PPARδ activation improves cardiac mitochondrial homeostasis in desmin deficient mice but does not alleviate systolic dysfunction.

Peroxisome proliferator-activated receptor (PPAR) δ is a major transcriptional regulator of cardiac energy metabolism with pleiotropic properties, including anti-inflammatory, anti-oxidative and cardioprotective action. In this study, we sought to investigate whether pharmacological activation of PPARδ via intraperitoneal administration of the selective ligand GW0742 could ameliorate heart failure and mitochondrial dysfunction that have been previously reported in a characterized genetic model of heart failure, the desmin null mice (Des-/-). Our studies demonstrate that treatment of Des-/- mice with the PPARδ agonist attenuated cardiac inflammation, fibrosis and cardiac remodeling. In addition, PPARδ activation alleviated oxidative stress in the failing myocardium as evidenced by decreased ROS levels. Importantly, PPARδ activation stimulated mitochondrial biogenesis, prevented mitochondrial and sarcoplasmic reticulum vacuolar degeneration and improved the mitochondrial intracellular distribution. Finally, PPARδ activation alleviated the mitochondrial respiratory dysfunction, prevented energy depletion and alleviated excessive autophagy and mitophagy in Des-/- hearts. Nevertheless, improvement of all these parameters did not suffice to overcome the significant structural deficiencies that desmin deletion incurs in cardiomyocytes and cardiac function did not improve significantly. In conclusion, pharmacological PPARδ activation in Des-/- hearts exerts protective effects during myocardial degeneration and heart failure by preserving the function and quality of the mitochondrial network. These findings implicate PPARδ agonists as a supplemental constituent of heart failure medications.

The role of glucose in cardiac physiology and pathophysiology.

PURPOSE OF REVIEW: Heart failure is one of the major causes of death worldwide and continues to increase despite therapeutics and pharmacology advances. Fatty acids and glucose are used as ATP-producing fuels in heart to meet its energy demands. However, dysregulation of metabolites' use plays a pivotal role in cardiac diseases. How glucose becomes toxic or drives cardiac dysfunction is incompletely understood. In the present review, we summarize the recent findings on cardiac cellular and molecular events that are driven by glucose during pathologic conditions and potential therapeutic strategies to tackle hyperglycemia-mediated cardiac dysfunction. RECENT FINDINGS: Several studies have emerged recently, demonstrating that excessive glucose utilization has been correlated with impairment of cellular metabolic homeostasis primarily driven by mitochondrial dysfunction and damage, oxidative stress, and abnormal redox signaling. This disturbance is associated with cardiac remodeling, hypertrophy, and systolic and diastolic dysfunction. Both human and animal heart failure studies, report that glucose is a preferable fuel at the expense of fatty acid oxidation during ischemia and hypertrophy, but the opposite happens in diabetic hearts, which warrants further investigation. SUMMARY: A better understanding of glucose metabolism and its fate during distinct types of heart disease will contribute to developing novel therapeutic options for the prevention and treatment of heart failure.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

Specificity Proteins (SP) and Krüppel-like Factors (KLF) in Liver Physiology and Pathology.

The liver acts as a central hub that controls several essential physiological processes ranging from metabolism to detoxification of xenobiotics. At the cellular level, these pleiotropic functions are facilitated through transcriptional regulation in hepatocytes. Defects in hepatocyte function and its transcriptional regulatory mechanisms have a detrimental influence on liver function leading to the development of hepatic diseases. In recent years, increased intake of alcohol and western diet also resulted in a significantly increasing number of people predisposed to the incidence of hepatic diseases. Liver diseases constitute one of the serious contributors to global deaths, constituting the cause of approximately two million deaths worldwide. Understanding hepatocyte transcriptional mechanisms and gene regulation is essential to delineate pathophysiology during disease progression. The current review summarizes the contribution of a family of zinc finger family transcription factors, named specificity protein (SP) and Krüppel-like factors (KLF), in physiological hepatocyte functions, as well as how they are involved in the onset and development of hepatic diseases.

Krüppel-like factor (KLF)5: An emerging foe of cardiovascular health.

