Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries.

BACKGROUND: Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway. METHODS: Type 2 diabetes models were established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein-coupled receptor-targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies. RESULTS: Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase-mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes Rip1, Rip3, and Mlkl. Under hyperglycemic conditions, high glucose induced CB2R internalization in a ß-arrestin 2-dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of Bach2 diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries. CONCLUSIONS: CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.

Endothelial Intracellular ANG (Angiogenin) Protects Against Atherosclerosis by Decreasing Endoplasmic Reticulum Stress.

BACKGROUND: ANG (angiogenin) is essential for cellular adaptation to endoplasmic reticulum (ER) stress, a process closely associated with cardiovascular diseases, including atherosclerosis. We aimed to investigate the role of ANG in the progression of atherosclerosis and elucidate its underlying molecular mechanisms. METHODS: We constructed adenoassociated virus 9 ANG overexpression vectors and endothelial ANG- and ApoE (apolipoprotein E)-deficient mice to determine the effects of ANG on ER stress and atherosclerotic lesions. RNA sequencing of endothelial ANG- and ApoE-deficient mice identified ANG-dependent downregulation of ST3GAL5 (ST3 beta-galactoside alpha-2,3-sialyltransferase 5) expression, and the direct regulation of ST3GAL5 by ANG was verified by chromatin immunoprecipitation sequencing and luciferase reporter assay results. RESULTS: Reanalysis of expression profiling datasets indicated decreased ANG levels in patients' atherosclerotic lesions, and these data were validated in aortas from ApoE-/- mice. ER stress marker and adhesion molecule levels, aortic root lesions and macrophage deposition were substantially reduced in ApoE-/- mice injected with an adenoassociated virus 9 ANG without signal peptide (ANG-ΔSP) overexpression vector compared with empty and full-length ANG overexpression vectors. Endothelial ANG deficiency significantly elevated ER stress and increased adhesion molecule expression, which aggravated atherosclerotic lesions and enhanced THP-1 monocyte adhesion to endothelial cells in vivo and in vitro, respectively. Furthermore, ANG-ΔSP overexpression significantly attenuated oxidized low-density lipoprotein-induced ER stress and THP-1 monocyte adhesion to endothelial cells, which were reversed by ST3GAL5 inhibition. CONCLUSIONS: These results suggest that endothelial intracellular ANG is a novel therapeutic against atherosclerosis and exerts atheroprotective effects via ST3GAL5-mediated ER stress suppression.

Oxidized LDL but not angiotensin II induces cardiomyocyte hypertrophic responses through the interaction between LOX-1 and AT1 receptors.

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

Dendritic cell-mediated chronic low-grade inflammation is regulated by the RAGE-TLR4-PKCß1 signaling pathway in diabetic atherosclerosis.

BACKGROUND: The unique mechanism of diabetic atherosclerosis has been a central research focus. Previous literature has reported that the inflammatory response mediated by dendritic cells (DCs) plays a vital role in the progression of atherosclerosis. The objective of the study was to explore the role of DCs in diabetes mellitus complicated by atherosclerosis. METHODS: ApoE-/- mice and bone marrow-derived DCs were used for in vivo and in vitro experiments, respectively. Masson's staining and Oil-red-O staining were performed for atherosclerotic lesion assessment. The content of macrophages and DCs in plaque was visualized by immunohistochemistry. The expression of CD83 and CD86 were detected by flow cytometry. The fluctuations in the RNA levels of cytokines, chemokines, chemokine receptors and adhesions were analyzed by quantitative RT-PCR. The concentrations of IFN-γ and TNF-α were calculated using ELISA kits and the proteins were detected using western blot. Coimmunoprecipitation was used to detect protein-protein interactions. RESULTS: Compared with the ApoE-/- group, the volume of atherosclerotic plaques in the aortic root of diabetic ApoE-/- mice was significantly increased, numbers of macrophages and DCs were increased, and the collagen content in plaques decreased. The expression of CD83 and CD86 were significantly upregulated in splenic CD11c+ DCs derived from mice with hyperglycemia. Increased secretion of cytokines, chemokines, chemokine receptors, intercellular cell adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) also were observed. The stimulation of advanced glycation end products plus oxidized low-density lipoprotein, in cultured BMDCs, further activated toll-like receptor 4, protein kinase C and receptor of AGEs, and induced immune maturation of DCs through the RAGE-TLR4-PKCß1 signaling pathway that was bound together by intrinsic structures on the cell membrane. Administering LY333531 significantly increased the body weight of diabetic ApoE-/- mice, inhibited the immune maturation of spleen DCs, and reduced atherosclerotic plaques in diabetic ApoE-/- mice. Furthermore, the number of DCs and macrophages in atherosclerotic plaques was significantly reduced in the LY333531 group, and the collagen content was increased. CONCLUSIONS: Diabetes mellitus aggravates chronic inflammation, and promotes atherosclerotic plaques in conjunction with hyperlipidemia, which at least in part through inducing the immune maturation of DCs, and its possible mechanism of action is through the RAGE-TLR4-pPKCß1 signaling pathway.

