ABSTRACT
BACKGROUND: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of heart failure. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells, and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helper and CD8+ cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post-myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8+ T cells. METHODS: We induced type 2 myocardial infarction-like ischemic injury in the heart by treatment with a single high dose of the ß-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and Clec9a-depleted mice lacking DC cross-priming function. RESULTS: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a-/- mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8+ T cells. CONCLUSION: Activation of cytotoxic CD8+ T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.
Subject(s)
Cross-Priming , Dendritic Cells/immunology , Myocardium/pathology , Animals , Antigen Presentation , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/metabolism , Disease Models, Animal , Female , Heart Failure/pathology , Humans , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Lymph Nodes/immunology , Lymph Nodes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Myocardial Infarction/immunology , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardium/immunology , Myocardium/metabolism , Receptors, Chemokine/metabolism , Receptors, Immunologic/deficiency , Receptors, Immunologic/geneticsABSTRACT
Uncontrolled inflammation is a major pathological factor underlying a range of diseases including autoimmune conditions, cardiovascular disease, and cancer. Improving localized delivery of immunosuppressive drugs to inflamed tissue in a non-invasive manner offers significant promise to reduce severe side effects caused by systemic administration. Here, a neutrophil-mediated delivery system able to transport drug-loaded nanocarriers to inflamed tissue by exploiting the inherent ability of neutrophils to migrate to inflammatory tissue is reported. This hybrid system (neutrophils loaded with liposomes ex vivo) efficiently migrates in vitro following an inflammatory chemokine gradient. Furthermore, the triggered release of loaded liposomes and reuptake by target macrophages is studied. The migratory behavior of liposome-loaded neutrophils is confirmed in vivo by demonstrating the delivery of drug-loaded liposomes to an inflamed skeletal muscle in mice. A single low-dose injection of the hybrid system locally reduces inflammatory cytokine levels. Biodistribution of liposome-loaded neutrophils in a human-disease-relevant myocardial ischemia reperfusion injury mouse model after i.v. injection confirms the ability of injected neutrophils to carry loaded liposomes to inflammation sites. This strategy shows the potential of nanocarrier-loaded neutrophils as a universal platform to deliver anti-inflammatory drugs to promote tissue regeneration in inflammatory diseases.
Subject(s)
Muscle, Skeletal/metabolism , Myocardial Ischemia/metabolism , Neutrophils/metabolism , Animals , Humans , Inflammation/metabolism , Liposomes , MiceABSTRACT
Ischemic heart disease remains the foremost cause of death globally, with survivors at risk for subsequent heart failure. Paradoxically, cell therapies to offset cardiomyocyte loss after ischemic injury improve long-term cardiac function despite a lack of durable engraftment. An evolving consensus, inferred preponderantly from non-human models, is that transplanted cells benefit the heart via early paracrine signals. Here, we tested the impact of paracrine signals on human cardiomyocytes, using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as the target of mouse and human cardiac mesenchymal stromal cells (cMSC) with progenitor-like features. In co-culture and conditioned medium studies, cMSCs markedly inhibited human cardiomyocyte death. Little or no protection was conferred by mouse tail tip or human skin fibroblasts. Consistent with the results of transcriptomic profiling, functional analyses showed that the cMSC secretome suppressed apoptosis and preserved cardiac mitochondrial transmembrane potential. Protection was independent of exosomes under the conditions tested. In mice, injecting cMSC-conditioned media into the infarct border zone reduced apoptotic cardiomyocytes > 70% locally. Thus, hPSC-CMs provide an auspicious, relevant human platform to investigate extracellular signals for cardiac muscle survival, substantiating human cardioprotection by cMSCs, and suggesting the cMSC secretome or its components as potential cell-free therapeutic products.
Subject(s)
Mesenchymal Stem Cells/cytology , Myocytes, Cardiac/cytology , Pluripotent Stem Cells/cytology , Stromal Cells/cytology , Animals , Coculture Techniques , Culture Media, Conditioned , Humans , MiceABSTRACT
Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival.