Krüppel-like factors (KLFs) are DNA-binding transcriptional factors, which regulate various pathways that pertain to development, metabolism and other cellular mechanisms. KLF5 was first cloned in 1993 and by 1999, it was reported as the intestinal-enriched KLF. Beyond findings that have associated KLF5 with normal development and cancer, it has been associated with various types of cardiovascular (CV) complications and regulation of metabolic pathways in the liver, heart, adipose tissue and skeletal muscle. Specifically, increased KLF5 expression has been linked with cardiomyopathy in diabetes, end-stage heart failure, and as well as in vascular atherosclerotic lesions. In this review article, we summarize research findings about transcriptional, post-transcriptional and post-translational regulation of KLF5, as well as the role of KLF5 in the biology of cells and organs that affect cardiovascular health either directly or indirectly. Finally, we propose KLF5 inhibition as an emerging approach for cardiovascular therapeutics.

Cardiomyocyte Krüppel-Like Factor 5 Promotes De Novo Ceramide Biosynthesis and Contributes to Eccentric Remodeling in Ischemic Cardiomyopathy.

BACKGROUND: We previously showed that cardiomyocyte Krϋppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. METHODS: Using real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5-/-). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography-tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. RESULTS: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post-permanent left coronary artery ligation. αMHC-KLF5-/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5-/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5-/- mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. CONCLUSIONS: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. METHODS: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. RESULTS: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. CONCLUSIONS: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

PKCδ causes sepsis-induced cardiomyopathy by inducing mitochondrial dysfunction.

Sepsis-induced cardiomyopathy (SIC) is associated with increased patient mortality. At present, there are no specific therapies for SIC. Previous studies have reported increased reactive oxygen species (ROS) and mitochondrial dysfunction during SIC. However, a unifying mechanism remains to be defined. We hypothesized that PKCδ is required for abnormal calcium handling and cardiac mitochondrial dysfunction during sepsis and that genetic deletion of PKCδ would be protective. Polymicrobial sepsis induced by cecal ligation and puncture (CLP) surgery decreased the ejection fraction of wild-type (WT) mice but not PKCδ knockout (KO) mice. Similarly, WT cardiomyocytes exposed to lipopolysaccharide (LPS) demonstrated decreases in contractility and calcium transient amplitude that were not observed in PKCδ KO cardiomyocytes. LPS treatment decreased sarcoplasmic reticulum calcium stores in WT cardiomyocytes, which correlated with increased ryanodine receptor-2 oxidation in WT hearts but not PKCδ KO hearts after sepsis. LPS exposure increased mitochondrial ROS and decreased mitochondrial inner membrane potential in WT cardiomyocytes. This corresponded to morphologic changes consistent with mitochondrial dysfunction such as decreased overall size and cristae disorganization. Increased cellular ROS and changes in mitochondrial morphology were not observed in PKCδ KO cardiomyocytes. These data show that PKCδ is required in the pathophysiology of SIC by generating ROS and promoting mitochondrial dysfunction. Thus, PKCδ is a potential target for cardiac protection during sepsis.NEW & NOTEWORTHY Sepsis is often complicated by cardiac dysfunction, which is associated with a high mortality rate. Our work shows that the protein PKCδ is required for decreased cardiac contractility during sepsis. Mice with deletion of PKCδ are protected from cardiac dysfunction after sepsis. PKCδ causes mitochondrial dysfunction in cardiac myocytes, and reducing mitochondrial oxidative stress improves contractility in wild-type cardiomyocytes. Thus, PKCδ is a potential target for cardiac protection during sepsis.

Loss of dynamic regulation of G protein-coupled receptor kinase 2 by nitric oxide leads to cardiovascular dysfunction with aging.

Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including ß-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2's desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD.NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.

The Glitazars Paradox: Cardiotoxicity of the Metabolically Beneficial Dual PPARα and PPARγ Activation.

The most common complications in patients with type-2 diabetes are hyperglycemia and hyperlipidemia that can lead to cardiovascular disease. Alleviation of these complications constitutes the major therapeutic approach for the treatment of diabetes mellitus. Agonists of peroxisome proliferator-activated receptor (PPAR) alpha and PPARγ are used for the treatment of hyperlipidemia and hyperglycemia, respectively. PPARs belong to the nuclear receptors superfamily and regulate fatty acid metabolism. PPARα ligands, such as fibrates, reduce circulating triglyceride levels, and PPARγ agonists, such as thiazolidinediones, improve insulin sensitivity. Dual-PPARα/γ agonists (glitazars) were developed to combine the beneficial effects of PPARα and PPARγ agonism. Although they improved metabolic parameters, they paradoxically aggravated congestive heart failure in patients with type-2 diabetes via mechanisms that remain elusive. Many of the glitazars, such as muraglitazar, tesaglitazar, and aleglitazar, were abandoned in phase-III clinical trials. The objective of this review article pertains to the understanding of how combined PPARα and PPARγ activation, which successfully targets the major complications of diabetes, causes cardiac dysfunction. Furthermore, it aims to suggest interventions that will maintain the beneficial effects of dual PPARα/γ agonism and alleviate adverse cardiac outcomes in diabetes.

