Phospholipid homeostasis and lipotoxic cardiomyopathy

Obesity has reached pandemic proportions globally and is often associated with lipotoxic heart diseases. In the obese state, caloric surplus is accommodated in the adipocytes as triglycerides. As the storage capacity of adipocytes is exceeded or malfunctioning, lipids begin to infiltrate and accumulate in non-adipose tissues, including the myocardium of the heart, leading to organ dysfunction. While the disruption of caloric homeostasis has been widely viewed as a principal mechanism in contributing to peripheral tissue steatosis and lipotoxicity, our recent studies in Drosophila have led to the novel finding that deregulation of phospholipid homeostasis may also significantly contribute to the pathogenesis of lipotoxic cardiomyopathy. Fly mutants that bear perturbations in phosphatidylethanolamine (PE) biosynthesis, such as the easily-shocked (eas) mutants defective in ethanolamine kinase, incurred aberrant activation of the sterol regulatory element binding protein (SREBP) pathway, thereby causing chronic lipogenesis and cardiac steatosis that culminates in the development of lipotoxic cardiomyopathy ¹. Here, we describe the potential relationship between SREBP and other eas-associated phenotypes, such as neuronal excitability defects. We will further discuss the additional implications presented by our work toward the effects of altered lipid metabolism on cellular growth and/or proliferation in response to defective phospholipid homeostasis.     

Lipid metabolism and signaling in cardiac lipotoxicity

The heart balances uptake, metabolism and oxidation of fatty acids (FAs) to maintain ATP production, membrane biosynthesis and lipid signaling. Under conditions where FA uptake 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 accumulate in cardiomyocytes and cause cardiomyopathy. Moreover, studies using mutant mice 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 adversely affect cardiomyocyte function. This review summarizes our current understanding of lipid uptake, metabolism and signaling pathways that have been implicated in the development of lipotoxic cardiomyopathy under conditions including obesity, diabetes, aging, and myocardial ischemia–reperfusion. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.     

Hormone-sensitive lipase protects adipose triglyceride lipase-deficient mice from lethal lipotoxic cardiomyopathy

Lipid droplets (LDs) are multifunctional organelles that regulate energy storage and cellular homeostasis. The first step of triacylglycerol hydrolysis in LDs is catalyzed by adipose triglyceride lipase (ATGL), deficiency of which results in lethal cardiac steatosis. Although hormone-sensitive lipase (HSL) functions as a diacylglycerol lipase in the heart, we hypothesized that activation of HSL might compensate for ATGL deficiency. To test this hypothesis, we crossed ATGL-KO (AKO) mice and cardiac-specific HSL-overexpressing mice (cHSL) to establish homozygous AKO mice and AKO mice with cardiac-specific HSL overexpression (AKO+cHSL). We found that cardiac triacylglycerol content was 160-fold higher in AKO relative to Wt mice, whereas that of AKO+cHSL mice was comparable to the latter. In addition, AKO cardiac tissues exhibited reduced mRNA expression of PPARα-regulated genes and upregulation of genes involved in inflammation, fibrosis, and cardiac stress. In contrast, AKO+cHSL cardiac tissues exhibited expression levels similar to those observed in Wt mice. AKO cardiac tissues also exhibited macrophage infiltration, apoptosis, interstitial fibrosis, impaired systolic function, and marked increases in ceramide and diacylglycerol contents, whereas no such pathological alterations were observed in AKO+cHSL tissues. Furthermore, electron microscopy revealed considerable LDs, damaged mitochondria, and disrupted intercalated discs in AKO cardiomyocytes, none of which were noted in AKO+cHSL cardiomyocytes. Importantly, the life span of AKO+cHSL mice was comparable to that of Wt mice. HSL overexpression normalizes lipotoxic cardiomyopathy in AKO mice and the findings highlight the applicability of cardiac HSL activation as a therapeutic strategy for ATGL deficiency-associated lipotoxic cardiomyopathies.     

Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy

Fatty acid oxidation (FAO) is a primary energy source for meeting the heart's energy requirements. Peroxisome proliferator-activated receptor-delta (PPAR-delta) may have important roles in FAO. But it remains unclear whether PPAR-delta is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-delta in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-delta deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-delta is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-delta is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.     

Brg1 regulates pro-lipogenic transcription by modulating SREBP activity in hepatocytes

Alteration of hepatic lipid metabolism contributes to a range of human diseases including steatosis. Sterol response element binding protein (SREBP) is the master regulator of lipid metabolism. The epigenetic mechanism whereby SREBP activity is regulated remains incompletely understood. We have previously shown that systemic knockdown of brahma-related gene 1 (Brg1), a chromatin remodeling protein, attenuates steatosis in mice by down-regulating the synthesis of pro-inflammatory mediators. Here we show that hepatocyte conditional Brg1 knockout (HepcKO) mice were largely protected from high-fat diet (HFD) induced steatosis as evidenced by decelerated weight gains, improved insulin sensitivity, ameliorated steatotic injuries, and diminished hepatic inflammation. Brg1 contributed to lipid metabolism by trans-activating the genes involved in fatty acid esterification. Mechanistically, Brg1 interacted with and was recruited by sterol response element binding protein (SREBP1c) to the promoters of SREBP target genes and optimized the chromatin structure to facilitate SREBP1c binding. Therefore, our data have identified a previously unrecognized role for Brg1 in hepatic lipid metabolism by portraying Brg1 as an essential epigenetic co-factor for SREBP1c.     

A critical role for eukaryotic elongation factor 1A-1 in lipotoxic cell death

The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide- and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease.     

A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the ¹²⁵I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.     

