Objective and methods
In this study, we investigated whether Ca2+ transients are altered in Purkinje cell aggregates dispersed from the subendocardium overlying the infarcted zone of the left ventricle (IZPCs) 48 h after coronary artery occlusion. To do so, we combined epifluorescent imaging with microelectrode recordings of IZPCs and normal canine Purkinje cell aggregates (NZPCs).
NZPCs respond to an action potential (AP) by a small Ca2+ transient at the cell surface immediately after the AP upstroke followed by a large [Ca2+ ] transient, which propagates to the cell core. In addition, focal Ca2+ waves can originate spontaneously later during the AP or during the diastolic interval (Circ Res 2000;86:448-55) and then propagate throughout the aggregate as ‘cell-wide Ca2+ waves’. Electrically-evoked Ca2+ transients in IZPCs arose significantly faster than those in NZPCs, and showed substantial spatiotemporal nonuniformity within an IZPC aggregate as well as between IZPC aggregates. IZPCs showed, hitherto undetected, low amplitude, micro Ca2+ transients (extent ≤5 μm) at a fivefold higher incidence than in NZPCs. Micro Ca2+ transients appeared to meander over distances ≤100 μm and reduced the local Ca2+ transient of the next paced beat. Micro Ca2+ transients nearly always preceded the cell-wide Ca2+ waves, which occurred more frequently in IZPCs than in NZPCs and caused non-driven electrical activity of the Purkinje aggregate.
Micro Ca2+ transients preceded cell-wide Ca2+ waves so often that it is probable that micro Ca2+ transients induced cell-wide Ca2+ waves. Cell-wide Ca2+ waves, in turn, clearly elicited spontaneous APs. We propose that the high incidence of micro Ca2+ transients in IZPCs is a fundamental element of the abnormal Ca2+ handling of diseased Purkinje cells, underlying arrhythmias originating in the subendocardial Purkinje network post myocardial infarction.
Arrhythmia (mechanisms); Calcium (cellular); Impulse formation; Infarction; Membrane potential; Purkinje fiber
The definitive cardiac outflow channels have three components: the intrapericardial arterial trunks; the arterial roots with valves; and the ventricular outflow tracts. We studied the normal and abnormal development of the most distal of these, the arterial trunks, comparing findings in mouse and man.
Methods and Results
Using lineage tracing and three-dimensional visualization by episcopic reconstruction and scanning electron microscopy, we studied embryonic day 9.5 to 12.5 mouse hearts, clarifying the development of the outflow tracts distal to the primordia of the arterial valves. We characterize a transient aortopulmonary foramen, located between the leading edge of a protrusion from the dorsal wall of the aortic sac and the distal margins of the two outflow cushions. The foramen is closed by fusion of the protrusion, with its cap of neural crest cells, with the neural crest cell-filled cushions; the resulting structure then functioning transiently as an aortopulmonary septum. Only subsequent to this closure is it possible to recognize, more proximally, the previously described aortopulmonary septal complex. The adjacent walls of the intrapericardial trunks are derived from the protrusion and distal parts of the outflow cushions, while the lateral walls are formed from intrapericardial extensions of pharyngeal mesenchyme derived from the second heart field.
We provide, for the first time, objective evidence of the mechanisms of closure of an aortopulmonary foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of aortopulmonary windows and the variants of common arterial trunk.
Mouse development; neural crest; second heart field; aortopulmonary window
Myeloperoxidase (MPO), a cardiovascular risk factor in humans, is an in vivo catalyst for lipoprotein modification via intermediate formation of reactive chlorinating species. Among the different lipoprotein classes, anti-atherogenic high-density lipoprotein (HDL) represents a major target for modification by hypochlorous acid (HOCl), generated from H2O2 by MPO in the presence of physiological chloride concentrations. As MPO was identified as an HDL-associated protein that could facilitate selective oxidative modification of its physiological carrier, the aim of the present study was to investigate whether and to what extent modification of HDL by HOCl affects the binding affinity of MPO in vitro.