HMGB1 in macrophage nucleus protects against pressure overload induced cardiac remodeling via regulation of macrophage differentiation and inflammatory response.

Pressure overload induced cardiac remodeling is associated with a complex spectrum of pathophysiological mechanisms. As inflammatory cells, macrophages maintain a critical position in mechanical stress-induced myocardial remodeling. HMGB1 is a highly conserved, ubiquitous protein in various types of cells whose biological roles are closely dependent on subcellular sites. However, whether HMGB1 expressed in macrophages performs the protective or pathological responses in cardiac remodeling is unknown. In this study, we generated the myeloid-specific HMGB1 knockout mice and detected the effects of macrophage HMGB1 in response to pathophysiological stress. Our data showed HMGB1 in macrophages played a protective role against the pressure overload induced cardiac pathophysiology. The deletion of HMGB1 in macrophages gains more differentiation of M1-type pro-inflammatory macrophage during the mechanical stress-induced myocardial remodeling, thereby aggravating the inflammatory response in whole heart, resulting in accelerated deterioration of cardiac function. Moreover, in vitro data also validated HMGB1 got involved in the process of macrophage polarization. Macrophages without HMGB1 are more inclined to differentiate into M1 during the stretch process. In summary, the present results indicated that loss of HMGB1 in macrophages can exacerbate heart failure through increased differentiation of pro-inflammatory macrophages and enhanced inflammatory response.

Inhibition of calpain reduces cell apoptosis by suppressing mitochondrial fission in acute viral myocarditis.

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.

AT1 receptor blocker inhibits HMGB1 expression in pressure overload-induced acute cardiac dysfunction by suppressing the MAPK/NF-κB signaling pathway.

BACKGROUND: High-mobility group box 1 (HMGB1) expression not only peaks during the early phase of pressure overload (PO), but also serves a role in the pathogenesis of PO-induced cardiac remodeling. Meanwhile, angiotensin II type 1 (AT1) receptor blockers reverse PO-induced cardiac remodeling and repress the secretion of inflammatory factors. However, whether AT1 receptor inhibitors decrease HMGB1 expression in the early stages of PO remains unknown. MATERIALS AND METHODS: PO mouse models were established using transverse aortic constriction (TAC), in which losartan was administrated. Transthoracic echocardiography was performed 3 days after the operation, and serum and cardiac HMGB1 expression, as well as the expression levels of related proteins were measured. RESULTS: PO-induced acute cardiac dysfunction was observed 3 days after TAC, and was subsequently slightly, but not significantly relieved by losartan. The expression levels of HMGB1, tumor necrosis factor-α and interleukin-6 in both the serum and myocardium were upregulated in response to TAC, while they were significantly reduced by losartan. Moreover, the phosphorylation of extracellular signal-regulated kinases, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in the myocardium were significantly increased under PO, and this was also prevented by losartan. CONCLUSION: These data suggest that losartan may downregulate the expression of HMGB1 in acute cardiac dysfunction induced by PO by inhibiting the MAPKs/NF-κB signaling pathway, which indicates a novel beneficial role of AT1 receptor antagonists in ameliorating cardiac remodeling under PO.

