Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1^T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1^T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1^T−/− hearts, control and Acsl1^T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function.     

A mouse model of inherited choline kinase β-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia

The CHKB gene encodes choline kinase β, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb^−/− mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb^+/− mice. Unlike wildtype mice, 60% of the Chkb^+/− and all Chkb^−/− mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb^+/− and Chkb^−/− hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb^−/− hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb^+/− and Chkb^−/− mice.     

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.     

Cardiac Per2 Functions as Novel Link between Fatty Acid Metabolism and Myocardial Inflammation during Ischemia and Reperfusion Injury of the Heart

Disruption of peripheral circadian rhyme pathways dominantly leads to metabolic disorders. Studies on circadian rhythm proteins in the heart indicated a role for Clock or Per2 in cardiac metabolism. In contrast to Clock^−/−, Per2^−/− mice have larger infarct sizes with deficient lactate production during myocardial ischemia. To test the hypothesis that cardiac Per2 represents an important regulator of cardiac metabolism during myocardial ischemia, we measured lactate during reperfusion in Per1^−/−, Per2^−/− or wildtype mice. As lactate measurements in whole blood indicated an exclusive role of Per2 in controlling lactate production during myocardial ischemia, we next performed gene array studies using various ischemia-reperfusion protocols comparing wildtype and Per2^−/− mice. Surprisingly, high-throughput gene array analysis revealed dominantly lipid metabolism as the differentially regulated pathway in wildtype mice when compared to Per2^−/−. In all ischemia-reperfusion protocols used, the enzyme enoyl-CoA hydratase, which is essential in fatty acid beta-oxidation, was regulated in wildtype animals only. Studies using nuclear magnet resonance imaging (NMRI) confirmed altered fatty acid populations with higher mono-unsaturated fatty acid levels in hearts from Per2^−/− mice. Unexpectedly, studies on gene regulation during reperfusion revealed solely pro inflammatory genes as differentially regulated ‘Per2-genes’. Subsequent studies on inflammatory markers showed increasing IL-6 or TNFα levels during reperfusion in Per2^−/− mice. In summary, these studies reveal an important role of cardiac Per2 for fatty acid metabolism and inflammation during myocardial ischemia and reperfusion, respectively.     

Haploinsufficiency of the Ammonia Transporter Rhcg Predisposes to Chronic Acidosis: Rhcg IS CRITICAL FOR APICAL AND BASOLATERAL AMMONIA TRANSPORT IN THE MOUSE COLLECTING DUCT*

Ammonia secretion by the collecting duct (CD) is critical for acid-base homeostasis and, when defective, causes distal renal tubular acidosis (dRTA). The Rhesus protein RhCG mediates NH₃ transport as evident from cell-free and cellular models as well as from Rhcg-null mice. Here, we investigated in a Rhcg mouse model the metabolic effects of Rhcg haploinsufficiency, the role of Rhcg in basolateral NH₃ transport, and the mechanisms of adaptation to the lack of Rhcg. Both Rhcg^+/+ and Rhcg^+/− mice were able to handle an acute acid load, whereas Rhcg^−/− mice developed severe metabolic acidosis with reduced ammonuria and high mortality. However, chronic acid loading revealed that Rhcg^+/− mice did not fully recover, showing lower blood HCO₃⁻ concentration and more alkaline urine. Microperfusion studies demonstrated that transepithelial NH₃ permeability was reduced by 80 and 40%, respectively, in CDs from Rhcg^−/− and Rhcg^+/− mice compared with controls. Basolateral membrane permeability to NH₃ was reduced in CDs from Rhcg^−/− mice consistent with basolateral Rhcg localization. Rhcg^−/− responded to acid loading with normal expression of enzymes and transporters involved in proximal tubular ammoniagenesis but reduced abundance of the NKCC2 transporter responsible for medullary accumulation of ammonium. Consequently, tissue ammonium content was decreased. These data demonstrate a role for apical and basolateral Rhcg in transepithelial NH₃ transport and uncover an incomplete dRTA phenotype in Rhcg^+/− mice. Haploinsufficiency or reduced expression of RhCG may underlie human forms of (in)complete dRTA.     

