bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelium-dependent contractions in rabbit pulmonary artery are mediated by thromboxane A2.
Authors: Buzzard C J , Pfister S L , Campbell W B
Published: June 2012

This study was designed to characterize the endothelium-dependent contracting factor (EDCF) released by arachidonic acid (AA) and methacholine (MeCH) in the rabbit pulmonary artery. AA and MeCH contract the rabbit pulmonary artery; however, the effects of both are blocked by denuding the vessels and by administration of indomethacin (a cyclooxygenase inhibitor), dazoxiben (a thromboxane [TX] synthase inhibitor), and SQ29548 (a TXA2/prostaglandin [PG] H2 receptor antagonist). When segments of rabbit pulmonary artery were incubated with [14C]AA and the [14C] metabolites were resolved by reverse-phase high-performance liquid chromatography (HPLC), radioactive products were observed that comigrated with 6-keto-PGF1 alpha and TXB2, the stable metabolites of prostacyclin and TXA2. The TXB2 radioactive peak was rechromatographed on normal-phase HPLC and again migrated with TXB2. Finally, the structures of derivatized [14C]6-keto-PGF1 alpha and [14C]TXB2 peaks were confirmed by gas chromatography/mass spectrometry. The synthesis of [14C]6-keto-PGF1 alpha and [14C]TXB2 was inhibited by removal of the endothelium and by indomethacin. Dazoxiben inhibited the synthesis of [14C]TXB2 but not [14C]6-keto-PGF1 alpha. Using specific radioimmunoassays, AA and MeCH stimulated 6-keto-PGF1 alpha and TXB2 release. Indomethacin blocked the production of both 6-keto-PGF1 alpha and TXB2, whereas dazoxiben only blocked TXB2. In a superfusion/bioassay system, AA stimulated an endothelium-intact donor vessel to release a labile substance that contracted an indomethacin-treated endothelium-denuded recipient vessel. The EDCF released by AA had an approximate half-life of 30 seconds. Cultured rabbit pulmonary arterial endothelial cells synthesized 6-keto-PGF1 alpha but not TXB2. Immunohistochemical studies indicated the presence of cyclooxygenase, but not TX synthase, in pulmonary artery endothelial cells. TXA2 appears to be the EDCF released by AA and MeCH in rabbit pulmonary artery; however, TXA2 is not produced by endothelial cells but may arise from cells that adhere to the luminal surfaces, such as platelets or macrophages.
Endocardial versus epicardial differences of intracellular free calcium under normal and ischemic conditions in perfused rat hearts.
Authors: Figueredo V M , Brandes R , Weiner M W , Massie B M , Camacho S A
Published: June 2012

Transmural heterogeneity of myocardial metabolism and function are present in the left ventricle under normal and ischemic conditions. To determine if endocardial versus epicardial differences of [Ca2+]i are also present, perfused rat heart studies using indo-1 fluorescence as an index of [Ca2+]i were performed in the left ventricular epicardium and endocardium. Hearts were studied during control conditions and low-flow ischemia. Results demonstrated the following: 1) At a pacing rate of 1.5 Hz, endocardial levels of diastolic and systolic [Ca2+]i (470 +/- 40 and 1,240 +/- 170 nM) were higher than epicardial levels (290 +/- 30 and 920 +/- 150 nM). 2) At a more physiological pacing rate of 5 Hz, endocardial levels of diastolic and systolic [Ca2+]i (680 +/- 50 and 1,230 +/- 70 nM) were also higher than epicardial levels (390 +/- 20 and 950 +/- 60 nM. 3) During low-flow ischemia, endocardial levels of diastolic [Ca2+]i rose to a greater degree (from 680 +/- 50 to 1,050 +/- 70 nM at 10% of control coronary flow) compared with epicardial levels (from 390 +/- 20 to 580 +/- 40 nM at 10% of control flow), suggesting that the endocardium is more susceptible to low-flow ischemia. 4) The amplitude of the [Ca2+]i transient was the same at the endocardium (540 +/- 50 nM) and epicardium (560 +/- 50 nM) and did not change during low-flow ischemia, despite marked contractile dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)
Increased capillary permeability to albumin in diabetic rat myocardium.
Authors: Yamaji T , Fukuhara T , Kinoshita M
Published: June 2012

To clarify the mechanism for the well-known increase in microvascular permeability that occurs with diabetes mellitus, we investigated capillary permeability to albumin in diabetic rat myocardium by electron microscopy using albumin-gold (Alb-Au) complexes as a tracer. Diabetes was induced by an intravenous injection of streptozotocin. After 24-32 weeks, hearts from diabetic rats and age-matched control rats were perfused with Krebs-Henseleit bicarbonate buffer containing Alb-Au for 5 or 20 minutes and then fixed and processed for electron microscopy. The binding and transport of Alb-Au by capillary endothelium was quantitatively evaluated. In control rats, Alb-Au particles were found preferentially bound to the luminal plasmalemmal vesicles. In diabetic rats, the labeling of luminal vesicles was more extensive and more pronounced after 5 minutes of perfusion when compared with control vesicles. The plasma membrane proper was also heavily labeled in diabetic rats. After 20 minutes, Alb-Au particles were transported across the capillary endothelium via plasmalemmal vesicles, but they did not penetrate the intercellular junctions in either control or diabetic rats. The vesicular transport of Alb-Au across the capillary endothelium was significantly increased in the diabetic myocardium when compared with control myocardium (percentage of abluminal labeled vesicles, 25.9 +/- 5.5% versus 1.3 +/- 0.5%; p < 0.01). The study on food-restricted rats with body weights close to those of diabetic rats suggested that caloric deficiency alone did not have much effect on capillary permeability. The data indicate that capillary permeability to albumin is markedly increased in diabetic myocardium because of enhanced vesicular transport. This may play an important role in the pathogenesis of diabetic cardiomyopathy.
Phosphatidylinositol metabolism in hypertrophic rat heart.
Authors: Kawaguchi H , Sano H , Iizuka K , Okada H , Kudo T , Kageyama K , Muramoto S , Murakami T , Okamoto H , Mochizuki N
Published: June 2012

