bluuebunny/crossref_metadata_2025 huggingface dataset:
Myocardial ischemic contracture. Metabolites affect rigor tension development and stiffness.
Authors: Ventura-Clapier R , Veksler V
Published: June 2012

Myocardial ischemia is characterized by a decrease in phosphocreatine (PCr) and Mg(2+)-ATP contents as well as an accumulation of myosin ATPase reaction products (inorganic phosphate [P(i)], protons, and Mg(2+)-ADP). The possibility that these metabolites play a role in rigor tension development was checked in rat ventricular Triton X-100-skinned fibers. Rigor tension was induced by stepwise decreasing [Mg(2+)-ATP] in the presence or in the absence of 12 mmol/L PCr. To mimic the diastolic ionic environment of the myofibrils, [free Ca2+] was set at 100 nmol/L (pCa 7); [free Mg2+], at 1 mmol/L; and ionic strength, at 160 mmol/L. In control conditions (pH 7.1, with no added P(i) or Mg(2+)-ADP), the pMg(2+)-ATP for half-maximal rigor tension (pMg(2+)-ATP50) was 5.07 +/- 0.03 in the presence of PCr. After withdrawal of PCr, the pMg2+)-ATP50 value was shifted toward higher Mg(2+)-ATP values (3.57 +/- 0.03). Addition of 20 mmol/L P(i) shifted the pMg(2+)-ATP50 to 3.71 +/- 0.04 (P < .05) in the absence of PCr and in the opposite direction to 4.98 +/- 0.02 (P < .01) in the presence of PCr. Acidic pH (6.6) strongly increased pMg(2+)-ATP50 in both the absence (3.90 +/- 0.03, P < .001) and presence (5.44 +/- 0.02, P < .001) of PCr. Conversely, Mg(2+)-ADP (250 mumol/L) decreased pMg(2+)-ATP50 to 3.26 +/- 0.06 (P < .001) in the absence of PCr; at pMg(2+)-ATP 4, no rigor tension was observed until PCr concentration was decreased to < 2 mmol/L. At acidic pH, maximal rigor tension was lower by 29% compared with control conditions, whereas in the presence of Mg(2+)-ADP, maximal rigor tension developed to 143% of the control value; P(i) had no effect. The tension-to-stiffness (measured by the quick length-change technique) ratio was lower in rigor (no PCr and pMg(2+)-ATP 6) than during Ca2+ activation in the presence of both PCr and ATP. Compared with control rigor conditions, this parameter was unchanged by Mg(2+)-ADP and decreased by acidic pH, suggesting a proton-induced decrease in the amount of force per crossbridge. In addition to their known effects on active tension, Mg(2+)-ADP and protons affect rigor tension and influence ischemic contracture development. It is concluded that ischemic contracture and increased myocardial stiffness may be mediated by a decreased PCr and local Mg(2+)-ADP accumulation. This emphasizes the importance of myofibrillar creatine kinase activity in preventing ischemic contracture.
Contractile arrest increases sarcoplasmic reticulum calcium uptake and SERCA2 gene expression in cultured neonatal rat heart cells.
Authors: Bassani J W , Qi M , Samarel A M , Bers D M
Published: June 2012

We developed protocols with intact cultured neonatal rat myocytes to directly evaluate the function of the sarcoplasmic reticulum (SR) Ca-ATPase (or SERCA2), Na-Ca exchange (Na-CaX), and slow Ca transport systems (mitochondria and sarcolemmal Ca-ATPase). Spontaneously beating control cells were compared with cells cultured for 2 days in the presence of verapamil (verapamil-arrested cells, VA). Intracellular calcium (Cai) transients were measured by use of indo-1 during (1) spontaneous twitches, (2) contractures induced by rapid application of caffeine (CafC, with and without Nao), and (3) twitches induced by brief depolarizations with high [K]o solution (K-twitches). We also measured mRNA levels for the SR Ca-ATPase and Na-CaX in the same experimental preparations. The t1/2 for [Ca]i decline when both the SR Ca uptake and Na-CaX were prevented was the same for control and VA cells (approximately 20 seconds), indicating unaltered slow Ca transport systems. Similarly, there was no significant difference in the t1/2 of CafC when Na-CaX was the main mechanism responsible for [Ca]i decline (t1/2 approximately 1.5 seconds), indicating unaltered Na-CaX. Conversely, we found nearly a twofold increase in the rate of [Ca]i decline during K-twitches (control t1/2, 0.84 +/- 0.05 seconds; VA t1/2, 0.48 +/- 0.06 second; P < .001), indicating an increase in SR Ca-pumping activity in VA cells. This was also reflected by a 56% increase in the peak [Ca]i reached during CafC used to assess maximal SR Ca content (427 +/- 49 nmol/L in control versus 665 +/- 75 nmol/L in VA cells).(ABSTRACT TRUNCATED AT 250 WORDS)
The cAMP response element binding protein is expressed and phosphorylated in cardiac myocytes.
Authors: Goldspink P H , Russell B
Published: June 2012

