β2-Adrenoreceptor immunoreactivity in cardiac ganglia of the guinea pig

Summary Previous pharmacological studies in co-culture systems have indicated, the presence of β-adrenoreceptors on intrinsic cardiac neurons of the guinea pig (Horackovaet al., 1993) but radiologand binding studies of tissue sections failed to provide a definite answer as to the presence of such receptors on cardiac neuronsin situ, due to the iodine-binding properties of cardiac nerve bundles and ganglia (Molenaaret al., 1992). We therefore addressed this question by immunohistochemistry, using antisera raised against synthetic peptides of the β2-adrenoreceptor. For comparison, cholinergic and catecholaminergic neurons were identified immunohistochemically by means of antibodies against the enzymes involved in the synthesis of acetylcholine (choline acetyltransferase), and of catecholamines (tyrosine hydroxylase). Virtually all intrinsic cardiac neurons contained both β2-adrenoreceptor- and choline acetyltransferase-immunoreactivities. In addition, some nerve fibre bundles exhibited β2-adrenoreceptor-immunoreactivity. Several ganglia were innervated by tyrosine hydroxylase-immunoreactive axons, but the majority of ganglia did not receive tyrosine hydroxylase-immunoreactive nerve terminals, and additional intraganglionic sources of catecholamine synthesis could not be identified. Thus, the results are in favour of β-adrenergic modulation of guinea pig cardiac ganglia by humorally and, partially, by locally released catecholamines.     

Curcumin reduces the cardiac ischemia–reperfusion injury: involvement of the toll-like receptor 2 in cardiomyocytes

Curcumin, a polyphenolic compound derived from turmeric, has protective effects on myocardial injury through attenuation of oxidative stress and inflammation. Toll-like receptor 2 (TLR2), a key mediator of the innate immune system, is involved in myocardial infarction and examined if controlled by curcumin. Rat cardiomyocytes (CMs) were stimulated with tumor necrosis factor (TNF)-α, peptidoglycan (PGN) or hypoxia/reoxygenation (H/R) with or without curcumin pretreatment. Sprague–Dawley rats were fed curcumin (300 mg/kg/day) 1 week before cardiac ischemia/reperfusion (I/R) injury. The expression level of TLR2 and cardiac function were assessed. Both mRNA and protein of TLR2 were up-regulated in infarcted myocardium, while TLR4 remained unchanged. In CMs, TLR2 and monocyte chemoattractant protein (MCP)-1 mRNAs were increased by TNF-α, PGN or H/R, whereas they were blunted by curcumin. Immunofluorescence staining of CMs also showed that TLR2 and MCP-1 were increased after H/R, whereas curcumin-pretreated CMs were not. In animal study, 2 weeks after I/R, TLR2 was increased in the infarct zone, whereas it stayed unchanged in the Cur+I/R group. Macrophage infiltration (CD68), high-mobility group box 1 and fibrosis were increased in the I/R group, whereas they were decreased in the Cur+I/R group. Connexin 43 was reduced in the I/R group, while it recovered significantly in the Cur+I/R group. Cardiac contractility in the Cur+I/R group was also improved compared with that in the I/R group (max dp/dt in Cur+I/R group: 9660±612 vs. I/R group: 8119±366, P<.05). These results suggest that selective inhibition of TLR2 by curcumin could be preventive and therapeutic for myocardial infarction.     

Identification of the cardiac sarcolemmal Na+−Ca2+ exchanger using monoclonal antibodies

Summary We have previously partially purified the sarcolemmal Na⁺–Ca²⁺ exchange protein and produced rabbit polyclonal antibodies to the exchanger (Philipson, K. D., Longoni, S., Ward, R. 1988.Biochim. Biophys.Acta 945:298–306). We now describe the generation of three stable murine hybridoma lines which secrete monoclonal antibodies (MAb's) to the exchanger. These MAb's immunoprecipitate 50–75% of solubilized Na⁺–Ca²⁺ exchange activity. The MAb's appear to be reactive with native conformation-dependent expitopes on the Na⁺–Ca²⁺ exchanger since they do not react on immunoblots. An indirect method was used to identify Na⁺–Ca²⁺ exchange proteins. A column containing Na⁺–Ca²⁺ exchanger immobilized by MAb's was used to affinity purify the rabbit polyclonal antibody. The affinity-purified polyclonal antibody reacted with proteinsof, apparent molecular weights of 70, 120, and 160 kDa on immunoblots of sarcolemma. The data provide strong support for our prevous association of Na⁺–Ca²⁺ exchange with these proteins.     