Methods and results
We show that binding affinity of 125I-labelled MPO to HDL markedly increases as a function of increasing extent of HOCl modification of HDL. In contrast to native HDL, HOCl–HDL potently inhibits MPO binding/uptake by endothelial cells and effectively attenuates metabolism of MPO by macrophages. Reduction of HDL-associated chloramines with methionine strongly impaired binding affinity of MPO towards HOCl–HDL. This indicates that N-chloramines generated by HOCl are regulators of the high-affinity interaction between HOCl–HDL and positively charged MPO. Most importantly, the presence of HOCl–HDL is almost without effect on the halogenating activity of MPO.
We propose that MPO-dependent modification of HDL and concomitant increase in the binding affinity for MPO could generate a vicious cycle of MPO transport to and MPO-dependent modification at sites of chronic inflammation.
Hypochlorous acid; ApoA-I; Inflammation; Neutrophils; MPO–hydrogen peroxide–halide system
An increased expression of adenosine receptors is a promising target for gene therapy aimed at protecting the myocardium against ischemic damage, but may alter cardiac electrophysiology. We therefore studied the effects of heart-directed overexpression of A3 adenosine receptors (A3ARs) at different gene doses on sinus and atrio-ventricular (AV) nodal function in mice.
Methods and results
Mice with heart-specific overexpression of A3AR at high (
A3high) or low (
A3low) levels and their wild-type littermates were studied. Telemetric electrocardiogram (ECG) recordings in adult freely moving
A3high mice showed profound sinus bradycardia resulting in either ventricular escape rhythms or an incessant bradycardia–tachycardia syndrome (minimal heart rate
A3high 217 ±25*; WT 406 ±21 beats/min, all values as mean ±S.E.M., n = 7 per genotype, *p < 0.05). Exercise attenuated bradycardia in
A3high mice (maximal heart rate
A3high 650 ±13*; WT 796 ±13 beats/min) and first-degree AV nodal block was present (PQ interval
A3high 36 ±4*; WT 23 ±5 ms). Isolated hearts showed complete heart block (10/17
A3high * vs. 1/17 WT). Atrial bradycardia and AV block were already present 3 weeks after birth. Doppler echocardiography revealed atrial dysfunction and progressive atrial enlargement that was moderate at 3 and 8 weeks, and progressed at 12 and 21 weeks of age (all p < 0.05 vs. WT). Atrial contractility and sarcoendoplasmic Ca2 + ATPase (SERCA) 2a protein expression were reduced in isolated left
A3high atria at the age of 14 weeks. Fibrosis was present in left
A3high atria at 14 weeks, but not at 5 weeks of age.
A3high mice had first-degree AV block without arrhythmias or structural changes.
Heart-directed overexpression of A3AR resulted in gene dose-dependent AV block and pronounced sinus nodal dysfunction in vivo. Profound bradycardia heralded atrial and ventricular dilatation, dysfunction, and fibrosis. In contrast to A1 adenosine receptors (A1AR), the effects of A3AR overexpression were attenuated during exercise. This may have implications for the physiology of sinus nodal regulation and for therapeutic attempts to increase the expression of adenosine receptors.
Integrative physiology; Heart rate regulation; Autonomous nervous system; Sinus node dysfunction; AV block; Atrial cardiomyopathy; A3 adenosine receptor; Transgenic mice
The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1+/− mice, having a 50% reduction in OPA1 protein expression in cardiac tissue.
Methods and Results
In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1+/+. Electron and fluorescence microscopy revealed altered morphology of the Opa1+/− mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibers, mitochondrial functional properties were maintained, but direct energy channeling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction (TAC), Opa1+/− hearts demonstrated hypertrophy almost two-fold higher (p<0.01) than in wild type mice with altered ejection fraction (decrease of 43% versus 22% in Opa1+/+ mice, p<0.05).
These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.
