Anthrax Lethal Toxin Kills Macrophages in a Strain-Specific Manner by Apoptosis or Caspase-1-Mediated Necrosis

Murine macrophages have been classified as either susceptible or nonsusceptible to killing by anthrax lethal toxin (LT) depending upon genetic background. While considered resistant to LT killing, we found that bone marrow-derived macrophages (BMMs) from DBA/2, AKR, and C57BL/6 mice were slowly killed by apoptosis following LT exposure. LT killing was not restricted to in vitro assays, as splenic macrophages were also depleted in LT-injected C57BL/6 mice. Human macrophages, also considered LT resistant, similarly underwent slow apoptosis in response to LT challenge. In contrast, LT triggered rapid necrosis and a broad protein release in BMMs derived from BALB/c and C3H/HeJ, but not C57BL/6 mice. Released proteins included processed interleukin-18, confirming reports of inflammasome and caspase-1 activation in LT-mediated necrosis in macrophages. Complete inhibition of caspase-1 activity was required to block LT-mediated necrosis. Strikingly, minimal residual caspase-1 activity was sufficient to trigger significant necrosis in LT-treated macrophages, indicating the toxicity of caspase-1 in this process. IL-18 release does not trigger cytolysis, as IL-18 is released late and only from LT-treated macrophages undergoing membrane perturbation. We propose that caspase-1-mediated macrophage necrosis is the source of the cytokine storm and rapid disease progression reported in LT-treated BALB/c mice.     

Erythropoiesis Suppression Is Associated with Anthrax Lethal Toxin-Mediated Pathogenic Progression

Anthrax is a disease caused by the bacterium Bacillus anthracis, which results in high mortality in animals and humans. Although some of the mechanisms are already known such as asphyxia, extensive knowledge of molecular pathogenesis of this disease is deficient and remains to be further investigated. Lethal toxin (LT) is a major virulence factor of B. anthracis and a specific inhibitor/protease of mitogen-activated protein kinase kinases (MAPKKs). Anthrax LT causes lethality and induces certain anthrax-like symptoms, such as anemia and hypoxia, in experimental mice. Mitogen-activated protein kinases (MAPKs) are the downstream pathways of MAPKKs, and are important for erythropoiesis. This prompted us to hypothesize that anemia and hypoxia may in part be exacerbated by erythropoietic dysfunction. As revealed by colony-forming cell assays in this study, LT challenges significantly reduced mouse erythroid progenitor cells. In addition, in a proteolytic activity-dependent manner, LT suppressed cell survival and differentiation of cord blood CD34⁺-derived erythroblasts in vitro. Suppression of cell numbers and the percentage of erythroblasts in the bone marrow were detected in LT-challenged C57BL/6J mice. In contrast, erythropoiesis was provoked through treatments of erythropoietin, significantly ameliorating the anemia and reducing the mortality of LT-treated mice. These data suggested that suppressed erythropoiesis is part of the pathophysiology of LT-mediated intoxication. Because specific treatments to overcome LT-mediated pathogenesis are still lacking, these efforts may help the development of effective treatments against anthrax.     

Nitric Oxide Synthase-3 Promotes Embryonic Development of Atrioventricular Valves

Nitric oxide synthase-3 (NOS3) has recently been shown to promote endothelial-to-mesenchymal transition (EndMT) in the developing atrioventricular (AV) canal. The present study was aimed to investigate the role of NOS3 in embryonic development of AV valves. We hypothesized that NOS3 promotes embryonic development of AV valves via EndMT. To test this hypothesis, morphological and functional analysis of AV valves were performed in wild-type (WT) and NOS3^−/− mice at postnatal day 0. Our data show that the overall size and length of mitral and tricuspid valves were decreased in NOS3^−/− compared with WT mice. Echocardiographic assessment showed significant regurgitation of mitral and tricuspid valves during systole in NOS3^−/− mice. These phenotypes were all rescued by cardiac specific NOS3 overexpression. To assess EndMT, immunostaining of Snail1 was performed in the embryonic heart. Both total mesenchymal and Snail1⁺ cells in the AV cushion were decreased in NOS3^−/− compared with WT mice at E10.5 and E12.5, which was completely restored by cardiac specific NOS3 overexpression. In cultured embryonic hearts, NOS3 promoted transforming growth factor (TGFβ), bone morphogenetic protein (BMP2) and Snail1expression through cGMP. Furthermore, mesenchymal cell formation and migration from cultured AV cushion explants were decreased in the NOS3^−/− compared with WT mice. We conclude that NOS3 promotes AV valve formation during embryonic heart development and deficiency in NOS3 results in AV valve insufficiency.     