Hypertrophic preconditioning attenuates post-myocardial infarction injury through deacetylation of isocitrate dehydrogenase 2.

Ischemic preconditioning induced by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischemic insult. In this study we investigated whether a short-term nonischemic stimulation of hypertrophy renders the heart resistant to subsequent ischemic injury. Male mice were subjected to transient transverse aortic constriction (TAC) for 3 days followed aortic debanding on D4 (T3D4), as well as ligation of the left coronary artery to induce myocardial infarction (MI). The TAC preconditioning mice showed markedly improved contractile function and significantly reduced myocardial fibrotic area and apoptosis following MI. We revealed that TAC preconditioning significantly reduced MI-induced oxidative stress, evidenced by increased NADPH/NADP ratio and GSH/GSSG ratio, as well as decreased mitochondrial ROS production. Furthermore, TAC preconditioning significantly increased the expression and activity of SIRT3 protein following MI. Cardiac-specific overexpression of SIRT3 gene through in vivo AAV-SIRT3 transfection partially mimicked the protective effects of TAC preconditioning, whereas genetic ablation of SIRT3 in mice blocked the protective effects of TAC preconditioning. Moreover, expression of an IDH2 mutant mimicking deacetylation (IDH2 K413R) in cardiomyocytes promoted myocardial IDH2 activation, quenched mitochondrial reactive oxygen species (ROS), and alleviated post-MI injury, whereas expression of an acetylation mimic (IDH2 K413Q) in cardiomyocytes inactivated IDH2, exacerbated mitochondrial ROS overload, and aggravated post-MI injury. In conclusion, this study identifies TAC preconditioning as a novel strategy for induction of an endogenous self-defensive and cardioprotective mechanism against cardiac injury. Therapeutic strategies targeting IDH2 are promising treatment approaches for cardiac ischemic injury.

Angiotensin II Increases HMGB1 Expression in the Myocardium Through AT1 and AT2 Receptors When Under Pressure Overload.

High-mobility group box 1 (HMGB1) is increased in the myocardium under pressure overload (PO) and is involved in PO-induced cardiac remodeling. The mechanisms of the upregulation of cardiac HMGB1 expression have not been fully elucidated. In the present study, a mouse transverse aortic constriction (TAC) model was used, and an angiotensin II (Ang II) type 1 (AT1) receptor inhibitor (losartan) or Ang II type 2 (AT2) receptor inhibitor (PD123319) was administrated to mice for 14 days. Cardiac myocytes were cultured and treated with Ang II for 5 minutes to 48 hours conditionally with the blockage of the AT1 or AT2 receptor. TAC-induced cardiac hypertrophy was observed at 14 days after the operation, which was partially reversed by losartan, but not by PD123319. Similarly, the upregulated HMGB1 expression levels observed in both the serum and myocardium induced by TAC were reduced by losartan. Elevated cardiac HMGB1 protein levels, but not mRNA or serum levels, were significantly decreased by PD123319. Furthermore, HMGB1 expression levels in culture media and cardiac myocytes were increased following Ang II treatment in vitro, positively associated with the duration of treatment. Similarly, Ang II-induced upregulation of HMGB1 in vitro was inhibited by both losartan and PD123319. These results suggest that upregulation of HMGB1 in serum and myocardium under PO, which are partially derived from cardiac myocytes, may be induced by Ang II via the AT1 and AT2 receptors. Additionally, amelioration of PO-induced cardiac hypertrophy following losartan treatment may be associated with the reduction of HMGB1 expression through the AT1 receptor.

Inhibition of Calpain Alleviates Apoptosis in Coxsackievirus B3-induced Acute Virus Myocarditis Through Suppressing Endoplasmic Reticulum Stress.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

Hyperlipidemia inhibits the protective effect of lisinopril after myocardial infarction via activation of dendritic cells.