Adaptation, Biological; Animals; Down-Regulation; GTP Phosphohydrolases; metabolism; Mice; Mice, Knockout; Mitochondria; genetics; metabolism; ultrastructure; Mitochondrial Membrane Transport Proteins; genetics; metabolism; Mitochondrial Membranes; metabolism; Mitochondrial Proteins; genetics; physiology; Myocytes, Cardiac; cytology; metabolism; Optic Atrophy, Autosomal Dominant; genetics; metabolism; physiopathology; Permeability; Pressure; cardiac energy metabolism; mitochondria; mitochondria dynamics; permeability transition pore; hypertrophy
This review focuses on target receptors that have been shown to have the potential to mimic the cardioprotective effect of ischemic preconditioning (IPC). There is an abundance of information concerning the intracellular mechanisms and membrane-bound receptors responsible for IPC. Important intracellular mediators of this cardioprotection likely reside in the activation of multiple kinase cascades. The major players in IPC are thought to include protein kinase C, tyrosine kinases, and members of the mitogen-activated protein kinase signaling family and these topics will be covered in more detail in other papers of this focused issue. However, many of these kinase-mediated mechanisms are triggered by the activation of transmembrane spanning receptors, some of which may be manipulated therapeutically to induce cardioprotection in humans with unstable angina or who are at risk for myocardial infarction. In this review, we will discuss the evidence supporting the possibility of manipulating several of these G protein-coupled receptors as potential therapeutic targets.
Stimulation of numerous receptors has been targeted as possible triggers for IPC. Some of those that have been identified include A1 adenosine, α1 adrenergic, M2 muscarinic, B2 bradykinin, δ1 opioid, AT1 angiotensin, and endothelin-1 receptors. In general, these receptors are thought to couple to inhibitory G proteins. In this review, we will focus on the most likely therapeutic candidates for cardioprotection, namely adenosine, opioid, and bradykinin receptors since selective agonists and antagonists, either alone or in combination, have most often been shown to mimic or block IPC in numerous animal models and man, respectively. This is not meant to completely rule out other receptors since it is clear that IPC is a phenomenon with multiple pathways that appear to be responsible for the cardioprotection observed.
Preconditioning; Infarction; Adenosine; Opioids; Bradykinin
The vascular endothelium starts to age at the first heartbeat. There is no longer a need to demonstrate that an increased resting heart rate—above 70 b.p.m.—is associated with the onset of cardiovascular events and reduces lifespan in humans. Each cardiac cycle imposes a mechanical constraint on the arteries, and we would like to propose that this mechanical stress damages the vascular endothelium, its dysfunction being the prerequisite for atherogenesis. Consequently, reducing heart rate could protect the endothelium and slow the onset of atherosclerosis. The potential mechanisms by which reducing heart rate could be beneficial to the endothelium are likely a combination of a reduction in mechanical stress and tissue fatigue and a prolongation of the period of steady laminar flow, and thus sustained shear stress, between each systole. With age, irreparable damage accumulates in endothelial cells and leads to senescence, which is characterized by a pro-atherogenic phenotype. In the body, the highest mechanical stress occurs in the coronary vessels, where blood only flows during diastole and even reverses during systole; thus, coronary arteries are the prime site of atherosclerosis. All classical risk factors for cardiovascular diseases add up, to accelerate atherogenesis, but hypertension, which further raises mechanical stress, is likely the most damaging. By inducing flow through the arteries, the heart rate determines shear stress and its stability: mechanical stress and the associated damage induced by each systole are efficiently counteracted by the repair capacities of a healthy endothelium. The maintenance of a physiological, low heart rate may be key to prolonging the endothelial healthy lifespan and thus, vascular health.
PMID: 19586943 CAMSID: cams3022
Endothelium; Resting heart rate; Mechanical stress; Cellular maintenance; Cellular repair; Atherosclerosis
Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1−/− mice.
Methods and results
Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT–PCR. Entpd1−/− mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1−/− mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y6 receptor and was independent of other uracil nucleotide-responding receptors (P2Y2 and P2Y4). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1−/− mice. In addition, pressurized mesenteric arteries from Entpd1−/− mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2.
NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.
PMID: 19640930 CAMSID: cams3021
NTPDase1; CD39; UTP; UDP; P2Y receptor; Vasoconstriction; Myogenic tone; Smooth muscle cell
We sought to determine whether interleukin (IL)-6 modulates myocardial infarction or the late phase of preconditioning (PC).
Wild-type and IL-6−/− mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion with or without six cycles of coronary occlusion/reperfusion 24 h earlier. Myocardial IL-6 protein expression, activation of Janus kinase (JAK) 1 and JAK2, and signal transducers and activators of transcription (STAT) 1 and STAT3 after ischemic PC protocol were examined. The expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined 24 h after the PC ischemia.
