bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane-bound nucleoside diphosphate kinase activity in atrial cells of frog, guinea pig, and human.
Authors: Heidbüchel H , Callewaert G , Vereecke J , Carmeliet E
Published: June 2012

Muscarinic K+ channels in inside-out patches of atrial cells from guinea pig or rabbit can be activated by Mg(2+)-ATP in the absence of acetylcholine and GTP or GDP. The ATP-dependent activation involves a phosphorylation and is postulated to be due to the association of a membrane-bound nucleoside diphosphate kinase (NDPK) with the G protein GK: direct phosphorylation of the GK-bound GDP into GTP, catalyzed by NDPK, would result in activation of the G protein and, hence, activation of the channels. The aim of this study was to identify the presence of NDPK activity in atrial membranes by investigating the phosphate transfer between tritium-labeled nucleotides. We show that frog, guinea pig, and human atrial membranes contain a substantial NDPK activity since they catalyze the conversion from [3H]GDP+nucleoside triphosphate (NTP or NTP gamma S) to [3H]GTP (or [3H]GTP gamma S), from [3H]ADP+NTP to [3H]ATP, and from [3H]GTP+nucleoside diphosphate (NDP) to [3H]GDP. The phosphate transfer rates for the [3H]GDP+ATP to [3H]GTP conversion are 1.8, 0.5, and 2.4 mumol inorganic phosphate formation/mg per 10 minutes at 37 degrees C in frog, guinea pig, and human, respectively. The order of substrate efficiency for different NTPs was ATP greater than ITP approximately equal to GTP greater than UTP greater than CTP, which parallels the efficiency of these nucleotides in their activation of the muscarinic K+ channels. Addition of other nucleotides blocked the transphosphorylation reaction, indicating that the NTP-NDP conversion mechanism is aspecific, as is expected for an NDPK-catalyzed reaction. In conclusion, the data support the concept of NDPK involvement in the atrial muscarinic signal transduction cascade.
Effects of 2,4-dinitrophenol or low [ATP]i on cell excitability and action potential propagation in guinea pig ventricular myocytes.
Authors: Morley G E , Anumonwo J M , Delmar M
Published: June 2012

Inhibition of aerobic metabolism leads to a major disruption of cardiac cell homeostasis. The purpose of the present study was twofold: 1) We determined the relative importance of junctional and nonjunctional membrane resistance (Rj and Rm, respectively) in the development of propagation failure during inhibition of aerobic metabolism in guinea pig ventricular cell pairs. 2) We used the patch-action potential clamp technique in single ventricular myocytes to study some of the properties of the membrane channels that are responsible for shortening of action potential duration and eventual failure of cell excitation after metabolic blockade. In most experiments, whole-cell patch pipettes were filled with a solution containing 1 mM EGTA, 5 mM HEPES, and 5 mM ATP. Our results in cell pairs showed that pharmacological inhibition of aerobic metabolism with the mitochondrial uncoupler 2,4-dinitrophenol (DNP) led to a drop in Rm followed by an increase in Rj. The increase in Rj was not sufficient to cause a measurable delay in cell-to-cell propagation, whereas the drop in Rm consistently led to failure of cell excitation. Similar results were obtained in additional experiments in which the EGTA concentration in the pipette was reduced to 50 microM. Similar results were also obtained by loading the recording patch pipettes with a solution containing only 0.1 mM ATP. Our patch-action potential clamp experiments, on the other hand, revealed that DNP induced the opening of time- and voltage-independent membrane channels, with a unitary conductance of 23 pS. The channels allowed for the passage of outward current in the voltage range of the action potential, and the increase in membrane patch conductance correlated with the observed shortening of action potential duration during DNP superfusion. Our experiments provide the first simultaneous recordings of action potentials and DNP-induced channel currents in guinea pig ventricular myocytes. Overall, the data provide new evidence for the understanding of the cellular and subcellular mechanisms involved in the development of slow conduction velocity and propagation block after metabolic blockade.
Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.
Authors: Scott-Burden T , Schini V B , Elizondo E , Junquero D C , Vanhoutte P M
Published: June 2012

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation. Platelet-derived growth factor-AB and -BB inhibited the production of nitric oxide (measured as nitrite levels in conditioned medium) by cells treated simultaneously with interleukin-1 beta and growth factor. However, platelet-derived growth factor-AA neither affected nitrite production nor thymidine incorporation by smooth muscle cells. Levels of cytokine-stimulated nitrite production by smooth muscle cells were increased synergistically by the presence of fibroblast growth factors or epidermal growth factor. The inhibition of thymidine incorporation and concomitant elevation of nitrite production was abolished in the presence of nitro-L-arginine. Cultures maintained in the presence of low levels of the cytokine for 9 days were growth-inhibited, and this was reversed when culture medium was supplemented with nitro-L-arginine. The treatment of smooth muscle cells, which were grown in coculture inserts with the cytokine to induce nitric oxide production, before their combination with other quiescent layers of cells resulted in the inhibition of thymidine incorporation by this second layer of cells regardless of the growth factor used for stimulation. Nitric oxide may act as an endogenous inhibitor of smooth muscle cell proliferation in the vessel wall, and impairment of its production may be one action of potent vascular mitogens such as platelet-derived growth factor.
Endothelium-derived relaxing factor modulates platelet aggregation in an in vivo model of recurrent platelet activation.
Authors: Golino P , Cappelli-Bigazzi M , Ambrosio G , Ragni M , Russolillo E , Condorelli M , Chiariello M
Published: June 2012