To investigate the prevention of cardiac remodelling and inflammatory immune response after myocardial infarction (MI) via ACEI regulating dendritic cells (DCs), we explored whether the protective effect of ACEI was repressed under hyperlipidemic environment. In vivo, the survival rate and left ventricular function of the mice were recorded on day 7 after MI. Tissue samples of the myocardium, spleen, bone marrow and peripheral blood were assessed for Ang II concentration, inflammatory cytokines and DCs expression. In vitro, DCs were treated with ox-LDL + Ang II, simulating the internal environment of MI in ApoE-/- mice to explore the mechanism involved in the DCs maturation and inflammation. Under hyperlipidemic circumstances, we found that the cardioprotective effect of ACEI was attenuated through regulating DCs maturation and inflammation after MI, affecting survival rate and left ventricular function. Effects of lisinopril on the release of spleen-derived DCs and myocardial infiltration were also reduced under hyperlipidemic conditions. In vitro, immune maturation and inflammation of DCs were further induced by ox-LDL on the basis of Ang II treatment, as indicated by the upregulation of CD83, CD86, and the expressions of cytokines and chemokines. Furthermore, ox-LDL could activate TLR4-MyD88 signalling pathway, promoting IRAK-4 and NF-κB. The present study demonstrated that ACEI reduced the recruitment of DCs to the infarct site, leading to a higher survival rate and improved function. However, this effect was inhibited under hyperlipidemic environment. TLR4-MyD88 signalling pathway may be responsible for the molecular mechanism involved in the immune maturation and inflammation of DCs induced by ox-LDL.

Heat shock transcription factor 1 regulates exercise-induced myocardial angiogenesis after pressure overload via HIF-1α/VEGF pathway.

Exercise training is believed to have a positive effect on cardiac hypertrophy after hypertension. However, its mechanism is still not fully understood. Herein, our findings suggest that heat shock transcription factor 1 (HSF1) improves exercise-initiated myocardial angiogenesis after pressure overload. A sustained narrowing of the diagonal aorta (TAC) and moderately- intense exercise training protocol were imposed on HSF1 heterozygote (KO) and their littermate wild-type (WT) male mice. After two months, the cardiac function was assessed using the adaptive responses to exercise training, or TAC, or both of them such as catheterization and echocardiography. The HE stains assessed the area of myocyte cross-sectional. The Western blot and real-time PCR measured the levels of expression for heat shock factor 1 (HSF1), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) in cardiac tissues. The anti-CD31 antibody immunohistochemical staining was done to examine how exercise training influenced cardiac ontogeny. The outcome illustrated that exercise training significantly improved the cardiac ontogeny in TAC mice, which was convoyed by elevated levels of expression for VEGF and HIF-1α and preserved the heart microvascular density. More importantly, HSF1 deficiency impaired these effects induced by exercise training in TAC mice. In conclusion, exercise training encourages cardiac ontogeny by means of HSF1 activation and successive HIF-1α/VEGF up-regulation in endothelial cells during continued pressure overload.

Lipoprotein receptor-related protein 6 is required to maintain intercalated disk integrity.

The intercalated disk (ID), a highly organized adhesion structure connecting neighboring cardiomyocytes, fulfills mechanical and electrical signaling communication to ensure normal heart function. Lipoprotein receptor-related protein 6 (LRP6) is a co-receptor inducing canonical Wnt/ß-catenin signaling. It was recently reported that LRP6 deficiency in cardiomyocytes predisposes to arrhythmia independent of Wnt signaling. However, whether LRP6 directly regulates the structure of IDs requires further investigation. The aim of the present study was to explore the role of LRP6 in IDs and the potential underlying mechanisms by inducible cardiac-specific LRP6 knockout mice. The results revealed that LRP6 was predominately expressed in the cell membrane, including the IDs of cardiomyocytes. Tamoxifen-inducible cardiac-specific LRP6 knockout mice displayed overt cardiac dysfunction and disruption of ID structure. Further analysis revealed that cardiac LRP6 deficiency induced the imbalance of ID component proteins, characterized by the sharply decreased expression of connexin 43 (Cx43) and the significantly increased expression of N-cadherin, desmoplakin and γ-catenin in tissue lysates or membrane fraction from the left ventricle. STRING database analysis indicated that ß-catenin, but no other ID-associated proteins, interacted with LRP6. Our immunoprecipitation analysis demonstrated that LRP6 strongly interacted with Cx43, N-cadherin and γ-catenin, and weakly interacted with ß-catenin, whereas there was no association with desmoplakin. In response to LRP6 deficiency, the recruitment of ß- or γ-catenin to N-cadherin was increased, but they displayed little interaction with Cx43. In conclusion, LRP6 is required to maintain the integrity of ID structure and the balance of ID proteins, and the interaction between LRP6 and Cx43, N-cadherin and γ-catenin may be involved in this process.

