Konstantinos Drosatos

MicroRNA-370 controls the expression of microRNA-122 and Cpt1alpha and affects lipid metabolism.

We previously observed that treatment of mice with a dominant negative form of cJun (dn-cJun) increased the expression of genes involved in lipid metabolism and modulated the expression of nine microRNAs (miR). To investigate the potential effect of these miRs on the expression of the genes of lipid metabolism, we performed studies in cultured HepG2 cells. Transfection of HepG2 cells with sense or antisense miR-370 or miR-122 upregulated and downregulated, respectively, the transcription factor sterol-regulatory element binding protein 1c (SREBP-1c) and the enzymes diacylglycerol acyltransferase-2 (DGAT2), fatty acid synthase (FAS), and acyl-CoA carboxylase 1 (ACC1) that regulate fatty acid and triglyceride biosynthesis. The other seven miRs identified by the miR array screening did not affect the expression of lipogenic genes. miR-370 upregulated the expression of miR-122. Furthermore, the effect of miR-370 on the expression of the lipogenic genes was abolished by antisense miR-122. miR-370 targets the 3′ untranslated region (UTR) of Cpt1α, and it downregulated the expression of the carnitine palmitoyl transferase 1α (Cpt1α) gene as well as the rate of β oxidation. Our data suggest that miR-370 acting via miR-122 may have a causative role in the accumulation of hepatic triglycerides by modulating initially the expression of SREBP-1c, DGAT2, and Cpt1α and, subsequently, the expression of other genes that affect lipid metabolism.

Ventricular Assist Device Implantation Corrects Myocardial Lipotoxicity, Reverses Insulin Resistance, and Normalizes Cardiac Metabolism in Patients With Advanced Heart Failure

Background— Heart failure is associated with impaired myocardial metabolism with a shift from fatty acids to glucose use for ATP generation. We hypothesized that cardiac accumulation of toxic lipid intermediates inhibits insulin signaling in advanced heart failure and that mechanical unloading of the failing myocardium corrects impaired cardiac metabolism. Methods and Results— We analyzed the myocardium and serum of 61 patients with heart failure (body mass index, 26.5±5.1 kg/m2; age, 51±12 years) obtained during left ventricular assist device implantation and at explantation (mean duration, 185±156 days) and from 9 control subjects. Systemic insulin resistance in heart failure was accompanied by decreased myocardial triglyceride and overall fatty acid content but increased toxic lipid intermediates, diacylglycerol, and ceramide. Increased membrane localization of protein kinase C isoforms, inhibitors of insulin signaling, and decreased activity of insulin signaling molecules Akt and Foxo were detectable in heart failure compared with control subjects. Left ventricular assist device implantation improved whole-body insulin resistance (homeostatic model of analysis–insulin resistance, 4.5±0.6–3.2±0.5; P<0.05) and decreased myocardial levels of diacylglycerol and ceramide, whereas triglyceride and fatty acid content remained unchanged. Improved activation of the insulin/phosphatidylinositol-3 kinase/Akt signaling cascade after left ventricular assist device implantation was confirmed by increased phosphorylation of Akt and Foxo, which was accompanied by decreased membrane localization of protein kinase C isoforms after left ventricular assist device implantation. Conclusions— Mechanical unloading after left ventricular assist device implantation corrects systemic and local metabolic derangements in advanced heart failure, leading to reduced myocardial levels of toxic lipid intermediates and improved cardiac insulin signaling.

Enhanced Efferocytosis of Apoptotic Cardiomyocytes Through Myeloid-Epithelial-Reproductive Tyrosine Kinase Links Acute Inflammation Resolution to Cardiac Repair After Infarction