In preconditioned wild-type mice, infarct size was reduced from 60.5 ± 2.6% of the risk region to 33.5 ± 3.6%, indicating a late PC effect. In nonpreconditioned IL-6−/− mice, infarct size was similar to that observed in wild-type mice (59.9 ± 3.8%), indicating that the deletion of IL-6 has no effect on infarct size. However, in preconditioned IL-6−/− mice, infarct size was not reduced (65.1 ± 3.1%), indicating that the infarct-sparing effect was completely abrogated. Ischemic PC increased the expression of IL-6 in the cytoplasm of cardiomyocytes in the ischemic/reperfused zone. In IL-6−/− mice, the ischemic PC-induced activation of JAK1 and JAK2 and STAT1 and STAT3 was significantly reduced, and the increase in iNOS and COX-2 protein expression 24 h after the PC ischemia was markedly attenuated.
IL-6 does not modulate myocardial infarct size in naïve myocardium. However, following a PC stimulus, IL-6 is obligatorily required for the activation of the JAK–STAT pathway, the ensuing upregulation of iNOS and COX-2 (co-mediators of late PC), and the development of a cardioprotective phenotype.
Interleukins; Preconditioning; Infarction; Signal transduction; Cyclooxygenase
Angiotensin II; Animals; Anti-Inflammatory Agents; pharmacology; Antigens, CD31; pharmacology; Aortic Aneurysm, Abdominal; chemically induced; genetics; immunology; metabolism; pathology; prevention & control; Aortic Diseases; chemically induced; genetics; immunology; metabolism; pathology; prevention & control; Apolipoproteins E; deficiency; genetics; Atherosclerosis; chemically induced; genetics; immunology; metabolism; pathology; prevention & control; Cells, Cultured; Chemotaxis, Leukocyte; drug effects; Disease Models, Animal; Dose-Response Relationship, Drug; Lymphocyte Activation; drug effects; Macrophage Activation; drug effects; Male; Mice; Mice, Knockout; Peptides; pharmacology; Receptors, Antigen, T-Cell; agonists; metabolism; T-Lymphocytes; drug effects; immunology; Time Factors; Atherosclerosis; CD31; Peptides; Angiotensin II; Aneurysm
Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway.
Methods and results
Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1pos CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105pos progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt.
We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.
Diabetes; Cardiac progenitor cells; Oxidative stress; Glucose
C-type natriuretic peptide (CNP) has recently been suggested to represent an endothelium-derived hyperpolarizing factor (EDHF) in the mammalian resistance vasculature, important in the regulation of local blood flow and systemic blood pressure. Additionally, this peptide has been shown to protect against ischaemia-reperfusion injury and inhibits leukocyte and platelet activation. Herein, we use a novel, selective natriuretic peptide receptor-C (NPR-C) antagonist (M372049) to highlight the pivotal contribution of CNP/NPR-C signalling in the EDHF-dependent regulation of vascular tone and investigate the mechanism(s) underlying the release and biological activity of CNP and EDHF.
In vitro pharmacological investigation was conducted in rat (Sprague-Dawley) aorta and mesenteric resistance arteries. Relaxant responses to CNP, atrial natriuretic peptide (ANP), the nitric oxide donor spermine-NONOate (SPER-NO) and the endothelium-dependent vasodilator, acetylcholine (ACh) were examined in the absence and presence of M372049 or inhibitor cocktails shown previously to block endothelium-dependent dilatation in the resistance vasculature. RT-PCR was employed to characterize the expression of NPR subtypes in the vessels studied.
M372049 produced concentration-dependent inhibition of the vasorelaxant activity of CNP in rat isolated mesenteric resistance arteries but not aorta; in contrast, M372049 did not affect relaxations to ANP or SPER-NO in either vessel. M372049 or ouabain alone produced small, significant inhibition of EDHF-dependent relaxations in mesenteric arteries and in combination acted synergistically to abolish such responses. A combination of M372049 with established inhibitors of EDHF-dependent relaxation revealed that multiple, distinct pathways coordinate the bioactivity of EDHF in the resistance vasculature, and that CNP/NPR-C signalling represents a major component.