Cardiac-specific LRP6 knockout induces lipid accumulation through Drp1/CPT1b pathway in adult mice.

We recently reported low-density lipoprotein receptor-related protein 6 (LRP6) decreased in dilated cardiomyopathy hearts, and cardiac-specific knockout mice displayed lethal heart failure through activation of dynamin-related protein 1 (Drp1). We also observed lipid accumulation in LRP6 deficiency hearts, but the detailed molecular mechanisms are unclear. Here, we detected fatty acids components in LRP6 deficiency hearts and explored the potential molecular mechanisms. Fatty acid analysis by GC-FID/MS revealed cardiac-specific LRP6 knockout induced the higher level of total fatty acids and some medium-long-chain fatty acids (C16:0, C18:1n9 and C18:2n6) than in control hearts. Carnitine palmitoyltransferase 1b (CPT1b), a rate-limiting enzyme of mitochondrial ß-oxidation in adult heart, was sharply decreased in LRP6 deficiency hearts, coincident with the activation of Drp1. Drp1 inhibitor greatly improved cardiac dysfunction and attenuated the increase in total fatty acids and fatty acids C16:0, C18:1n9 in LRP6 deficiency hearts. It also greatly inhibited the decrease in the cardiac expression of CPT1b and the transcriptional factors CCCTC-binding factor (CTCF) and c-Myc induced by cardiac-specific LRP6 knockout in mice. C-Myc but not CTCF was identified to regulate CPT1b expression and lipid accumulation in cardiomyocytes in vitro. The present study indicated cardiac-specific LRP6 knockout induced lipid accumulation by Drp1/CPT1b pathway in adult mice, and c-Myc is involved in the process. It suggests that LRP6 regulates fatty acid metabolism in adult heart.

The diagonal branches and outcomes in patients with anterior ST- elevation myocardial infarction.

BACKGROUND: The management of diagonal branch (D) occlusion is still controversary. The association between the flow loss of D and the prognosis remains unclear. We aim to detect the impact of D flow on cardiac function and clinical outcomes in patients with anterior ST-segment elevation myocardial infarction (STEMI). METHODS: Patients with anterior STEMI undergoing primary percutaneous coronary intervention (PCI) at our clinic between October 2015 and October 2018were reviewed. Anterior STEMI due to left anterior descending artery (LAD) occlusion with or without loss of the main D flow (TIMI grade 0-1 or 2-3) was enrolled in the analysis. The short- and long-term incidence of major adverse cardiac events (MACEs, a composite of all-cause death, target vessel revascularization and reinfarction) and left ventricular ejection fraction (LVEF) were analyzed. RESULTS: A total of 392 patients (mean age of 63.9 years) with anterior STEMI treated with primary PCI was enrolled in the study. They were divided into two groups, loss (TIMI grade 0-1, n = 69) and no loss (TIMI grade2-3, n = 323) of D flow, before primary PCI. Compared with the group without loss of D flow, the group with loss of D flow showed a lower LVEF post PCI (41.0% vs. 48.8%, p = 0.003). Meanwhile, loss of D flow resulted in the higher in-hospital, one-month, and 18-month incidence of MACEs, especially in all-cause mortality (all p < 0.05). Landmark analysis further indicated that the significant differences in 18-month outcomes between the two groups mainly resulted from the differences during the hospitalization. In addition, multivariate Cox proportional hazards analysis found that D flow loss before primary PCI was independent factor predicting short- and long-term outcomes in patients with anterior STEMI. CONCLUSION: Loss of the main D flow in anterior STEMI patients was independently associated with the higher in-hospital incidences of MACEs and all-cause death as well as the lower LVEF.