Acid Sphingomyelinase Gene Knockout Ameliorates Hyperhomocysteinemic Glomerular Injury in Mice Lacking Cystathionine-β-Synthase

Acid sphingomyelinase (ASM) has been implicated in the development of hyperhomocysteinemia (hHcys)-induced glomerular oxidative stress and injury. However, it remains unknown whether genetically engineering of ASM gene produces beneficial or detrimental action on hHcys-induced glomerular injury. The present study generated and characterized the mice lacking cystathionine β-synthase (Cbs) and Asm mouse gene by cross breeding Cbs^+/− and Asm^+/− mice. Given that the homozygotes of Cbs^−/−/Asm^−/− mice could not survive for 3 weeks. Cbs^+/−/Asm^+/+, Cbs^+/−/Asm^+/− and Cbs^+/−/Asm^−/− as well as their Cbs wild type littermates were used to study the role of Asm^−/− under a background of Cbs^+/− with hHcys. HPLC analysis revealed that plasma Hcys level was significantly elevated in Cbs heterozygous (Cbs^+/−) mice with different copies of Asm gene compared to Cbs^+/+ mice with different Asm gene copies. Cbs^+/−/Asm^+/+ mice had significantly increased renal Asm activity, ceramide production and O₂.⁻ level compared to Cbs^+/+/Asm^+/+, while Cbs^+/−/Asm^−/− mice showed significantly reduced renal Asm activity, ceramide production and O₂.⁻ level due to increased plasma Hcys levels. Confocal microscopy demonstrated that colocalization of podocin with ceramide was much lower in Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice, which was accompanied by a reduced glomerular damage index, albuminuria and proteinuria in Cbs^+/−/Asm^−/− mice. Immunofluorescent analyses of the podocin, nephrin and desmin expression also illustrated less podocyte damages in the glomeruli from Cbs^+/−/Asm^−/− mice compared to Cbs^+/−/Asm^+/+ mice. In in vitro studies of podocytes, hHcys-enhanced O₂.⁻ production, desmin expression, and ceramide production as well as decreases in VEGF level and podocin expression in podocytes were substantially attenuated by prior treatment with amitriptyline, an Asm inhibitor. In conclusion, Asm gene knockout or corresponding enzyme inhibition protects the podocytes and glomeruli from hHcys-induced oxidative stress and injury.     

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.     

Inducible Deletion of CD28 Prior to Secondary Nippostrongylus brasiliensis Infection Impairs Worm Expulsion and Recall of Protective Memory CD4+ T Cell Responses

IL-13 driven Th2 immunity is indispensable for host protection against infection with the gastrointestinal nematode Nippostronglus brasiliensis. Disruption of CD28 mediated costimulation impairs development of adequate Th2 immunity, showing an importance for CD28 during the initiation of an immune response against this pathogen. In this study, we used global CD28^−/− mice and a recently established mouse model that allows for inducible deletion of the cd28 gene by oral administration of tamoxifen (CD28^−/loxCre^+/−+TM) to resolve the controversy surrounding the requirement of CD28 costimulation for recall of protective memory responses against pathogenic infections. Following primary infection with N. brasiliensis, CD28^−/− mice had delayed expulsion of adult worms in the small intestine compared to wild-type C57BL/6 mice that cleared the infection by day 9 post-infection. Delayed expulsion was associated with reduced production of IL-13 and reduced serum levels of antigen specific IgG1 and total IgE. Interestingly, abrogation of CD28 costimulation in CD28^−/loxCre^+/− mice by oral administration of tamoxifen prior to secondary infection with N. brasiliensis resulted in impaired worm expulsion, similarly to infected CD28^−/− mice. This was associated with reduced production of the Th2 cytokines IL-13 and IL-4, diminished serum titres of antigen specific IgG1 and total IgE and a reduced CXCR5⁺ T_FH cell population. Furthermore, total number of CD4⁺ T cells and B220⁺ B cells secreting Th1 and Th2 cytokines were significantly reduced in CD28^−/− mice and tamoxifen treated CD28^−/loxCre^+/− mice compared to C57BL/6 mice. Importantly, interfering with CD28 costimulatory signalling before re-infection impaired the recruitment and/or expansion of central and effector memory CD4⁺ T cells and follicular B cells to the draining lymph node of tamoxifen treated CD28^−/loxCre^+/− mice. Therefore, it can be concluded that CD28 costimulation is essential for conferring host protection during secondary N. brasiliensis infection.     