A link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen Cryptococcus neoformans

Fungal pathogens of humans require molecular oxygen for several essential biochemical reactions, yet virtually nothing is known about how they adapt to the relatively hypoxic environment of infected tissues. We isolated mutants defective in growth under hypoxic conditions, but normal for growth in normoxic conditions, in Cryptococcus neoformans, the most common cause of fungal meningitis. Two regulatory pathways were identified: one homologous to the mammalian sterol-response element binding protein (SREBP) cholesterol biosynthesis regulatory pathway, and the other a two-component-like pathway involving a fungal-specific hybrid histidine kinase family member, Tco1. We show that cleavage of the SREBP precursor homolog Sre1-which is predicted to release its DNA-binding domain from the membrane-occurs in response to hypoxia, and that Sre1 is required for hypoxic induction of genes encoding for oxygen-dependent enzymes involved in ergosterol synthesis. Importantly, mutants in either the SREBP pathway or the Tco1 pathway display defects in their ability to proliferate in host tissues and to cause disease in infected mice, linking for the first time to our knowledge hypoxic adaptation and pathogenesis by a eukaryotic aerobe. SREBP pathway mutants were found to be a hundred times more sensitive than wild-type to fluconazole, a widely used antifungal agent that inhibits ergosterol synthesis, suggesting that inhibitors of SREBP processing could substantially enhance the potency of current therapies.     

Ceramide is a cardiotoxin in lipotoxic cardiomyopathy

Ceramide is among a number of potential lipotoxic molecules that are thought to modulate cellular energy metabolism. The heart is one of the tissues thought to become dysfunctional due to excess lipid accumulation. Dilated lipotoxic cardiomyopathy, thought to be the result of diabetes and severe obesity, has been modeled in several genetically altered mice, including animals with cardiac-specific overexpression of glycosylphosphatidylinositol (GPI)-anchored human lipoprotein lipase (LpL(GPI)). To test whether excess ceramide was implicated in cardiac lipotoxicity, de novo ceramide biosynthesis was inhibited pharmacologically by myriocin and genetically by heterozygous deletion of LCB1, a subunit of serine palmitoyltransferase (SPT). Inhibition of SPT, a rate-limiting enzyme in ceramide biosynthesis, reduced fatty acid and increased glucose oxidation in isolated perfused LpL(GPI) hearts, improved systolic function, and prolonged survival rates. Our results suggest a critical role for ceramide accumulation in the pathogenesis of lipotoxic cardiomyopathy.     

Lipotoxicity and cardiac dysfunction in mammals and Drosophila

The lipotoxic effects of obesity are important contributing factors in cancer, diabetes, and cardiovascular disease (CVD), but the genetic mechanisms, by which lipotoxicity influences the initiation and progression of CVD are poorly understood. Hearts, of obese and diabetic individuals, exhibit several phenotypes in common, including ventricular remodeling, prolonged QT intervals, enhanced frequency of diastolic and/or systolic dysfunction, and decreased fractional shortening. High systemic lipid concentrations are thought to be the leading cause of lipid-related CVD in obese or diabetic individuals. However, an alternative possibility is that obesity leads to cardiac-specific steatosis, in which lipids and their metabolites accumulate within the myocardial cells themselves and thereby disrupt normal cardiovascular function. Drosophila has recently emerged as an excellent model to study the fundamental genetic mechanisms of metabolic control, as well as their relationship to heart function. Two recent studies of genetic and diet-induced cardiac lipotoxicity illustrate this. One study found that alterations in genes associated with membrane phospholipid metabolism may play a role in the abnormal lipid accumulation associated with cardiomyopathies. The second study showed that Drosophila fed a diet high in saturated fats, developed obesity, dysregulated insulin and glucose homeostasis, and severe cardiac dysfunction. Here, we review the current understanding of the mechanisms that contribute to the detrimental effects of dysregulated lipid metabolism on cardiovascular function. We also discuss how the Drosophila model could help elucidate the basic genetic mechanisms of lipotoxicity- and metabolic syndrome-related cardiomyopathies in mammals.     

Lipotoxicity and cardiac dysfunction in mammals and Drosophila

The lipotoxic effects of obesity are important contributing factors in cancer, diabetes, and cardiovascular disease (CVD), but the genetic mechanisms, by which lipotoxicity influences the initiation and progression of CVD are poorly understood. Hearts, of obese and diabetic individuals, exhibit several phenotypes in common, including ventricular remodeling, prolonged QT intervals, enhanced frequency of diastolic and/or systolic dysfunction, and decreased fractional shortening. High systemic lipid concentrations are thought to be the leading cause of lipid-related CVD in obese or diabetic individuals. However, an alternative possibility is that obesity leads to cardiac-specific steatosis, in which lipids and their metabolites accumulate within the myocardial cells themselves and thereby disrupt normal cardiovascular function. Drosophila has recently emerged as an excellent model to study the fundamental genetic mechanisms of metabolic control, as well as their relationship to heart function. Two recent studies of genetic and diet-induced cardiac lipotoxicity illustrate this. One study found that alterations in genes associated with membrane phospholipid metabolism may play a role in the abnormal lipid accumulation associated with cardiomyopathies. The second study showed that Drosophila fed a diet high in saturated fats, developed obesity, dysregulated insulin and glucose homeostasis, and severe cardiac dysfunction. Here, we review the current understanding of the mechanisms that contribute to the detrimental effects of dysregulated lipid metabolism on cardiovascular function. We also discuss how the Drosophila model could help elucidate the basic genetic mechanisms of lipotoxicity- and metabolic syndrome-related cardiomyopathies in mammals.