Subject(s)
Doxorubicin/pharmacology , Infarction/drug therapy , Infarction/pathology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Female , Humans , Hydrogen Peroxide/pharmacology , Induced Pluripotent Stem Cells/cytology , Infarction/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myocytes, Cardiac/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Structure-Activity RelationshipABSTRACT
Cysteine and glycine rich protein 3 (CSRP3) encodes Muscle LIM Protein (MLP), a well-established disease gene for Hypertrophic Cardiomyopathy (HCM). MLP, in contrast to the proteins encoded by the other recognised HCM disease genes, is non-sarcomeric, and has important signalling functions in cardiomyocytes. To gain insight into the disease mechanisms involved, we generated a knock-in mouse (KI) model, carrying the well documented HCM-causing CSRP3 mutation C58G. In vivo phenotyping of homozygous KI/KI mice revealed a robust cardiomyopathy phenotype with diastolic and systolic left ventricular dysfunction, which was supported by increased heart weight measurements. Transcriptome analysis by RNA-seq identified activation of pro-fibrotic signalling, induction of the fetal gene programme and activation of markers of hypertrophic signalling in these hearts. Further ex vivo analyses validated the activation of these pathways at transcript and protein level. Intriguingly, the abundance of MLP decreased in KI/KI mice by 80% and in KI/+ mice by 50%. Protein depletion was also observed in cellular studies for two further HCM-causing CSRP3 mutations (L44P and S54R/E55G). We show that MLP depletion is caused by proteasome action. Moreover, MLP C58G interacts with Bag3 and results in a proteotoxic response in the homozygous knock-in mice, as shown by induction of Bag3 and associated heat shock proteins. In conclusion, the newly generated mouse model provides insights into the underlying disease mechanisms of cardiomyopathy caused by mutations in the non-sarcomeric protein MLP. Furthermore, our cellular experiments suggest that protein depletion and proteasomal overload also play a role in other HCM-causing CSPR3 mutations that we investigated, indicating that reduced levels of functional MLP may be a common mechanism for HCM-causing CSPR3 mutations.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Cardiomyopathy, Hypertrophic/genetics , Heart/physiopathology , LIM Domain Proteins/genetics , Muscle Proteins/genetics , Animals , Cardiomyopathy, Hypertrophic/physiopathology , Disease Models, Animal , Gene Knock-In Techniques , Humans , Mice , Mutation , Sarcomeres/geneticsABSTRACT
AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.
Subject(s)
AMP-Activated Protein Kinases/genetics , Bradycardia/genetics , Calcium/metabolism , Heart Rate/genetics , Sarcolemma/metabolism , Sinoatrial Node/metabolism , Adult , Animals , Bradycardia/metabolism , Electrocardiography, Ambulatory , Exercise , Heart/diagnostic imaging , Humans , Magnetic Resonance Imaging, Cine , Magnetic Resonance Spectroscopy , Mice , Microscopy, Electron, Transmission , Mutation , Myocardium/metabolism , Myocardium/pathology , Myocardium/ultrastructure , Physical Conditioning, Animal , Physical Endurance , Ryanodine Receptor Calcium Release Channel/metabolism , Sinoatrial Node/pathologyABSTRACT
AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory ß (ß1 and ß2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 µM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Benzimidazoles/pharmacology , Benzoates/pharmacology , Enzyme Activators/pharmacology , Glucose/metabolism , Hypoglycemic Agents/pharmacology , Muscle, Skeletal/drug effects , Ribonucleotides/pharmacology , AMP-Activated Protein Kinases/drug effects , Aminoimidazole Carboxamide/pharmacology , Animals , Antibodies, Blocking/pharmacology , Humans , In Vitro Techniques , Isoenzymes , Mice , Mice, Knockout , Muscle Contraction/drug effectsABSTRACT
Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.