Recombination hot spots within the I region of the mouse H-2 complex map to the E β and E α genes

Recombinant mouse strains with crossovers in the I region of the H-2 major histocompatibility complex were examined by restriction fragment analysis for the presence of polymorphic restriction sites within the E and E genes. Nine recombinant mouse strains were shown to have crossed over within a 5 kb DNA segment that contains the large intron between the second and third exons of the E gene. These results are in accord with previous studies mapping a recombination hot spot within this gene. Seven recombinant mouse strains between the p and k haplotypes were shown to have crossed over in a 6 kb segment within the E gene. These results show the existence of a recombination hot spot within the E gene. Comparison of the H-2 haplotypes involved in these two recombination hot spots suggests that a specific DNA sequence in b, s, f, and q haplotypes may act to promote recombination in the E gene and a specific DNA sequence in the p haplotype may act to promote recombination in the E gene.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

Fifty years of cardiac pacing: the dark side of the moon?

Fifty years after its introduction, cardiac pacing has evolved from an experimental medical treatment to an expanding field in today's cardiology. Only recently there is accumulating evidence that prolonged stimulation of the right ventricular apex is associated with clinically significant adverse effects. In this commentary, the potential adverse effects are summarised and potential modifications in contemporary pacing are discussed. (Neth Heart J 2008;16(suppl 1):S12-S14.)     

Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair

Wnts are required for cardiogenesis but the role of specific Wnts in cardiac repair remains unknown. In this report, we show that a dynamic Wnt1/βcatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. Acute ischaemic cardiac injury upregulates Wnt1 that is initially expressed in the epicardium and subsequently by cardiac fibroblasts in the region of injury. Following cardiac injury, the epicardium is activated organ-wide in a Wnt-dependent manner, expands, undergoes epithelial-mesenchymal transition (EMT) to generate cardiac fibroblasts, which localize in the subepicardial space. The injured regions in the heart are Wnt responsive as well and Wnt1 induces cardiac fibroblasts to proliferate and express pro-fibrotic genes. Disruption of downstream Wnt signalling in epicardial cells decreases epicardial expansion, EMT and leads to impaired cardiac function and ventricular dilatation after cardiac injury. Furthermore, disruption of Wnt/βcatenin signalling in cardiac fibroblasts impairs wound healing and decreases cardiac performance as well. These findings reveal that a pro-fibrotic Wnt1/βcatenin injury response is critically required for preserving cardiac function after acute ischaemic cardiac injury.     

Role for sulfur-containing groups in the Na+−Ca2+ exchange of cardiac sarcolemmal vesicles

Summary Different amino acid residues in cardiac sarcolemmal vesicles were modified by incubation with various chemical reagents. The effects of these modifications on sarcolemmal Na⁺–Ca²⁺ exchange were examined. Dithiothreitol, an agent that maintains sulfur-containing residues in a reduced state, caused a time- and concentration-dependent decrease in Na⁺–Ca²⁺ exchange. The treatment with dithiothreitol resulted in a decrease inV _max values but did not alter theK _m for Ca²⁺ for the Na²⁺–Ca²⁺ exchange reaction. If Na⁺ replaced K⁺ as the ion present during the modification of sarcolemmal membranes with dithiothreitol, there was substantially less of an inhibitor effect on Na⁺–Ca²⁺ exchange. Similar results were obtained with reduced glutathione, a reagent that also maintains sulfur-containing residues in a reduced state. Two sulfhydryl modifying reagents, methylmethanethiosulfonate and N-ethylmaleimide, were capable of altering Na⁺–Ca²⁺ exchange, and the type of ion present during modification significantly affected the extent of this alteration. Almost all of the chemical reagents investigated that modified other amino acid resides (carboxyl, lysyl, histidyl, tyrosyl, tryptophanyl, arginyl and hydroxyl) had the capacity to alter Na⁺–Ca²⁺ exchange after preincubation with the sarcolemmal membrane vesicles. However, the sulfur residue-modifying reagents were the only compounds to exhibit significant differences in their action on Na⁺–Ca²⁺ exchange, depending on whether Na⁺ or K⁺ was present in the preincubation modification medium. The tryptophan modifier, N-bromosuccinimide, was the sole reagent that elicited a substantial increase in membrane permeability. The evidence is consistent with the hypothesis that sulfurcontaining residues interact with a Na⁺-binding site for Na⁺–Ca²⁺ exchange in cardiac sarcolemmal vesicles.     

