Ablation of Toll-like receptor 9 attenuates myocardial ischemia/reperfusion injury in mice.

In myocardial ischemia/reperfusion injury, the innate immune and subsequent inflammatory responses play a crucial role in the extension of myocardial damage. Toll-like receptor 9 (TLR9) is a critical receptor for recognizing unmethylated CpG motifs that mitochondria contain in their DNA, and induces inflammatory responses. The aim of this study was to elucidate the role of TLR9 in myocardial ischemia/reperfusion injury. Isolated hearts from TLR9-deficient and control wild-type mice were subjected to 35 min of global ischemia, followed by 60 min of reperfusion with Langendorff apparatus. Furthermore, wild-type mouse hearts were infused with DNase I and subjected to ischemia/reperfusion. Ablation of TLR9-mediated signaling pathway attenuates myocardial ischemia/reperfusion injury and inflammatory responses, and digestion of extracellular mitochondrial DNA released from the infarct heart partially improved myocardial ischemia/reperfusion injury with no effect on inflammatory responses. TLR9 could be a therapeutic target to reduce myocardial ischemia/reperfusion injury.

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

Generation of Induced Pluripotent Stem Cells From Patients With Duchenne Muscular Dystrophy and Their Induction to Cardiomyocytes.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene which encodes dystrophin protein. Dystrophin defect affects cardiac muscle as well as skeletal muscle. Cardiac dysfunction is observed in all patients with DMD over 18 years of age, but there is no curative treatment for DMD cardiomyopathy. To establish novel experimental platforms which reproduce the cardiac phenotype of DMD patients, here we established iPS cell lines from T lymphocytes donated from two DMD patients, with a protocol using Sendai virus vectors. We successfully conducted the differentiation of the DMD patient-specific iPS cells into beating cardiomyocytes. DMD patient-specific iPS cells and iPS cell-derived cardiomyocytes would be a useful in vitro experimental system with which to investigate DMD cardiomyopathy.

Lysophosphatidylserine induces necrosis in pressure overloaded male mouse hearts via G protein coupled receptor 34.

Heart failure is a leading cause of mortality in developed countries. Cell death is a key player in the development of heart failure. Calcium-independent phospholipase A2ß (iPLA2ß) produces lipid mediators by catalyzing lipids and induces nuclear shrinkage in caspase-independent cell death. Here, we show that lysophosphatidylserine generated by iPLA2ß induces necrotic cardiomyocyte death, as well as contractile dysfunction mediated through its receptor, G protein-coupled receptor 34 (GPR34). Cardiomyocyte-specific iPLA2ß-deficient male mice were subjected to pressure overload. While control mice showed left ventricular systolic dysfunction with necrotic cardiomyocyte death, iPLA2ß-deficient mice preserved cardiac function. Lipidomic analysis revealed a reduction of 18:0 lysophosphatidylserine in iPLA2ß-deficient hearts. Knockdown of Gpr34 attenuated 18:0 lysophosphatidylserine-induced necrosis in neonatal male rat cardiomyocytes, while the ablation of Gpr34 in male mice reduced the development of pressure overload-induced cardiac remodeling. Thus, the iPLA2ß-lysophosphatidylserine-GPR34-necrosis signaling axis plays a detrimental role in the heart in response to pressure overload.

Rubicon-regulated beta-1 adrenergic receptor recycling protects the heart from pressure overload.

Heart failure has high morbidity and mortality in the developed countries. Autophagy is important for the quality control of proteins and organelles in the heart. Rubicon (Run domain Beclin-1-interacting and cysteine-rich domain-containing protein) has been identified as a potent negative regulator of autophagy and endolysosomal trafficking. The aim of this study was to investigate the in vivo role of Rubicon-mediated autophagy and endosomal trafficking in the heart. We generated cardiomyocyte-specific Rubicon-deficient mice and subjected the mice to pressure overload by means of transverse aortic constriction. Rubicon-deficient mice showed heart failure with left ventricular dilatation, systolic dysfunction and lung congestion one week after pressure overload. While autophagic activity was unchanged, the protein amount of beta-1 adrenergic receptor was decreased in the pressure-overloaded Rubicon-deficient hearts. The increases in heart rate and systolic function by beta-1 adrenergic stimulation were significantly attenuated in pressure-overloaded Rubicon-deficient hearts. In isolated rat neonatal cardiomyocytes, the downregulation of the receptor by beta-1 adrenergic agonist was accelerated by knockdown of Rubicon through the inhibition of recycling of the receptor. Taken together, Rubicon protects the heart from pressure overload. Rubicon maintains the intracellular recycling of beta-1 adrenergic receptor, which might contribute to its cardioprotective effect.

mTOR Hyperactivation by Ablation of Tuberous Sclerosis Complex 2 in the Mouse Heart Induces Cardiac Dysfunction with the Increased Number of Small Mitochondria Mediated through the Down-Regulation of Autophagy.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.

Apolipoprotein B-48 to triglyceride ratio is a novel and useful marker for detection of type III hyperlipidemia after antihyperlipidemic intervention.

