Genetic ablation of Lmp2 increases the susceptibility for impaired cardiac function.

Proteasome degradation is an integral part of cellular growth and function. Proteasomal intervention may mitigate adverse myocardial remodeling, but is associated with the onset of heart failure. Previously, we have demonstrated that increasing abundance of cardiac Lmp2 and its incorporation into proteasome complexes is an endogenous mechanism for proteasome regulation during hypertrophic remodeling of the heart induced by chronic ß-adrenoreceptor stimulation. Here, we investigated whether Lmp2 is required for myocardial remodeling not driven by inflammation and show that Lmp2 is a tipping element for growth and function in the heart but not for proteasome insufficiency. While it has no apparent impact under unchallenged conditions, myocardial remodeling without Lmp2 exacerbates hypertrophy and restricts cardiac function. Under chronic ß-adrenoreceptor stimulation, as seen in the development of cardiovascular disease and the manifestation of heart failure, genetic ablation of Lmp2 in mice caused augmented concentric hypertrophy of the left ventricle. While the heart rate was similarly elevated as in wildtype, myocardial contractility was not maintained without Lmp2, and apparently uncoupled of the ß-adrenergic response. Normalized to the exacerbated myocardial mass, contractility was reduced by 41% of the pretreatment level, but would appear preserved at absolute level. The lack of Lmp2 interfered with elevated 26S proteasome activities during early cardiac remodeling reported previously, but did not cause bulk proteasome insufficiency, suggesting the Lmp2 containing proteasome subpopulation is required for a selected group of proteins to be degraded. In the myocardial interstitium, augmented collagen deposition suggested matrix stiffening in the absence of Lmp2. Indeed, echocardiography of left ventricular peak relaxation velocity (circumferential strain rate) was reduced in this treatment group. Overall, targeting Lmp2 in a condition mimicking chronic ß-adrenoreceptor stimulation exhibited the onset of heart failure. Anticancer therapy inhibiting proteasome activity, including Lmp2, is associated with adverse cardiac events, in particular heart failure. Sparing Lmp2 may be an avenue to reduce adverse cardiac events when chronic sympathetic nervous system activation cannot be excluded.

Unsupervised mRNA-seq classification of heart transplant endomyocardial biopsies.

BACKGROUND: Endomyocardial biopsy (EMB) is currently considered the gold standard for diagnosing cardiac allograft rejection. However, significant limitations related to histological interpretation variability are well-recognized. We sought to develop a methodology to evaluate EMB solely based on gene expression, without relying on histology interpretation. METHODS: Sixty-four EMBs were obtained from 47 post-heart transplant recipients, who were evaluated for allograft rejection. EMBs were subjected to mRNA sequencing, in which an unsupervised classification algorithm was used to identify the molecular signatures that best classified the EMBs. Cytokine and natriuretic peptide peripheral blood profiling was also performed. Subsequently, we performed gene network analysis to identify the gene modules and gene ontology to understand their biological relevance. We correlated our findings with the unsupervised and histological classifications. RESULTS: Our algorithm classifies EMBs into three categories based solely on clusters of gene expression: unsupervised classes 1, 2, and 3. Unsupervised and histological classifications were closely related, with stronger gene module-phenotype correlations for the unsupervised classes. Gene ontology enrichment analysis revealed processes impacting on the regulation of cardiac and mitochondrial function, immune response, and tissue injury response. Significant levels of cytokines and natriuretic peptides were detected following the unsupervised classification. CONCLUSION: We have developed an unsupervised algorithm that classifies EMBs into three distinct categories, without relying on histology interpretation. These categories were highly correlated with mitochondrial, immune, and tissue injury response. Significant cytokine and natriuretic peptide levels were detected within the unsupervised classification. If further validated, the unsupervised classification could offer a more objective EMB evaluation.

Clinical phenomapping and outcomes after heart transplantation.