Subject(s)
AMP-Activated Protein Kinases/metabolism , B-Lymphocytes/cytology , Cardiomyopathies/metabolism , Carrier Proteins/genetics , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Proteins/metabolism , AMP-Activated Protein Kinases/genetics , Animals , B-Lymphocytes/enzymology , B-Lymphocytes/metabolism , Cardiomyopathies/genetics , Carrier Proteins/metabolism , Cell Count , Humans , Mice , Mice, Inbred C57BL , Mutation , Proto-Oncogene Proteins/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Despite significant advances in our understanding of the biology determining systemic energy homeostasis, the treatment of obesity remains a medical challenge. Activation of AMP-activated protein kinase (AMPK) has been proposed as an attractive strategy for the treatment of obesity and its complications. AMPK is a conserved, ubiquitously expressed, heterotrimeric serine/threonine kinase whose short-term activation has multiple beneficial metabolic effects. Whether these translate into long-term benefits for obesity and its complications is unknown. Here, we observe that mice with chronic AMPK activation, resulting from mutation of the AMPK γ2 subunit, exhibit ghrelin signaling-dependent hyperphagia, obesity, and impaired pancreatic islet insulin secretion. Humans bearing the homologous mutation manifest a congruent phenotype. Our studies highlight that long-term AMPK activation throughout all tissues can have adverse metabolic consequences, with implications for pharmacological strategies seeking to chronically activate AMPK systemically to treat metabolic disease.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Insulin-Secreting Cells/enzymology , Insulin-Secreting Cells/pathology , Obesity/enzymology , Adiposity/genetics , Adult , Aging/pathology , Agouti-Related Protein/metabolism , Animals , Arcuate Nucleus of Hypothalamus/metabolism , Energy Metabolism/genetics , Enzyme Activation , Feeding Behavior , Female , Heterozygote , Humans , Hyperphagia/complications , Hyperphagia/enzymology , Hyperphagia/genetics , Hyperphagia/pathology , Hypothalamus/metabolism , Insulin/metabolism , Male , Mice , Mitochondria/metabolism , Mutation/genetics , Neurons/metabolism , Obesity/blood , Obesity/complications , Obesity/pathology , Oxidative Phosphorylation , Receptors, Ghrelin/metabolism , Ribosomes/metabolism , Signal Transduction/genetics , Transcriptome/genetics , Up-Regulation/geneticsABSTRACT
We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.
Subject(s)
Biopolymers/physiology , Dynamins/physiology , Heart Failure/etiology , Mitophagy , Myocarditis/etiology , Animals , Biopolymers/genetics , Biopolymers/metabolism , Cells, Cultured , Dynamins/genetics , Dynamins/metabolism , Heart Failure/physiopathology , Mice , Mutation , Myocarditis/physiopathology , Oxidative PhosphorylationABSTRACT
Energy depletion has been highlighted as an important contributor to the pathology of hypertrophic cardiomyopathy (HCM), a common inherited cardiac disease. Pharmacological reversal of energy depletion appears an attractive approach and the use of perhexiline has been proposed as it is thought to shift myocardial metabolism from fatty acid to glucose utilisation, increasing ATP production and myocardial efficiency. We used the Mybpc3-targeted knock-in mouse model of HCM to investigate changes in the cardiac metabolome following perhexiline treatment. Echocardiography indicated that perhexiline induced partial improvement of some, but not all hypertrophic parameters after six weeks. Non-targeted metabolomics, applying ultra-high performance liquid chromatography-mass spectrometry, described a phenotypic modification of the cardiac metabolome with 272 unique metabolites showing a statistically significant change (p < 0.05). Changes in fatty acids and acyl carnitines indicate altered fatty acid transport into mitochondria, implying reduction in fatty acid beta-oxidation. Increased glucose utilisation is indirectly implied through changes in the glycolytic, glycerol, pentose phosphate, tricarboxylic acid and pantothenate pathways. Depleted reduced glutathione and increased production of NADPH suggest reduction in oxidative stress. These data delineate the metabolic changes occurring during improvement of the HCM phenotype and indicate the requirements for further targeted interventions.