Chemically synthesized Secoisolariciresinol diglucoside (LGM2605) improves mitochondrial function in cardiac myocytes and alleviates septic cardiomyopathy.

Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular metabolic health, we tested if the synthetic anti-oxidant lignan secoisolariciresinol diglucoside (SDG; LGM2605) would alleviate septic cardiac dysfunction and investigated the underlying mechanism. Using the cecal ligation and puncture (CLP) mouse model of peritonitis-induced sepsis, we observed impairment of cardiac function beginning at 4 h post-CLP surgery. Treatment of mice with LGM2605 (100 mg/kg body weight, i.p.) 6 h post-CLP surgery reduced cardiac ROS accumulation and restored cardiac function. Assessment of mitochondrial respiration (Seahorse XF) in primary cardiomyocytes obtained from adult C57BL/6 mice that had undergone CLP and treatment with LGM2605 showed restored basal and maximal respiration, as well as preserved oxygen consumption rate (OCR) associated with spare capacity. Further analyses aiming to identify the cellular mechanisms that may account for improved cardiac function showed that LGM2605 restored mitochondria abundance, increased mitochondrial calcium uptake and preserved mitochondrial membrane potential. In addition to protecting against cardiac dysfunction, daily treatment with LGM2605 and antibiotic ertapenem (70 mg/kg) protected against CLP-associated mortality and reversed hypothermia when compared against mice receiving ertapenem and saline. Therefore, treatment of septic mice with LGM2605 emerges as a novel pharmacological approach that reduces cardiac ROS accumulation, protects cardiac mitochondrial function, alleviates cardiac dysfunction, and improves survival.

MicroRNA-195 Regulates Metabolism in Failing Myocardium Via Alterations in Sirtuin 3 Expression and Mitochondrial Protein Acetylation.

BACKGROUND: Heart failure leads to mitochondrial dysfunction and metabolic abnormalities of the failing myocardium coupled with an energy-depleted state and cardiac remodeling. The mitochondrial deacetylase sirtuin 3 (SIRT3) plays a pivotal role in the maintenance of mitochondrial function through regulating the mitochondrial acetylome. It is interesting to note that unique cardiac and systemic microRNAs have been shown to play an important role in cardiac remodeling by modulating key signaling elements in the myocardium. METHODS: Cellular signaling was analyzed in human cardiomyocyte-like AC16 cells, and acetylation levels in rodent models of SIRT3-/-and transgenic microRNA-195 (miR-195) overexpression were compared with wild type. Luciferase assays, Western blotting, immunoprecipitation assays, and echocardiographic analysis were performed. Enzymatic activities of pyruvate dehydrogenase (PDH) and ATP synthase were measured. RESULTS: In failing human myocardium, we observed induction of miR-195 along with decreased expression of the mitochondrial deacetylase SIRT3 that was associated with increased global protein acetylation. We further investigated the role of miR-195 in SIRT3-mediated metabolic processes and its impact on regulating enzymes involved in deacetylation. Proteomic analysis of the total acetylome showed increased overall acetylation, and specific lysine acetylation of 2 central mitochondrial metabolic enzymes, PDH and ATP synthase, as well. miR-195 downregulates SIRT3 expression through direct 3'-untranslated region targeting. Treatments with either sirtuin inhibitor nicotinamide, small interfering RNA-mediated SIRT3 knockdown or miR-195 overexpression enhanced acetylation of PDH complex and ATP synthase. This effect diminished PDH and ATP synthase activity and impaired mitochondrial respiration.SIRT3-/- and miR-195 transgenic mice consistently showed enhanced global protein acetylation, including PDH complex and ATP synthase, associated with decreased enzymatic activity. CONCLUSIONS: Altogether, these data suggest that increased levels of miR-195 in failing myocardium regulate a novel pathway that involves direct SIRT3 suppression and enzymatic inhibition via increased acetylation of PDH and ATP synthase that are essential for cardiac energy metabolism.

Nutriepigenetics and cardiovascular disease.