BACKGROUND: Survival after heart transplantation (HTx) is limited by complications related to alloreactivity, immune suppression, and adverse effects of pharmacologic therapies. We hypothesize that time-dependent phenomapping of clinical and molecular data sets is a valuable approach to clinical assessments and guiding medical management to improve outcomes. METHODS: We analyzed clinical, therapeutic, biomarker, and outcome data from 94 adult HTx patients and 1,557 clinical encounters performed between January 2010 and April 2013. Multivariate analyses were used to evaluate the association between immunosuppression therapy, biomarkers, and the combined clinical end point of death, allograft loss, retransplantation, and rejection. Data were analyzed by K-means clustering (K = 2) to identify patterns of similar combined immunosuppression management, and percentile slopes were computed to examine the changes in dosages over time. Findings were correlated with clinical parameters, human leucocyte antigen antibody titers, and peripheral blood mononuclear cell gene expression of the AlloMap (CareDx, Inc., Brisbane, CA) test genes. An intragraft, heart tissue gene coexpression network analysis was performed. RESULTS: Unsupervised cluster analysis of immunosuppressive therapies identified 2 groups, 1 characterized by a steeper immunosuppression minimization, associated with a higher likelihood for the combined end point, and the other by a less pronounced change. A time-dependent phenomap suggested that patients in the group with higher event rates had increased human leukocyte antigen class I and II antibody titers, higher expression of the FLT3 AlloMap gene, and lower expression of the MARCH8 and WDR40A AlloMap genes. Intramyocardial biomarker-related coexpression network analysis of the FLT3 gene showed an immune system-related network underlying this biomarker. CONCLUSIONS: Time-dependent precision phenotyping is a mechanistically insightful, data-driven approach to characterize patterns of clinical care and identify ways to improve clinical management and outcomes.

HSPA5 Gene encoding Hsp70 chaperone BiP in the endoplasmic reticulum.

The HSPA5 gene encodes the binding immunoglobulin protein (BiP), an Hsp70 family chaperone localized in the ER lumen. As a highly conserved molecular chaperone, BiP assists in a wide range of folding processes via its two structural domains, a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). BiP is also an essential component of the translocation machinery for protein import into the ER, a regulator for Ca2+ homeostasis in the ER, as well as a facilitator of ER-associated protein degradation (ERAD) via retrograde transportation of aberrant proteins across the ER membrane. When unfolded/misfolded proteins in the ER overwhelm the capacity of protein folding machinery, BiP can initiate the unfolded protein response (UPR), decrease unfolded/misfolded protein load, induce autophagy, and crosstalk with apoptosis machinery to assist in the cell survival decision. Post-translational modifications (PTMs) of BiP have been shown to regulate BiP's activity, turnover, and availability upon different extrinsic or intrinsic stimuli. As a master regulator of ER function, BiP is associated with cancer, cardiovascular disease, neurodegenerative disease, and immunological diseases. BiP has been targeted in cancer therapies and shows promise for application in other relevant diseases.

Protein kinetic signatures of the remodeling heart following isoproterenol stimulation.

Protein temporal dynamics play a critical role in time-dimensional pathophysiological processes, including the gradual cardiac remodeling that occurs in early-stage heart failure. Methods for quantitative assessments of protein kinetics are lacking, and despite knowledge gained from single-protein studies, integrative views of the coordinated behavior of multiple proteins in cardiac remodeling are scarce. Here, we developed a workflow that integrates deuterium oxide (2H2O) labeling, high-resolution mass spectrometry (MS), and custom computational methods to systematically interrogate in vivo protein turnover. Using this workflow, we characterized the in vivo turnover kinetics of 2,964 proteins in a mouse model of ß-adrenergic-induced cardiac remodeling. The data provided a quantitative and longitudinal view of cardiac remodeling at the molecular level, revealing widespread kinetic regulations in calcium signaling, metabolism, proteostasis, and mitochondrial dynamics. We translated the workflow to human studies, creating a reference dataset of 496 plasma protein turnover rates from 4 healthy adults. The approach is applicable to short, minimal label enrichment and can be performed on as little as a single biopsy, thereby overcoming critical obstacles to clinical investigations. The protein turnover quantitation experiments and computational workflow described here should be widely applicable to large-scale biomolecular investigations of human disease mechanisms with a temporal perspective.

New frontiers in myocardial protection: a systems biology approach.

Myocardial ischemic injury and cardioprotection are characterized by a cascade of molecular changes, which includes gene expression, protein expression, protein localization, interactions, and posttranslational modifications (PTMs). A systems biology approach allows the study of these genes and proteins on a large scale; the omics technologies have led to new discoveries that further enhance our understanding of these molecular events. The complexity of the prosurvival signaling networks in cardiac cells is increasingly recognized; they afford beneficial effects on the integrity and functionality of a common effector, the mitochondrion. Mitochondrial proteome undergoes dynamic modifications in the course of ischemic injury; depending on the degree of injury, a variety of functional clusters are being affected including the changes in their protein properties (eg, PTMs), which consequently impact their function. The mitochondrial proteome appears to have inherent molecular machinery that initiates a versatile prosurvival mode, resisting environmental challenges. The molecular features in these mitochondrial pathways enabling adaptations involve distinct phosphorylation sites, S-nitrosylation cysteine residues, and other important amino acid domains subjected to PTMs. They become critical players in the determination of cell death and survival. Cardioprotective protein kinases, such as protein kinase C∈, can activate these PTMs, and provide a unique therapeutic platform for the use of small peptide regulators. Combining genomics and metabolomics discovery with that of proteomics information allows biological insights into cardioprotection at an integrated systems level. The current review discusses the systems biology concepts of myocardial ischemic injury and cardioprotection, as well as outlines the interrelationships of proteomics, genomics, and metabolomics in the quest to comprehend the prosurvival cell-signaling networks.

Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria.

Mitochondrial functions are dynamically regulated in the heart. In particular, protein phosphorylation has been shown to be a key mechanism modulating mitochondrial function in diverse cardiovascular phenotypes. However, site-specific phosphorylation information remains scarce for this organ. Accordingly, we performed a comprehensive characterization of murine cardiac mitochondrial phosphoproteome in the context of mitochondrial functional pathways. A platform using the complementary fragmentation technologies of collision-induced dissociation (CID) and electron transfer dissociation (ETD) demonstrated successful identification of a total of 236 phosphorylation sites in the murine heart; 210 of these sites were novel. These 236 sites were mapped to 181 phosphoproteins and 203 phosphopeptides. Among those identified, 45 phosphorylation sites were captured only by CID, whereas 185 phosphorylation sites, including a novel modification on ubiquinol-cytochrome c reductase protein 1 (Ser-212), were identified only by ETD, underscoring the advantage of a combined CID and ETD approach. The biological significance of the cardiac mitochondrial phosphoproteome was evaluated. Our investigations illustrated key regulatory sites in murine cardiac mitochondrial pathways as targets of phosphorylation regulation, including components of the electron transport chain (ETC) complexes and enzymes involved in metabolic pathways (e.g. tricarboxylic acid cycle). Furthermore, calcium overload injured cardiac mitochondrial ETC function, whereas enhanced phosphorylation of ETC via application of phosphatase inhibitors restored calcium-attenuated ETC complex I and complex III activities, demonstrating positive regulation of ETC function by phosphorylation. Moreover, in silico analyses of the identified phosphopeptide motifs illuminated the molecular nature of participating kinases, which included several known mitochondrial kinases (e.g. pyruvate dehydrogenase kinase) as well as kinases whose mitochondrial location was not previously appreciated (e.g. Src). In conclusion, the phosphorylation events defined herein advance our understanding of cardiac mitochondrial biology, facilitating the integration of the still fragmentary knowledge about mitochondrial signaling networks, metabolic pathways, and intrinsic mechanisms of functional regulation in the heart.

Differential regulation of proteasome function in isoproterenol-induced cardiac hypertrophy.

RATIONALE: Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation. OBJECTIVE: The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart. METHOD AND RESULTS: Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by ß-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible ß subunits. CONCLUSIONS: Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.

Proteomic and metabolomic analysis of cardioprotection: Interplay between protein kinase C epsilon and delta in regulating glucose metabolism of murine hearts.

We applied a combined proteomic and metabolomic approach to obtain novel mechanistic insights in PKCvarepsilon-mediated cardioprotection. Mitochondrial and cytosolic proteins from control and transgenic hearts with constitutively active or dominant negative PKCvarepsilon were analyzed using difference in-gel electrophoresis (DIGE). Among the differentially expressed proteins were creatine kinase, pyruvate kinase, lactate dehydrogenase, and the cytosolic isoforms of aspartate amino transferase and malate dehydrogenase, the two enzymatic components of the malate aspartate shuttle, which are required for the import of reducing equivalents from glycolysis across the inner mitochondrial membrane. These enzymatic changes appeared to be dependent on PKCvarepsilon activity, as they were not observed in mice expressing inactive PKCvarepsilon. High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy confirmed a pronounced effect of PKCvarepsilon activity on cardiac glucose and energy metabolism: normoxic hearts with constitutively active PKCvarepsilon had significantly lower concentrations of glucose, lactate, glutamine and creatine, but higher levels of choline, glutamate and total adenosine nucleotides. Moreover, the depletion of cardiac energy metabolites was slower during ischemia/reperfusion injury and glucose metabolism recovered faster upon reperfusion in transgenic hearts with active PKCvarepsilon. Notably, inhibition of PKCvarepsilon resulted in compensatory phosphorylation and mitochondrial translocation of PKCdelta. Taken together, our findings are the first evidence that PKCvarepsilon activity modulates cardiac glucose metabolism and provide a possible explanation for the synergistic effect of PKCdelta and PKCvarepsilon in cardioprotection.