GPR55 Deletion in Mice Leads to Age-Related Ventricular Dysfunction and Impaired Adrenoceptor-Mediated Inotropic Responses

G protein coupled receptor 55 (GPR55) is expressed throughout the body, and although its exact physiological function is unknown, studies have suggested a role in the cardiovascular system. In particular, GPR55 has been proposed as mediating the haemodynamic effects of a number of atypical cannabinoid ligands; however this data is conflicting. Thus, given the incongruous nature of our understanding of the GPR55 receptor and the relative paucity of literature regarding its role in cardiovascular physiology, this study was carried out to examine the influence of GPR55 on cardiac function. Cardiac function was assessed via pressure volume loop analysis, and cardiac morphology/composition assessed via histological staining, in both wild-type (WT) and GPR55 knockout (GPR55^−/−) mice. Pressure volume loop analysis revealed that basal cardiac function was similar in young WT and GPR55^−/− mice. In contrast, mature GPR55^−/− mice were characterised by both significant ventricular remodelling (reduced left ventricular wall thickness and increased collagen deposition) and systolic dysfunction when compared to age-matched WT mice. In particular, the load-dependent parameter, ejection fraction, and the load-independent indices, end-systolic pressure-volume relationship (ESPVR) and E _max, were all significantly (P<0.05) attenuated in mature GPR55^−/− mice. Furthermore, GPR55^−/− mice at all ages were characterised by a reduced contractile reserve. Our findings demonstrate that mice deficient in GPR55 exhibit maladaptive adrenergic signalling, as evidenced by the reduced contractile reserve. Furthermore, with age these mice are characterised by both significant adverse ventricular remodelling and systolic dysfunction. Taken together, this may suggest a role for GPR55 in the control of adrenergic signalling in the heart and potentially a role for this receptor in the pathogenesis of heart failure.     

Proteome analysis of mouse macrophages treated with anthrax lethal toxin

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

Glutaredoxin-2 Is Required to Control Oxidative Phosphorylation in Cardiac Muscle by Mediating Deglutathionylation Reactions

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2^−/−) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2^−/− hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2^+/−) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2^+/− mice were far less hypertensive than Grx2^−/− mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.     

MAIR‐II deficiency ameliorates cardiac remodelling post‐myocardial infarction by suppressing TLR9‐mediated macrophage activation

Macrophages are fundamental components of inflammation in post‐myocardial infarction (MI) and contribute to adverse cardiac remodelling and heart failure. However, the regulatory mechanisms in macrophage activation have not been fully elucidated. Previous studies showed that myeloid‐associated immunoglobulin–like receptor II (MAIR‐II) is involved in inflammatory responses in macrophages. However, its role in MI is unknown. Thus, this study aimed to determine a novel role and mechanism of MAIR‐II in MI. We first identified that MAIR‐II–positive myeloid cells were abundant from post‐MI days 3 to 5 in infarcted hearts of C57BL/6J (WT) mice induced by permanent left coronary artery ligation. Compared to WT, MAIR‐II–deficient (Cd300c2 ^−/−) mice had longer survival, ameliorated cardiac remodelling, improved cardiac function and smaller infarct sizes. Moreover, we detected lower pro‐inflammatory cytokine and fibrotic gene expressions in Cd300c2 ^−/−‐infarcted hearts. These mice also had less infiltrating pro‐inflammatory macrophages following MI. To elucidate a novel molecular mechanism of MAIR‐II, we considered macrophage activation by Toll‐like receptor (TLR) 9–mediated inflammation. In vitro, we observed that Cd300c2 ^−/− bone marrow–derived macrophages stimulated by a TLR9 agonist expressed less pro‐inflammatory cytokines compared to WT. In conclusion, MAIR‐II may enhance inflammation via TLR9‐mediated macrophage activation in MI, leading to adverse cardiac remodelling and poor prognosis.     

Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.     

Mannose-Binding Lectin Regulates Host Resistance and Pathology during Experimental Infection with Trypanosoma cruzi

Mannose-binding lectin (MBL) is a humoral pattern-recognition molecule important for host defense. Although recent genetic studies suggest an involvement of MBL/MASP2-associated pathways in Chagas’ disease, it is currently unknown whether MBL plays a role in host resistance to the intracellular protozoan Trypanosoma cruzi, the causative agent of Chagas’ disease. In this study we employed MBL^−/− mice to assess the role of MBL in resistance to experimental infection with T. cruzi. T. cruzi infection enhanced tissue expression of MBL both at the mRNA and protein level. Similarly, symptomatic acute Chagas’ disease patients displayed increased serum concentrations of MBL compared to patients with indeterminate, asymptomatic forms of the disease. Furthermore, increased parasite loads in the blood and/or tissue were observed in MBL^−/− mice compared to WT controls. This was associated with reduced systemic levels of IL-12/23p40 in MBL^−/− mice. Importantly, MBL^−/− mice infected with a cardiotropic strain of T. cruzi displayed increased myocarditis and cardiac fibrosis compared to WT controls. The latter was accompanied by elevated hydroxyproline content and mRNA levels of collagen-1 and -6 in the heart. These observations point to a previously unappreciated role for MBL in regulating host resistance and cardiac inflammation during infection with a major human pathogen.     

Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2?/?CX3CR1GFP/GFP Mice

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2^−/−CX3CR1^GFP/GFP mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2^−/−CX3CR1^GFP/GFP mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17∼60% of 12-month, and 30∼100% of 18-month CCL2^−/−CX3CR1^GFP/GFP mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2^−/−CX3CR1^GFP/GFP mice respectively, but not in wild-type mice. All CCL2^−/−CX3CR1^GFP/GFP mice exposed to extra-light (∼800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20–25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2^−/−CX3CR1^GFP/GFP mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2^−/−CX3CR1^GFP/GFP mice. GABA expression was reduced in the inner retina of aged CCL2^−/−CX3CR1^GFP/GFP mice. Significantly increased Müller glial and microglial activation was observed in CCL2^−/−CX3CR1^GFP/GFP mice compared to age-matched WT mice. Macrophages from CCL2^−/−CX3CR1^GFP/GFP mice were less phagocytic, but expressed higher levels of iNOS, IL-1β, IL-12 and TNF-α under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.     

Nuclear Recruitment of Neuronal Nitric-oxide Synthase by α-Syntrophin Is Crucial for the Induction of Mitochondrial Biogenesis

Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSβ) does not localize in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis. Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular diseases associated with mitochondrial impairment.     

Generation and Characterization of a Mouse Model Harboring the Exon-3 Deletion in the Cardiac Ryanodine Receptor

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del^+/−) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del^+/− mice are not susceptible to CPVT. Ex3-del^+/− cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca²⁺ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del^+/− mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del^+/− mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del^+/− mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.     

Partial Deletion of eNOS Gene Causes Hyperinsulinemic State,Unbalance of Cardiac Insulin Signaling Pathways and Coronary Dysfunction Independently of High Fat Diet

Abnormalities in eNOS gene, possibly interacting with high fat diet (HFD), affect peripheral vascular function and glucose metabolism. The relative role of eNOS gene, HFD and metabolic derangement on coronary function has not been fully elucidated. We test whether eNOS gene deficiency per se or in association with HFD modulates coronary function through mechanisms involving molecular pathways related to insulin signaling. Wild type (WT), eNOS^−/− and eNOS^+/− mice were studied. WT and eNOS^+/− mice were fed with either standard or HF diet for 16 weeks and compared with standard diet fed eNOS^−/−. Glucose and insulin tolerance tests were performed during the last week of diet. Coronary resistance (CR) was measured at baseline and during infusions of acetylcholine (Ach) or sodium-nitroprusside (SNP) to evaluate endothelium-dependent or independent vasodilation, in the Langendorff isolated hearts. Cardiac expression of Akt and ERK genes as evaluation of two major insulin-regulated signaling pathways involved in the control of vascular tone were assessed by western blot. HFD-fed mice developed an overt diabetic state. Conversely, chow-fed genetically modified mice (in particular eNOS^−/−) showed a metabolic pattern characterized by normoglycemia and hyperinsulinemia with a limited degree of insulin resistance. CR was significantly higher in animals with eNOS gene deletions than in WT, independently of diet. Percent decrease in CR, during Ach infusion, was significantly lower in both eNOS^−/− and eNOS^+/− mice than in WT, independently of diet. SNP reduced CR in all groups except eNOS^−/−. The cardiac ERK1-2/Akt ratio, increased in animals with eNOS gene deletions compared with WT, independently of diet. These results suggest that the eNOS genetic deficiency, associated or not with HFD, has a relevant effect on coronary vascular function, possibly mediated by increase in blood insulin levels and unbalance in insulin-dependent signaling in coronary vessels, consistent with a shift towards a vasoconstrictive pattern.     

The Role of Interferon in Influenza Virus Tissue Tropism

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1−/− animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1−/− mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT−/− mice, and mice lacking gamma interferon (IFNγ−/− mice) or the IFN-α receptor (IFNαR−/− mice). Influenza A/WSN/33 virus replicates to high titers in STAT1−/− or IFNαR−/− fibroblasts, while cells derived from WT or IFNγ−/− animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.     