These data substantiate CNP/NPR-C signalling as a fundamental pathway underlying EDHF-dependent regulation of vascular tone in the rat mesenteric resistance vasculature. An increased understanding of the physiological roles of CNP/NPR-C signalling in the vasculature (now facilitated by the identification of a selective NPR-C antagonist) should aid determination of the (patho)physiological importance of EDHF and might provide the rationale for the design of novel therapeutics.
Blood flow; natriuretic peptide; endothelium-derived hyperpolarizing factor; microcirculation; vasodilation
The art and science of the use of deposition markers for the estimation of blood flow distributions throughout the body and within organs is reviewed. Development of diffusible tracer techniques started 50 years ago. Twenty years later, radioactive 15 micron microspheres became the standard marker. Early studies on small animals, fetal sheep in 1967 and rats in 1976, provoked much of the technical development. Needs for avoiding the use of radioactivity, for having long lasting labels, and for providing higher spatial resolution, are driving the continuing exploration of newer techniques using colored and fluorescent microspheres and molecular deposition markers. Strengths and weaknesses of the various methods are compared.
Blood flow; Flow heterogeneity; Cardiac output; Deposition markers; Rats; Mice; Dogs
Traditional concepts of vascular inflammation are considered “inside-out” responses centered on the monocyte adhesion and lipid oxidation hypotheses. These mechanisms likely operate in concert, holding the central tenet that the inflammatory response is initiated at the luminal surface. However, growing evidence supports a new paradigm of an “outside-in” hypothesis, in which vascular inflammation is initiated in the adventitia and progresses inward toward the intima. Hallmarks of the outside-in hypothesis include population of the adventitia with exogenous cell types, including monocytes, macrophages, and lymphocytes, the phenotypic switch of adventitial fibroblasts into migratory myofibroblasts, and increased vasa vasorum neovascularization. The resident and migrating cells deposit collagen and matrix components, respond to and upregulate inflammatory chemokines and/or antigens, and regulate the local redox state of the adventitia. B cells and T cells generate local humoral immune responses against local antigen presentation by foam cells and antigen presenting cells. These events result in increased local expression of cytokines and growth factors, evoking an inflammatory response that propagates inward toward the intima. Ultimately, it appears that the basic mechanisms of cellular activation and migration in vascular inflammation are highly conserved across a variety of cardiovascular disease states and that major inflammatory events begin in the adventitia.
Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene.
To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR.
Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression.
In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.
Gap junctions; Cx40; Nkx2-5; GATA4; Tbx5; Muscle gene regulation
More than 10 years after its discovery, the function of cyclooxygenase-2 (COX-2) in the cardiovascular system remains largely an enigma. Many scholars have assumed that the allegedly detrimental effects of COX-2 in other systems (e.g. proinflammatory actions and tumorigenesis) signify a detrimental role of this protein in cardiovascular homeostasis as well. This view, however, is ill-founded. Recent studies have demonstrated that ischemic preconditioning (PC) upregulates the expression and activity of COX-2 in the heart, and that this increase in COX-2 activity mediates the protective effects of the late phase of PC against both myocardial stunning and myocardial infarction. An obligatory role of COX-2 has been observed in the setting of late PC induced not only by ischemia but also by δ-opioid agonists and physical exercise, supporting the view that the recruitment of this protein is a central mechanism whereby the heart protects itself from ischemia. The beneficial actions of COX-2 appear to be mediated by the synthesis of PGE2 and/ or PGI2. Since inhibition of iNOS in preconditioned myocardium blocks COX-2 activity whereas inhibition of COX-2 does not affect iNOS activity, COX-2 appears to be downstream of iNOS in the protective pathway of late PC. The results of these studies challenge the widely accepted paradigm that views COX-2 activity as detrimental. The discovery that COX-2 plays an indispensable role in the anti-stunning and anti-infarct effects of late PC demonstrates that the recruitment of this protein is a fundamental mechanism whereby the heart adapts to stress, thereby revealing a novel, hitherto unappreciated cardioprotective function of COX-2. From a practical standpoint, the recognition that COX-2 is an obligatory co-mediator (together with iNOS) of the protection afforded by late PC has implications for the clinical use of COX-2 selective inhibitors as well as nonselective COX inhibitors. For example, the possibility that inhibition of COX-2 activity may augment myocardial cell death by obliterating the innate defensive response of the heart against ischemia/reperfusion injury needs to be considered and is the object of much current debate. Furthermore, the concept that the COX-2 byproducts, PGE2 and/ or PGI2, play a necessary role in late PC provides a basis for novel therapeutic strategies designed to enhance the biosynthesis of these cytoprotective prostanoids in the ischemic myocardium. From a conceptual standpoint, the COX-2 hypothesis of late PC expands our understanding of the function of this enzyme in the cardiovascular system and impels a critical reassessment of current thinking regarding the biologic significance of COX-2.