Cardiac cells grow in response to a number of stimuli that activate intracellular signaling pathways. The cAMP-signaling pathway mediates the activation of gene transcription in other cell types by the cAMP response element binding protein (CREB-P). Our aim was to explore the physiological role of CREB-P in response to elevated cAMP in cardiac cells by determining if phosphorylation of CREB-P (to phosphoCREB-P) rapidly induces transcription in culture. Primary embryonic chick heart cultures were used in which cAMP was raised by forskolin (5 mumol/L) or isoproterenol (10 mumol/L) treatment. Since both these agents have inotropic effects, tension production was controlled with 2,3-butanedione monoxime (BDM). This allowed us to determine whether the cAMP-signaling pathway or the contractile state was regulating phosphorylation and transcription. The responses for time periods up to 2 hours were assayed with antibodies to detect phosphoCREB-P and by quantitative filter hybridization for creb gene expression. The staining intensity of the phosphoprotein increased in myocyte nuclei after 10 minutes and persisted for 1 hour with either forskolin or isoproterenol treatment. An increase in creb mRNA abundance was also detected, with the maximum level of expression being at 1 hour with forskolin treatment. These changes are independent of the contractile state, because BDM itself caused no change. BDM plus forskolin induced the same pattern of creb expression as observed with forskolin alone. Therefore, we conclude that elevation of cAMP leads to phosphorylation of CREB-P and an increase in creb mRNA abundance.
Transfer of human myocarditis into severe combined immunodeficiency mice.
Authors: Schwimmbeck P L , Badorff C , Schultheiss H P , Strauer B E
Published: June 2012

Severe combined immunodeficiency (SCID) mice possess neither T nor B lymphocytes and are thus suitable recipients for lymphocytes of different species. Because autoimmune mechanisms are suspected in the pathogenesis of myocarditis (MC), we attempted to determine whether peripheral blood lymphocytes (PBLs) from patients with MC could be transferred into SCID mice and whether they had an autoimmunologic effect. Groups of three mice each were injected intraperitoneally with up to 50 million PBLs from five MC patients with autoantibodies against the adenine nucleotide translocator (ANT), a myocardial autoantigen. The PBLs from three healthy blood donors were used as controls. After 60 days, human PBLs could be demonstrated in the peripheral blood of the SCID mice transfused with the PBLs of MC patients, representing up to 9.9% of the peripheral blood mononuclear cells. The transfused SCID mice sera showed human immunoglobulin levels of up to 3 mg/mL, both IgG and IgM. Autoantibodies against ANT were present in the mice receiving PBLs from MC patients but not from the control subjects. In addition, infiltrating human lymphocytes were present in the hearts of the SCID mice transfused with PBLs from MC patients. The presence of an ongoing autoimmune process in the SCID mice transfused with PBLs from MC patients is suggested by increased levels of soluble interleukin-2 receptor in the serum in contrast to SCID mice transfused with PBLs from healthy blood donors. We conclude that the autoimmune reactions seen in human MC can be transferred to SCID mice by the transfer of PBLs from MC patients. These findings stress the significance of autoimmune mechanisms in the pathogenesis of human MC.
Two independent sets of monoclonal antibodies define neoepitopes linked to soluble ligand binding and leukocyte adhesion functions of activated alpha M beta 2.
Authors: Elemer G S , Edgington T S
Published: June 2012

The integrin alpha M beta 2 mediates a variety of events, adhesive, phagocytic, and inflammatory. Evidence has suggested that the functional events may be mediated by the "activated" conformational forms of alpha M beta 2 produced by appropriate stimulation of myeloid and monocytic lineage. The activation of alpha M beta 2 may be associated with new epitopes on alpha M beta 2, sites that may be related to the acquired receptor functions. Monoclonal antibodies were produced that preferentially bind neoepitopes expressed by activated alpha M beta 2. These anti-neo antibodies each inhibited three activation-associated specific receptor alpha M beta 2 functions, though to different extents. One set of anti-neo antibodies inhibited in a concordant manner the binding of factor X and of fibrinogen by > 90%, abolished the alpha M beta 2-initiated cellular coagulant response, and inhibited monocyte adhesion to unstimulated endothelial monolayers. A second set of anti-neo antibodies only diminished factor X and fibrinogen binding by approximately 40% to 50% but markedly suppressed Xa generation and only partially inhibited monocyte adherence to unstimulated endothelium. Concordance was observed between binding of factor X or fibrinogen and competence for leukocyte adhesion to unstimulated endothelium. Antibody competition assays segregated the anti-neo antibodies into the same two distinct sets, consistent with recognition of separate neoepitopes that are linked to alpha M beta 2 function. These data support the conclusion that the activated conformer of alpha M beta 2 that binds fibrinogen and factor X also mediates monocyte-endothelial interactions as well as the alternative cellular coagulation pathway.
Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure. Marked upregulation of genes encoding extracellular matrix components.
Authors: Boluyt M O , O'Neill L , Meredith A L , Bing O H , Brooks W W , Conrad C H , Crow M T , Lakatta E G
Published: June 2012