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.     

Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6+/? Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6^+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6^+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6^+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6^+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6^+/− heterozygotes but BrdU-LRCs were also present in Pax6^+/− corneas. It seems likely that Pax6^+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6^+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6^+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6^+/− than WT mice. This implies that epithelial cell loss is higher in Pax6^+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6^+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.     

Mitophagy‐regulated mitochondrial health strongly protects the heart against cardiac dysfunction after acute myocardial infarction

Autophagy including mitophagy serves as an important regulatory mechanism in the heart to maintain the cellular homeostasis and to protect against heart damages caused by myocardial infarction (MI). The current study aims to dissect roles of general autophagy and specific mitophagy in regulating cardiac function after MI. By using Beclin1^+/−, Fundc1 knockout (KO) and Fundc1 transgenic (TG) mouse models, combined with starvation and MI models, we found that Fundc1 KO caused more severe mitochondrial and cardiac dysfunction damages than Beclin1^+/− after MI. Interestingly, Beclin1^+/− caused notable decrease of total autophagy without detectable change to mitophagy, and Fundc1 KO markedly suppressed mitophagy but did not change the total autophagy activity. In contrast, starvation increased total autophagy without changing mitophagy while Fundc1 TG elevated total autophagy and mitophagy in mouse hearts. As a result, Fundc1 TG provided much stronger protective effects than starvation after MI. Moreover, Beclin1^+/−/Fundc1 TG showed increased total autophagy and mitophagy to a level comparable to Fundc1 TG per se, and completely reversed Beclin1^+/−‐caused aggravation of mitochondrial and cardiac injury after MI. Our results reveal that mitophagy but not general autophagy contributes predominantly to the cardiac protective effect through regulating mitochondrial function.     

An Enhanced Immune Response of Mclk1 +/? Mutant Mice Is Associated with Partial Protection from Fibrosis,Cancer and the Development of Biomarkers of Aging

The immune response is essential for survival by destroying microorganisms and pre-cancerous cells. However, inflammation, one aspect of this response, can result in short- and long-term deleterious side-effects. Mclk1 ^+/− mutant mice can be long-lived despite displaying a hair-trigger inflammatory response and chronically activated macrophages as a result of high mitochondrial ROS generation. Here we ask whether this phenotype is beneficial or simply tolerated. We used models of infection by Salmonella serovars and found that Mclk1 ^+/− mutants mount a stronger immune response, control bacterial proliferation better, and are resistant to cell and tissue damage resulting from the response, including fibrosis and types of oxidative damage that are considered to be biomarkers of aging. Moreover, these same types of tissue damage were found to be low in untreated 23 months-old mutants. We also examined the initiation of tumour growth after transplantation of mouse LLC1 carcinoma cells into Mclk1 ^+/− mutants, as well as during spontaneous tumorigenesis in Mclk1 ^+/− Trp53 ^+/− double mutants. Tumour latency was increased by the Mclk1 ^+/− genotype in both models. Furthermore, we used the transplantation model to show that splenic CD8+ T lymphocytes from Mclk1 ^+/− graft recipients show enhanced cytotoxicity against LLC1 cells in vitro. Mclk1 ^+/− mutants thus display an association of an enhanced immune response with partial protection from age-dependent processes and from pathologies similar to those that are found with increased frequency during the aging process. This suggests that the immune phenotype of these mutants might contribute to their longevity. We discuss how these findings suggest a broader view of how the immune response might impact the aging process.     