Subject(s)
Cardiomyopathy, Hypertrophic/drug therapy , Cardiomyopathy, Hypertrophic/metabolism , Metabolome , Myocardium/metabolism , Perhexiline/therapeutic use , Animals , Cardiomyopathy, Hypertrophic/diagnostic imaging , Cardiomyopathy, Hypertrophic/pathology , Chromatography, High Pressure Liquid , Disease Models, Animal , Male , Mass Spectrometry , Metabolome/drug effects , Metabolomics , Mice, Inbred C57BL , Myocardium/pathology , Perhexiline/pharmacology , Phenotype , Principal Component Analysis , UltrasonographyABSTRACT
SCFA are produced in the gut by bacterial fermentation of undigested carbohydrates. Activation of the Gαi-protein-coupled receptor GPR41 by SCFA in ß-cells and sympathetic ganglia inhibits insulin secretion and increases sympathetic outflow, respectively. A possible role in stimulating leptin secretion by adipocytes is disputed. In the present study, we investigated energy balance and glucose homoeostasis in GPR41 knockout mice fed on a standard low-fat or a high-fat diet. When fed on the low-fat diet, body fat mass was raised and glucose tolerance was impaired in male but not female knockout mice compared to wild-type mice. Soleus muscle and heart weights were reduced in the male mice, but total body lean mass was unchanged. When fed on the high-fat diet, body fat mass was raised in male but not female GPR41 knockout mice, but by no more in the males than when they were fed on the low-fat diet. Body lean mass and energy expenditure were reduced in male mice but not in female knockout mice. These results suggest that the absence of GPR41 increases body fat content in male mice. Gut-derived SCFA may raise energy expenditure and help to protect against obesity by activating GPR41.
Subject(s)
Adipose Tissue/metabolism , Body Composition/genetics , Dietary Fats/pharmacology , Energy Metabolism/genetics , Fatty Acids, Volatile/metabolism , Obesity/genetics , Receptors, G-Protein-Coupled/genetics , Adipose Tissue/drug effects , Animals , Bacteria/metabolism , Body Fluid Compartments/drug effects , Body Fluid Compartments/metabolism , Diet, Fat-Restricted , Diet, High-Fat , Dietary Fats/metabolism , Female , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/microbiology , Glucose Intolerance/genetics , Heart/drug effects , Insulin/metabolism , Insulin Secretion , Leptin/metabolism , Male , Mice , Mice, Knockout , Muscle, Skeletal/drug effects , Obesity/etiology , Obesity/metabolism , Obesity/prevention & control , Organ Size , Receptors, G-Protein-Coupled/metabolism , Sex FactorsABSTRACT
The citric acid cycle (CAC) metabolite fumarate has been proposed to be cardioprotective; however, its mechanisms of action remain to be determined. To augment cardiac fumarate levels and to assess fumarate's cardioprotective properties, we generated fumarate hydratase (Fh1) cardiac knockout (KO) mice. These fumarate-replete hearts were robustly protected from ischemia-reperfusion injury (I/R). To compensate for the loss of Fh1 activity, KO hearts maintain ATP levels in part by channeling amino acids into the CAC. In addition, by stabilizing the transcriptional regulator Nrf2, Fh1 KO hearts upregulate protective antioxidant response element genes. Supporting the importance of the latter mechanism, clinically relevant doses of dimethylfumarate upregulated Nrf2 and its target genes, hence protecting control hearts, but failed to similarly protect Nrf2-KO hearts in an in vivo model of myocardial infarction. We propose that clinically established fumarate derivatives activate the Nrf2 pathway and are readily testable cytoprotective agents.
Subject(s)
Antioxidants/metabolism , Fumarates/therapeutic use , NF-E2-Related Factor 2/metabolism , Animals , Dimethyl Fumarate , Fumarate Hydratase/deficiency , Fumarate Hydratase/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Myocardial Infarction/genetics , Myocardial Infarction/prevention & control , NF-E2-Related Factor 2/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
RATIONALE: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. OBJECTIVE: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. METHODS AND RESULTS: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. CONCLUSIONS: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Myocardial Contraction , Myocytes, Cardiac/enzymology , Troponin I/metabolism , Ventricular Function, Left , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/genetics , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Calcium Signaling , Enzyme Activation , Enzyme Activators/pharmacology , Heart Ventricles/enzymology , Humans , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/drug effects , Myosins/drug effects , Myosins/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Ribonucleotides/pharmacology , Serine , Time Factors , Troponin I/genetics , Two-Hybrid System Techniques , Ventricular Function, Left/drug effectsABSTRACT
GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Galpha(i). Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Galpha(i) signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Galpha(i) signalling mediated by GPR43 in SCFA-stimulated leptin secretion.