Mast cells: the missing source of cardiac renin?

The renin-angiotensin system (RAS) acts to regulate blood volume and arterial pressure, and has direct effects on the heart. Renin, released by the kidney, circulates and acts-in the rate-limiting step of angiotensin II (Ang II) production-to convert angiotensinogen to inactive angiotensin I (Ang I). Ang II constricts vessels, leading to increased arterial pressure, among other effects. Components of the RAS have been found in a number of extra-renal tissues. Recent research indicates that mast cells in the heart may produce renin, creating a cardiac-specific RAS that acts locally to produce Ang II. These results, however, are not without controversy. Others have searched for sites of renin production and have found no other significant source that was physiologically important or that could not be completely ruled out as a possible contaminant. How important is mast cell-synthesized renin for direct cardiac-related effects?     

Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction?

Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulin-dependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location 11p11.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC.     

Prostaglandin E2 Stimulates the Production of Amyloid-�� Peptides through Internalization of the EP4 Receptor

Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E₂ (PGE₂), a strong inducer of inflammation, stimulates the production of Aβ through EP₂ and EP₄ receptors, and here we have examined the molecular mechanism. Activation of EP₂ and EP₄ receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP₂, but not EP₄, receptor-mediated stimulation of the Aβ production. In contrast, inhibitors of endocytosis suppressed EP₄, but not EP₂, receptor-mediated stimulation. Activation of γ-secretase was observed with the activation of EP₄ receptors but not EP₂ receptors. PGE₂-dependent internalization of the EP₄ receptor was observed, and cells expressing a mutant EP₄ receptor lacking the internalization activity did not exhibit PGE₂-stimulated production of Aβ. A physical interaction between the EP₄ receptor and PS-1, a catalytic subunit of γ-secretases, was revealed by immunoprecipitation assays. PGE₂-induced internalization of PS-1 and co-localization of EP₄, PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP₄ receptor null mice. These results suggest that PGE₂-stimulated production of Aβ involves EP₄ receptor-mediated endocytosis of PS-1 followed by activation of the γ-secretase, as well as EP₂ receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.Alzheimer disease (AD)² is the most common neurodegenerative disorder of the central nervous system and the leading cause of adult onset dementia. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β (Aβ) peptides Aβ40 and Aβ42 (1, 2). To generate Aβ, β-amyloid precursor protein (APP) is first cleaved by β-secretase and then by γ-secretase. Cleavage of APP by α-secretase produces non-amyloidogenic peptides (3, 4). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5). Early onset familial AD is linked to three genes, APP, PS1, and PS2 (5, 6), strongly suggesting that γ-secretase-dependent production of Aβ is a key factor in the pathogenesis of AD. Therefore, cellular factors that affect the γ-secretase-dependent production of Aβ may be good targets for the development of drugs to prevent and treat AD.Both APP and PS-1 are transmembrane proteins, and their intracellular localization is controlled by secretory and endocytic pathways. These proteins are modified in the endoplasmic reticulum and trafficked to the cell surface through the trans-Golgi network (TGN). Then, they are internalized again and trafficked to early endosomes. Next, they are trafficked to late endosomes and lysosomes (LEL), which are recycling endosomes that are targeted to the cell surface or the TGN (7–11). The production of Aβ seems to occur in a broad range of cellular compartments including the cell surface, TGN, and endosomes (12). Abnormalities of secretory and endocytic pathways have been observed in the brains of AD patients (9, 13). Importantly, factors that control these vesicle transport systems affect the production of Aβ. For example, overproduction of Rab5, a factor essential for traffic of vesicles to early endosomes, has been shown to stimulate the production of Aβ (14), and SorL1 has been shown to reduce the production of Aβ by stimulating the traffic of APP in early endosomes to the TGN (15, 16).It has been suggested that inflammation is important in the pathogenesis of AD; chronic inflammation has been observed in the brains of AD patients, and trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (17–19). Cyclooxygenase (COX) is essential for the synthesis of prostaglandin E₂ (PGE₂), a potent inducer of inflammation and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively, whereas