bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells.
Authors: Cook C L , Weiser M C , Schwartz P E , Jones C L , Majack R A
Published: June 2012

Little is known about the phenotypic changes that occur in vascular smooth muscle cells (SMCs) as the developing aorta undergoes the transition from a loosely organized, highly replicative tissue to a morphologically mature, quiescent tissue. In the present study, we have characterized the in vivo pattern of SMC replication during intrauterine and neonatal aortic development in the rat and have cultured and assessed the in vitro growth properties of embryonic, fetal, and neonatal vascular SMCs. Embryonic SMCs, which exhibited a very high in vivo replication rate (75% to 80% per day), demonstrated a significant potential for self-driven replication, as assessed by the ability to proliferate under serum-deprived conditions. Several lines of evidence suggest that the autonomous growth of SMCs in the "embryonic growth phenotype" may be driven by a unique mechanism independent of known adult SMC mitogens: embryonic SMC replication was not associated with the detectable secretion of mitogenic activity capable of stimulating adult SMCs, and embryonic SMCs were mitogenically unresponsive to a variety of known adult SMC growth factors. The capacity for self-driven growth was lost by embryonic day 20, suggesting that important changes in gene expression and phenotype occur in developing SMCs between embryonic days 18 and 20. Taken together, the data describe a unique embryonic growth phenotype of vascular SMCs and suggest that the replication of aortic SMCs during intrauterine development is self driven, self regulated, and controlled by a developmental timing mechanism. The conversion of SMCs from the embryonic to the late fetal/adult growth phenotype will likely be found to be an important component of a developmental system controlling vascular morphogenesis.
Expression of prostaglandin H2-mediated mechanism of vascular contraction in hypertensive rats. Relation to lipoxygenase and prostacyclin synthase activities.
Authors: Lin L , Balazy M , Pagano P J , Nasjletti A
Published: June 2012

We tested the hypothesis that a prostanoid-mediated mechanism of vascular contraction is expressed in rats with aortic coarctation-induced hypertension. Rings of descending thoracic aorta taken from normotensive and hypertensive rats were contrasted in terms of constrictor responsiveness to arachidonic acid (AA), AA-induced release of eicosanoids, and ability to convert exogenous prostaglandin (PG) H2 to PGI2. AA (10(-8) to 10(-5) mol/L) increased isometric tension in aortic rings (bathed in Krebs' bicarbonate buffer) of hypertensive but not normotensive rats. AA (10(-5) mol/L) also elicited the release of PGI2, PGE2, thromboxane (TX) A2, and monohydroxyeicosatetraenoic acids (HETEs); this release from the aortic rings of hypertensive rats exceeded the corresponding release from the aortic rings of normotensive rats. However, the rate of conversion of exogenous PGH2 to PGI2 by aortic rings of hypertensive rats was < 50% the rate of conversion by aortic rings of normotensive rats. The constrictor effect of AA in aortic rings of hypertensive rats was abolished by an inhibitor of cyclooxygenase (indomethacin, 10 mumol/L) and a blocker of TXA2-PGH2 receptors (SQ29548, 1 mumol/L) but was not affected by an inhibitor of TXA2 synthesis (CGS13080, 10 mumol/L), suggesting mediation by PGH2. The lipoxygenase inhibitor baicalein (75 mumol/L) also attenuated the constrictor effect of AA in aortic rings of hypertensive rats while decreasing the associated release of HETEs and correcting the impairment in the conversion of PGH2 to PGI2.(ABSTRACT TRUNCATED AT 250 WORDS)
Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro.
Authors: Liaw L , Almeida M , Hart C E , Schwartz S M , Giachelli C M
Published: June 2012

Osteopontin is an Arg-Gly-Asp-containing acidic phosphoprotein recently shown to be upregulated in vascular smooth muscle during rat arterial neointima formation and in human atherosclerotic plaques. Functional studies showed that osteopontin promoted adhesion of both cultured aortic endothelial cells and aortic smooth muscle cells. Adhesion of vascular cells to osteopontin was dose dependent and half maximal when solutions containing 7 and 30 nmol/L osteopontin were used to coat wells for endothelial and smooth muscle cells, respectively. Smooth muscle cells adherent to osteopontin were spread after 60 minutes, whereas endothelial cells remained round, although flattened, at this time point but were spread at 90 minutes. Cell spreading on osteopontin was accompanied by the formation of focal adhesion plaques. A newly developed anti-osteopontin antibody completely inhibited adhesion of both cell types to osteopontin but not to fibronectin or vitronectin. In addition, the peptide GRGDSP blocked adhesion to osteopontin, suggesting that integrins mediate Arg-Gly-Asp-dependent adhesion. Indeed, an antibody against the alpha v beta 3 integrin neutralized adhesion of both endothelium and smooth muscle cells to osteopontin by approximately 50%, demonstrating that alpha v beta 3 is one osteopontin receptor on vascular cells. Osteopontin also promoted the migration of smooth muscle cells in a Boyden-type chamber, with half-maximal effects observed at 77 nmol/L osteopontin. Checkerboard analysis demonstrated that this stimulus was chemotactic in nature. Our findings suggest that osteopontin may be functionally important as an adhesive and chemotactic molecule for vascular cells, particularly when levels of osteopontin are dramatically increased, as is the case after arterial angioplasty and in atherosclerotic plaques.
Enhanced Na(+)-Ca2+ exchange activity in cardiomyopathic Syrian hamster.
Authors: Hatem S N , Sham J S , Morad M
Published: June 2012