It has been shown that endothelium-derived relaxing factor (EDRF) may inhibit platelet aggregation in vitro through activation of platelet-soluble guanylate cyclase. To assess whether EDRF may also affect platelet function in vivo, intravascular platelet aggregation was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries. Carotid blood flow velocity was measured continuously by a Doppler flow probe placed proximal to the constrictor. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent platelet aggregation, developed at the site of the stenosis. After CFRs were observed for 30 minutes, a solution of authentic nitric oxide (NO, n = 10) was infused into the carotid artery via a small catheter placed proximally to the stenosis. Before infusion of NO, CFR frequency averaged 18.3 +/- 2.9 cycles per hour, and CFR severity (lowest carotid blood flow as percentage of baseline values) was 6 +/- 1%. NO completely inhibited CFRs in all animals, as shown by the normal and constant pattern of carotid blood flow (CFR frequency, 0 cycles per hour, p < 0.001; carotid blood flow, 92 +/- 5%, p = NS versus baseline). These effects were transient; CFRs were restored spontaneously within 10 minutes after cessation of NO infusion. After CFRs returned, S-nitroso-cysteine (S-NO-cys), a proposed form of EDRF, was infused into the carotid artery. S-NO-cys also abolished CFRs in all animals but at a significantly lower dose than NO (0.3 +/- 0.1 versus 12 +/- 4 nmol/min). The role of endogenously released EDRF in modulating in vivo platelet function was then tested in additional experiments. In 10 animals, endogenous release of EDRF was stimulated by infusing acetylcholine into the aortic root during CFRs. Infusion of acetylcholine was also associated with a complete inhibition of CFRs, similar to that observed during exogenous infusion of NO or S-NO-cys. These antithrombotic effects of acetylcholine were completely lost when EDRF synthesis was prevented by administration of the L-arginine analogue NG-monomethyl L-arginine (L-NMMA). Furthermore, in six additional rabbits the basal release of EDRF was blocked by L-NMMA after CFRs had been previously abolished with aspirin or the combination of aspirin and ketanserin, a serotonin S2 receptor antagonist. L-NMMA caused restoration of CFRs in all animals, indicating that even the basal release of EDRF is important in modulating platelet reactivity in vivo. Taken together, the data of the present study demonstrate that endogenous EDRF might importantly contribute to the modulation of platelet function in vivo.
Mechanism of preconditioning. Ionic alterations.
Authors: Steenbergen C , Perlman M E , London R E , Murphy E
Published: June 2012

The mechanism by which preconditioning (brief intermittent periods of ischemia and reflow) improves recovery of function and reduces enzyme release after a subsequent 30-minute period of ischemia was investigated in perfused rat hearts. Specifically, it was hypothesized that ischemia after preconditioning would result in a decreased production of H+ and therefore a smaller rise in [Na+]i and [Ca2+]i via Na(+)-H+ and Na(+)-Ca2+ exchange. To test this hypothesis we measured pHi, [Na+]i, [Ca2+]i, and cell high-energy phosphates during ischemia and reflow, and we correlated this with recovery of contractile function and release of creatine kinase during reflow. 31P nuclear magnetic resonance (NMR) was used to measure pHi and cell phosphates. [Na+]i was measured by 23Na NMR using the shift reagent thulium 1,4,7,10-tetraazacyclododecane-N,N,'N",N"'-tetramethylenephosph onate to distinguish intracellular from extracellular sodium. [Ca2+]i was measured by 19F NMR using hearts loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, termed 5F-BAPTA. Basal time-averaged levels of pHi, [Na+]i, and [Ca2+]i were 7.07 +/- 0.08, 9.4 +/- 0.8 mM, and 715 +/- 31 nM, respectively. After 30 minutes of ischemia, in preconditioned hearts, pHi was 6.5 +/- 0.06, [Na+]i was 2.09 +/- 4.4 mM, [Ca2+]i was 2.1 +/- 0.4 microM, and ATP was negligible. In untreated hearts, after 30 minutes of ischemia, pHi was 6.3 +/- 0.08, [Na+]i was 26.7 +/- 3.8 mM, [Ca2+]i was 3.2 +/- 0.6 microM, and ATP was undetectable. During reperfusion after 30 minutes of ischemia, preconditioned hearts had significantly better recovery of contractile function than untreated hearts (71 +/- 9% versus 36 +/- 8% initial left ventricular developed pressure), and after 60 minutes of ischemia, preconditioned hearts had significantly less release of the intracellular enzyme creatine kinase (102 +/- 12 versus 164 +/- 17 IU/g dry wt). We also found that unpreconditioned hearts arrested with 16 mM MgCl2 (to inhibit calcium entry via calcium channels and Na(+)-Ca2+ exchange) before 30 minutes of ischemia recover function on reflow to the same extent as preconditioned hearts with or without magnesium arrest. Thus, preconditioning has no additional benefit in addition to magnesium arrest. In addition, in hearts that received 16 mM MgCl2 just before the 30-minute period of ischemia, preconditioning had no effect on the rise in [Ca2+]i during the 30-minute period of ischemia. These data support the hypothesis that preconditioning attenuates the increase in [Ca2+]i, [Na+]i, and [H+]i during ischemia, most likely because of reduced stimulation of Na(+)-H+ and Na(+)-Ca2+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
Epicardial mapping of ventricular defibrillation with monophasic and biphasic shocks in dogs.
Authors: Zhou X , Daubert J P , Wolf P D , Smith W M , Ideker R E
Published: June 2012