The failing heart is characterized by impaired cardiac muscle function and increased interstitial fibrosis. Our purpose was to determine whether the functional impairment of the failing heart is associated with changes in levels of mRNA encoding proteins that modulate parameters of contraction and relaxation and whether the increased fibrosis observed in the failing heart is related to elevated expression of genes encoding extracellular matrix components. We studied hearts of 18- to 24-month-old spontaneously hypertensive rats with signs and symptoms of heart failure (SHR-F) or without evidence of failure (SHR-NF) and of age-matched normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, SHR-NF exhibited left ventricular (LV) hypertrophy (2.2-fold) and right ventricular (RV) hypertrophy (1.5-fold), whereas SHR-F were characterized by comparable LV hypertrophy (2.1-fold) and augmented RV hypertrophy (2.4-fold; all P < .01). Total RNA was isolated from ventricles and subjected to Northern blot analysis. In SHR-F hearts, the level of alpha-myosin heavy chain mRNA was decreased in both ventricles to 1/3 and 1/5 of the SHR-NF and WKY values, respectively (both P < .01). Levels of beta-myosin heavy chain, alpha-cardiac actin, and myosin light chain-2 mRNAs were not significantly altered in hearts of SHR-NF or SHR-F. Levels of alpha-skeletal actin were twofold greater in SHR-NF hearts compared with WKY hearts and were intermediate in SHR-F hearts. Levels of atrial natriuretic factor (ANF) mRNA were elevated threefold in the LV of SHR-NF (P < .05) but were not significantly increased in the RV of SHR-NF compared with WKY rats. During the transition to failure (SHR-F versus SHR-NF), ANF mRNA levels increased an additional 1.6-fold in the LV and were elevated 4.7-fold in the RV (both P < .05). Levels of sarcoplasmic reticulum Ca(2+)-ATPase (SRCA) mRNA were maintained in the LV of hypertensive and failing hearts at levels not significantly different from WKY values. In contrast, the level of RV SRCA mRNA was 24% less in SHR-NF compared with WKY rats, and during the transition to failure, this difference was not significantly exacerbated (29% less than the WKY value). The levels of fibronectin and pro-alpha 1(I) and pro-alpha 1(III) collagen mRNAs were not significantly elevated in either ventricle of the SHR-NF group but were fourfold to fivefold higher in both ventricles of SHR-F (all P < .05).(ABSTRACT TRUNCATED AT 400 WORDS)
Hypoxia stimulates prostacyclin synthesis in newborn pulmonary artery endothelium by increasing cyclooxygenase-1 protein.
Authors: North A J , Brannon T S , Wells L B , Campbell W B , Shaul P W
Published: June 2012

In newborn lambs, pulmonary prostacyclin (PGI2) production increases acutely in response to low oxygen. We tested the hypothesis that decreased oxygenation directly stimulates PGI2 synthesis in arterial segments and cultured endothelial cells from newborn lamb intrapulmonary arteries. In segments studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded prostaglandin E2 (PGE2) by 73%. Endothelium removal lowered PGI2 by 77% and PGE2 by 66%. At low oxygen tension (PO2, 40 mm Hg), PGI2 and PGE2 synthesis rose by 96% and 102%, respectively. Similarly, in endothelial cells studied at PO2 of 680 mm Hg, the synthesis of PGI2 exceeded PGE2 by 50%, and at low oxygen tension both PGI2 and PGE2 increased (89% and 64%, respectively). Endothelial cell PGI2 synthesis maximally stimulated by bradykinin, A23187, or arachidonic acid was also increased at low PO2 by 50%, 66%, and 48%, respectively. PGE2 synthesis was similarly altered, increasing by 33%, 37%, and 41%, respectively. In contrast, lowering oxygen had minimal effect on PGI2 and PGE2 synthesis with exogenous PGH2, which is the product of cyclooxygenase. Immunoblot analyses revealed that there was a 2.6-fold greater abundance of cyclooxygenase-1 protein at PO2 of 40 versus 680 mm Hg, and the increase at lower oxygen tension was inhibited by cycloheximide. The cyclooxygenase-2 isoform was not detected. Thus, attenuated oxygenation directly stimulates PGI2 and PGE2 synthesis in intrapulmonary arterial segments and endothelial cells from newborn lambs. This process is due to enhanced cyclooxygenase activity related to increased abundance of the cyclooxygenase-1 protein, and this effect may be due to increased synthesis of the enzyme protein.
Effects of oxidant exposure on substrate utilization and high-energy phosphates in isolated rat hearts.
Authors: Burton K P , Jones J G , Le T H , Sherry A D , Malloy C R
Published: June 2012

The effects of a xanthine oxidase-mediated free radical-generating system containing purine and iron-loaded transferrin or solutions containing hydrogen peroxide and iron-loaded transferrin on substrate utilization and high-energy phosphates were evaluated by nuclear magnetic resonance (NMR) spectroscopy in isolated perfused rat hearts. Hearts were supplied with lactate, acetate, and glucose, and the contribution of each substrate to acetyl coenzyme A was measured in control hearts and in the presence of a free radical-generating system. Perfused hearts were monitored by 31P NMR, and tissue extracts were analyzed by 13C NMR. Free radicals decreased the phosphocreatine and beta-ATP peak areas and reduced contractile function. Under control conditions, lactate, acetate, and endogenous sources were the major contributors of acetyl coenzyme A units, with only 5% originating from glucose. In the presence of a xanthine oxidase-mediated free radical-generating system, the glucose contribution increased to 54%, while contributions from acetate and endogenous sources were significantly reduced. Both 13C and 31P NMR analyses showed no significant accumulation of glycolytic sugar phosphates, suggesting little inhibition of glyceraldehyde-3-phosphate dehydrogenase. The increased contribution of glucose to the tricarboxylic acid cycle relative to acetate and endogenous sources is consistent with activation of pyruvate dehydrogenase. In contrast, hearts exposed to a hydrogen peroxide-based free radical-generating system showed an increase in lactate utilization, a decrease in acetate utilization, and no change in glucose utilization compared with control hearts. Glycolytic sugar phosphates were found to accumulate, suggesting possible inhibition of glyceraldehyde-3-phosphate. Thus, different radicals or their metabolites may have varying effects on myocardial metabolism.
Active cytomegalovirus infection of arterial smooth muscle cells in immunocompromised rats. A clue to herpesvirus-associated atherogenesis?
Authors: Persoons M C , Daemen M J , Bruning J H , Bruggeman C A
Published: June 2012