PURPOSE OF REVIEW: We present a current perspective of epigenetic alterations that can lead to cardiovascular disease (CVD) and the potential of dietary factors to counteract their actions. In addition, we discuss the challenges and opportunities of dietary treatments as epigenetic modifiers for disease prevention and therapy. RECENT FINDINGS: Recent epigenome-wide association studies along with candidate gene approaches and functional studies in cell culture and animal models have delineated mechanisms through which nutrients, food compounds and dietary patterns may affect the epigenome. Several risk factors for CVD, including adiposity, inflammation and oxidative stress, have been associated with changes in histone acetylation, lower global DNA methylation levels and shorter telomere length. A surplus of macronutrients such as in a high-fat diet or deficiencies of specific nutrients such as folate and other B-vitamins can affect the activity of DNA methyltransferases and histone-modifying enzymes, affecting foetal growth, glucose/lipid metabolism, oxidative stress, inflammation and atherosclerosis. Bioactive compounds such as polyphenols (resveratrol, curcumin) or epigallocatechin may activate deacetylases Sirtuins (SIRTs), histone deacetylases or acetyltransferases and in turn the response of inflammatory mediators. Adherence to cardioprotective dietary patterns, such as the Mediterranean diet (MedDiet), has been associated with altered methylation and expression of genes related to inflammation and immuno-competence. SUMMARY: The mechanisms through which nutrients and dietary patterns may alter the cardiovascular epigenome remain elusive. The research challenge is to determine which of these nutriepigenetic effects are reversible, so that novel findings translate into effective dietary interventions to prevent CVD or its progression.

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.

Cardiac Myocyte KLF5 Regulates Ppara Expression and Cardiac Function.

RATIONALE: Fatty acid oxidation is transcriptionally regulated by peroxisome proliferator-activated receptor (PPAR)α and under normal conditions accounts for 70% of cardiac ATP content. Reduced Ppara expression during sepsis and heart failure leads to reduced fatty acid oxidation and myocardial energy deficiency. Many of the transcriptional regulators of Ppara are unknown. OBJECTIVE: To determine the role of Krüppel-like factor 5 (KLF5) in transcriptional regulation of Ppara. METHODS AND RESULTS: We discovered that KLF5 activates Ppara gene expression via direct promoter binding. This is blocked in hearts of septic mice by c-Jun, which binds an overlapping site on the Ppara promoter and reduces transcription. We generated cardiac myocyte-specific Klf5 knockout mice that showed reduced expression of cardiac Ppara and its downstream fatty acid metabolism-related targets. These changes were associated with reduced cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction. Diabetic mice showed parallel changes in cardiac Klf5 and Ppara expression levels. CONCLUSIONS: Cardiac myocyte KLF5 is a transcriptional regulator of Ppara and cardiac energetics.

Lipolysis, and not hepatic lipogenesis, is the primary modulator of triglyceride levels in streptozotocin-induced diabetic mice.

OBJECTIVE: Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. APPROACH AND RESULTS: To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin-deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin-induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signaling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial triglycerides clearance, which was associated with decreased lipoprotein lipase and peroxisome proliferator-activated receptor α mRNA levels in peripheral tissues and decreased lipoprotein lipase activity in skeletal muscle, heart, and brown adipose tissue. Diabetic heterozygous lipoprotein lipase knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial triglycerides clearance. CONCLUSIONS: Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic triglycerides production and secretion.

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Sequestration of fatty acids in triglycerides prevents endoplasmic reticulum stress in an in vitro model of cardiomyocyte lipotoxicity.

We used human cardiomyocyte-derived cells to create an in vitro model to study lipid metabolism and explored the effects of PPARγ; ACSL1 and ATGL on fatty acid-induced ER stress. Compared to oleate, palmitate treatment resulted in less intracellular accumulation of lipid droplets and more ER stress, as measured by upregulation of CHOP, ATF6 and GRP78 gene expression and phosphorylation of eukaryotic initiation factor 2a (EIF2a). Both ACSL1 and PPARγ adenovirus-mediated expression augmented neutral lipid accumulation and reduced palmitate-induced upregulation of ER stress markers to levels similar to those in the oleate and control treatment groups. This suggests that increased channeling of non-esterified free fatty acids (NEFA) towards storage in the form of neutral lipids in lipid droplets protects against palmitate-induced ER stress. Overexpression of ATGL in cells incubated with oleate-containing medium increased NEFA release and stimulated expression of ER stress markers. Thus, inefficient creation of lipid droplets as well greater release of stored lipids induces ER stress.

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 Ly6c(LO) 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.