To study the mechanism of defibrillation and the reason for the increased defibrillation efficacy of biphasic waveforms, the potential gradient in a 32 x 30-mm region of the right ventricle in 15 dogs was progressively lowered in four steps while a strong potential gradient field was maintained throughout the rest of the ventricular myocardium. The volume of right ventricle beneath the plaque was 10 +/- 2% of the total ventricular mass. A 10-msec monophasic (eight dogs) or 5/5-msec biphasic (seven dogs) truncated exponential shock 30% above the defibrillation threshold voltage was given via electrodes on the left ventricular apex and right atrium to create the strong potential gradient field. Simultaneously, a weaker shock with the same waveform but opposite polarity was given via mesh electrodes on either side of the small right ventricular region to cancel part of the potential difference in the region and to create one of the four levels of potential gradient fields. Shock potentials and activations were recorded from 117 epicardial electrodes in the small region, and in one dog global epicardial activations and potentials were recorded from a sock containing 72 electrodes. Each gradient field was tested 10 times for successful defibrillation after 10 seconds of electrically induced fibrillation. For both monophasic and biphasic shocks, the percentage of successful defibrillation attempts decreased (p < 0.05) as the potential gradient decreased in the small region. Defibrillation was successful approximately 80% of the time for a mean +/- SD potential gradient of 5.4 +/- 0.8 V/cm for monophasic shocks and 2.7 +/- 0.3 V/cm for biphasic shocks (p < 0.05). No postshock activation fronts arose from the small region for eight waveform when the gradient was more than 5 V/cm. For both waveforms, the postshock activation fronts after the shocks were markedly different from those just before the shock and exhibited either a focal origin or unidirectional conduction.(ABSTRACT TRUNCATED AT 400 WORDS)
Pacemaker current exists in ventricular myocytes.
Authors: Yu H , Chang F , Cohen I S
Published: June 2012

I(f), the "cardiac pacemaker current," is a nonselective cation channel activated on hyperpolarization in primary and secondary pacemaker regions of the mammalian heart. The cardiac pacing rate can be modulated by shifting the activation of I(f) to more positive (faster pacing) or more negative (slower pacing) voltages. We report for the first time the presence of this pacemaker current in ventricular myocytes. The potential importance of this observation to the mechanism of differentiation of nonpacing regions in the heart is discussed.
Dissociation between cellular K+ loss, reduction in repolarization time, and tissue ATP levels during myocardial hypoxia and ischemia.
Authors: Yan G X , Yamada K A , Kléber A G , McHowat J , Corr P B
Published: June 2012

The mechanisms underlying the marked increase in [K+]o in response to ischemia are not fully understood. Accordingly, the present study was performed to assess the contribution of ATP-regulated K+ channels by using simultaneous measurements of cellular K+ efflux, [K+]o, transmembrane action potentials, and tissue ATP, ADP, phosphocreatine, and creatine content in a unique isolated, blood-perfused papillary muscle preparation during hypoxia compared with ischemia. During 15 minutes of hypoxic perfusion (PO2, 6.1 +/- 0.9 mm Hg) with normal [K+]o of 4.1 +/- 0.1 mM, action potential duration (APD) was not altered even though tissue ATP levels decreased markedly from 33.5 +/- 1.8 to 14.7 +/- 2.0 nmol.mg protein-1 (p < 0.01). Net cellular K+ efflux, based on measured differences of [K+] between the venous effluent and the perfusate, was 13.23 +/- 0.79 mumol.g wet wt-1 during hypoxia. In contrast, after 15 minutes of zero-flow ischemia, APD at 80% of repolarization (APD80) decreased by 47% from 171 +/- 5 to 92 +/- 5 msec (p < 0.01), but integrated net cellular K+ efflux over 15 minutes of ischemia was 8.4-fold less (1.57 +/- 0.13 mumol.g wet wt-1) than during hypoxia. Tissue ATP levels, however, decreased by only 35.2% to 21.7 +/- 2.1 nmol.mg protein-1, which was significantly less than that induced by 15 minutes of hypoxia. Perfusion with hypoxic blood containing high [K+]o of 10.3 +/- 0.3 mM resulted in APD shortening similar to that observed during ischemia. Cellular K+ loss, however, was inhibited markedly by high [K+]o perfusion (only 4.51 +/- 0.28 mumol.g wet wt-1). Pretreatment with glibenclamide (5 microM), a drug that has been reported to inhibit ATP-regulated K+ channels and accelerate glycolysis in normoxic tissue, partially inhibited cellular K+ efflux during hypoxic perfusion with normal [K+]o (7.35 +/- 0.71 versus 13.23 +/- 0.79 mumol.g wet wt-1, p < 0.01) but had no significant influence on repolarization time or tissue ATP levels. Although glibenclamide partially prevented action potential shortening induced by hypoxic perfusion in the presence of elevated [K+]o, the proportion of cellular K+ efflux reduced by glibenclamide was less (23%) than that observed with glibenclamide in hypoxic perfusion with normal [K+]o (44%).(ABSTRACT TRUNCATED AT 400 WORDS)
Coronary venular responses to flow and pressure.
Authors: Kuo L , Arko F , Chilian W M , Davis M J
Published: June 2012