Cardiomyocyte-specific Loss of Diacylglycerol Acyltransferase 1 (DGAT1) Reproduces the Abnormalities in Lipids Found in Severe Heart Failure

Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triglyceride synthesis, the conversion of diacylglycerol (DAG) to triglyceride. Dgat1^−/− mice exhibit a number of beneficial metabolic effects including reduced obesity and improved insulin sensitivity and no known cardiac dysfunction. In contrast, failing human hearts have severely reduced DGAT1 expression associated with accumulation of DAGs and ceramides. To test whether DGAT1 loss alone affects heart function, we created cardiomyocyte-specific DGAT1 knock-out (hDgat1^−/−) mice. hDgat1^−/− mouse hearts had 95% increased DAG and 85% increased ceramides compared with floxed controls. 50% of these mice died by 9 months of age. The heart failure marker brain natriuretic peptide increased 5-fold in hDgat1^−/− hearts, and fractional shortening (FS) was reduced. This was associated with increased expression of peroxisome proliferator-activated receptor α and cluster of differentiation 36. We crossed hDgat1^−/− mice with previously described enterocyte-specific Dgat1 knock-out mice (hiDgat1^−/−). This corrected the early mortality, improved FS, and reduced cardiac ceramide and DAG content. Treatment of hDgat1^−/− mice with the glucagon-like peptide 1 receptor agonist exenatide also improved FS and reduced heart DAG and ceramide content. Increased fatty acid uptake into hDgat1^−/− hearts was normalized by exenatide. Reduced activation of protein kinase Cα (PKCα), which is increased by DAG and ceramides, paralleled the reductions in these lipids. Our mouse studies show that loss of DGAT1 reproduces the lipid abnormalities seen in severe human heart failure.     

Altered Lipid and Salt Taste Responsivity in Ghrelin and GOAT Null Mice

Taste perception plays an important role in regulating food preference, eating behavior and energy homeostasis. Taste perception is modulated by a variety of factors, including gastric hormones such as ghrelin. Ghrelin can regulate growth hormone release, food intake, adiposity, and energy metabolism. Octanoylation of ghrelin by ghrelin O-acyltransferase (GOAT) is a specific post-translational modification which is essential for many biological activities of ghrelin. Ghrelin and GOAT are both widely expressed in many organs including the gustatory system. In the current study, overall metabolic profiles were assessed in wild-type (WT), ghrelin knockout (ghrelin^−/−), and GOAT knockout (GOAT^−/−) mice. Ghrelin^−/− mice exhibited decreased food intake, increased plasma triglycerides and increased ketone bodies compared to WT mice while demonstrating WT-like body weight, fat composition and glucose control. In contrast GOAT^−/− mice exhibited reduced body weight, adiposity, resting glucose and insulin levels compared to WT mice. Brief access taste behavioral tests were performed to determine taste responsivity in WT, ghrelin^−/− and GOAT^−/− mice. Ghrelin and GOAT null mice possessed reduced lipid taste responsivity. Furthermore, we found that salty taste responsivity was attenuated in ghrelin^−/− mice, yet potentiated in GOAT^−/− mice compared to WT mice. Expression of the potential lipid taste regulators Cd36 and Gpr120 were reduced in the taste buds of ghrelin and GOAT null mice, while the salt-sensitive ENaC subunit was increased in GOAT^−/− mice compared with WT mice. The altered expression of Cd36, Gpr120 and ENaC may be responsible for the altered lipid and salt taste perception in ghrelin^−/− and GOAT^−/− mice. The data presented in the current study potentially implicates ghrelin signaling activity in the modulation of both lipid and salt taste modalities.     

Mice with Tissue Inhibitor of Metalloproteinases 4 (Timp4) Deletion Succumb to Induced Myocardial Infarction but Not to Cardiac Pressure Overload

Tissue inhibitor of metalloproteinases 4 (TIMP4) is expressed highly in heart and found dysregulated in human cardiovascular diseases. It controls extracellular matrix remodeling by inhibiting matrix metalloproteinases (MMPs) and is implicated in processes including cell proliferation, apoptosis, and angiogenesis. Timp4-deficient mice (Timp4^−/−) were generated to assess TIMP4 function in normal development and in models of heart disease. We deleted exons 1–3 of the Timp4 gene by homologous recombination. Timp4^−/− mice are born healthy, develop normally, and produce litters of normal size and gender distribution. These mice show no compensation by overexpression of Timp1, Timp2, or Timp3 in the heart. Following cardiac pressure overload by aortic banding, Timp4^−/− mice have comparable survival rate, cardiac histology, and cardiac function to controls. In this case, Timp4 deficiency is compensated by increased cardiac Timp2 expression. Strikingly, the induction of myocardial infarction (MI) leads to significantly increased mortality in Timp4^−/− mice primarily due to left ventricular rupture. The post-MI mortality of Timp4^−/− mice is reduced by administration of a synthetic MMP inhibitor. Furthermore, combining the genetic deletion of Mmp2 also rescues the higher post-MI mortality of Timp4^−/− mice. Finally, Timp4^−/− mice suffer reduced cardiac function at 20 months of age. Timp4 is not essential for murine development, although its loss moderately compromises cardiac function with aging. Timp4^−/− mice are more susceptible to MI but not to pressure overload, and TIMP4 functions in its capacity as a metalloproteinase inhibitor after myocardial infarction.