Rationale: Efficient clearance of apoptotic cells (efferocytosis) is a prerequisite for inflammation resolution and tissue repair. After myocardial infarction, phagocytes are recruited to the heart and promote clearance of dying cardiomyocytes. The molecular mechanisms of efferocytosis of cardiomyocytes and in the myocardium are unknown. The injured heart provides a unique model to examine relationships between efferocytosis and subsequent inflammation resolution, tissue remodeling, and organ function. Objective: We set out to identify mechanisms of dying cardiomyocyte engulfment by phagocytes and, for the first time, to assess the causal significance of disrupting efferocytosis during myocardial infarction. Methods and Results: In contrast to other apoptotic cell receptors, macrophage myeloid-epithelial-reproductive tyrosine kinase was necessary and sufficient for efferocytosis of cardiomyocytes ex vivo. In mice, Mertk was specifically induced in Ly6cLO myocardial phagocytes after experimental coronary occlusion. Mertk deficiency led to an accumulation of apoptotic cardiomyocytes, independently of changes in noncardiomyocytes, and a reduced index of in vivo efferocytosis. Importantly, suppressed efferocytosis preceded increases in myocardial infarct size and led to delayed inflammation resolution and reduced systolic performance. Reduced cardiac function was reproduced in chimeric mice deficient in bone marrow Mertk; reciprocal transplantation of Mertk+/+ marrow into Mertk−/− mice corrected systolic dysfunction. Interestingly, an inactivated form of myeloid-epithelial-reproductive tyrosine kinase, known as solMER, was identified in infarcted myocardium, implicating a natural mechanism of myeloid-epithelial-reproductive tyrosine kinase inactivation after myocardial infarction. Conclusions: These data collectively and directly link efferocytosis to wound healing in the heart and identify Mertk as a significant link between acute inflammation resolution and organ function.

Cardiac Lipotoxicity: Molecular Pathways and Therapeutic Implications

Diabetes and obesity are both associated with lipotoxic cardiomyopathy exclusive of coronary artery disease and hypertension. Lipotoxicities have become a public health concern and are responsible for a significant portion of clinical cardiac disease. These abnormalities may be the result of a toxic metabolic shift to more fatty acid and less glucose oxidation with concomitant accumulation of toxic lipids. Lipids can directly alter cellular structures and activate downstream pathways leading to toxicity. Recent data have implicated fatty acids and fatty acyl coenzyme A, diacylglycerol, and ceramide in cellular lipotoxicity, which may be caused by apoptosis, defective insulin signaling, endoplasmic reticulum stress, activation of protein kinase C, MAPK activation, or modulation of PPARs.

Cardiomyocyte lipids impair β-adrenergic receptor function via PKC activation.

Normal hearts have increased contractility in response to catecholamines. Because several lipids activate PKCs, we hypothesized that excess cellular lipids would inhibit cardiomyocyte responsiveness to adrenergic stimuli. Cardiomyocytes treated with saturated free fatty acids, ceramide, and diacylglycerol had reduced cellular cAMP response to isoproterenol. This was associated with increased PKC activation and reduction of β-adrenergic receptor (β-AR) density. Pharmacological and genetic PKC inhibition prevented both palmitate-induced β-AR insensitivity and the accompanying reduction in cell surface β-ARs. Mice with excess lipid uptake due to either cardiac-specific overexpression of anchored lipoprotein lipase, PPARγ, or acyl-CoA synthetase-1 or high-fat diet showed reduced inotropic responsiveness to dobutamine. This was associated with activation of protein kinase C (PKC)α or PKCδ. Thus, several lipids that are increased in the setting of lipotoxicity can produce abnormalities in β-AR responsiveness. This can be attributed to PKC activation and reduced β-AR levels.

Human Aldose Reductase Expression Accelerates Atherosclerosis in Diabetic Apolipoprotein E−/− Mice

Objective—There are several pathways that mediate the aberrant metabolism of glucose and that might induce greater vascular damage in the setting of diabetes. The polyol pathway mediated by aldose reductase (AR) has been postulated to be one such pathway. However, it has been reported that AR reduces toxic lipid aldehydes and, under some circumstances, might be antiatherogenic. Methods and Results—Atherosclerosis development was quantified in 2 lines of transgenic mice expressing human AR (hAR) crossed on the apolipoprotein E knockout background. The transgenes were used to increase the normally low levels of this enzyme in wild-type mice. Both generalized hAR overexpression and hAR expression via the Tie 2 promoter increased lesion size in streptozotocin diabetic mice. In addition, pharmacological inhibition of AR reduced lesion size. Conclusion—Although in some settings AR expression might reduce levels of toxic aldehydes, transgenic expression of this enzyme within the artery wall leads to greater atherosclerosis.