In previous studies, we demonstrated that both endothelium-dependent flow-induced vasodilation and endothelium-independent myogenic responses occur in porcine coronary arterioles. However, it was not established whether these responses are present in the coronary venular microcirculation. The aim of this study was to test the hypotheses that 1) coronary venules, like arterioles, exhibit flow-induced dilation and myogenic responsiveness, and 2) venular flow-induced dilation is endothelium-dependent and is mediated by the release of a nitrovasodilator. Experiments were performed in porcine subepicardial coronary venules, 80-120 microns in diameter, by using cannulated isolated vessel techniques to allow intraluminal pressure and flow to be independently controlled. Flow was initiated by simultaneously moving two perfusion reservoirs connected to the cannulating pipettes in equal amounts but in opposite directions. In the absence of flow, i.e., zero pressure gradient (delta P) between the two reservoirs, venules developed spontaneous tone to 75-80% of maximum diameter at 10 cm H2O intraluminal pressure. Venules gradually dilated in response to stepwise increases in flow (i.e., delta P). The threshold for the flow-induced dilation occurred at delta P = 1 cm H2O (flow = 3.5 nl/sec), and the maximal response (dilation to 93 +/- 2% of maximum diameter) occurred when delta P was elevated to > or = 6 cm H2O (flow = 21 nl/sec at delta P = 6 cm H2O). Flow-induced dilation was abolished after the endothelium was damaged by perfusion of an air bolus through the lumen. Vasoconstriction was observed when denuded venules were subjected to relatively high luminal flows (> or = 21 nl/sec).(ABSTRACT TRUNCATED AT 250 WORDS)
Intracellular H+ and Ca2+ modulation of trypsin-modified ATP-sensitive K+ channels in rabbit ventricular myocytes.
Authors: Fan Z , Makielski J C
Published: June 2012

ATP-sensitive K+ current (IK.ATP) channels are thought to play a role in the K+ efflux observed in cardiac ischemia. Intracellular acidosis is a prominent early effect in ischemia; therefore, the effects of acidosis on IK.ATP may have certain pathophysiological implications. Increased intracellular proton concentration (pHi) is known to regulate IK.ATP in frog skeletal muscle by increasing open probability. The pHi effect on IK.ATP is not clearly understood in heart because, unlike frog skeletal muscle, low pHi causes IK.ATP run-down in inside-out patches. This would tend to mask any opening effect of low pHi if it exists. Trypsin modification of IK.ATP has recently been shown to prevent run-down in inside-out patches. We used single channel recordings in inside-out patches to study IK.ATP after exposure to trypsin. After trypsin treatment, the open probability of IK.ATP was not sensitive to pHi in the absence of ATP. In the presence of ATP, however, a decrease in pHi consistently increased the open probability of trypsin-modified IK.ATP by reducing ATP inhibition. In the absence of ATP the mean open probability was 0.43 +/- 0.07 at pHi 7.4, and 0.5 mM ATP decreased the mean open probability to 0.03 +/- 0.04 at pHi 7.4, but mean open probability was significantly increased to 0.20 +/- 0.07 at pHi 6.3 (n = 7, p < 0.01). Ca2+ did not affect the activity of trypsin-modified IK.ATP in either the absence or presence of ATP at pHi 7.4. However, Ca2+ was able to antagonize the low pHi effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation. Is the hydroxyl radical really the most damaging radical species?
Authors: Khalid M A , Ashraf M
Published: June 2012

Isolated adult rat cardiac myocytes were subjected to anoxia and substrate deprivation for 15, 30, 60, 90, and 120 minutes and reoxygenation for 120 seconds. The supernatant and cell extract were analyzed for hydroxyl radicals (.OH) with high-performance liquid chromatography using salicylate as a trapping agent. The production of intracellular H2O2 as a possible precursor of .OH was also documented using the fluorescent probe dichlorofluorescein diacetate. The release of the cytosolic enzyme lactate dehydrogenase (LDH) and malondialdehyde (MDA) formation were used as cell injury markers. Trypan blue and horseradish peroxidase stains were used as markers for altered membrane permeability. Maximum formation of .OH was observed in myocytes subjected to 15 minutes of anoxia/reoxygenation (2.83 +/- 0.27 nmol/mg protein), at which time no injury was observed at light and ultramicroscopic levels. On the other hand, there was no correlation between the amount of .OH production and different parameters of cell injury in myocytes subjected to anoxia/reoxygenation longer than 15 minutes. Myocytes developed extensive blebbing, loss of cell membrane permeability, and ultrastructural damage. The enzyme leakage was minimal at 15 minutes (0.094 +/- 0.021 units/mg protein) and increased fivefold after 120 minutes (0.428 +/- 0.069 units/mg protein). Similarly, MDA increased from 0.78 +/- 0.14 nmol/mg protein at 15 minutes to 1.65 +/- 0.35 nmol/mg protein at 120 minutes. Incubation with 1 mM deferoxamine reduced the .OH production at all anoxic intervals, most significantly at 15 minutes, but did not decrease LDH and MDA release or provide ultrastructural preservation. However, preincubation with 2.5 microM diphenylphenylenediamine markedly reduced both LDH and MDA release and offered prominent ultrastructural protection. These results suggest that 1) myocytes were able to generate .OH endogenously; 2) maximum .OH was produced at 15 minutes after anoxic reoxygenation without compromising cell viability; 3) prolongation of the anoxic period exacerbated cell damage without parallel increase in .OH generation; 4) there was no significant production of .OH after 15 minutes of anoxia/reoxygenation with or without treatment of deferoxamine, suggesting that prolonged anoxia/reoxygenation does not induce additional .OH formation and thus mediate cell injury; and 5) it is likely that the damage to myocytes in this system was still mediated by free radicals other than .OH, as indicated by the protection by diphenylphenylenediamine against the cellular injury.
Computer simulations of three-dimensional propagation in ventricular myocardium. Effects of intramural fiber rotation and inhomogeneous conductivity on epicardial activation.
Authors: Pollard A E , Burgess M J , Spitzer K W
Published: June 2012