Cyclin-dependent kinase 2 signaling regulates myocardial ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury to the heart is accompanied by the upregulation and posttranslational modification of a number of proteins normally involved in regulating cell cycle progression. Two such proteins, cyclin-dependent kinase-2 (Cdk2) and its downstream target, the retinoblastoma gene product (Rb), also play a critical role in the control of apoptosis. Myocardial ischemia activates Cdk2, resulting in the phosphorylation and inactivation of Rb. Blocking Cdk2 activity reduces apoptosis in cultured cardiac myocytes. Genetic or pharmacological inhibition of Cdk2 activity in vivo during I/R injury led to a 36% reduction in infarct size (IFS), when compared to control mice, associated with a reduction in apoptotic myocytes. To confirm that Rb was the critical target in Cdk2-mediated I/R injury, we determined the consequences of I/R injury in cardiac-specific Rb-deficient mice (CRb(L/L)). IFS was increased 140% in CRb(L/L) mice compared to CRb+/+ controls. TUNEL positive nuclei and caspase-3 activity were augmented by 92% and 36%, respectively, following injury in the CRb(L/L) mice demonstrating that loss of Rb in the heart significantly exacerbates I/R injury. These data suggest that Cdk2 signaling pathways are critical regulators of cardiac I/R injury in vivo and support a cardioprotective role for Rb.

Ischemic preconditioning modulates mitochondrial respiration, irrespective of the employed signal transduction pathway.

We tested in the in vivo rat heart the hypothesis that although ischemic preconditioning can employ different signal transduction pathways, these pathways converge ultimately at the level of the mitochondrial respiratory chain. Infarct size produced by a 60-min coronary artery occlusion (69%+/-2% of the area at risk) was limited by a preceding 15-min coronary occlusion (48%+/-4%). Cardioprotection by this stimulus was triggered by adenosine receptor stimulation, which was followed by protein kinase C and tyrosine kinase activation and then mitochondrial K(+)(ATP)-channel opening. In contrast, cardioprotection by 3 cycles of 3-min coronary occlusions (infarct size 27%+/-5% of the area at risk) involved the release of reactive oxygen species, which was followed by protein kinase C and tyrosine kinase activation, but was independent of adenosine receptor stimulation and K(+)(ATP)-channel activation. However, both pathways decreased respiratory control index (RCI; state-3/state-2, using succinate as complex-II substrate) from 3.1+/-0.2 in mitochondria from sham-treated hearts to 2.4+/-0.2 and 2.5+/-0.1 in hearts subjected to a single 15-min and triple 3-min coronary occlusions, respectively (both P<0.05). The decreases in RCI were due to an increase in state-2 respiration, whereas state-3 respiration was unchanged. Abolition of cardioprotection by blockade of either signal transduction pathway was paralleled by a concomitant abolition of mitochondrial uncoupling. These observations are consistent with the concept that mild mitochondrial uncoupling contributes to infarct size limitation by various ischemic preconditioning stimuli, despite using different signal transduction pathways. In conclusion, in the in vivo rat heart, different ischemic preconditioning (IPC) stimuli can activate highly different signal transduction pathways, which seem to converge at the level of the mitochondria where they increase state-2 respiration.

Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis.

Myocardium tolerant to an adenosine-dependent ischemic preconditioning stimulus can still be protected by stimuli that employ alternative signaling pathways.

Clinical studies on cardioprotection by preinfarct angina are ambiguous, which may involve development of tolerance to repeated episodes of ischemia. Not all preconditioning stimuli use identical signaling pathways, and because patients likely experience varying numbers of episodes of preinfarct angina of different degrees and durations, it is important to know whether myocardium tolerant to a particular preconditioning stimulus can still be protected by stimuli employing alternative signaling pathways. We tested the hypothesis that development of tolerance to a particular stimulus does not affect cardioprotection by stimuli that employ different signaling pathways. Anesthetized rats underwent classical, remote or pharmacological preconditioning. Infarct size (IS), produced by a 60-min coronary artery occlusion (CAO), was determined after 120 min of reperfusion. Preconditioning by two 15-min periods of CAO (2CAO15, an adenosine-dependent stimulus) limited IS from 69 +/- 2% to 37 +/- 6%, but when 2CAO15 was preceded by 4CAO15, protection by 2CAO15 was absent (IS = 68 +/- 1%). This development of tolerance coincided with a loss of cardiac interstitial adenosine release, whereas two 15-min infusions of adenosine (200 microg/min i.v.) still elicited cardioprotection (IS = 40 +/- 4%). Furthermore, cardioprotection was produced when 4CAO15 was followed by the adenosine-independent stimulus 3CAO3 (IS = 50 +/- 8%) or the remote preconditioning stimulus of two 15-min periods of mesenteric artery occlusion (IS = 49 +/- 6%). In conclusion, development of tolerance to cardioprotection by an adenosine-dependent preconditioning stimulus still allows protection by pharmacological or ischemic stimuli intervention employing different signaling pathways.

Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition.

Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a powerful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg i.v. each) 24 h before 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size was significantly reduced in DETA/NO-treated mice (30 +/- 2% of risk region in treated mice vs. 50 +/- 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg i.v.), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 +/- 1 vs. 30 +/- 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca(2+)-induced swelling by 20 micromol/l CaCl(2) or by the higher concentration of 200 micromol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 micromol/l CaCl(2), thereby confirming that Ca(2+) induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca(2+)-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator.

The tyrosine phosphatase inhibitor bis(maltolato)oxovanadium attenuates myocardial reperfusion injury by opening ATP-sensitive potassium channels.

Vanadate has been shown to inhibit tyrosine phosphatase, leading to an increased tyrosine phosphorylation state. The latter has been demonstrated to be involved in the signal transduction pathway of ischemic preconditioning, the most potent endogenous mechanism to limit myocardial infarct size. Furthermore, there is evidence that phosphatase inhibition may be cardioprotective when given late after the onset of ischemia, but the mechanism of protection is unknown. We tested the hypothesis that the organic vanadate compound bis(maltolato)oxovanadium (BMOV) limits myocardial infarct size by attenuating reperfusion injury and investigated the underlying mechanism. Myocardial infarction was produced in 112 anesthetized rats by a 60-min coronary artery occlusion, and infarct size was determined histochemically after 180 min of reperfusion. Intravenous infusion of BMOV in doses of 3.3, 7.5, and 15 mg/kg i.v. decreased infarct size dose-dependently from 70 +/- 2% of the area at risk in vehicle-treated rats down to 41 +/- 5% (P < 0.05 versus control), when administered before occlusion. Administration of the low dose just before reperfusion was ineffective, but administration of the higher doses was equally cardioprotective as compared with administration before occlusion. The cardioprotection by BMOV was abolished by the tyrosine kinase inhibitor genistein and by the ATP-sensitive potassium (K(+)(ATP)) channel blocker glibenclamide but was not affected by the ganglion blocker hexamethonium. We conclude that BMOV afforded significant cardioprotection principally by limiting reperfusion injury. The mode of action appears to be by opening of cardiac K(+)(ATP) channels via increased tyrosine phosphorylation.

Sites of action of adenosine in interorgan preconditioning of the heart.

The mechanism underlying interorgan preconditioning of the heart remains elusive, although a role for adenosine and activation of a neurogenic pathway has been postulated. We tested in rats the hypothesis that adenosine released by the remote ischemic organ stimulates local afferent nerves, which leads to activation of myocardial adenosine receptors. Preconditioning with a 15-min mesenteric artery occlusion (MAO15) reduced infarct size produced by a 60-min coronary artery occlusion (60-min CAO) from 68 +/- 2% to 48 +/- 4% (P < 0.05). Pretreatment with the ganglion blocker hexamethonium or 8-(p-sulfophenyl)theophylline (8-SPT) abolished the protection by MAO15. Intramesenteric artery (but not intraportal vein) infusion of adenosine (10 microg/min) was as cardioprotective as MAO15, which was also abolished by hexamethonium. Whereas administration of hexamethonium at 5 min of reperfusion following MAO15 had no effect, 8-SPT at 5 min of reperfusion abolished the protection. Permanent reocclusion of the mesenteric artery before the 60-min CAO enhanced the cardioprotection by MAO15 (30 +/- 5%), but all protection was abolished when 8-SPT was administered after reocclusion of the mesenteric artery. Together, these findings demonstrate the involvement of myocardial adenosine receptors. We therefore conclude that locally released adenosine during small intestinal ischemia stimulates afferent nerves in the mesenteric bed during early reperfusion, initiating a neurogenic pathway that leads to activation of myocardial adenosine receptors.