The susceptibility of medial and neointimal arterial smooth muscle cells (SMCs) to acute cytomegalovirus (CMV) infection was investigated in immunocompetent and immunocompromised rats. The left common carotid artery of all the rats was injured by balloon catheterization. On days 14 and 17 after injury, rats were either intravenously infected with a rat CMV (RCMV) or mock-infected. Active RCMV infection was shown in the neointima of the injured arteries of immunosuppressed rats, characterized by specific cytopathological changes in hematoxylin-eosin-stained sections and by the presence of early viral antigens and the presence of the virus at different stages of maturation at the electron-microscopic level. Viral genome was shown as well by use of in situ hybridization procedures. However, medial cells were hardly ever infected, and RCMV did not infect neointimal parts that were recovered by endothelial cells. No infection was seen in control right carotid arteries or in the injured arteries of immunocompetent rats. Acute intimal RCMV infection was accompanied by infiltration of inflammatory cells, predominantly mononuclear cells that stained positive with an ED-1 monoclonal antibody. The majority of infected neointimal cells were SMCs containing smooth muscle actin. No changes were found in medial or neointimal cross-sectional areas of the infected arteries. In the present study, an active RCMV infection of arterial SMCs was established in vivo, and it was concluded that neointimal SMCs are far more susceptible than are medial SMCs and that the absence of endothelium and immunosuppression are necessary conditions for arterial RCMV infection.
Differential regulation of acidic and basic fibroblast growth factor gene expression in fibroblast growth factor-treated rat aortic smooth muscle cells.
Authors: Alberts G F , Hsu D K , Peifley K A , Winkles J A
Published: June 2012

The acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) proteins are potent vascular smooth muscle cell (SMC) mitogens that are expressed by endothelial cells and SMCs in vivo. Overexpression of these proteins in transfected cell lines can result in autocrine transformation; therefore, the precise control of fibroblast growth factor gene expression in the vessel wall may be an important mechanism regulating vascular cell growth. In the present study, we demonstrate that bFGF can induce bFGF mRNA expression, but not aFGF mRNA expression, in serum-starved rat aortic SMCs. bFGF autoinduction is maximal at 4 hours, requires de novo RNA and protein synthesis, and is mediated predominantly by a protein kinase C-dependent signaling pathway. Furthermore, aFGF treatment of rat SMCs also increases bFGF mRNA and protein expression; however, aFGF mRNA levels are only slightly modulated. These results suggest that the local release of aFGF or bFGF within the vessel wall could promote a prolonged period of elevated bFGF synthesis. This, in turn, could be of importance in the SMC hyperplasia that occurs in response to vascular injury and during atherosclerotic plaque formation.
Endocytosis and uptake of lucifer yellow by cultured atrial myocytes and isolated intact atria from adult rats. Regulation and subcellular localization.
Authors: Page E , Goings G E , Upshaw-Earley J , Hanck D A
Published: June 2012

The time course of endocytic uptake of Lucifer yellow (LY) was followed by fluorescence and electron microscopy after exposure of primary cultures of atrial myocytes from adult rats to LY under conditions that prevented transplasmalemmal LY entry via channels or carriers. After a 2-minute exposure to LY at 37 degrees C, electron microscopy revealed classic clathrin-coated vesicles fused to endosomes, which were absent in LY-free medium or at 2 degrees C, suggesting that LY turns on endocytosis or accelerates a slow constitutive endocytosis. Fluorescence microscopy, which detected no LY entry at 2 minutes in LY, showed punctate cytoplasmic fluorescent densities after 10 minutes, which were readily distinguishable from intrinsic perinuclear fluorescence. Fluorescence microscopy after immunostaining with antibodies against clathrin, vacuolar H(+)-ATPase, atrial peptide, or a marker for acidified compartments suggested LY sorting into an acidified prelysosomal pathway. Using absence of punctate fluorescence after 10 minutes in LY as a criterion for inhibition of endocytosis, we showed that endocytosis was inhibited by inhibitors of protein phosphatases 1 and 2A or inhibitors of cAMP-dependent protein kinases 1 and 2, by effects of caffeine on sarcoplasmic reticulum Ca2+ release, and by temperatures below 18 degrees C, but not by staurosporine, phorbol esters, pertussis toxin, thapsigargin, preventing contractions with nifedipine, ryanodine and low [Ca2+]o, or raising cytosolic cAMP concentrations. Both phosphatase inhibitors and caffeine also inhibited a fraction of LY uptake by intact rat atria. We conclude that endocytic uptake of LY is an energy-dependent, specifically regulated process, whose understanding and control are potentially important for the medically relevant problem of introducing drugs and macromolecules into atrial heart muscle cells.
Heterogeneity of red blood cell perfusion in capillary networks supplied by a single arteriole in resting skeletal muscle.
Authors: Ellis C G , Wrigley S M , Groom A C
Published: June 2012

Flow heterogeneity within capillary beds may have two sources: (1) unequal distribution of red blood cell (RBC) supply among arterioles and (2) unique properties of RBC flow in branching networks of capillaries. Our aim was to investigate the capillary network as a source of both spatial and temporal heterogeneity of RBC flow. Five networks, each supplied by a single arteriole, were studied in frog sartorius muscle (one network per frog) by intravital video microscopy. Simultaneous data on RBC velocity (millimeters per second), lineal density (RBCs per millimeter), and supply rate (RBCs per second) were measured continuously (10 samples per second) from video recordings in 5 to 10 capillary segments per network for 10 minutes by use of automated computer analysis. To quantify heterogeneity, mean values from successive 10-second intervals were tabulated for each flow parameter in each capillary segment (ie, portion of capillary between successive bifurcations), and percent coefficient of variation (SD/mean.100%) was calculated for (1) spatial heterogeneity among vessels (CVs) every 10 seconds and for the entire 10-minute sample and (2) temporal heterogeneity within vessels for every capillary segment and for the mean flow parameter. Analysis of these data indicates that (1) capillary networks are a significant source of both spatial and temporal flow heterogeneity, and (2) continuous redistributions of flow occur within networks, resulting in substantial temporal changes in CVs, although a persistent spatial heterogeneity of perfusion still exists on a 10-minute basis. In most networks, CVs decreased as supply rate within the network increased, thus indicating that rheology plays a significant role in determining the perfusion heterogeneity.
Gene expression of the cardiac Na(+)-Ca2+ exchanger in end-stage human heart failure.
Authors: Studer R , Reinecke H , Bilger J , Eschenhagen T , Böhm M , Hasenfuss G , Just H , Holtz J , Drexler H
Published: June 2012