The accumulation of inositol 1,4,5-trisphosphate (IP3) after hormonal stimulation has a physiological role, possibly by alteration of Ca2+ levels in cardiac myocyte. However, this accumulation has not been studied under pathophysiological conditions. In this report, we examine phosphatidylinositol metabolism during cellular response to norepinephrine in pressure-overloaded hypertrophic rat heart. After stimulation with norepinephrine, the accumulations of IP3 and diacylglyceride significantly increased in isolated myocytes from stroke-prone spontaneously hypertensive rat (SHRSP) heart, indicating phosphatidylinositol-specific phospholipase C activity increased in SHRSP heart cells. Protein kinase C activity was also enhanced in SHRSP, with a marked increase in particulate activity. We determined the intracellular calcium concentration and found it to be higher in SHRSP than in Wistar-Kyoto (WKY) rats at 30-40 weeks of age. Ca2+ influx was also elevated in SHRSP stimulated by norepinephrine. In SHRSP heart, cytosolic Ca2+ concentration may rise quickly in response to some stimuli, such as alpha 1-adrenergic stimulation, which is shown to be one of the pathways that increases cytosolic Ca2+ levels in hypertrophied rat heart. These data suggest that a part of the phosphatidylinositol-turnover pathway, such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway, may play an important role in the development of hypertrophy in SHRSP heart.
Mechanosensitive gating of atrial ATP-sensitive potassium channels.
Authors: Van Wagoner D R
Published: June 2012

Cell-attached and inside-out excised-patch recording techniques were used to search for mechanosensitive ion channels in neonatal and adult rat atrial myocytes. A channel activated by negative pressure applied to the patch, with a single-channel conductance of 52 pS in symmetric potassium solutions, was frequently observed. This channel has been identified as the atrial ATP-sensitive potassium (KATP) channel on the basis of its potassium selectivity, as well as its inhibition by ATP or tolbutamide in the inside-out excised patch. Mechanosensitive modulation of the KATP channel has not previously been reported. In the presence of 1 mM ATP, 10-50 microM pinacidil (a specific KATP channel agonist) does not significantly increase basal KATP channel activity; however, these concentrations of pinacidil potentiated the mechanosensitive modulation of the KATP channel. A hypotonic swelling protocol (a mechanical stimulus) was used in an effort to determine whether mechanosensitive modulation of this channel can generate significant whole-cell currents. Under perforated-patch whole-cell recording conditions, superfusion of atrial myocytes with a 240 mosm/kg solution (control solution, 290 mosm/kg) stimulated whole-cell currents with a magnitude similar to those activated by 10 microM pinacidil. These results demonstrate that the gating of the atrial KATP channel is mechanosensitive and suggest that mechanosensitive modulation may be an additional and significant mechanism, modulating channel activity under both physiological and pathological conditions.
Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts.
Authors: Schorb W , Booz G W , Dostal D E , Conrad K M , Chang K C , Baker K M
Published: June 2012

Angiotensin II has been reported to be a hormonal stimulus of cardiac growth, a response that may involve myocyte hypertrophy as well as growth of nonmyocytes. This study was designed to determine whether neonatal rat cardiac fibroblasts have an angiotensin II receptor that is coupled with hypertrophic and/or proliferative growth. Competitive radioligand binding studies showed that cardiac fibroblasts have a single class of high-affinity (IC50, 1.0 nM) angiotensin II binding sites (Bmax, 778 fmol/mg protein) that are sensitive to the competitive nonpeptide AT1 receptor antagonist losartan (IC50, 13 nM). Other angiotensin peptides competed for [125I]angiotensin II binding in the following rank order: angiotensin II > angiotensin III > angiotensin I > > [des-Asp1-des-Arg2]angiotensin II. A nonhydrolyzable analogue of guanosine triphosphate increased the dissociation rate of bound [125I]angiotensin II and decreased hormone binding to the receptor at equilibrium. The angiotensin II receptor was coupled with increases in intracellular calcium. Incorporation of precursors into protein, DNA, and RNA in response to angiotensin II was determined. In serum-deprived cultures, a 24-hour exposure to 1 microM [Sar1]angiotensin II increased rates of phenylalanine, thymidine, and uridine incorporation by 58%, 103%, and 118%, respectively. These increases were blocked by the noncompetitive AT1 receptor antagonist EXP3174. After 48 hours, [Sar1]angiotensin II increased total protein and DNA of cardiac fibroblasts by 23% and 15%, respectively, with no change in the protein/DNA ratio. [Sar1]Angiotensin II increased cell number by 138% after a 24-hour exposure, without affecting cell area. In summary, cardiac fibroblasts have G protein-linked AT1 receptors that are coupled with proliferative growth. These results suggest that angiotensin II-induced cardiac hypertrophy is, in part, secondary to stimulated increases in nonmyocyte cellular growth.
Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.
Authors: Fedida D , Wible B , Wang Z , Fermini B , Faust F , Nattel S , Brown A M
Published: June 2012