Inhibition of c-Jun-N-terminal Kinase Increases Cardiac Peroxisome Proliferator-activated Receptor α Expression and Fatty Acid Oxidation and Prevents Lipopolysaccharide-induced Heart Dysfunction

Septic shock results from bacterial infection and is associated with multi-organ failure, high mortality, and cardiac dysfunction. Sepsis causes both myocardial inflammation and energy depletion. We hypothesized that reduced cardiac energy production is a primary cause of ventricular dysfunction in sepsis. The JNK pathway is activated in sepsis and has also been implicated in impaired fatty acid oxidation in several tissues. Therefore, we tested whether JNK activation inhibits cardiac fatty acid oxidation and whether blocking JNK would restore fatty acid oxidation during LPS treatment. LPS treatment of C57BL/6 mice and adenovirus-mediated activation of the JNK pathway in cardiomyocytes inhibited peroxisome proliferator-activated receptor α expression and fatty acid oxidation. Surprisingly, none of the adaptive responses that have been described in other types of heart failure, such as increased glucose utilization, reduced αMHC:βMHC ratio or induction of certain microRNAs, occurred in LPS-treated mice. Treatment of C57BL/6 mice with a general JNK inhibitor (SP600125) increased fatty acid oxidation in mice and a cardiomyocyte-derived cell line. JNK inhibition also prevented LPS-mediated reduction in fatty acid oxidation and cardiac dysfunction. Inflammation was not alleviated in LPS-treated mice that received the JNK inhibitor. We conclude that activation of JNK signaling reduces fatty acid oxidation and prevents the peroxisome proliferator-activated receptor α down-regulation that occurs with LPS.

Peroxisome Proliferator–Activated Receptor-γ Activation Prevents Sepsis-Related Cardiac Dysfunction and Mortality In Mice

Impaired cardiac contractility contributes to the hypotension and increased mortality that occur with sepsis1. A possible cause of sepsis-mediated cardiac dysfunction is reduced energy production due in part to compromised fatty acid oxidation (FAO)2-5 and glucose catabolism3, 6. Thus, it is likely that sepsis compromises cardiac energy production, which might be the major cause of cardiac dysfunction. Alternatively, sepsis induces the production of inflammatory cytokines, such as tumor necrosis factor (TNF) α, interleukin (IL)-1 and IL-6, and these might directly alter heart function7-9. Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) has been extensively used to model many of the clinical features of sepsis, including elevated inflammation and cardiac dysfunction10. LPS leads to production of inflammatory cytokines7-9, 11 and also reduces cardiac energy utilization2, 3, 12. Nuclear receptors, particularly peroxisomal proliferator-activated receptors (PPARs), regulate cardiac FAO. The PPAR family consists of three members, PPARα, PPARδ and PPARγ. PPARα increases FA storage in triglycerides13 and FAO in heart14 and induces expression of peroxisomal and mitochondrial enzymes. Besides PPARα, cardiac FAO can be increased by activation of PPARγ15 or PPARδ16. Cardiomyocyte-specific overexpression of PPARα14 or PPARγ17 leads to cardiac lipid accumulation, an indication that lipid uptake exceeds FAO. PPARγ-coactivator-1 (PGC-1) α and β18 enhance FAO and mitochondrial biogenesis19. Both PPARα and PGC-1 mRNA levels are markedly reduced in the heart by LPS administration2, 3, 12, 20, while PPARγ is not affected2. Our group showed that maintenance of normal cardiac FAO via c-Jun-N-terminal kinase (JNK) inhibitor-mediated prevention of PPARα downregulation rescued cardiac function in septic mice despite elevated expression of cardiac inflammatory markers. In a similar context constitutive cardiac expression of PGC-1β prevented cardiac dysfunction that was caused by LPS-mediated sepsis3, an observation that was proposed to be due to improvement in cardiac FAO and attenuation of reactive oxygen species production. In the current study we show that constitutive cardiomyocyte-specific expression of PPARγ or systemic administration of the PPARγ agonist, rosiglitazone, increased cardiac FAO and prevented cardiac dysfunction in mice with LPS-induced sepsis, despite increased expression of cardiac inflammatory markers. In addition, we show that rosiglitazone-mediated activation of PPARγ prevents the loss of cardiac mitochondria that occurs in sepsis. Moreover, we show that restoration of cardiac FAO by rosiglitazone not only prevents but also treats LPS-induced heart dysfunction and improves survival. Thus the use of rosiglitazone is proposed as a potential treatment for septic cardiac dysfunction.Background—Cardiac dysfunction with sepsis is associated with both inflammation and reduced fatty acid oxidation. We hypothesized that energy deprivation accounts for sepsis-related cardiac dysfunction. Methods and Results—Escherichia coli lipopolysaccharide (LPS) administered to C57BL/6 mice (wild type) induced cardiac dysfunction and reduced fatty acid oxidation and mRNA levels of peroxisome proliferator–activated receptor (PPAR)-&agr; and its downstream targets within 6–8 hours. Transgenic mice in which cardiomyocyte-specific expression of PPAR&ggr; is driven by the &agr;-myosin heavy chain promoter (&agr;MHC-PPAR&ggr;) were protected from LPS-induced cardiac dysfunction. Despite a reduction in PPAR&agr;, fatty acid oxidation and associated genes were not decreased in hearts of LPS-treated &agr;MHC-PPAR&ggr; mice. LPS treatment, however, continued to induce inflammation-related genes, such as interleukin-1&agr;, interleukin-1&bgr;, interleukin-6, and tumor necrosis factor-&agr; in hearts of &agr;MHC-PPAR&ggr; mice. Treatment of wild-type mice with LPS and the PPAR&ggr; agonist, rosiglitazone, but not the PPAR&agr; agonist (WY-14643), increased fatty acid oxidation, prevented LPS-mediated reduction of mitochondria, and treated cardiac dysfunction, as well as it improved survival, despite continued increases in the expression of cardiac inflammatory markers. Conclusions—Activation of PPAR&ggr; in LPS-treated mice prevented cardiac dysfunction and mortality, despite development of cardiac inflammation and PPAR&agr; downregulation.

