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Pathways Underlying Impaired Cardiac Preconditioning Potential in Subjects with the Metabolic Syndrome, and its Restoration by ACE-inhibition

ABSTRACT: Aims. The goal of the study was to identify the possible transcriptional alterations responsible for the restoration of the second window preconditioning in the metabolic syndrome heart. Methods. LDLR-/-;ob/ob (DKO) and C57Bl6/J (WT) mice were injected with ACE-inhibitor, captopril (10 mg/kg/day) or NaCl 0.9% as controls. Preconditioning (HPC) was induced by five 6-min cycles at 6% oxygen interposed with 6 min at 21% oxygen. Non-preconditioned mice underwent a sham procedure without hypoxia. Transcriptome analysis (RNA-seq) from the whole heart RNA samples, differential gene expression with Cufdiff, pathway and network analysis with Ingenuity Pathway Analysis (IPA), qRT-PCR, and transcription factor motif enrichment analysis with i-cisTarget were performed. In separate groups, the infarct size was determined histologically after a 30-min occlusion of LAD, followed by 60-min reperfusion. Results. Preconditioning could not reduce infarct size in the untreated DKO mice (1+2%, p>.05). Preconditioning reduced infarct size to 28+2% in WT-HPC and to 23+1% in WTACE-I-HPC, both p<.05. In DKOACE-I hearts, preconditioning potential was partially restored with a reduction of infarct size by 16%. Preconditioning induced low number of differentially expressed genes (DEG) in DKO hearts (29 genes, p<.05) when compared WT and WTACE-I hearts (1634 and 2215 genes respectively, p<.05). In DKOACE-I, the number of DEG after preconditioning increased to 59, p<.05. In the three groups that could phenotypically be protected, preconditioning triggered shared pathways associated with the cell cycle quiescence, such as mTORC2 signaling, eIF2 signaling, regulation of eIF4 and p70S6K signaling, and actin cytoskeleton pathway signaling. The model-specific pathways and transcription factors differed between the groups. In the WT hearts, preconditioning enriched the pathways of mitochondrial biogenesis, oxidative phosphorylation, and anti-apoptotic signalling. In the WTACE-I hearts, preconditioning enriched the pathways of immune system signaling, metabolism, and cell cycle regulation. In the DKOACE-I hearts, pathways associated with negative inotropic effect and inhibition of myocyte growth were enriched after preconditioning. Conclusion. Our findings explain the molecular mechanism behind preconditioning in the healthy and the metabolic syndrome hearts. The phenotypically cardioprotective effect of HPC was absent in the metabolic syndrome hearts and was partially reinstated after ACE-I therapy. Transcriptional response to preconditioning differs between DKO and WT hearts and is model-specific with some degree of overlap.

ORGANISM(S): Mus musculus

PROVIDER: GSE135198 | GEO | 2019/08/01

REPOSITORIES: GEO

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Project description:Coronary heart disease is the leading cause of death worldwide. After an acute myocardial infarction, early reperfusion reduces infarct size, which correlates with improved clinical outcomes. Paradoxically, reperfusion although relieving ischemia, accelerates apoptosis in injured cardiomyocytes, which has led to the view that myocardial salvage is futile beyond the first few hours of reperfusion. In murine hearts subjected to 90 min of coronary artery occlusion and then 48 h of reperfusion, we show transient activation of intrinsic prosurvival insulin-like growth factor-1 (IGF-1) signaling. In these hearts, acute IGF-1 receptor inhibition decreases the abundance of prosurvival signaling molecules, and markedly activates caspase-3, a potent effector of apoptosis, in infarct border zone cardiomyocytes. We found that mouse mast cell protease-4 (MMCP-4) degraded IGF-1 in vitro by a novel catalytic activity of chymases. In vivo, this degradation, which is triggered by mast cell infiltration into the peri-infarct region and MMCP-4 extravasation, between 48 and 72 h post-ischemia/reperfusion (I/R), attenuates IGF-1 prosurvival signaling. In MMCP-4-deficient mice, while infarct size is not reduced at 24 h post-I/R, at 72 h post-I/R myocardial IGF-1 levels and signaling are increased, resulting in activation of the survival kinases Akt and ERK, inhibition of caspase-3, and reduced myocardial cell death. As a consequence, I/R-mediated loss of viable myocardium, adverse cardiac remodeling and contractile impairment are markedly reduced. Cardiomyocyte survival with consequent myocardial salvage may thus be possible even days after an ischemic insult, making them a novel therapeutic target for delayed cardioprotective therapy. Group 1 is wild type C57Bl6 uninjured hearts. These mice were not undergone any surgery and used as controls. Group 2 are wild type C57Bl6 72 h post-ischemia reperfusion (IR) injury hearts. These mice for subjected to ischemia reperfusion (IR) involving 90 min of left anterior descending coronary artery occlusion followed by reperfusion for 3 days or 72 h.