In human myocardium, the nature of the K+ currents mediating repolarization of the action potential is still speculative. Delayed rectifier channels have recently been cloned from human myocardium, but it is unclear whether or not these currents are involved in the termination of the cardiac action potential plateau. In intact human atrial myocytes, we have identified a rapid delayed rectifier K+ current with properties and kinetics identical to those expressed by a K+ channel clone (fHK) isolated from human heart and stably incorporated into a human cell line for the first time. The myocyte current amplitude was 3.6 +/- 0.2 pA/pF (at +20 mV, n = 15) and activated with a time constant of 13.1 +/- 2 milliseconds at 0 mV (n = 15). The half-activation potential (V0.5) was -6 +/- 2.5 mV (n = 10) with a slope factor (k) of 8.6 +/- 2.2 (n = 10). The heterologously expressed fHK current amplitude was 136 pA/pF (at +20 mV, n = 9) with an activation time constant of 11.8 +/- 4.6 milliseconds at 0 mV; V0.5 was 4.1 +/- 2.4 mV (mean +/- SEM, n = 8); and k was 7.0. The conductance of single fHK channels was 16.9 picosiemens in 5 mM bath K+. Both native and cloned channel currents inactivated partially during sustained depolarizing pulses. Both currents were blocked by micromolar concentrations of 4-aminopyridine and were relatively insensitive to tetraethylammonium ions and class III antiarrhythmic agents.(ABSTRACT TRUNCATED AT 250 WORDS)
Crossbridge scheme and the kinetic constants of elementary steps deduced from chemically skinned papillary and trabecular muscles of the ferret.
Authors: Kawai M , Saeki Y , Zhao Y
Published: June 2012

Elementary steps of the crossbridge cycle in chemically skinned ferret myocardium were investigated with sinusoidal analysis. The muscle preparations were activated at pCa 4.82 and an ionic strength of 200 mM, and the effects of the change in the MgATP (S) and phosphate (Pi) concentrations on three exponential processes were studied at 20 degrees C. Results are consistent with the following crossbridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and Det includes all detached states (MS and MDP) and weakly attached states (AMS and AMDP). From our studies, we obtained K1a = 0.99 mM-1 (MgATP association), k1b = 270 s-1 (ATP isomerization), k-1b = 280 s-1 (reverse isomerization), K1b = k1b/k-1b = 0.95, k2 = 48 s-1 (crossbridge detachment), k-2 = 14 s-1 (reverse detachment), K2 = 3.5, k4 = 11 s-1 (crossbridge attachment), k-4 = 107 s-1 (reverse attachment), K4 = 0.11, and K5 = 0.06 mM-1 (Pi association). K6 is the rate-limiting step, and it is the slowest forward reaction in the cycle, which results in the rigor-like AM state. K1a (MgATP binding) is four times that of rabbit psoas, and K5 (Pi binding) is 0.3 times that of psoas, demonstrating that crossbridges in myocardium bind MgATP more and Pi less than psoas. The rate constants of ATP isomerization (k1b, k-1b), crossbridge detachment (k2, k-2), and crossbridge attachment (k4) steps are generally an order of magnitude slower than rabbit psoas. The reverse attachment step (k-4) is similar to that in psoas, indicating that this step may occur irrespective of the myosin type and possibly spontaneously. The above scheme with the deduced kinetic constants predicts the following crossbridge distributions at 5 mM MgATP2- and 8 mM Pi:AM (3%), AM S (15%), AM*S (14%), Det (50%), AM*DP (6%), and AM*D (12%). The actual number of attached crossbridges was measured to be 51 +/- 4% by the stiffness ratio during activation and after rigor induction, and a strong correlation was seen with the prediction. Our results are consistent with the hypothesis that force generation occurs at the Det-->AM*DPi transition, and the same force is maintained after the release of Pi.
Delayed rectifier outward current and repolarization in human atrial myocytes.
Authors: Wang Z , Fermini B , Nattel S
Published: June 2012

Previous work has suggested that the primary time-dependent repolarizing current in human atrium is the transient outward current (Ito), but interventions known to alter the magnitude of the delayed rectifier current (IK) affect atrial electrophysiology and arrhythmias in humans. To explore the potential role of IK in human atrial tissue, we used the whole-cell configuration of the patch-clamp technique to record action potentials and ionic currents in isolated myocytes from human atrium. A delayed outward current was present in the majority of myocytes, activating with a time constant ranging from 348 +/- 61 msec (mean +/- SEM) at -20 mV to 129 +/- 25 msec at +60 mV. The reversal potential of tail currents was linearly related to log [K+]o with a slope of 55 mV per decade, and fully activated tail currents showed inward rectification. The potassium selectivity, kinetics, and voltage dependence were similar to those reported for IK in other cardiac preparations. In cells with both Ito and IK, IK greatly exceeded both components of Ito (Ito1 and Ito2) within 50 msec of a voltage step from -70 to +20 mV. Based on the relative magnitude of Ito and IK, three types of cells could be distinguished: type 1 (58% [73/126] of the cells) displayed a large Ito together with a clear IK, type 2 (13% [17/126] of the cells) displayed only IK, and type 3 (29% [36/126] of the cells) was characterized by a prominent Ito and negligible IK. Consistent differences in action potential morphology were observed, with type 2 cells having a higher plateau and steeper phase 3 slope and type 3 cells showing a triangular action potential and lesser phase 3 slope compared with type 1 cells. We conclude that IK is present in a majority of human atrial myocytes and may play a significant role in their repolarization and that previously observed variability in human atrial action potential morphology may be partially due to differences in the relative magnitude of time-dependent outward currents.
Block of transient outward-type cloned cardiac K+ channel currents by quinidine.
Authors: Yatani A , Wakamori M , Mikala G , Bahinski A
Published: June 2012