The regulation of cytosolic Ca2+ concentration during excitation-contraction coupling is altered in the failing human heart. Previous studies have focused on disturbances in Ca2+ release and reuptake from the sarcoplasmic reticulum (SR), whereas functional studies of the cardiac Na(+)-Ca2+ exchanger, another important determinant of myocyte homeostasis, are lacking for the failing human heart. Using a cardiac Na(+)-Ca2+ exchanger cDNA recently cloned from a guinea pig cDNA library, we investigated the gene expression of the cardiac Na(+)-Ca2+ exchanger in relation to the SR Ca(2+)-ATPase. Expression of both genes was quantified in left ventricular myocardium from 24 failing human cardiac explants and 7 control heart samples in relation to beta-myosin heavy chain mRNA by slot blot analysis. Compared with patients with nonfailing hearts, patients with dilated cardiomyopathy (DCM, n = 13) showed a 55% increase in Na(+)-Ca2+ exchanger mRNA levels (P < .05 versus control value) and a 41% increase in patients with coronary artery disease (CAD, n = 11). In the same hearts, SR Ca(2+)-ATPase mRNA levels were decreased by 50% in DCM and by 45% in CAD (P < .05 for both versus control value). There was a positive correlation between Na(+)-Ca2+ exchanger and SR Ca(2+)-ATPase mRNA levels both in normal and failing human hearts, albeit with different slopes and intercepts of the regression line. The Na(+)-Ca2+ exchanger protein levels as assessed by Western blot analysis and normalized to beta-myosin heavy chain protein were increased in DCM and CAD (P < .05 and P < .01 versus control value, respectively), whereas SR Ca(2+)-ATPase protein levels were reduced (P < .05 for both groups versus control values). Thus, the Na(+)-Ca2+ exchanger gene expression is enhanced in failing human hearts and may, in part, compensate for the depressed SR function with regard to diastolic Ca2+ removal.
Discrepancy between plasma and lung angiotensin-converting enzyme activity in experimental congestive heart failure. A novel aspect of endothelium dysfunction.
Authors: Huang H , Arnal J F , Llorens-Cortes C , Challah M , Alhenc-Gelas F , Corvol P , Michel J B
Published: June 2012

The renin-angiotensin and cardiac natriuretic systems play an important role in the pathophysiology of congestive heart failure (CHF). The status of the membrane-bound pulmonary and renal activities of three ectoenzymes involved in the regulation of these systems-angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and aminopeptidase A (APA)-was investigated in Wistar rats 3 months after induction of myocardial infarction (MI) and in sham-operated (control) rats. Plasma renin activity and ACE activity, plasma angiotensin II (Ang II) levels, and atrial natriuretic factor levels were simultaneously determined. The lung ACE activity was decreased in MI rats compared with control rats (P < .0001), and this decrease depended on the severity of the heart failure. In contrast, plasma ACE activity was increased in MI rats (P < .01), and this increase was also proportional to the severity of MI. Northern blot analysis showed that the lung ACE mRNA level in severe MI rats was half that of the control rats. Renal ACE activity of the MI rats was not affected, and neither renal or pulmonary NEP nor pulmonary APA activities were altered. Thus, lung ACE gene expression appears to be both organ- and enzyme-specifically regulated during CHF. Whereas plasma renin was increased in heart failure rats, plasma Ang II levels were not different from those of control rats. Thus, decreased lung ACE activity could possibly contribute to keeping plasma Ang II levels in the normal range. The decrease in lung ACE activity and mRNA levels, combined with increased plasma ACE activity, represents a novel aspect of endothelial dysfunction in CHF.(ABSTRACT TRUNCATED AT 250 WORDS)
Selective dye and ionic permeability of gap junction channels formed by connexin45.
Authors: Veenstra R D , Wang H Z , Beyer E C , Brink P R
Published: June 2012

Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the gating of an aqueous pore permeable to ions and molecules of up to 1 kD or 8 to 14 A in diameter. We performed ion-substitution and dye-transfer experiments to determine the relative Cl-/K+ conductance and dye permeability of anionic fluorescein derivatives in chick connexin45 (Cx45) channels. We demonstrate that Cx45 forms a 26 +/- 6-picosiemen (pS) channel with a maximum detectable Cl- permeability of 0.2 relative to K+ or Cs+. Although homogeneous channel conductances were observed in multichannel recordings, the open probability estimates were indicative of nonhomogeneous gating behavior and occasional cooperativity. A second conductance state of 19 +/- 4 pS begins to predominate at higher voltages. Cx45 gap junctions are permeable to 2',7'-dichlorofluorescein but are not permeable to the more polar 6-carboxyfluorescein dye. These observations suggest that the Cx45 pore diameter is approximately 10 A and is associated with a fixed negative charge within the junctional channel.
Voltage-independent effects of extracellular K+ on the Na+ current and phase 0 of the action potential in isolated cardiac myocytes.
Authors: Whalley D W , Wendt D J , Starmer C F , Rudy Y , Grant A O
Published: June 2012