Gut microbe-derived metabolite trimethylamine N-oxide accelerates fibroblast-myofibroblast differentiation and induces cardiac fibrosis.

BACKGROUND: Trimethylamine N-oxide (TMAO), a gut microbe-derived metabolite of dietary choline and other trimethylamine-containing nutrients, has been associated with poor prognosis in coronary heart disease. However, the role and underlying mechanisms of TMAO in the cardiac fibrosis after myocardial infarction (MI) remains unclear. METHODS: We used mouse MI models and primary cardiac fibroblasts cultures to study the role of TMAO in the heart and in cardiac fibroblasts. C57BL/6 mice were fed a control diet, high choline (1.2%) or/and DMB diet or a diet containing TMAO (0.12%) starting 3 weeks before MI. DMB, a structural analogue of choline, inhibited microbial TMA lyases and reduced the level of TMAO in mice. Cardiac function was measured 7 days after MI using echocardiography. One week post MI, myocardial tissues were collected to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. The expression of TGF-ß receptor, P-Smad2, α-SMA or collagen I in myocardial tissues and fibroblasts were analyzed by western blot or immunocytochemistry. RESULTS: We demonstrated that cardiac function and cardiac fibrosis were significantly deteriorated in mice fed either TMAO or high choline diets compared with the control diet, and DMB reversed the cardiac function damage of high choline diet (p < .05). Cardiomyocyte necrosis, apoptosis and macrophage infiltration after MI was significantly increased after treatment with TMAO or high choline diets. The size and migration of fibroblasts were increased after TMAO treatment compared with non-treated fibroblasts in vitro. Furthermore, TMAO increased TGF-ß receptor I expression, which promoted the phosphorylation of Smad2 and up-regulated the expression of α-SMA and collagen I. The ubiquitination of TGF-ßRI was decreased in neonatal mouse fibroblasts after TMAO treatment. TMAO also inhibited the expression of smurf2. Inhibition of TGF-ß1 receptor with the small molecule inhibitor SB431542 decreased TGF-ß receptor I expression, reduced the phosphorylation of Smad2, down-regulated TMAO-induced α-SMA and collagen I expression in cardiac fibroblasts. CONCLUSIONS: Cardiac function and cardiac fibrosis were significantly exacerbated in mice fed diets supplemented with either choline or TMAO, probably through accelerating the transformation of fibroblasts into myofibroblasts, indicating activation of TGF-ßRI/Smad2 pathway.

FTY720 alleviates coxsackievirus B3-induced myocarditis and inhibits viral replication through regulating sphingosine 1-phosphate receptors and AKT/caspase-3 pathways.

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis.

HSF1 deficiency accelerates the transition from pressure overload-induced cardiac hypertrophy to heart failure through endothelial miR-195a-3p-mediated impairment of cardiac angiogenesis.

Heat shock transcription factor 1 (HSF1) deficiency aggravates cardiac remodeling under pressure overload. However, the mechanism is still unknown. Here we employed microRNA array analysis of the heart tissue of HSF1-knockout (KO) mice to investigate the potential roles of microRNAs in pressure overload-induced cardiac remodeling under HSF-1 deficiency, and the profiles of 478 microRNAs expressed in the heart tissues of adult HSF1-KO mice were determined. We found that the expression of 5 microRNAs was over 2-fold higher expressed in heart tissues of HSF1-KO mice than in those of wild-type (WT) control mice. Of the overexpressed microRNAs, miR-195a-3p had the highest expression level in HSF1-null endothelial cells (ECs). Induction with miR-195a-3p in ECs significantly suppressed CD31 and VEGF, promoted AngII-induced EC apoptosis, and impaired capillary-like tube formation. In vivo, the upregulation of miR-195a-3p accentuated cardiac hypertrophy, increased the expression of ß-MHC and ANP, and compromised systolic function in mice under pressure overload induced by transverse aortic constriction (TAC). By contrast, antagonism of miR-195a-3p had the opposite effect on HSF1-KO mice. Further experiments confirmed that AMPKα2 was the direct target of miR-195a-3p. AMPKα2 overexpression rescued the reduction of eNOS and VEGF, and the impairment of angiogenesis that was induced by miR-195a-3p. In addition, upregulation of AMPKα2 in the myocardium of HSF1-null mice by adenovirus-mediated gene delivery enhanced CD31, eNOS and VEGF, reduced ß-MHC and ANP, alleviated pressure overload-mediated cardiac hypertrophy and restored cardiac function. Our findings revealed that the upregulation of miR-195a-3p due to HSF1 deficiency impaired cardiac angiogenesis by regulating AMPKα2/VEGF signaling, which disrupted the coordination between the myocardial blood supply and the adaptive hypertrophic response and accelerated the transition from cardiac hypertrophy to heart failure in response to pressure overload.