The antiarrhythmic drug quinidine has been shown to block several types of K+ channel currents in cardiac preparations including the transient outward current (Ito). To characterize the molecular mechanism of quinidine block, a cloned Ito-type cardiac K+ channel (RHK1) was expressed in Xenopus oocytes, and drug effects were examined on whole-cell and single-channel currents. Extracellular application of quinidine reduced whole-cell RHK1 current amplitude in a concentration-dependent manner. The block was voltage dependent, with an IC50 of 1.69 mM at 0 mV, and the value decreased to 875 microM at +60 mV. Quinidine significantly slowed the current inactivation time course during voltage-clamp pulses without changing the rate of activation or the steady-state inactivation. To test the channel-state dependence of quinidine block, the cells were "rested" in the presence of quinidine (500 microM) for 2 to 3 minutes before applying depolarizing pulses to +60 mV. During the first pulse, the current inactivation rate was slower than control, but the peak current was only reduced by less than 5%. Subsequent pulses reduced the peak current amplitude to approximately 50% of control. These results suggest that quinidine blocks the open channel and that the drug must first dissociate before the channel can close, thereby causing a crossover in current tracings. In measurements of single-channel current from cell-attached patches, open time was reduced by quinidine in a concentration-dependent manner. Single-channel current amplitude was not altered by quinidine. Application of quinidine to the intracellular side of inside-out patches had an effect similar to that obtained from cell-attached patches but at 10-fold lower concentrations. External quinidine may therefore have to pass into or through the cell membrane to reach its blocking site.
Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure.
Authors: Näbauer M , Beuckelmann D J , Erdmann E
Published: June 2012

A variety of outward currents exists in ventricular myocardium of different species influencing action potential duration and electrical activity. Transient outward currents have been reported in ventricular tissue of some animals but are small or absent in others. This study was conducted to investigate whether a transient outward current exists in human ventricular myocardium and to characterize its basic electrophysiological properties. Currents were recorded from enzymatically isolated human ventricular myocytes obtained from explanted hearts of 22 patients with terminal heart failure. In almost all cells studied, a transient outward current could be recorded on depolarization to between -20 and +80 mV. The size of the transient outward current was usually large enough to mask the Ca2+ current. It could be recorded under conditions in which Ca2+ influx and intracellular Ca2+ transients were suppressed. Basic current characteristics were similar to transient outward currents observed in other species. Inactivation of the transient outward current was monoexponential, with a time constant of 54.8 +/- 3.7 milliseconds at +40 mV. Half-maximal activation occurred at 16.7 +/- 1.6 mV; half-maximal steady-state inactivation occurred at -34.5 +/- 2.3 mV. Frequency-dependent reduction of peak transient outward current was 29.8 +/- 1.4% at 2 Hz compared with resting conditions. Recovery from inactivation was voltage dependent and had a biexponential time course; the faster time constant (41.0 +/- 6.5 milliseconds at -80 mV) accounted for 86.0 +/- 5.2% of total current. The transient outward current was sensitive to 4-aminopyridine (IC50, 1.15 mM). These results indicate that a large Ca(2+)-independent transient outward K+ current is present in human ventricular myocytes that might be regulated by physiological or pathological events and is a potential site for pharmacological intervention.
Molecular characterization of angiotensin II--induced hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. Critical role of the AT1 receptor subtype.
Authors: Sadoshima J , Izumo S
Published: June 2012

Increasing evidence suggests that angiotensin II (Ang II) may act as a growth factor for the heart. However, direct effects of Ang II on mammalian cardiac cells (myocytes and nonmyocytes), independent of secondary hemodynamic and neurohumoral effects, have not been well characterized. Therefore, we analyzed the molecular phenotype of cultured cardiac cells from neonatal rats in response to Ang II. In addition, we examined the effects of selective Ang II receptor subtype antagonists in mediating the biological effects of Ang II. In myocyte culture, Ang II caused an increase in protein synthesis without changing the rate of DNA synthesis. In contrast, Ang II induced increases in protein synthesis, DNA synthesis, and cell number in nonmyocyte cultures (mostly cardiac fibroblasts). The Ang II-induced hypertrophic response of myocytes and mitogenic response of fibroblasts were mediated primarily by the AT1 receptor. Ang II caused a rapid induction of many immediate-early genes (c-fos, c-jun, jun B, Egr-1, and c-myc) in myocyte and nonmyocyte cultures. Ang II induced "late" markers for cardiac hypertrophy, skeletal alpha-actin and atrial natriuretic factor expression, within 6 hours in myocytes. Ang II also caused upregulation of the angiotensinogen gene and transforming growth factor-beta 1 gene within 6 hours. Induction of immediate-early genes, late genes, and growth factor genes by Ang II was fully blocked by an AT1 receptor antagonist but not by an AT2 receptor antagonist. These results indicate that: (1) Ang II causes hypertrophy of cardiac myocytes and mitogenesis of cardiac fibroblasts, (2) the phenotypic changes of cardiac cells in response to Ang II in vitro closely mimic those of growth factor response in vitro and of load-induced hypertrophy in vivo, (3) all biological effects of Ang II examined here are mediated primarily by the AT1 receptor subtype, and (4) Ang II may initiate a positive-feedback regulation of cardiac hypertrophic response by inducing the angiotensinogen gene and transforming growth factor-beta 1 gene.
Effects of brief repetitive ischemia on contractility, relaxation, and coronary flow. Exaggerated postischemic diastolic dysfunction in pressure-overload hypertrophy.
Authors: Mochizuki T , Eberli F R , Ngoy S , Apstein C S , Lorell B H
Published: June 2012