Subject(s)
Acetates/metabolism , Adipocytes/metabolism , Butyrates/metabolism , Leptin/metabolism , Propionates/metabolism , Animals , Fatty Acids, Volatile/metabolism , Mice , Mice, Knockout , Pertussis Toxin/metabolism , Signal TransductionABSTRACT
MAPKAPK-2 (MK2) is a protein kinase activated downstream of p38-MAPK which phosphorylates the small heat shock proteins HSP27 and alphaB crystallin and modulates p38-MAPK cellular distribution. p38-MAPK activation is thought to contribute to myocardial ischemic injury; therefore, we investigated MK2 effects on ischemic injury and p38 cellular localization using MK2-deficient mice (KO). Immunoblotting of extracts from Langendorff-perfused hearts subjected to aerobic perfusion or global ischemia or reperfusion showed that the total and phosphorylated p38 levels were significantly lower in MK2(-/-) compared to MK2(+/+) hearts at baseline, but the ratio of phosphorylated/total p38 was similar. These results were confirmed by cellular fractionation and immunoblotting for both cytosolic and nuclear compartments. Furthermore, HSP27 and alphaB crsytallin phosphorylation were reduced to baseline in MK2(-/-) hearts. On semiquantitative immunofluorescence laser confocal microscopy of hearts during aerobic perfusion, the mean total p38 fluorescence was significantly higher in the nuclear compared to extranuclear (cytoplasmic, sarcomeric, and sarcolemmal compartments) in MK2(+/+) hearts. However, although the increase in phosphorylated p38 fluorescence intensity in all compartments following ischemia in MK2(+/+) hearts was lost in MK2(-/-) hearts, it was basally elevated in nuclei of MK2(-/-) hearts and was similar to that seen during ischemia in MK2(+/+) hearts. Despite these differences, similar infarct volumes were recorded in wild-type MK2(+/+) and MK2(-/-) hearts, which were decreased by the p38 inhibitor SB203580 (1 microM) in both genotypes. In conclusion, p38 MAPK-induced myocardial ischemic injury is not modulated by MK2. However, the absence of MK2 perturbs the cellular distribution of p38. The preserved nuclear distribution of active p38 MAPK in MK2(-/-) hearts and the conserved response to SB203580 suggests that activation of p38 MAPK may contribute to injury independently of MK2.
Subject(s)
Heat-Shock Proteins, Small/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Myocardial Ischemia/metabolism , Protein Serine-Threonine Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Enzyme Activation/drug effects , Imidazoles/pharmacology , Intracellular Signaling Peptides and Proteins/deficiency , Mice , Mice, Knockout , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/deficiency , Protein Transport , Pyridines/pharmacology , Signal Transduction , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
OBJECTIVE: Brief reversible ischemic episodes (ischemic preconditioning, IPC) protect the heart against arrhythmias during a subsequent prolonged low-flow ischemia. We have recently shown that this protection involves release of bradykinin, activation of bradykinin B2 receptors followed by opening of sarcolemmal, but not mitochondrial ATP-sensitive K+ channels. The goal of this study was to clarify a trigger and/or mediator role of bradykinin in the antiarrhythmic effects of IPC during low-flow ischemia. METHODS: Isolated perfused rat hearts underwent 60 minutes of low-flow ischemia induced by reducing perfusion pressure followed by 60 minutes of reperfusion. Preconditioning was induced by 2 x 5 minutes episodes of zero-flow ischemia. In yet other groups, preconditioned or non-preconditioned hearts were treated either with bradykinin (10 nmol/L) or with HOE 140 (bradykinin B2 receptor antagonist, 100 nmol/L). RESULTS: IPC reduced the number of ventricular premature beats, as well as the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia. In addition, this protection was abolished by HOE 140 given during low-flow ischemia. Pharmacological preconditioning using short bradykinin perfusion instead of IPC did not show antiarrhythmic effects. However, bradykinin administered during low-flow ischemia and reperfusion reduced the number of ventricular premature beats and the incidence of ventricular tachycardia and of ventricular fibrillation during low-flow ischemia. CONCLUSION: Bradykinin is a mediator, but unlikely a trigger, of antiarrhythmic effects of IPC during low-flow ischemia.