expression of COX-2 is induced under inflammatory conditions and is responsible for the progression of inflammation (20–22). The following evidences of the involvement of PGE₂ (and COX-2) in the progression of AD suggest that they are good targets for the development of AD drugs: (i) Elevated levels of PGE₂ and overexpression of COX-2 have been observed in the brains of AD patients (23–25); (ii) the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (26); (iii) transgenic mice constitutively overexpressing COX-2 show aging-dependent neural apoptosis and memory dysfunction (27); (iv) prolonged use of nonsteroidal anti-inflammatory drugs, inhibitors of COX, delays the onset and reduces the risk of AD (28); (v) PGE₂ stimulates the production of reactive oxygen species in microglia cells, resulting in activation of β-secretase (29).We recently reported that PGE₂ stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells that stably express a form of APP with two mutations (K651N/M652L) (APPsw) that elevate cellular and secreted levels of Aβ (30). Similar results were reported by another group (31). Using agonists and antagonists specific for each of the four PGE₂ receptors (EP₁, EP₂, EP₃, and EP₄), we found that EP₄ receptors alone and also both EP₂ and EP₄ receptors are involved in PGE₂-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively (30). Furthermore, experiments with transgenic mice suggest that EP₂ and EP₄ receptors are involved in the production of Aβ in vivo (30). Based on these results, we propose that antagonists of the EP₂ and/or EP₄ receptors may be therapeutically beneficial for the treatment of AD. Understanding the mechanism governing EP₂ and EP₄ receptor-mediated stimulation of production of Aβ by PGE₂ will be important for such drug development.Activation of EP₂ and EP₄ receptors causes activation of adenylate cyclase and an increase in the cellular level of cAMP (32). We have shown that an EP₄ receptor agonist or both EP₂ and EP₄ receptor agonists increase the cellular level of cAMP in HEK293 or SH-SY5Y cells, respectively, and that a cAMP analogue, 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP), increases the level of Aβ in HEK293 cells (30). These findings suggest that the cellular level of cAMP is important for PGE₂-stimulated production of Aβ. An increase in the cellular level of cAMP is known to activate protein kinase A (PKA), which is important for cAMP-regulated intracellular signal transduction (33). However, an inhibitor of PKA, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide (H-89), does not block PGE₂-stimulated production of Aβ in HEK293 cells (30). Other cAMP-regulated signal transduction factors, such as phosphatidylinositol 3-kinase and Epac (exchange protein directly activated by cAMP), were also shown not to be involved in PGE₂-stimulated production of Aβ in HEK293 cells (30). Thus, the mechanism whereby the activation of EP₂ and EP₄ receptors stimulates the production of Aβ has remained unknown. In this study, by using inhibitors of adenylate cyclase and PKA, we found that activation of the EP₂ receptor stimulates production of Aβ through activation of adenylate cyclase and PKA. We also propose that activation of the EP₄ receptor causes its co-internalization with PS-1 (γ-secretase) into endosomes and that this co-internalization is important for EP₄ receptor-mediated stimulation of Aβ production by PGE₂ through the activation of γ-secretase.     

Does the voltage dependent anion channel modulate cardiac ischemia-reperfusion injury?

The voltage dependent anion channel (VDAC) provides exchange of metabolites, anions, and cations across the outer mitochondrial membrane. VDAC provides substrates and adenine nucleotides necessary for electron transport and therefore plays a key role in regulating mitochondrial bioenergetics. VDAC has also been suggested to regulate the response to cell death signaling. Emerging data show that VDAC is regulated by protein-protein interactions as well as by post-translational modifications. This review will focus on the regulation of VDAC and its potential role in regulating cell death in cardiac ischemia-reperfusion. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Sudden cardiac death: what should we tell the family?

The sudden unexpected death of a child or a young person can have enormous implications for the surviving family members in addition to the immediate shock, sadness and sense of loss. The sudden loss of a sibling without any prior medical history may confront the family members with the question whether the sudden death was caused by a genetic condition and the fear that other family members may suffer the same fate.     

E2→E1 transition and Rb(+) release induced by Na(+) in the Na(+)/K(+)-ATPase. Vanadate as a tool to investigate the interaction between Rb(+) and E2

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).