Ischemia; Nitric oxide; Preconditioning; Reperfusion
In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signals. From a yeast two-hybrid assay, we have identified Enigma Homolog 1 (ENH1) as a new binding partner of PKD1. Since in neurons, ENH1, associated with protein kinase Cε, was shown to modulate the activity of N-type calcium channels, and the pore-forming subunit of the cardiac L-type voltage-gated calcium channel, α1C, possesses a potential phosphorylation site for PKD1, we studied here a possible role of ENH1 and PKD1 in the regulation of the cardiac L-type voltage-gated calcium channel.
Methods and results
PKD1-interacting proteins were searched by yeast two-hybrid screening. In vivo protein interactions in cardiomyocytes isolated from heart ventricles of newborn rats were tested by co-immunoprecipitation. Small interfering RNA and a dominant negative mutant of PKD1 were delivered into cardiomyocytes by use of an adenovirus. Calcium currents were measured by the patch-clamp technique. Both ENH1 and PKD1 interact with α1C in cardiomyocytes. This interaction is increased upon stimulation. Silencing of ENH1 prevented the binding of PKD1 to α1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the α-adrenergic-induced increase of L-type calcium currents.
We found a new binding partner, ENH1, and a new target, α1C, for PKD1 in neonatal rat cardiomyocytes. We propose a model where ENH1 scaffolds PKD1 to α1C in order to form a signalling complex that regulates the activity of cardiac L-type voltage-gated Ca2+ channels.
Protein kinases; Ca-channel; Signal transduction
Reducing the heart's temperature by 2–5°C is a potent cardioprotective treatment in animal models of coronary artery occlusion. The anti-infarct benefit depends upon the target temperature and the time at which cooling is instituted. Protection primarily results from cooling during the ischaemic period, whereas cooling during reperfusion or beyond offers little protection. In animal studies, protection is proportional to both the depth and duration of cooling. An optimal cooling protocol must appreciably shorten the normothermic ischaemic time to effectively salvage myocardium. Patients presenting with acute myocardial infarction could be candidates for mild hypothermia since the current door-to-balloon time is typically 90 min. But they would have to be cooled quickly shortly after their arrival. Several strategies have been proposed for ultra-fast cooling, but most like liquid ventilation and pericardial perfusion are too invasive. More feasible strategies might include cutaneous cooling, peritoneal lavage with cold solutions, and endovascular cooling with intravenous thermodes. This last option has been investigated clinically, but the results have been disappointing possibly because the devices lacked capacity to cool the patient quickly or cooling was not implemented soon enough. The mechanism of hypothermia's protection has been assumed to be energy conservation. However, whereas deep hypothermia clearly preserves ATP, mild hypothermia has only a modest effect on ATP depletion during ischaemia. Some evidence suggests that intracellular signalling pathways might be responsible for the protection. It is unknown how cooling could trigger these pathways, but, if true, then it might be possible to duplicate cooling's protection pharmacologically.
Cardioprotection; Cooling; Hypothermia; Infarction; Ischaemia
Adenosine Triphosphate; physiology; Animals; Cardiotonic Agents; therapeutic use; Humans; Hypothermia; chemically induced; physiopathology; Models, Animal; Myocardial Infarction; physiopathology; Myocardial Reperfusion Injury; physiopathology; prevention & control; Signal Transduction; physiology; Cardioprotection; Cooling; Hypothermia; Infarction; Ischemia
Knockout of the neural and cardiac expressed transcription factor HF-1b causes electrophysiological abnormalities including fatal ventricular arrhythmias that occur with increasing frequency around the 4th week of postnatal life. This study addresses factors that may contribute to conduction disturbance in the ventricle of the HF-1b knockout mouse. Disruptions to gap junctional connexin40 (Cx40) have been reported in distal (i.e., apically located), but not proximal His–Purkinje conduction tissues of the HF-1b knockout mouse. This abnormality in myocardial Cx40 led us to address whether 4-week-old HF-1b knockout postnates display other disruptions to ventricular structure and function.