A rise in [K+]o, by depolarizing the resting membrane potential and partially inactivating the inward Na+ current (INa), is believed to play a critical role in slowing conduction during myocardial ischemia. In multicellular ventricular preparations, elevation of [K+]o has been suggested to decrease Vmax to a greater extent than expected from membrane depolarization alone. The mechanism of this voltage-independent effect of [K+]o is currently unknown, and its significance in single cardiac cells has not been determined. We have examined the voltage-independent effects of elevated [K+]o on INa and the action potential upstroke in isolated rabbit atrial and ventricular myocytes under voltage- and current-clamp conditions. Superfusate [K+] was varied from 5 mmol/L to 14 or 24 mmol/L, whereas [Na+] was maintained at 150 mmol/L. In cultured atrial cells and excised outside-out patches from freshly isolated atrial and ventricular cells, the amplitude and kinetics of INa were unchanged by elevation of [K+]o. In atrial cells, action potentials elicited from a holding potential of -70 mV had a similar Vmax (114.9 +/- 5.7 versus 112.2 +/- 4.8 V/s, mean +/- SEM, n = 6) and action potential amplitude (115.0 +/- 2.4 versus 113.4 +/- 3.9 mV) in 5 and 24 mmol/L [K+]o. In contrast, in ventricular cells at a holding potential of -70 mV, increasing [K+]o fro 5 to 14 mmol/L decreased Vmax from 161.8 +/- 18.0 to 55.3 +/- 5.0 V/s (n = 7, P < .001) and action potential amplitude from 128.1 +/- 1.3 to 86.6 +/- 5.4 mV (P < .001). This voltage-independent decrease in Vmax and action potential amplitude induced by elevated [K+]o was abolished in the presence of 1 mmol/L Ba2+, suggesting that it is attributable to an increased background K+ conductance. We conclude that elevation of [K+]o to levels expected during ischemia causes a marked voltage-independent depression of Vmax in ventricular cells, which may, in turn, contribute to the slowing of myocardial conduction characteristic of early ischemia.
Developmental and tissue-specific regulation of rabbit skeletal and cardiac muscle calcium channels involved in excitation-contraction coupling.
Authors: Brillantes A M , Bezprozvannaya S , Marks A R
Published: June 2012

Two types of calcium channels signal excitation-contraction (E-C) coupling in striated muscle: dihydropyridine receptors (DHPRs, voltage-gated L-type calcium channels on the transverse tubule) and ryanodine receptors (RyRs, calcium release channels on the sarcoplasmic reticulum). Sarcolemmal depolarization activates the DHPR; subsequently, the RyR is activated and releases calcium that activates muscle contraction. We show in the present study that expression of the E-C coupling calcium channels is upregulated during myogenic development in the rabbit. Skeletal and cardiac muscle isoforms of the following genes were examined: the DHPR alpha 1, alpha 2, beta, and gamma subunits and the RyR. Distinct cardiac and skeletal muscle-specific cDNAs were isolated, encoding each of the DHPR subunits and the RyR. The skeletal muscle DHPR alpha 1, alpha 2, beta, and gamma subunits and the cardiac DHPR alpha 1 subunit mRNA levels increased on the day of birth and at the adult stage compared with fetal levels. The skeletal and cardiac RyR mRNA levels increased on the day of birth and at adult stages compared with fetal levels. Ryanodine binding sites increased in both skeletal and cardiac muscle. We now provide a molecular explanation for the physiological "maturation" of the E-C coupling apparatus observed at the day of birth and during early postnatal development in both skeletal and cardiac muscles. Low levels of calcium channel expression in fetal cardiac and skeletal muscle make these tissues more sensitive to pharmacological therapy with calcium channel blockers, a phenomenon that has been reported in human neonates.
Voltage dependence of beta-adrenergic modulation of conduction in the canine Purkinje fiber.
Authors: Munger T M , Johnson S B , Packer D L
Published: June 2012

Although recent voltage-clamp and microelectrode studies have demonstrated beta-adrenergic modulation of Na+ current (INa) the modulation of conduction by catecholamines and the voltage dependence of that process have not been elucidated. To determine whether voltage-dependent modulation of conduction occurs in the presence of a beta-adrenergic agonist, the effect of 1 mumol/L isoproterenol on impulse propagation in canine Purkinje fibers was examined by using a dual-microelectrode technique. At physiological membrane potentials ([K+]o 5.4 mmol/L), isoproterenol increased squared conduction velocity [theta 2, 0.39 +/- 0.25 (m/s)2 (mean +/- SD)] from 3.46 +/- 0.86 to 3.85 +/- 0.98 (m/s)2 (P < .011), an 11% change, without altering the maximum first derivative of the upslope of phase 0 of the action potential (Vmax, 641 +/- 50 versus 657 +/- 47 V/s, P = NS). At transmembrane potential of -65 mV, produced by 12 mmol/L [K+]o titration, theta 2 declined 79% to 0.73 +/- 0.44 (m/s)2 as Vmax decreased 85% to 95 +/- 43 V/s (P < .02). The addition of isoproterenol further decreased theta 2 to 0.49 +/- 0.33 (m/s)2 (P = .02) in parallel with a further decline in Vmax to 51 +/- 25 V/s (P < .05). Isoproterenol produced a 3-mV hyperpolarizing shift of apparent Na+ channel availability curves generated from both theta 2 and Vmax, used as indexes of the fast inward INa, without changing the slopes of the relation. The relation between normalized theta 2 and Vmax over a range of depolarized potentials was linear and was not appreciably altered by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)
Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat.
Authors: Bendeck M P , Zempo N , Clowes A W , Galardy R E , Reidy M A
Published: June 2012