Three-dimensional membrane-based simulations of action potential propagation in ventricular myocardium were performed. Specifically, the effects of the intramural rotation of the fiber axes and inhomogeneous conductivity on the timing and pattern of epicardial activation were examined. Models were built, with approximately 400,000 microscopic elements arranged in rectangular parallelepipeds in each model. Simulations used the nonlinear Ebihara and Johnson membrane equations for the fast sodium current. Constructed models had histological features of ventricular myocardium. All models were anisotropic. In a subset of the models, an abrupt intramural rotation of the fiber axes was included. This feature was also combined with randomly distributed inhomogeneous conductivity and regions of high transverse resistance to represent nonuniform anisotropy in a further subset of the models. Epicardial stimuli were applied for each simulation. Three-dimensional activation patterns and epicardial isochron maps were constructed from the simulations. We noted that the rotation of fiber axes accelerated epicardial activation distant from the stimulus site. The inhomogeneous conductivity caused regional acceleration and deceleration of activation spread. We also noted features of epicardial activation that resulted from the fiber rotation, and the inhomogeneous conductivity corresponded to that observed in maps from experimental animals.
Myocardial fibrin deposition in experimental viral myocarditis that progresses to dilated cardiomyopathy.
Authors: Schnitt S J , Stillman I E , Owings D V , Kishimoto C , Dvorak H F , Abelmann W H
Published: June 2012

Myocardial fibrosis is a characteristic late feature in cases of viral myocarditis that progress to dilated cardiomyopathy. However, the pathogenesis of the myocardial fibrosis in such cases is unknown. Prior studies have shown that in healing wounds and tumor stroma generation, interstitial fibrin deposition precedes the development of fibrosis. Therefore, interstitial fibrin deposition in the myocardium was investigated in a murine model of myocarditis in which dilated cardiomyopathy develops. Inbred male C3H/He mice inoculated with coxsackievirus B3 were killed 0, 3, 7, 14, 21, 30, and 60 days after infection. Paraffin sections of hearts were stained with hematoxylin-eosin, Masson's trichrome stain, and antibodies to fibrinogen/fibrin by use of an immunoperoxidase technique. Pretreatment of all mice with anticoagulants and antifibrinolytics 5 minutes before death was used to prevent artifactual fibrin deposition and fibrinolysis during tissue manipulation. Tissue fixation in formalin supplemented with acetic acid served to extract non-cross-linked fibrin, fibrinogen, and fibrinogen and fibrin degradation products, thus ensuring that clotted and cross-linked fibrin was the major immunoreactant. Myocardial fibrin deposition and fibrosis were each quantitated by computer-assisted image analysis. Myocardial fibrin deposition first appeared on day 3, was maximal on day 14, and disappeared by day 30. Conversely, myocardial fibrosis was not detectable until day 14 and was maximal at day 60. Thus, as in healing wounds and developing tumor stroma, fibrin deposition preceded fibrosis in this murine model of myocarditis that progresses to dilated cardiomyopathy.
Selective contribution of two types of carotid sinus baroreceptors to the control of blood pressure.
Authors: Seagard J L , Hopp F A , Drummond H A , Van Wynsberghe D M
Published: June 2012

This study was performed to determine if selective elimination of afferent input from two different types of previously described baroreceptors altered the ability of the dog to regulate blood pressure (BP), examining specifically if there was differential loss of baroreceptor control of tonic levels of baseline pressure versus dynamic changes in pressure. In the first series of experiments in this study, anodal block of the carotid sinus nerve was used to selectively block afferent input in a sequence from large-diameter A-fiber carotid baroreceptors (mostly type I) to smaller A-fiber and nonmyelinated C-fiber baroreceptors (mostly type II). In the second series of experiments, anesthetic block of the carotid sinus nerve with bupivacaine was used to selectively eliminate afferent input in reverse order from anodal block, first blocking input from baroreceptors with small afferent fibers and then additionally eliminating input from the larger-diameter A-fiber baroreceptors. The effects of selective elimination of each baroreceptor type were determined by monitoring baseline BP during constant carotid sinus pressure (CSP) perfusion of a vascularly isolated carotid sinus (tonic control) and obtaining baroreflex sensitivity (slope) during ramp pressure stimulations of the carotid sinus (dynamic control) under various blocking conditions. Low levels of anodal block significantly attenuated baroreflex sensitivity (-0.84 +/- 0.11 versus -0.63 +/- 0.10 mm Hg BP/mm Hg CSP) at levels of block that had no effect on tonic baseline BP (158.41 +/- 9.5 versus 160.7 +/- 9.5 mm Hg BP). In contrast, low levels of bupivacaine block produced significant increases in tonic BP (158.8 +/- 6.4 versus 169.0 +/- 6.5 mm Hg BP), whereas there was no effect on dynamic baroreflex sensitivity (-0.85 +/- 0.08 versus -0.73 +/- 0.08 mm Hg BP/mm Hg CSP). Thus, blocking large A-fiber baroreceptors resulted in significant decreases in baroreflex sensitivity without changes in baseline levels of BP, indicating primarily an attenuation in dynamic baroreflex regulation. Blocking of smaller A-fiber and unmyelinated C-fiber baroreceptors resulted in smaller decreases in baroreflex sensitivity and significant elevations in baseline BP, indicating a loss of tonic control of pressure. These results suggest that the two types of baroreceptors contribute differently to the regulation of blood pressure.
Distinct behavior of cardiac myosin heavy chain gene constructs in vivo. Discordance with in vitro results.
Authors: Buttrick P M , Kaplan M L , Kitsis R N , Leinwand L A
Published: June 2012