Heterozygosity for Fibrinogen Results in Efficient Resolution of Kidney Ischemia Reperfusion Injury

Fibrinogen (Fg) has been recognized to play a central role in coagulation, inflammation and tissue regeneration. Several studies have used Fg deficient mice (Fg^−/−) in comparison with heterozygous mice (Fg^+/−) to point the proinflammatory role of Fg in diverse pathological conditions and disease states. Although Fg^+/− mice are considered ‘normal’, plasma Fg is reduced to ∼75% of the normal circulating levels present in wild type mice (Fg^+/+). We report that this reduction in Fg protein production in the Fg^+/− mice is enough to protect them from kidney ischemia reperfusion injury (IRI) as assessed by tubular injury, kidney dysfunction, necrosis, apoptosis and inflammatory immune cell infiltration. Mechanistically, we observed binding of Fg to ICAM-1 in kidney tissues of Fg^+/+ mice at 24 h following IRI as compared to a complete absence of binding observed in the Fg^+/− and Fg^−/− mice. Raf-1 and ERK were highly activated as evident by significantly higher phosphorylation in the Fg^+/+ kidneys at 24 h following IRI as compared to Fg^+/− and Fg^−/− mice kidneys. On the other hand Cyclin D1 and pRb, indicating higher cell proliferation, were significantly increased in the Fg^+/− and Fg^−/− as compared to Fg^+/+ kidneys. These data suggest that Fg heterozygosity allows maintenance of a critical balance of Fg that enables regression of initial injury and promotes faster resolution of kidney damage.     

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.     

Resistance to Diet-Induced Obesity and Associated Metabolic Perturbations in Haploinsufficient Monocarboxylate Transporter 1 Mice

The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1 ^+/− mice developed normally. However, when fed high fat diet (HFD), MCT1 ^+/− mice displayed resistance to development of diet-induced obesity (24.8% lower body weight after 16 weeks of HFD), as well as less insulin resistance and no hepatic steatosis as compared to littermate MCT1 ^+/+ mice used as controls. Body composition analysis revealed that reduced weight gain in MCT1 ^+/− mice was due to decreased fat accumulation (50.0% less after 9 months of HFD) notably in liver and white adipose tissue. This phenotype was associated with reduced food intake under HFD (12.3% less over 10 weeks) and decreased intestinal energy absorption (9.6% higher stool energy content). Indirect calorimetry measurements showed ∼ 15% increase in O₂ consumption and CO₂ production during the resting phase, without any changes in physical activity. Determination of plasma concentrations for various metabolites and hormones did not reveal significant changes in lactate and ketone bodies levels between the two genotypes, but both insulin and leptin levels, which were elevated in MCT1 ^+/+ mice when fed HFD, were reduced in MCT1 ^+/− mice under HFD. Interestingly, the enhancement in expression of several genes involved in lipid metabolism in the liver of MCT1 ^+/+ mice under high fat diet was prevented in the liver of MCT1 ^+/− mice under the same diet, thus likely contributing to the observed phenotype. These findings uncover the critical role of MCT1 in the regulation of energy balance when animals are exposed to an obesogenic diet.     

Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT^+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT^+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT^+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT^+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT^+/− EPCs. In AGT^+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.     

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.     

Sialylation and Muscle Performance: Sialic Acid Is a Marker of Muscle Ageing

Sialic acids (Sia) are widely expressed as terminal monosaccharides on eukaryotic glycoconjugates. They are involved in many cellular functions, such as cell–cell interaction and signal recognition. The key enzyme of sialic acid biosynthesis is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), which catalyses the first two steps of Sia biosynthesis in the cytosol. In this study we analysed sialylation of muscles in wild type (C57Bl/6 GNE ^+/+) and heterozygous GNE-deficient (C57Bl/6 GNE ^+/−) mice. We measured a significantly lower performance in the initial weeks of a treadmill exercise in C57Bl/6 GNE ^+/− mice compared to wild type C57Bl/6 GNE ^+/+animals. Membrane bound Sia of C57Bl/6 GNE ^+/− mice were reduced by 33–53% at week 24 and by 12–15% at week 80 in comparison to C57Bl/6 GNE ^+/+mice. Interestingly, membrane bound Sia concentration increased with age of the mice by 16–46% in C57Bl/6 GNE ^+/+, but by 87–207% in C57Bl/6 GNE ^+/−. Furthermore we could identify specific morphological changes in aged muscles. Here we propose that increased Sia concentrations in muscles are a characteristic feature of ageing and could be used as a marker for age-related changes in muscle.