AIM: Remnant lipoproteins are atherogenic and are accumulated in patients with type III hyperlipidemia (HL). Although type III HL is diagnosed by phenotyping apolipoprotein (apo) E, this procedure is time-consuming and inconvenient for routine clinical use. Clinical indices for screening type III HL in untreated HL patients have been proposed; however, in clinical settings, HL patients are promptly treated with lipid-lowering agents without diagnosing the underlying cause. We investigated whether existing clinical indices for screening type III HL as well as the apo B-48/triglyceride (TG) ratio, which was suggested to be related to the accumulation of small chylomicron (CM) remnants, are useful after the initiation of lipid-lowering therapies. METHODS: In 25 normolipidemic subjects and 191 treated HL patients (type I, n =6; IIa, 62; IIb, 66; III, 12; IV, 22; and V, 23) from Osaka University Hospital and related hospitals, fasting low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TG, and apolipoproteins were measured and clinical indices were evaluated statistically. RESULTS: Apo B-48 levels were significantly higher in patients with type I, III, and V HL, and TG levels were significantly higher in patients with type I and V HL. The apo B-48/TG ratio was significantly higher only in patients with type III HL compared with other types of HL (p<0.001), and was statistically significant among the other clinical indices (AUC-ROC value, 0.895; cut-off value, 0.110). CONCLUSION: The apo B-48/TG ratio is a novel and useful marker for detecting type III HL even after the initiation of lipid-lowering interventions.

Establishment of chemiluminescence enzyme immunoassay for apolipoprotein B-48 and its clinical applications for evaluation of impaired chylomicron remnant metabolism.

BACKGROUND: Apolipoprotein B-48 (apoB-48) is a constituent of chylomicron remnants synthesized in the small intestines. The serum concentration of apoB-48 at fasting has been reported to be a marker of postprandial hyperlipidemia, a presumed risk factor for atherosclerosis. METHODS: We evaluated the basal performance of a recently developed chemiluminescent enzyme immunoassay (CLEIA). We also examined the correlations between serum apoB-48 concentrations and other lipid concentrations or life style patterns, including smoking and drinking. We analyzed the data of 273 clinical samples by multiple regression analysis to examine the influence of other serum lipid values, age, sex, smoking, drinking status and BMI on serum apoB-48 values. RESULTS: Within-run and between-run precision was obtained with 1.7-2.7% and 1.2-7.3%, respectively. The correlativity of enzyme-linked immunosorbent assay was correlation coefficient r=0.953, and regression y=1.02×-1.59. Serum apoB-48 concentrations were higher in males than in females, and were correlated with the status of smoking as well as with remnant-like particle-cholesterol (RLP-C) concentrations. Patients with the metabolic syndrome showed higher values of serum apoB-48 compared with control subjects. CONCLUSION: Serum apoB-48 measurement by CLEIA was satisfactory for clinical use to assess abnormalities in the chylomicron remnant metabolism.

Serum adiponectin level is correlated with the size of HDL and LDL particles determined by high performance liquid chromatography.

OBJECTIVE: Adiponectin (APN) improves insulin resistance and prevents atherosclerosis, and HDL removes cholesterol from atherosclerotic lesions. We have demonstrated that serum HDL-cholesterol (HDL-C) and APN concentrations are positively correlated and that APN accelerates reverse cholesterol transport (RCT) by increasing HDL synthesis in the liver and cholesterol efflux from macrophages. We previously reported that APN reduced apolipoprotein (apo) B secretion from the liver. It is well-known that insulin resistance influences the lipoprotein profile. In this study, we investigated the clinical significance of APN levels and insulin resistance in lipoprotein metabolism. MATERIAL/METHOD: We investigated the correlation between serum APN concentration, HOMA-R, the lipid concentrations and lipoprotein particle size by high-performance liquid chromatography (HPLC) in 245 Japanese men during an annual health checkup. RESULTS: Serum APN level was positively correlated with the cholesterol content in large LDL and HDL particles, but inversely correlated with the cholesterol content in large VLDL and small LDL particles. HOMA-R was negatively correlated with the cholesterol content in large LDL and HDL particles and positively correlated with the cholesterol content in large VLDL and small LDL particles. By multivariate analysis, APN was correlated with the particle size of LDL-C and HDL-C independently of age, BMI and HOMA-R. CONCLUSIONS: APN may be associated with the formation of both HDL and LDL particles, reflecting the enhancement of RCT and the improvement in TG-rich lipoprotein metabolism and insulin resistance.

Fasting serum apolipoprotein B-48 can be a marker of postprandial hyperlipidemia.

AIM: Postprandial hyperlipidemia (PH) is thought to be caused by the impaired postprandial metabolism of triglycerides (TG)-rich lipoproteins in both endogenous and exogenous pathways; however, there is no consensus. It is difficult to estimate the presence of PH without performing a time-consuming oral fat loading (OFL) test, so postprandial lipoprotein metabolism was analyzed by measuring the postprandial levels of apolipoprotein (apo) B-48 and apo B-100, and the correlation between postprandial TG increase and fasting apoB-48 levels was assessed to establish a good marker of PH without performing an OFL test. METHODS: Ten male normolipidemic subjects were loaded with a high-fat (HF, 1045 kcal) or standard (ST, 566 kcal) meal, and the lipids, apolipoproteins and lipoprotein profiles were analyzed after each meal. RESULTS: TG, apo B-48, remnant-like particles (RLP)-cholesterol and RLP-TG levels were increased and their levels were significantly higher after intake of the HF meal than the ST meal; however, there was no postprandial increase in apo B-100 and LDL-C levels. Postprandial increases in TG levels of CM, VLDL, LDL and HDL were significantly higher after intake of the HF meal than the ST meal. Fasting apo B-48 levels were strongly correlated with the incremental area under the curve of TG after intake of the HF meal, but not the ST meal. CONCLUSION: Postprandial TG increase was mainly due to increased CM and CM-R, but not VLDL. Measurement of fasting serum apo B-48 may be a simple and useful method for assessment of the existence of PH.