Transcriptional thyroid hormone responsiveness of the cardiac alpha-myosin heavy chain (alpha-MHC) gene has been demonstrated in transfections into fetal and neonatal cardiomyocytes and in transgenic mice. However, the correspondence between the regulation of MHC expression in dissociated cells with that in the intact heart is unclear. Given the cost and time involved in generating multiple transgenic lines for the characterization of gene regulatory elements, we used direct cardiac gene transfer to identify elements regulating both basal and thyroid hormone responsive cardiac alpha-MHC gene expression in the adult heart in vivo. Sequences upstream of the rat alpha-MHC gene linked to a luciferase reporter gene were injected into the hearts of adult rats subjected to various thyroid manipulations. The 161-bp sequence upstream of the transcription start site, which contains a TATA box, a CCAATT box, and a thyroid hormone response element, was transcriptionally active but not thyroid hormone responsive. The expression of a construct containing 388 bp of upstream sequence was increased by thyroid hormone administration, a response that required an intact thyroid hormone response element. However, expression of this construct failed to decrease to basal levels in a hypothyroid state. To confer complete (positive and negative) thyroid hormone regulation, 2,936 bp of upstream sequence was sufficient. These results demonstrate that, although necessary, the thyroid hormone response element is not sufficient for complete thyroid hormone regulation of this gene in vivo. In addition, DNA sequences regulating the quantitative expression of cardiac alpha-MHC in the euthyroid state have been demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of shear stress and endothelial prostaglandins in flow- and viscosity-induced dilation of arterioles in vitro.
Authors: Koller A , Sun D , Kaley G
Published: June 2012

We have studied the effect of changes in shear stress on diameter of isolated arterioles of rat cremaster muscle. The steady-state active diameter of arterioles at a constant perfusion pressure (60 mm Hg) was 80 +/- 1.2 microns. The vessels' passive diameter (Ca(2+)-free solution) was 156 +/- 1.8 microns. Changes in shear stress were induced either by an increase in flow (velocity) or by an increase in viscosity of the perfusion solution. At a constant perfusion pressure, the stepwise increase in perfusion flow (0-80 microliters/min in 10-microliters/min steps) elicited, with a delay of approximately 20 seconds, a gradual increase in diameter up to 46%. At a constant 20-microliters/min flow rate, increases in viscosity of the perfusate (2%, 4%, and 6% dextran [molecular weight, 77,800]) caused a gradual vasodilation up to 22%. Varying flow and viscosity of the perfusate simultaneously resulted in an upward shift of the flow-diameter curve. Both flow- and viscosity-induced dilations were eliminated by the removal of the endothelium of arterioles (by air) or were inhibited by indomethacin (10(-5) M). The efficacy and specificity of these inhibitory treatments were assessed with vasoactive agents whose action, with regard to endothelial mediation, has been determined previously. The arteriolar dilation maintained calculated wall shear stress close to control values during increases in flow and/or viscosity of the perfusate, but when the dilation was inhibited by removal of the endothelium or by indomethacin, wall shear stress increased significantly in a cumulative manner.(ABSTRACT TRUNCATED AT 250 WORDS)
High alpha 1-adrenoceptor occupancy decreases relaxing potency of nifedipine by increasing myosin light chain phosphorylation.
Authors: Ratz P H
Published: June 2012

It has been proposed that the decreased potency of Ca2+ channel blockers to produce relaxation in vascular tissues contracted at high alpha 1-adrenoceptor occupancy may be caused by activation of spare receptors. Results from the present study using isolated strips of rabbit renal arteries contracted with the alpha 1-adrenoceptor agonist phenylephrine (PhE) support this hypothesis and provide a cellular explanation for the phenomenon. PhE at 1 microM activated only 23% of the total alpha 1-adrenoceptor pool but produced maximum stress while increasing the extent of myosin light chain phosphorylation to 35%. Activation of additional (spare) alpha 1-adrenoceptors with 100 microM PhE produced an additional increase in the extent of myosin light chain phosphorylation, which reached 45%, but did not produce an additional increase in stress above that produced by 1 microM PhE. A complete [PhE]-response curve revealed a quasihyperbolic dependency of stress on myosin light chain phosphorylation; i.e., a roughly linear relation existed at [PhE] values below that producing maximum stress, and at higher concentrations, phosphorylation was further increased but stress was not. Interestingly, the Ca2+ channel blocker nifedipine (0.1 microM) reduced the increases in myosin light chain phosphorylation produced by both 1 and 100 microM PhE by the same amount, approximately 12%. Nifedipine also reduced the increases in [Ca2+]i produced by 1 and 100 microM PhE by the same amount. However, nifedipine reduced the level of stress produced by 100 microM PhE by only 17%, whereas the level of stress produced by 1 microM PhE was reduced by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)
Heteromultimeric assembly of human potassium channels. Molecular basis of a transient outward current?
Authors: Po S , Roberds S , Snyders D J , Tamkun M M , Bennett P B
Published: June 2012