The recovery of systolic and diastolic function during unstable angina may be modified by the repetition of brief episodes of ischemia and by the presence of left ventricular hypertrophy (LVH). We studied the effects of six consecutive 5-minute cycles of no-flow ischemia and reperfusion followed by 25 minutes of recovery in isovolumic red blood cell-perfused hearts from aortic-banded rats with chronic LVH (n = 8) and sham-operated control rats (n = 8). At baseline (left ventricular end-diastolic pressure [LVEDP], 10 mm Hg), left ventricular developed pressure (123 +/- 5 versus 114 +/- 5 mm Hg/g) and coronary flow [2.5 +/- 0.3 versus 2.2 +/- 0.2 (mL/min)/g] were similar in LVH versus control rats. Repetitive ischemia was associated with progressive depression of postischemic recovery of left ventricular systolic function, and the recovery of left ventricular developed pressure after the final 25-minute reperfusion period was similar in LVH versus control rats (61 +/- 6% versus 72 +/- 4% of baseline, P = NS). Although there was no increase in isovolumic LVEDP during the initial cycle of transient ischemia, both groups showed a rapid and similar rise in LVEDP during subsequent ischemic cycles (delta 82 +/- 8 versus delta 89 +/- 7 mm Hg/g in response to the final ischemia cycle for LVH versus control rats, respectively; P = NS). The control hearts showed complete restoration of LVEDP to baseline during final reperfusion, whereas the LVH hearts showed prolonged and severe postischemic diastolic dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)
Catecholamines can induce adenosine receptor-mediated protection of the myocardium but do not participate in ischemic preconditioning in the rabbit.
Authors: Thornton J D , Daly J F , Cohen M V , Yang X M , Downey J M
Published: June 2012

The role of catecholamines in ischemic preconditioning is unclear. Accordingly, the effects of tyramine-induced norepinephrine release and alpha 1-receptor blockade were examined. Ischemic preconditioning with a 5-minute coronary occlusion 10 minutes before a 30-minute ischemic interval resulted in only 7.7 +/- 3.1% infarction of the risk area, significantly less than that in control rabbits with isolated 30-minute coronary occlusions (34.4 +/- 3.2%, P < .01). Intravenous infusion of tyramine 10 minutes before 30 minutes of ischemia also protected the heart from infarction to an extent similar to that seen with ischemic preconditioning (6.9 +/- 2.4% infarction). This protection observed with tyramine infusion was eliminated by alpha 1-receptor blockade with BE 2254 (36.8 +/- 2.6% infarction) but was unaffected by beta-blockade with propranolol (10.5 +/- 2.4% infarction). Furthermore, the protection was unaffected when the tyramine-induced hypertension was attenuated by allowing blood to flow into a volume reservoir (3.9 +/- 0.8% infarction, P < .01 vs control value). The nonselective adenosine-receptor blocker PD 115,199 also eliminated tyramine-induced protection (40.2 +/- 5.6% infarction), indicating that adenosine is involved in adrenergic-mediated protection. BE 2254 could not block ischemic preconditioning (3.9 +/- 1.1% infarction, P < .01 vs control value). Therefore, catecholamine release before prolonged ischemia can protect the heart from infarction via the alpha 1-receptor, but adenosine receptor stimulation is also involved. alpha-Adrenergic stimulation does not appear to be critical to the protection observed after ischemic preconditioning.
Preconditioning against myocardial dysfunction after ischemia and reperfusion by an alpha 1-adrenergic mechanism.
Authors: Banerjee A , Locke-Winter C , Rogers K B , Mitchell M B , Brew E C , Cairns C B , Bensard D D , Harken A H
Published: June 2012

Preconditioning may find ready applicability in humans facing scheduled global cardiac ischemia-reperfusion (IR) during bypass or transplantation, where such a maneuver is feasible before arrest. Our objective was to delineate and exploit the endogenous preconditioning mechanism triggered by transient ischemia (TI) and thereby attenuate myocardial postischemic mechanical dysfunction by clinically acceptable means. Preconditioning by 2 minutes of TI followed by 10 minutes of normal perfusion protected isolated rat left ventricle function assessed after 20 minutes of global, 37 degrees C ischemia and 40 minutes of reperfusion. Final recovery of developed pressure (DP) was improved (91.5 +/- 1.9% of equilibration DP versus unconditioned IR control, 57.4 +/- 2.4%, P < .01) and was accompanied by increased contractility (+/- dP/dt). Norepinephrine release increased after TI, and reserpine pretreatment abolished TI preconditioning. This suggests that endogenous norepinephrine mediates functional preconditioning in rat. Brief pretreatment (2 minutes) with exogenous norepinephrine reproduced the protection (89.1 +/- 1.4%) of postischemic function. Functional protection persisted after the hemodynamic effects had resolved. Norepinephrine-induced preconditioning was simulated by phenylephrine and blocked by alpha 1-adrenergic receptor antagonist. TI preconditioning was similarly lost after selective alpha 1-adrenergic receptor blockade. We conclude that transient ischemic preconditioning is mediated by the sympathetic neurotransmitter release and alpha 1-adrenergic receptor stimulation. Although the postreceptor mechanism remains unclear, functional protection after IR does not seem related to the magnitude of ATP depletion and elevation of resting pressure during ischemia. Rather, the endogenous mechanisms facilitate both recovery of mechanical function and ATP repletion during reperfusion.
Transforming growth factor-beta in cardiac ontogeny and adaptation.
Authors: MacLellan W R , Brand T , Schneider M D
Published: June 2012