Project description:Background The axon guidance cue Slit2 recently has been found to regulate calcium homeostasis and molecular signaling in various stress events in different organs. However, whether Slit2 plays a role in cardiac ischemia-reperfusion (IR) injury has not been reported. Here, we aimed to investigate the role of Slit2 and the underlying mechanisms in cardiac IR injury. Methods Langendorff-perfused isolated hearts from Slit2-overexpressing (Slit2-Tg) mice and their background strain C57BL/6J mice were subjected to 20 min of global ischemia followed by 40 min of reperfusion. Left ventricular function of isolated hearts was monitored. Infarct size of post-IR hearts was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and histological changes of cardiac tissues and cells were determined with hematoxylin-eosin (HE) staining and transmission electron microscopy. Transcriptomic analysis was used to predict the biological processes and signaling pathways affected by Slit2 overexpression in the post-IR myocardium. Pro-Q staining and Western blotting was used to assess the phosphorylation levels of cardiac myofilaments and expression levels of myofilament-associated protein kinase and phosphatases. Results Slit2 overexpression increased post-IR left ventricular developed pressure (LVDP) by 35% and reduced infarct size by 53%, along with decreased myofibrillar disruption, mitochondrial swelling, and mitochondrial cristae dissolution. Slit2 overexpression significantly changed post-IR gene expression profiles. Functional products of these genes include regulation of cation transmembrane transport, cation homeostasis, collagen fibril organization, and regulation of heart rate. And post-IR myocardial KEGG pathways upregulated by Slit2 overexpression include ECM-receptor interaction, PI3K-Akt signaling pathway, and adrenergic signaling. Slit2 overexpression impacted myofilament phosphorylation together with myofilament-associated protein kinase C (PKC) isoforms and protein phosphatases (PPs). IR in C57BL/6J hearts upregulated phosphorylation of cardiac troponin-I (cTnI), which was suppressed by Slit2 overexpression. Myofilament‐associated PKCε, PKCδ, and PP2A were significantly increased post‐IR in C57BL/6J hearts, but in Slit2‐Tg hearts, myofilament‐associated PKCε and PP2A were increased and PKCδ was suppressed. Conclusions Our results demonstrate that Slit2 overexpression protects cardiac function and reduces IR injury, which is associated with Slit2‐induced gene profile shifts. The suppression of MyBP‐C and troponin‐I phosphorylation, and myofilament‐associated PKCδ levels induced by Slit2 overexpression could contribute to the cardioprotection of Slit2 in post-IR myocardium.

Project description:OBJECTIVE: To understand the molecular pathways underlying the cardiac preconditioning effect of short-term caloric restriction (CR). BACKGROUND: Lifelong CR has been suggested to reduce the incidence of cardiovascular disease through a variety of mechanisms. However, prolonged adherence to a CR life-style is difficult. Here we show how short-term CR protects the mouse heart from ischemia. METHODS: Male 10-12 wk old C57bl/6 mice were randomly assigned to an ad libitum (AL) diet with free access to regular chow, or CR, receiving 30% less food over a period of 7 days (d), prior to myocardial infarction (MI) via permanent coronary ligation. Prior to MI (d8), the left ventricles (LV) of AL and CR mice were collected for Western blot, DNA and microRNA (miR) analyses. In separate groups, infarct size, cardiac hemodynamics and protein abundance of caspase 3 was measured at d2 post-MI. RESULTS: This short-term model of CR was associated with cardio-protection, as evidenced by decreased infarct size (18.5±2.4% vs. 26.6±1.7%, N=10/group; P=0.01). cDNA and miR profiles pre-MI (N=5/group) identified genes modulated by short-term CR to be associated with circadian clock, oxidative stress, immune function, apoptosis, metabolism, angiogenesis, cytoskeleton and extracellular matrix (ECM). Western blots pre-MI revealed CR-associated increases in phosphorylated Akt and GSK3ß, reduced levels of phosphorylated AMPK and mitochondrial related proteins PGC-1?, cytochrome C and cyclooxygenase (COX) IV, with no differences in the levels of phosphorylated eNOS or MAPK (ERK1/2; p38). CONCLUSIONS: Short-term CR for only 7d represents a preconditioning strategy that limits infarct size. It is associated with a unique gene and miR signature, including the activation of specific pro-survival kinases. These findings may have implications for therapeutic use of short-term CR. . Male 12 wk-old C57Bl/6 mice were obtained from Charles River (Montreal, PQ, Canada) and maintained for at least 2 weeks before experimentation. Animals were randomly assigned to AL, CR and Lira groups with no initial differences in body weights. AL group mice were allowed ad libitum diet. Lira group mice were injected with Liraglutide (Glucagon-like peptide-1 agonist) at a dose of 200 µg/Kg i.p., twice daily for 7 days which is a mild weight-loss inducing dose and had access to ad libitum food and water. CR group mice were put on a Caloric restriction diet (30% less than normal ad libitum caloric intake) for 7 days to induce weight loss comparable to Lira group mice. Mice for CR and AL were individually caged and their food intake was measured everyday between 10-11 am for 1 week to calculate the average daily food intake. CR mice were fed 30% less than the calculated mean daily AL food consumption. CR cages were inspected daily for any remaining food, and transferred to a new cage every day. For CR group, pre-weighed mouse chow pellets were placed in cages between 10-11 am every day. Body weights were recorded between 10-11 am before and after the 7 day study period. For pre-ischemic assessments, animals (n=5/group/assessment) were sacrificed on day-8 (pre-MI), hearts were immediately extracted, washed in sterile phosphate-buffered saline (PBS), with LV free walls dissected, snap frozen in liquid nitrogen and stored at -80o C.

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Pathways Underlying Impaired Cardiac Preconditioning Potential in Subjects with the Metabolic Syndrome, and its Restoration by ACE-inhibition

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