To gain insight into the molecular basis of cardiac repolarization, we have expressed K+ channels cloned from ventricular myocardium in Xenopus oocytes. A recently identified human cardiac K+ channel isoform (human Kv1.4) has properties similar to the 4-aminopyridine-sensitive calcium-insensitive component of the cardiac transient outward current. However, these channels recovered from inactivation much slower than native channels. Hybrid channels consisting of subunits from different K+ channel clones (delayed rectifier channels [Kv1.1, Kv1.2, and Kv1.5] and Kv1.4) were created by coinjection of cRNAs in oocytes. Multimeric channels consisting of Kv1.4:Kv1.1, Kv1.4:Kv1.2, and Kv1.4:Kv1.5 were expressed and compared. The hybrid channels displayed characteristics of heterotetrameric channels with kinetics that more closely resembled a native cardiac transient outward current. The inactivation and recovery from inactivation of the heteromeric channels indicated that the presence of a single inactivating subunit (Kv1.4) was probably sufficient to cause channel inactivation. The results demonstrate that expression of different K+ channel genes can produce channel protein subunits that assemble as heteromultimers with unique properties. It is shown that certain combinations of voltage-gated K+ channels probably do not contribute to native transient outward current. However, one combination of subunits could not be excluded. Therefore, this mechanism of channel assembly may underlie some of the functional diversity of potassium channels found in the cardiovascular system.
Smooth muscle cell proliferation. Expression and kinase activities of p34cdc2 and mitogen-activated protein kinase homologues.
Authors: Watson M H , Venance S L , Pang S C , Mak A S
Published: June 2012

Rat vascular smooth muscle cells were synchronized to the quiescent state (G0) by serum deprivation and then stimulated to enter the cell cycle by serum refeeding. At various times of the cell cycle, cells were analyzed for the expression of p34cdc2 and mitogen-activated protein kinase homologues by immunoblotting and for kinase activity toward histone H1, myelin basic protein, and caldesmon. A small amount of p34cdc2 was expressed in the G0/G1 phase (0 to 8 hours). At the G1/S transition (12 hours), the level of p34cdc2 started to accumulate and increased by 60-fold at G2/M (18 hours), accompanied by a more slowly migrating band. Histone H1 kinase activity was undetectable in anti-p34cdc2 immunoprecipitates in the G0/G1 cells but appeared around the G1/S boundary and peaked at G2/M (18 hours). The caldesmon kinase activity exhibited two distinct phases: the first appeared at G0/G1 (0 to 8 hours), and the second appeared at G1/S and continued through G2/M. Two mitogen-activated protein kinase isoforms were expressed throughout the cell cycle. Anti-mitogen-activated protein kinase immunoprecipitates possessed kinase activities toward myelin basic protein and caldesmon, which were activated within 15 minutes after serum stimulation and declined within a few hours. These findings suggest that p34cdc2 and mitogen-activated protein kinase homologues may play significant roles in regulating the progression of the cell cycle of smooth muscle cells, the former at the G2/M transition and the latter at the G0/G1 transition.
Gene targeting. The precise manipulation of the mammalian genome.
Authors: Robbins J
Published: June 2012

Genetics is a powerful tool for studying the function of different gene products as well as the physiological consequences of a normal or aberrant polypeptide. Until recently, the most powerful genetic applications have been restricted to relatively simple organisms whose genomes are more easily manipulated. For a number of years, in lower organisms, it has been possible to create defined genetic changes that are targeted to a specific locus or even to a defined codon or transcriptional regulatory sequence. With the development of gene targeting using embryonic stem cells derived from the preimplantation blastocyst of a mouse, it has become possible to extend these experiments to a mammalian system. Via homologous recombination, one can ablate, or "knock out," a defined genetic locus or mutate a particular set of nucleotides that encodes a peptide domain of interest. These techniques, when applied to genes that underlie normal cardiovascular function, promise to define the exact role(s) different proteins play during the development, growth, and maintenance of the heart. The ability to generate defined animal models of human disease in which the primary genetic defect is known should lead to fundamental advances in the study of the normal and failing heart.
Connexin expression in the developing avian cardiovascular system.
Authors: Minkoff R , Rundus V R , Parker S B , Beyer E C , Hertzberg E L
Published: June 2012