The transforming growth factor-beta (TGF-beta) superfamily comprises a set of regulatory peptides with multiple effects on cell growth and differentiation. The elaborate regulation of TGF-beta s during embryonic development of the heart, the upregulation of TGF-beta after hemodynamic stress, and the impact of TGF-beta on cardiac gene expression together imply a prominent functional role for this family of growth factors in cardiac organogenesis and hypertrophy. Basal and TGF-beta-induced expression of skeletal alpha-actin, one of several genes specifically associated with developing or hypertrophied myocardium, each are contingent on transcriptional activation by serum response factor. A truncated form of the type II TGF-beta receptor, created by deletion of the cytoplasmic kinase domain, acts as a dominant suppressor of TGF-beta signal transduction in cultured cardiac muscle cells and may provide a suitable means to establish the functions of TGF-beta in vivo.
Sarcoplasmic reticulum Ca2+ uptake and thapsigargin sensitivity in permeabilized rabbit and rat ventricular myocytes.
Authors: Hove-Madsen L , Bers D M
Published: June 2012

Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were measured simultaneously with indo 1 and a Ca(2+)-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes (approximately 10 mg/mL protein). In the presence of 25 mumol/L ruthenium red and 10 mmol/L oxalate, the Km for Ca2+ uptake by the SR was approximately 250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary to inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 mumol/kg wet wt for rabbit and 54.6 mumol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to < 150 nmol/L from a peak value > 10 mumol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in < 15 seconds and reduced the uptake rate by 95% in 5 seconds.(ABSTRACT TRUNCATED AT 250 WORDS)
Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart.
Authors: Kroll K , Decking U K , Dreikorn K , Schrader J
Published: June 2012

The intracellular flux rate through adenosine kinase (adenosine-->AMP) in the well-oxygenated heart was investigated, and the relation of the AMP-adenosine metabolic cycle (AMP<-->adenosine) to transmethylation (S-adenosylhomocysteine [SAH]-->adenosine) and coronary flow was determined. Adenosine kinase was blocked in isolated guinea pig hearts by infusion of iodotubercidin in the presence of the adenosine deaminase blocker erythro-9-(2-hydroxy-3-nonyl)adenine (5 mumol/L). Iodotubercidin (1 nmol/L to 4 mumol/L) caused graded increases in venous effluent concentrations of adenosine, from 8 +/- 3 to 145 +/- 32 nmol/L (mean +/- SEM, n = 3), and in coronary flow, which increased to maximal levels. Flow increases were completely abolished by adenosine deaminase (5 to 10 U/mL). Interstitial adenosine concentrations, estimated using a mathematical model, increased from 22 nmol/L during control conditions to 420 nmol/L during maximal vasodilation. The possibility that iodotubercidin caused increased venous adenosine by interfering with myocardial energy metabolism was ruled out in separate 31P nuclear magnetic resonance experiments. To estimate total normoxic myocardial production of adenosine (AMP-->adenosine<--SAH), the time course of coronary venous adenosine release was measured during maximal inhibition of adenosine kinase with 30 mumol/L iodotubercidin. Adenosine release increased more than 15-fold over baseline, reaching a new steady-state value of 3.4 +/- 0.3 nmol.min-1 x g-1 (n = 5) after 4 minutes. In parallel experiments, the relative roles of AMP hydrolysis and transmethylation (SAH hydrolysis) were determined, using adenosine dialdehyde (10 mumol/L) to block SAH hydrolase. In these experiments, adenosine release increased to similar levels of 3.4 +/- 0.5 nmol.min-1 x g-1 (n = 6) during inhibition of adenosine deaminase and adenosine kinase. It is concluded that (1) maximal increases in coronary flow are elicited by increases in interstitial adenosine concentration to approximately 400 nmol/L, (2) more than 90% of the adenosine produced in the heart is normally rephosphorylated to AMP without escaping into the venous effluent, (3) AMP hydrolysis is the dominant pathway for cardiac adenosine production under normoxic conditions, and (4) the high rate of adenosine salvage is due to rapid turnover of a metabolic cycle between AMP and adenosine. Rapid cycling may serve to amplify the relative importance of AMP hydrolysis over transmethylation in controlling cytosolic adenosine concentrations.
Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells.
Authors: Nugent M A , Karnovsky M J , Edelman E R
Published: June 2012