We have characterized matrix metalloproteinase expression in the rat carotid artery after two forms of arterial injury, balloon catheter denudation and nylon filament denudation. Gelatinolytic enzymes with molecular masses of 70 and 62 kD were produced constitutively in the rat carotid. Production of an 88-kD gelatinase was induced after balloon catheter injury, and proteinase production continued during the period of migration of smooth muscle cells from the media to the intima, from 6 hours to 6 days after balloon catheter injury. In addition, a marked increase in 62-kD gelatinolytic activity was observed between 4 and 14 days after arterial injury. Gelatinase activities (88 and 62 kD) were also increased after nylon filament denudation but were markedly less after this injury than after balloon catheter injury. These results suggested a correlation between gelatinase activity and smooth muscle cell migration after arterial injury. Administration of a metalloproteinase inhibitor after balloon catheter injury resulted in a 97% reduction in the number of smooth muscle cells migrating into the intima. Therefore, we hypothesize that gelatinase expression directly facilitates smooth muscle cell migration within the media and into the intima. These results suggest that gelatinases are involved in the vascular smooth muscle cell activation and neointimal formation that characterize arterial tissue remodeling after injury.
Protein kinase C. Its role in ischemic preconditioning in the rat.
Authors: Speechly-Dick M E , Mocanu M M , Yellon D M
Published: June 2012

The present study investigated whether protein kinase C (PKC) plays a role in ischemic preconditioning in the rat heart. Chelerythrine, a specific antagonist of PKC, and 1,2-dioctanoyl-sn-glycerol (DOG), a diacylglycerol analogue and specific antagonist of PKC, were used to determine whether preconditioning could be blocked or triggered, respectively. Sprague-Dawley rats were anesthetized and instrumented for coronary occlusion and reperfusion. All animals were subjected to 45 minutes of regional ischemia (ISC) followed by 2.5 hours of reperfusion. The preconditioning protocol consisted of 5 minutes of ischemia and then 10 minutes of reperfusion. There were six groups: (1) control (group C, n = 5), (2) preconditioned and ISC (group PC, n = 6), (3) chelerythrine given 2 minutes before ISC (group CC, n = 5), (4) preconditioned and chelerythrine given 2 minutes before ISC (group PCC, n = 6), (5) DOG (dissolved in dimethylsulfoxide [DMSO]) given 10 minutes before ISC (group CD, n = 5), and (6) DMSO given 10 minutes before ISC (group DMSO, n = 3). The end point was infarct size measured using triphenyl tetrazolium chloride and expressed as a percentage of the volume at risk (I/R), measured with fluorescent particles. I/R was significantly reduced by preconditioning (group C, 58.6 +/- 5.0%; group PC, 32.7 +/- 6.3%; P < .01) and by the PKC agonist DOG, which reduced I/R to a similar extent as preconditioning (group C, 58.6 +/- 5.0%; group CD, 28.0 +/- 7.0%; P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)
Hemodynamics and wall shear rate in the abdominal aorta of dogs. Effects of vasoactive agents.
Authors: White K C , Kavanaugh J F , Wang D M , Tarbell J M
Published: June 2012

Vasoactive drugs are known to affect impedance (pressure/flow) and vessel wall motion in arteries. The nonlinear theory of oscillatory flow in straight elastic vessels indicates that wall shear rate is affected by changes in impedance phase angle and wall motion. To test whether wall shear rate depends on impedance phase angle and wall motion in vivo, wall shear rate was measured in the abdominal aorta of anesthetized dogs by using a flush-mounted hot-film anemometer, and the hemodynamic state was characterized by pressure, flow, and vessel dimension measurements. Vasodilators (nitroprusside and isoproterenol) and vasoconstrictors (angiotensin II and norepinephrine) were administered acutely, and the responses of wall shear rate and hemodynamics were determined. In the control state (no drugs), peak wall shear rate was 1835 +/- 153 s-1 (mean +/- SEM). The vasodilators induced large increases in impedance phase angle and wall motion concomitant with large increases in peak wall shear rate (62.4 +/- 20.4% for nitroprusside and 68.9 +/- 28.3% for isoproterenol), which were not predicted accurately by Womersley's theory of oscillatory flow in a rigid vessel or the nonlinear theory of oscillatory flow in an elastic vessel, with measured flow and vessel dimension used as inputs. The vasoconstrictors induced small decreases in impedance phase angle and wall motion and small changes in peak wall shear rate (increase, 30.5 +/- 8.0% for norepinephrine; decrease, 18.2 +/- 7.1% for angiotensin II), which were predicted accurately by Womersley's theory. The present study shows that vasoactive drugs, particularly vasodilators, can have significant effects on wall shear rate (stress) in the abdominal aorta that appear to be related to changes in impedance phase angle and vessel wall motion. However, the effects on wall shear rate are not predicted accurately by straight-tube theory.
Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.
Authors: Graier W F , Myers P R , Rubin L J , Adams H R , Parker J L
Published: June 2012