Recent observations have suggested that the patterns of expression of the gap junction protein connexin43 in the developing cardiovascular system of the avian embryo diverge significantly from the patterns previously seen in mammalian species. Therefore, a detailed analysis of connexin43 expression in the chicken embryo was performed by use of immunofluorescent localization with two different connexin43-specific antipeptide antibodies as well as Western and Northern blot analysis. Connexin43 protein was not detected in the avian myocardium, the venous system, or the smaller vessels of the arterial system. Rather, it was limited exclusively to the vessels of the arterial outflow tract in a concentric pattern that became evident by embryonic day 8. Double staining with anti-alpha-smooth muscle actin and connexin43 demonstrated colocalization in the media of outflow tract vessel walls. The developmental expression of connexin43 was found to mirror the spatial patterning of secondary actin; connexin43, however, preceded the expression of secondary actin by a period of 1-2 days. In contrast, antibodies to a related gap junction protein (connexin42) revealed an absence of immunostaining in the avian outflow tract. Double staining with anti-connexin42 and anti-A-cell adhesion molecule (specific for avian intercalated discs) demonstrated colocalization between cardiac myocytes, indicating that connexin42 is a constituent of avian myocardial gap junctions. In light of these findings, developmental expression of differing myocardial connexins may reconcile previous studies showing different physiological properties of avian and mammalian cardiac gap junctions.
Cyclooxygenase-dependent vasoconstrictor alters vascular function in the vitamin E-deprived rat.
Authors: Davidge S T , Hubel C A , McLaughlin M K
Published: June 2012

We tested the hypothesis that increased production of lipid peroxides, mediated by a dietary vitamin E deprivation, would alter the cyclooxygenase pathway of arachidonate metabolism, resulting in impaired endothelium-dependent vascular function. Mesenteric arteries from control (n = 12) and vitamin E-deprived (n = 12) Sprague-Dawley rats were studied in a myograph. Endothelium-dependent relaxations to methacholine were similar in the arteries from vitamin E-deprived rats compared with control arteries (EC50, 0.057 +/- 0.006 versus 0.065 +/- 0.009 microM). However, in the arteries from the vitamin E-deprived rats, this response was potentiated in the presence of a cyclooxygenase inhibitor (1 microM meclofenamate; EC50, 0.035 +/- 0.003 versus 0.057 +/- 0.006 microM; P < .05) or thromboxane A2/prostaglandin H2 receptor blocker (1 microM SQ 29548; EC50 0.029 +/- 0.002 versus 0.057 +/- 0.006 microM; P < .05) but had no effect on the arteries from the control rats. Endothelium-independent relaxations to sodium nitroprusside were not effected by vitamin E deprivation. Arachidonic acid increased tension twofold more in the arteries from the vitamin E-deprived rats compared with the control rats (at 1 microM; 0.43 +/- 0.05 versus 0.23 +/- 0.03 mN/mm; P < .05). The enhanced vasoconstriction was blunted, and the group difference was eliminated by a cyclooxygenase inhibitor (0.15 +/- 0.02 versus 0.43 +/- 0.05 mN/mm, P < .05) or a thromboxane A2/prostaglandin H2 receptor blocker (0.17 +/- 0.04 versus 0.43 +/- 0.05 mN/mm, P < .05). Prostaglandin endoperoxide synthase expression, determined by Western immunoblotting on aortas from the same rats, was increased in the vitamin E-deprived rats (3.14 +/- 0.8 versus 1.06 +/- 0.4 fmol/ng DNA, P < .05). In summary, mesenteric arteries from the vitamin E-deprived rats demonstrated altered endothelium-dependent responses that were in part due to a cyclooxygenase-dependent vasoconstrictor binding to the thromboxane A2/prostaglandin H2 receptor. In the aorta, there was an associative increase in the expression of prostaglandin endoperoxide synthase. We speculate that, in some vascular diseases, increased lipid peroxidation may influence endothelium-dependent vascular function by modulating the cyclooxygenase pathway of arachidonate metabolism.
Bursting of cardiac sodium channels after acute exposure to 3,5,3'-triiodo-L-thyronine.
Authors: Dudley S C , Baumgarten C M
Published: June 2012

Physiological concentrations of 3,5,3'-triiodo-L-thyronine (T3) acutely increased burst-mode gating of Na+ channels in rabbit ventricular myocytes. Bursting was measured as the ratio of long events to the total number of events multiplied by 100 (%LE); a long event was defined as a set of openings or a single opening with a total duration greater than or equal to five times the control mean open time (MOT) for cell-attached patches. In the cell-attached configuration, adding either 5 or 50 nM T3 to the pipette increased the %LE. %LE had a biphasic voltage dependence and peaked at -50 mV, although the largest percentage change from control occurred between -30 and -40 mV. Neither unitary conductance nor the overall MOT was altered by T3-induced bursting. However, the MOT of openings within bursts increased, implying a kinetically distinct mode of channel gating during bursts. Long events sometimes were grouped into runs, but the more usual pattern suggested that modal shifts occurred in approximately 1 second. Similar behavior was observed with triiodothyroacetic acid, a T3 analogue that does not elicit protein synthesis. To investigate involvement of soluble second messengers, cell-attached recordings were made with and without T3 in the bath. Placed outside the pipette, 50 and 100 nM T3 failed to alter MOT, unitary current, or %LE. Na+ channel gating also was unaffected by patch excision and by exposing the cytoplasmic face of inside-out patches to 50 nM T3. Nevertheless, excision to the inside-out configuration with 5 nM T3 in the pipette dramatically increased the %LE and lengthened MOT. These results suggest that T3 induced Na+ channel bursting by an extranuclear mechanism that requires proximity of T3 to the extracellular face of the Na+ channel. Furthermore, T3 was not membrane permeant on the time scale of these experiments. Na+ channel bursting may contribute to the propensity for arrhythmias in hyperthyroidism and to the positive inotropic effect of acute T3 administration in the stunned and ischemic myocardium.