Basic fibroblast growth factor (bFGF) has been previously shown to be mitogenic for vascular smooth muscle cells (VSMCs) in vivo, but only after vascular injury. We show in the present study that the regulation of bFGF-stimulated VSMC proliferation, by vascular cell-secreted heparin-like compounds, correlates with inhibition of bFGF binding to cell-associated heparin sulfate proteoglycans. The stimulation of cultured VSMC proliferation by bFGF was markedly reduced when these cells were cocultured with confluent endothelial cells or confluent VSMCs (100.8 +/- 8.4% and 55.6 +/- 2.3% inhibition, respectively) or with conditioned media from these two cell types. Balb/c3T3 fibroblasts had no statistically significant effect on bFGF-stimulated VSMC proliferation. Vascular cell-conditioned media also inhibited bFGF binding to heparan sulfate proteoglycans on VSMCs, and the inhibition of binding correlated linearly with the inhibition of proliferation after a critical amount of binding was inhibited (44%) (r = .952, P < .0001). Heparinase or heparitinase treatment of conditioned media removed the bFGF-inhibitory effects, presumably by degrading heparin-like compounds. Indeed, heparin itself mimicked the inhibitory effects of conditioned media on bFGF-mediated proliferation and binding to heparin sulfate proteoglycans. These results suggest a bFGF regulatory role for vascular cell-produced heparin-like compounds, linking the mitogenic effects with binding to heparan sulfate proteoglycans for this heparin-binding growth factor.
Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents.
Authors: Wang Z , Fermini B , Nattel S
Published: June 2012

Depolarization of human atrial myocytes activates a transient outward current that rapidly inactivates, leaving a sustained outward current after continued depolarization. To evaluate the ionic mechanism underlying this sustained current (Isus), we applied whole-cell voltage-clamp techniques to single myocytes isolated from right atrial specimens obtained from patients undergoing coronary bypass surgery. The magnitude of Isus was constant for up to 10 seconds at +30 mV and was unaffected by 40 mmol/L tetraethylammonium, 100 nmol/L dendrotoxin, 1 mmol/L Ba2+, 0.1 mumol/L atropine, or removal of Cl- in the superfusate. Isus could be distinguished from the 4-aminopyridine (4AP)-sensitive transient outward current (Ito1) by differences in voltage-dependent inactivation (1000-millisecond prepulse to -20 mV reduced Ito1 by 91.7 +/- 0.1% [mean +/- SEM], P < .001, versus 9.4 +/- 0.4% reduction of Isus) and 4AP sensitivity (IC50 for block of Ito1, 1.96 mmol/L; for Isus, 49 mumol/L). Isus activation had a voltage threshold near -30 mV, a half-activation voltage of -4.3 mV, and a slope factor of 8.0 mV. Isus was not inactivated by 1000-millisecond prepulses but was reduced by 16 +/- 8% (P < .05) at a holding potential of -20 mV relative to values at a holding potential of -80 mV. Isus activated very rapidly, with time constants (tau) at 25 degrees C ranging from 18.2 +/- 1.8 to 2.1 +/- 0.2 milliseconds at -10 to +50 mV, two orders of magnitude faster than previously described kinetics of the rapid component of the delayed rectifier K+ current. At 16 degrees C, Isus activation was greatly slowed (tau at +10 mV, 46.7 +/- 4.1 milliseconds; tau at 25 degrees C, 7.1 +/- 0.8 milliseconds; P < .01), and the envelope of tails test was satisfied. The reversal potential of Isus tail currents changed linearly with log [K+]o (slope, 55.3 +/- 2.9 mV per decade), and the fully activated current-voltage relation showed substantial outward rectification. Selective inhibition of Isus with 50 mumol/L 4AP increased human atrial action potential duration by 66 +/- 11% (P < .01). In conclusion, Isus in human atrial myocytes is due to a very rapidly activating delayed rectifier K+ current, which shows limited slow inactivation, is insensitive to tetraethylammonium, Ba2+, and dendrotoxin, and is highly sensitive to 4AP. These properties resemble the characteristics of channels encoded by the Kv1.5 group of cardiac cDNAs and may represent a physiologically significant manifestation of such channels in human atrium.
Gap junction protein connexin40 is preferentially expressed in vascular endothelium and conductive bundles of rat myocardium and is increased under hypertensive conditions.
Authors: Bastide B , Neyses L , Ganten D , Paul M , Willecke K , Traub O
Published: June 2012

Gap junction channels consisting of connexin protein mediate electrical coupling between cardiac cells. Expression of two connexins, connexin40 (Cx40) and connexin43 (Cx43), has been studied in ventricular myocytes from normal and hypertensive rats. Polyclonal affinity-purified rabbit antibodies to Cx43 and Cx40 have been used for immunohistochemical analysis on frozen sections from rat heart. These studies revealed coexpression of Cx43 and Cx40 in ventricular myocytes. In addition, Cx40 is preferentially expressed in three distinct regions: first, in the endothelial layer of the heart blood vessels but not in the smooth muscle layer of the arteries; second, in the ventricular conductive myocardium, particularly in the atrioventricular bundle and bundle branches, where Cx43 is not observed; and third, in the myocyte layers close to the ventricular cavities. These results suggest that Cx40 is preferentially expressed in the fast conducting areas of myocardial tissue. Expression of both Cx40 and Cx43 was also found in immunoblots from normal and hypertensive rat myocardiocytes. Under hypertensive conditions (ie, in spontaneous hypertensive rats and in transgenic rats that exhibit hypertension due to expression of an exogenous renin gene), we found a 3.1-fold increase in Cx40 expression, compared with normal myocardium. Furthermore, we detected a 3.3-fold decrease in Cx43 protein level in transgenic hypertensive rats. The coexpression of Cx40 and Cx43 proteins in rat myocytes, their spatial distribution, and the increased amount of Cx40 protein during cardiac hypertrophy suggest that Cx40 may be involved in mediating fast conduction under normal and pathological conditions. The increased expression of Cx40 in hypertrophic heart may be a compensatory mechanism to increase conduction velocity.
Role of nitric oxide, muscarinic receptors, and the ATP-sensitive K+ channel in mediating the effects of acetylcholine to mimic preconditioning in dogs.
Authors: Yao Z , Gross G J
Published: June 2012

