CoA CGI-58 PLIN5

TAG

Acyl-CoA

Lipid peroxides

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Acyl-CoA

Acylcarnitines

ROS, peroxynitrite

Ceramides

FA uptake FA oxidation Lipid storage

CerS5 SPT1 ER

FoxO1 PPARα/δ/γ Nucleus

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Mitochondria

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β-oxidation

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S1P

DGAT1

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Figure 2. Schematic representation of lipids and proteins involved in lipid uptake and metabolism that have been implicated in cardiac lipotoxicity. Excess FA delivery and uptake in cardiomyocytes, impaired or incomplete FA oxidation, lipid peroxidation, as well as ceramide, glycerolipid, and phospholipid metabolism have been linked to cardiac lipotoxicity. Lipid classes and metabolites that have been associated with lipotoxicity in the heart include long chain acylCoAs, FAs, acylcarnitines, lipid peroxides, ceramides and other sphingolipids, DAGs, lyso-PLs, and eicosanoids (highlighted in bold letters).

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Insulin

FAs

TNFα IR

SMase

p38 MAPK

IRS

MLK3

NFκB

JNK

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PI3K

PKCδ

NFκB

PP2A Caspase 9

Akt

Apoptosis

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Caspase 3/8

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classical/novel PKCs

Ceramide JNK

Sarcolemma Insulin resistance

IκB Nucleus

NFκB

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TNFR

DAG

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TLR4

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Figure 3. Proposed mechanisms of cardiac lipotoxicity induced by DAGs and ceramides.

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DAGs induce lipotoxicity through the activation of classical and novel protein kinase C (PKC) isoforms and subsequent inhibition of insulin signaling at the level of insulin receptor (IRS) as well as through activation of nuclear factor κB (NFκB) and inflammatory cytokine signaling. Additionally, NFκB activation can lead to increased ceramide synthesis. Ceramides are also mediators of toll-like receptor 4 signaling and can induce insulin resistance via the activation of MAPK signaling and IRS inhibition or inhibition of Akt via protein kinase Cδ (PKCδ) and protein phosphatase 2A (PP2A). By stimulating caspases ceramides can also induce cellular apoptosis.

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Conflicts of Interest

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We have no conflicts of interest to declare.

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Highlights Cardiomyocyte lipid overload can have toxic effects and cause cardiac dysfunction.

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Cardiac lipotoxicity is observed during pathophysiological conditions including obesity,

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insulin resistance, diabetes, aging, and myocardial ischemia-reperfusion. Studies using animal models have uncovered mechanisms of cardiac lipotoxicity.

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Proteins involved in FA uptake and oxidation as well as TAG, sphingolipid, and

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

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phospholipid metabolism have been implicated in cardiac lipotoxicity.

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The role of ER stress in lipid metabolism and lipotoxicity.

A novel physiological role for cardiac myoglobin in lipid metabolism.

Lipid signaling in adipose tissue: Connecting inflammation & metabolism.

Fetuin A promotes lipotoxicity in β cells through the TLR4 signaling pathway and the role of pioglitazone in anti-lipotoxicity.

Cardiac-specific ablation of ARNT leads to lipotoxicity and cardiomyopathy.

Fetuin-A aggravates lipotoxicity in podocytes via interleukin-1 signaling.

BHBA influences bovine hepatic lipid metabolism via AMPK signaling pathway.

Aspirin regulates hepatocellular lipid metabolism by activating AMPK signaling pathway.

Lipids and lipid metabolism.

Lipid absorption and metabolism.

Polydatin improves glucose and lipid metabolism in experimental diabetes through activating the Akt signaling pathway.

Metabolomics insights into activated redox signaling and lipid metabolism dysfunction in chronic kidney disease progression.

CGI-58, a key regulator of lipid homeostasis and signaling in plants, also regulates polyamine metabolism.

Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism.

Effects of probucol on impaired cardiac performance and lipid metabolism in streptozotocin-induced diabetic rats.

Role of cardiac lymph and interstitial fluid in lipid metabolism of canine heart.

Decoration of intramyocellular lipid droplets with PLIN5 modulates fasting-induced insulin resistance and lipotoxicity in humans.

Lipid Signaling in Tumorigenesis.

Insulin Signaling in Bupivacaine-induced Cardiac Toxicity: Sensitization during Recovery and Potentiation by Lipid Emulsion.

Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal.

Integrated analysis of chronic lipotoxicity on muscle metabolism and stress and its reversal by antioxidants.

Beta-blockers and lipid metabolism.

Lipotoxicity in the liver.

Histone variants and lipid metabolism.