Adipose-specific Lipoprotein Lipase Deficiency More Profoundly Affects Brown than White Fat Biology

Background: Lipoprotein lipase (LpL) is rate-limiting for plasma triglyceride lipolysis, but its importance in adipose development is uncertain. Results: Adipocyte LpL knock-out affected brown but not white fat composition. White fat was reduced when muscle LpL expression was increased. Conclusion: LpL distribution and adipose metabolism affect adipogenesis. Significance: All fat depots are not equally dependent on triglyceride uptake. Adipose fat storage is thought to require uptake of circulating triglyceride (TG)-derived fatty acids via lipoprotein lipase (LpL). To determine how LpL affects the biology of adipose tissue, we created adipose-specific LpL knock-out (ATLO) mice, and we compared them with whole body LpL knock-out mice rescued with muscle LpL expression (MCK/L0) and wild type (WT) mice. ATLO LpL mRNA and activity were reduced, respectively, 75 and 70% in gonadal adipose tissue (GAT), 90 and 80% in subcutaneous tissue, and 84 and 85% in brown adipose tissue (BAT). ATLO mice had increased plasma TG levels associated with reduced chylomicron TG uptake into BAT and lung. ATLO BAT, but not GAT, had altered TG composition. GAT from MCK/L0 was smaller and contained less polyunsaturated fatty acids in TG, although GAT from ATLO was normal unless LpL was overexpressed in muscle. High fat diet feeding led to less adipose in MCK/L0 mice but TG acyl composition in subcutaneous tissue and BAT reverted to that of WT. Therefore, adipocyte LpL in BAT modulates plasma lipoprotein clearance, and the greater metabolic activity of this depot makes its lipid composition more dependent on LpL-mediated uptake. Loss of adipose LpL reduces fat accumulation only if accompanied by greater LpL activity in muscle. These data support the role of LpL as the “gatekeeper” for tissue lipid distribution.

Lipid Use and Misuse by the Heart

The heart utilizes large amounts of fatty acids as energy providing substrates. The physiological balance of lipid uptake and oxidation prevents accumulation of excess lipids. Several processes that affect cardiac function, including ischemia, obesity, diabetes mellitus, sepsis, and most forms of heart failure lead to altered fatty acid oxidation and often also to the accumulation of lipids. There is now mounting evidence associating certain species of these lipids with cardiac lipotoxicity and subsequent myocardial dysfunction. Experimental and clinical data are discussed and paths to reduction of toxic lipids as a means to improve cardiac function are suggested.