Altered release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) has been proposed as a final common pathway underlying the abnormal vasodilator responses to gram-negative lipopolysaccharide (endotoxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarified. We evaluated direct effects of Escherichia coli endotoxin on agonist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cultured bovine and porcine aortic endothelial cells (ECs). Two methods were used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct quantitative measurement of NO release (chemiluminescence method). Cytosolic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence methodology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 microgram/mL, 1 hour plus 1-hour wash) significantly inhibited bradykinin (100 nmol/L)- and ADP (10 mumol/L)-mediated increases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase in cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mumol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation with endotoxin inhibited ADP-stimulated increases in NO release and EDRF bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivity. Endotoxin produced significant decreases in both transient and sustained [Ca2+]i responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+]i in response to bradykinin was reduced to 31% of the initial increases in control cells, and the secondary plateau phase was reduced to only 3% of respective control responses. Concentration-response relation to endotoxin (10(-3)) to 10(0) micrograms/mL) indicated high correlation and similar IC50 values (0.025 and 0.021 micrograms/mL, respectively) for inhibitory effects on cGMP and [Ca2+]i. Endotoxin had no effect on inositol trisphosphate formation ([3H]myo-inositol incorporation) and intracellular Ca2+ release ([Ca2+]i responses in Ca(2+)-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was significantly attenuated by endotoxin treatment. These studies demonstrate that endotoxin directly decreases agonist (bradykinin and ADP)-mediated biosynthesis and release of EDRF/NO from ECs. These effects can be explained by altered [Ca2+]i mobilization mechanisms, which in turn produce subsequent decreases in activity of the Ca(2+)-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of agonist-mediated NO release and endothelium-dependent vasodilation.
Mechanisms for vagal modulation of ventricular repolarization and of coronary occlusion-induced lethal arrhythmias in cats.
Authors: Rosenshtraukh L , Danilo P , Anyukhovsky E P , Steinberg S F , Rybin V , Brittain-Valenti K , Molina-Viamonte V , Rosen M R
Published: June 2012

Our goal was to better understand the mechanisms underlying muscarinic receptor actions on the ventricle in vivo. Therefore, we studied the effects of vagal stimulation on ventricular repolarization and of vagal tone on lethal arrhythmias induced by 30 minutes of left anterior descending coronary artery ligation in anesthetized cats. Experimental groups included normal control cats subjected only to coronary ligation and cats pretreated with atropine, pertussis toxin (PTX), or propranolol. All cats received bilateral cervical vagal stimulation (Vstim) at 1, 3, and 5 Hz for 1 minute at 10-minute intervals. Before coronary ligation, Vstim slowed sinus rate, prolonged the PR interval, and lowered blood pressure. Most important from the point of view of electrophysiological function was a vagally induced acceleration of ventricular repolarization in paced and unpaced hearts, which could be explained by the effects of acetylcholine (ie, shortening the subepicardial muscle action potentials). The effect on repolarization was blocked by atropine or PTX but not by propranolol. The extent of sinus slowing and acceleration of repolarization was directly related to the level of functional PTX-sensitive G protein (P < .05). Coronary occlusion was performed during atrial pacing such that the heart rate in all groups was equal. The incidence of ventricular fibrillation (VF) was 10% in the control group and 50% and 54% in atropine and PTX groups, respectively (P < .05). During atrial pacing before coronary occlusion, a vagal index was calculated as percent QTc shortening during Vstim. When the vagal index was 13% to 26%, the incidence of VF during occlusion was zero. When the vagal index was 0% to 12%, VF was 52% (P < .01). Conclusions are as follows: (1) Vstim accelerates ventricular repolarization in cats via a pathway that incorporates a PTX-sensitive G protein and involves an altered gradient between epicardium and endocardium. (2) Removal of vagal tone during ischemia favors VF, as predicted by a vagal index.
Alteration of the cytosolic-mitochondrial distribution of high-energy phosphates during global myocardial ischemia may contribute to early contractile failure.
Authors: Rauch U , Schulze K , Witzenbichler B , Schultheiss H P
Published: June 2012

Depending on its duration, temporary myocardial ischemia leads to a disturbance of myocardial function before irreversible cellular necrosis is developed. Mechanical, electrical, and metabolic disturbances were suggested to be possible mechanisms accounting for the altered mechanical performance in ischemic hearts. To further investigate the alteration of myocardial energy metabolism on the subcellular level, we determined, by means of nonaqueous fractionation, the cytosolic-mitochondrial distribution of high-energy phosphates and other metabolites (ATP, ADP, phosphocreatine, creatine, and inorganic phosphate) in ischemic (zero-flow) guinea pig hearts after isolated perfused working heart preparation. Additional experiments using 31P nuclear magnetic resonance spectroscopy were performed to determine pHi and [Mg2+]i changes during global ischemia. The total ATP content of myocardial tissue dropped only slowly to 76% of control ATP at 10 minutes and to 51% at 30 minutes and reached almost zero at 60 minutes of ischemia. However, striking differences were observed on the subcellular level: While cytosolic phosphocreatine was almost completely consumed after 3 minutes of ischemia (from 19.1 +/- 1.6 to 3.3 +/- 0.5 mmol/L), ATP concentration in the cytosol decreased within 30 minutes from 8.4 +/- 0.6 to only 5.4 +/- 0.9 mmol/L. Mitochondrial ATP was rapidly and linearly reduced to 60% after 5 minutes of ischemia and was nearly unmeasurable after a further 20 minutes. Thus, in contrast to the breakdown of phosphocreatine in cytosol, the only slight alteration of cytosolic ATP reveals a reduction in cytosolic ATP utilization. Moreover, the unaffected cytosolic-mitochondrial difference in the phosphorylation potential of ATP demonstrates the intact function of the ADP/ATP carrier during early ischemia. These results might indicate a disturbance of the functional coupling between carrier and phosphocreatine kinase (phosphocreatine shuttle), which could be of importance for the early contractile failure in myocardial ischemia.