The aims of the current study were to examine the efficacy of acetylcholine (ACh) to mimic ischemic preconditioning in dogs and to determine the role of nitric oxide (NO), muscarinic receptors, and ATP-sensitive K+ (KATP) channels in mediating its effects. Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 4 hours of reperfusion. Preconditioning was elicited by 10 minutes of LAD occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. ACh (3 or 10 micrograms/min) or an equivalent volume of saline was infused into the LAD for 10 minutes, followed by a 10-minute drug-free period before the 60-minute ischemic insult. In other groups, the specific NO synthesis inhibitor NG-monomethyl-L-arginine (L-NMMA, 4 mg/min), the muscarinic receptor antagonist-NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 3 mg/min), or the specific KATP channel blocker 5-hydroxydecanoate (5-HD, 3 mg/min) was infused with ACh into the LAD for 10 minutes. The infusion of L-NAME, L-NMMA, or 5-HD was started 2 minutes before ACh infusion. Transmural myocardial blood flow was measured at 5 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. There were no significant differences in collateral blood flow or the area at risk between groups. Preconditioning produced a marked reduction (P < .05) in infarct size (6.2 +/- 3.0% versus 26.1 +/- 5.7% in the control group).(ABSTRACT TRUNCATED AT 250 WORDS)
Stereoselective actions of thiadiazinones on canine cardiac myocytes and myofilaments.
Authors: Solaro R J , Gambassi G , Warshaw D M , Keller M R , Spurgeon H A , Beier N , Lakatta E G
Published: June 2012

Thiadiazinones are cardiotonic agents that have potent, direct, and stereoselective actions on the myofilament response to Ca2+ in intact myocardium. Their mechanism of action is unknown. We studied the effects of racemic thiadiazinone, EMD 53998 (5-[1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydro-6-quinolyl]-6-meth yl-3,6- dihydro-2H-1,3,4-thiadiazin-2-one), and its enantiomers on Ca2+ signaling in myocytes, myofilaments, and myofilament proteins. Intact canine ventricular myocytes responded to the positive enantiomer, EMD 57033, with an increase in the extent of shortening during twitch contractions without increasing the peak amplitude of the Ca2+ transient. The negative enantiomer, EMD 57439, also increased the extent of shortening, but in this case there was a concentration-dependent increase in the peak amplitude of the Ca2+ transient. This is predicted from in vitro data showing that this enantiomer is a relatively potent inhibitor of phosphodiesterase activity. There was no effect of EMD 57439 on the relation between pCa and actomyosin Mg-ATPase activity of canine heart myofibrils. In contrast, EMD 57033 shifted the pCa-Mg-ATPase activity relation to the left. There was no effect of either enantiomer on Ca2+ binding to myofilament troponin C. Moreover EMD 57033, but not EMD 57439, stimulated actomyosin ATPase activity of myofilament preparations in which either troponin or troponin-tropomyosin had been extracted. EMD 57033 had no effect on Mg-ATPase activity of pure ventricular myosin. EMD 57033 also stimulated the velocity of actin filament sliding on myosin heads adhered to nitrocellulose-coated glass coverslips. We propose that the action of EMD 57033 is at the actin-myosin interface on a "receptor" that may be on actin or the crossbridge. Drug binding to this domain appears to reverse the inhibition of actin-myosin interactions by troponin-tropomyosin and also to promote transition of crossbridges from weak to strong force-generating states.
A monoclonal antibody against rabbit tissue factor inhibits thrombus formation in stenotic injured rabbit carotid arteries.
Authors: Pawashe A B , Golino P , Ambrosio G , Migliaccio F , Ragni M , Pascucci I , Chiariello M , Bach R , Garen A , Konigsberg W K
Published: June 2012

Tissue factor (TF) is a transmembrane protein that binds factor VII/VIIa, thus activating the extrinsic blood coagulation pathway. Since this pathway appears to be involved in the formation of intravascular thrombi, the anti-rabbit TF monoclonal antibody, AP-1, was produced and tested as an antithrombotic agent in a rabbit model of recurrent intravascular thrombosis. In this model, a plastic constrictor is positioned around the injured rabbit carotid arteries, and flow is monitored with a Doppler flow probe. This produces cyclic flow variation (CFV) in the carotid artery, which is caused by recurrent formation and dislodgment of thrombi at the site of the stenosis. After monitoring CFV pattern for 30 minutes, AP-1 was infused intravenously into nine rabbits at doses of 0.05 to 1.5 mg/kg body weight, and a control monoclonal antibody that does not react with rabbit TF was infused into four additional rabbits. In all rabbits receiving AP-1, CFV was abolished, and a steady normal blood flow was restored, indicating that thrombus formation had been blocked by AP-1. By contrast, in all rabbits that received the control monoclonal antibody, CFV continued unaltered. There was no change in the partial thromboplastin time and ex vivo platelet aggregation to several different agonists after infusion of AP-1, indicating an absence of systemic effects on the coagulation process. We conclude that activation of the extrinsic coagulation pathway has a key role in triggering intravascular thrombosis and that an anti-TF monoclonal antibody is an effective antithrombotic agent that could have therapeutic potential for humans.