Riboflavin attenuates myocardial injury via LSD1-mediated crosstalk between phospholipid metabolism and histone methylation in mice with experimental myocardial infarction.

The underlying mechanisms responsible for the cardioprotective effects of riboflavin remain elusive. Current study tested the hypothesis that riboflavin protects injured myocardium via epigenetic modification of LSD1. Here we showed that myocardial injury was attenuated and cardiac function was improved in riboflavin-treated mice with experimental myocardial infarction (MI), while these protective effects of riboflavin could be partly blocked by cotreatment with LSD1 inhibitor. Riboflavin also reduced apoptosis in hypoxic (1% oxygen) H9C2 cell lines. Results of ChIP-seq for H9C2 cells showed that riboflavin activated LSD1, as verified by decreased H3K4me2 levels of target genes. Subsequent LEGO bioinformatics analysis indicated that phospholipid metabolism genes Lpcat2 and Pld1 served as the potential target genes responsible for the LSD1 mediated protective effects. Overexpressions of Lpcat2 and Pld1 aggravated hypoxic injury in H9C2 cells, while these detrimental effects could be attenuated by overexpression of LSD1. We thus propose that riboflavin alleviates myocardial hypoxic/ischemic injury by activating LSD1 cellular activity and modulating the expression of phospholipid metabolism genes. LSD1-mediated crosstalk between phospholipid metabolism and histone methylation might thus be an important mechanism for the cardioprotective effects of riboflavin.

Heat-shock transcription factor 1 is critically involved in the ischaemia-induced cardiac hypertrophy via JAK2/STAT3 pathway.

Cardiac hypertrophy after myocardial infarction (MI) is an independent risk factor for heart failure. Regression of cardiac hypertrophy has emerged as a promising strategy in the treatment of MI patients. Here, we have been suggested that heat-shock transcription factor 1 (HSF1) is a novel repressor of ischaemia-induced cardiac hypertrophy. Ligation of left anterior descending coronary was used to produce MI in HSF1-deficient heterozygote (KO), HSF1 transgenic (TG) mice and their wild-type (WT) littermates, respectively. Neonatal rat cardiomyocytes (NRCMs) were treated by hypoxia to mimic MI in vitro. The HSF1 phosphorylation was significantly reduced in the infarct border zone of mouse left ventricles (LVs) 1 week after MI and in the hypoxia-treated NRCMs. HSF1 KO mice showed more significant maladaptive cardiac hypertrophy and deteriorated cardiac dysfunction 1 week after MI compared to WT MI mice. Deficiency of HSF1 by siRNA transfection notably increased the hypoxia-induced myocardial hypertrophy in NRCMs. Mechanistically, Janus kinase 2 (JAK2) and its effector, signal transducer and activator of transcription 3 (STAT3) were found to be significantly increased in the LV infarct border zone of WT mice after MI as well as the NRCMs treated by hypoxia. These alterations were more significant in HSF1 KO mice and NRCMs transfected with HSF1 SiRNA. Inversely, HSF1 TG mice showed significantly ameliorated cardiac hypertrophy and heart failure 1 week after LAD ligation compared to their WT littermates. Our data collectively demonstrated that HSF1 is critically involved in the pathological cardiac hypertrophy after MI via modulating JAK2/STAT3 signalling and may constitute a potential therapeutic target for MI patients.