Subject(s)
Bradykinin/metabolism , Heart Ventricles/metabolism , Ischemic Preconditioning, Myocardial/methods , Tachycardia, Ventricular/etiology , Ventricular Fibrillation/etiology , Animals , Bradykinin/analogs & derivatives , Bradykinin/drug effects , Bradykinin/pharmacology , Bradykinin Receptor Antagonists , Disease Models, Animal , Disease Progression , Electrocardiography , Heart Rate/physiology , Heart Ventricles/drug effects , Heart Ventricles/physiopathology , Male , Pilot Projects , Prognosis , Rats , Rats, Sprague-Dawley , Tachycardia, Ventricular/metabolism , Tachycardia, Ventricular/prevention & control , Ventricular Fibrillation/metabolism , Ventricular Fibrillation/prevention & controlABSTRACT
OBJECTIVES: The purpose of this study was to determine whether p38 mitogen-activated protein kinase (p38-MAPK) contributes to tumor necrosis factor-alpha (TNFalpha)-induced contractile depression. BACKGROUND: Tumor necrosis factor has both beneficial and detrimental consequences that may result from the activation of different downstream pathways. Tumor necrosis factor activates p38-MAPK, a stress-responsive kinase implicated in contractile depression and cardiac injury. METHODS: In isolated hearts from mice lacking the p38-MAPK activator, MAPK kinase 3 (MKK3), perfused at constant coronary pressure or flow, we measured the left ventricular developed pressure (LVDP) and the relationship between end-diastolic volume and LVDP in the presence and absence of 10 ng/ml TNFalpha. RESULTS: Within 15 min at constant pressure, TNFalpha significantly reduced LVDP and coronary flow in outbred and mkk3(+/+) mice. This early negative inotropic effect was associated with a marked phosphorylation of both p38-MAPK and its indirect substrate, HSP27. In hearts lacking MKK3, TNFalpha failed to activate p38-MAPK or to cause significant contractile dysfunction. The actions of TNFalpha were similarly attenuated in MAPK-activated protein kinase 2 (MK2)-deficient hearts, which have a marked reduction in myocardial p38-MAPK protein content, and by the p38-MAPK catalytic site inhibitor SB203580 (1 micromol/l). Under conditions of constant coronary flow, the p38-MAPK activation and contractile depression induced by TNFalpha, though attenuated, remained sensitive to the absence of MKK3 or the presence of SB203580. The role of p38-MAPK in TNFalpha-induced contractile depression was confirmed in isolated murine cardiac myocytes exposed to SB203580 or lacking MKK3. CONCLUSIONS: Tumor necrosis factor activates p38-MAPK in the intact heart and in isolated cardiac myocytes through MKK3. This activation likely contributes to the early cardiodepressant action of TNFalpha.
Subject(s)
Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase 3/deficiency , Male , Mice , Mice, Knockout , Myocardium/enzymology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Protein Kinases/deficiency , Protein Serine-Threonine Kinases , Pyridines/pharmacology , Stroke Volume/drug effectsABSTRACT
To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on protein kinase Cepsilon (PKCepsilon), isolated buffer-perfused mouse hearts were randomized to four groups: 1) antimycin A (AA) (0.1 microg/ml) for 3 min followed by 10 min washout and then 30 min global ischemia (I) and 2 h reperfusion (R); 2) controls of I/R alone; 3) AA bracketed with 13 min of N-2-mercaptopropionyl- glycine (MPG) followed by I/R; and 4) MPG (200 microM) alone, followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1 microg/ml), and lucigenin was used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1 microg/ml) with or without MPG, and PKCepsilon translocation was measured by cell fractionation and subsequent Western blot analysis. Finally, the dependence of AA protection on PKCepsilon was determined by the use of knockout mice (-/-) lacking PKCepsilon. AA exposure caused ROS production, which was abolished by the mitochondrial uncoupler mesoxalonitrile 4-trifluoromethoxyphenylhydrazone. In addition, AA significantly reduced the percent infarction-left ventricular volume compared with control I/R (26 +/- 4 vs. 43 +/- 2%; P < 0.05). Bracketing AA with MPG caused a loss of protection (52 +/- 7 vs. 26 +/- 4%; P < 0.05). AA caused PKCepsilon translocation only in the absence of MPG, and protection was lost on the pkcepsilon(-/-) background (38 +/- 3 vs. 15 +/- 4%; P < 0.001). AA causes ROS production, on which protection and PKCepsilon translocation depend. In addition, protection is absent in PKCepsilon null hearts. Our results imply that, in common with ischemic preconditioning, PKCepsilon is crucial to ROS-mediated protection.