Western blotting and immunoconfocal quantification of Cx43 and coronary arteriole density and function were undertaken in the ventricle. Electrical activation was described by optical mapping.
Western blotting and immunoconfocal microscopy indicated that overall levels of Cx43 (p <0.001) and percent of Cx43 localized in intercalated disks (p <0.001) were significantly decreased in the ventricular myocardium of knockouts relative to wildtype littermate controls. Analysis of the reduction in Cx43 level by basal and apical territories revealed that the decrease was most pronounced in the lower, apical half of the ventricle of knockouts relative to controls (p <0.001). Myocyte size also showed a significant decrease in the knockout, that was more marked within the apical half of the ventricle (p <0.05). Optical recordings of ventricular activation indicated apically localized sectors of slowed conduction in knockout ventricles not occurring in controls that could be correlated directly to tissues showing reduced Cx43. These discrete sectors of abnormal conduction in the knockout heart were resolved following point stimulation of the ventricular epicardium and thus were not explained by dysfunction of the His–Purkinje system. To further probe base-to-apex abnormalities in the HF-1b knockout ventricle, we analyzed coronary arterial structure and function. These analyses indicated that relative to controls, the apical ventricular territory of the HF-1b knockout had reductions in the density of small resistance vessels (p <0.01) and deficits in arterial function as assayed by bead perfusion (p <0.01).
The HF-1b knockout ventricle displays abnormalities in Cx43 level, myocyte size, activation spread and coronary arterial structure and function. These abnormalities tend to be more pronounced in the apical territory of the ventricle and seem likely to be factors contributing to the pathological disturbance of cardiac conduction that characterizes the heart of the HF-1b knockout mouse.
Transgenic animal models; Arrhythmia mechanisms; Arteries; Sudden death; Gap junctions
Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart.
Methods and results
We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 μg/kg iv in vivo. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial KATP channels (5-HD), and sarcolemmal KATP channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 μg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period.
A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and KATP channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.
Ischaemia; thrombopoietin; protein kinases; infarction; K-ATP channel
We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion.
Methods and results
Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV15′ groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32–34°C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV15′ vs. Control and Normo-TLV (15.1 ± 3.3%, 16.4 ± 2.3%, 1.8 ± 0.6%, and 1.1 ± 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV15′ vs. Control and Normo-TLV (4 ± 1%, 11 ± 5%, 39 ± 2%, and 42 ± 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 ± 12 vs. 86 ± 12 µM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control.
Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.
Cooling; Contractile function; Mitochondria; Infarction; Total liquid ventilation
VEGFs (VEGFs) are key regulators of permeability. The principal evidence behind how they increase vascular permeability in vivo and in vitro, and the consequences of that increase, are here addressed. Detailed analysis of the published literature showed that in vivo and in vitro data on VEGF mediated permeability differed in its time course, but had common involvement of many specific signalling pathways, in particular VEGF-Receptor-2 activation, calcium influx through transient receptor potential channels, activation of phospholipase C gamma and downstream activation of nitric oxide synthase. Downstream of endothelial-nitric oxide synthase appears to involve the guanylyl cyclase mediated activation of the rho-rac pathway and subsequent involvement of junctional signalling proteins such as vascular endothelial-cadherin and the tight junctional proteins zona occludens and occludin linked to the actin cytoskeleton. The signalling appears to be co-ordinated through spatial organisation of the cascade into a signalplex, and arguments for why this may be important are considered. Many proteins have been identified to be involved in the regulation of vascular permeability by VEGF, but still the mechanisms through which these are thought to interact to control permeability are dependent on the experimental system, and a synthesis of existing data reveals that in intact vessels the co-ordination of the pathways is still not understood.
VEGF; vascular permeability; calcium; capillary; endothelium
Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell–cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis.
Methods and results
Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (~35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice.
This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.
Vascular smooth muscle; Apoptosis; Atherosclerosis; Matrix-degrading metalloproteinase-7; N-cadherin