The signaling of contraction by Ca2+ in the Syrian hamster (BIO 14.6) heart in the late stage of the cardiomyopathy (220 to 300 days old) was compared with that in age-matched healthy hamster hearts. Membrane current and cell shortening or intracellular Ca2+ transients were measured simultaneously in isolated whole-cell-clamped myocytes. The density of the L-type Ca2+ current was smaller in myopathic than in normal myocytes (2.13 +/- 0.3 versus 3.21 +/- 0.4 pA/pF at 0 mV, P < .05). In both control and myopathic myocytes, the L-type Ca2+ current gated the release of Ca2+ and activation of contraction. In myopathic myocytes, activation of contraction also activated a slowly inactivating inward current of 1.73 +/- 0.2 pA/pF. The Na(+)-Ca2+ exchanger generated this current (INa-Ca), because it was suppressed by rapid replacement of Na+ with Li+ and depletion of the intracellular Ca2+ pool by caffeine. INa-Ca, activated by rapid application of caffeine, was not significantly different in both groups (3.7 +/- 0.5 pA/pF in control hearts versus 3.9 +/- 0.5 pA/pF in cardiomyopathic hearts). The activation of the inward exchanger current in myopathic myocytes coincided with a significant prolongation of contraction and the intracellular Ca2+ transient and a delay in the onset of relaxation. These results suggest that the enhanced activity of the Na(+)-Ca2+ exchanger may be related to compromised sequestration of Ca2+ in these animals.
Protein kinase C isoform expression and regulation in the developing rat heart.
Authors: Rybin V O , Steinberg S F
Published: June 2012

To determine whether age-dependent differences in cardiac responses to autonomic agonists could result from developmental changes in protein kinase C (PKC) isoform expression, we probed extracts from the fetal, neonatal, and adult heart as well as cultured neonatal and isolated adult ventricular myocytes with specific antisera to calcium-dependent (alpha and beta) and calcium-independent (delta, epsilon and zeta) isoforms of the enzyme. Although PKC-beta immunoreactivity could not be detected in cultured neonatal or isolated adult ventricular myocytes, adult and neonatal myocytes expressed multiple other isoforms of PKC. Our studies revealed an age-dependent decline in the immunoreactivity for three PKC isoforms. PKC-alpha was detected in extracts from the fetal and 2-day-old neonatal heart as well as cultured neonatal rat ventricular myocytes. Only faint PKC-alpha immunoreactivity was detected in extracts from the adult heart, and PKC-alpha was not detected in extracts from isolated adult ventricular myocytes, suggesting that PKC-alpha resides in nonmyocyte elements in the adult heart. PKC-delta also was detected in greater abundance in fetal and neonatal than in adult myocardial extracts. The decline in PKC-alpha and PKC-delta expression occurred during the first 2 postnatal weeks. PKC-zeta was detected in greatest abundance in extracts from the fetal heart. PKC-zeta expression declined markedly by the second postnatal day, and only faint PKC-zeta immunoreactivity was detected in extracts from adult myocardium. Failure to detect PKC-zeta in extracts from isolated adult ventricular myocytes suggests that PKC-zeta resides primarily in nonmyocyte elements in the adult heart. PKC-epsilon was detected in all preparations, but it was detected in greatest abundance in extracts from neonatal hearts. In vitro sympathetic innervation of previously noninnervated neonatal ventricular myocytes or in vivo chemical sympathectomy of the neonatal heart did not modulate PKC isoform expression, suggesting that sympathetic innervation does not significantly regulate PKC isoform expression. PKC-alpha partitioned to the soluble fraction of unstimulated myocytes and was selectively translocated to the particulate fraction by Ca2+. In contrast, a major portion of the novel PKC isoforms partitioned to the particulate fraction of unstimulated myocytes. The subcellular distribution of novel PKC isoforms was not influenced by Ca2+. 12-O-Tetradecanoylphorbol 13-acetate (TPA, 300 nmol/L) induced translocation of soluble PKC-alpha, PKC-delta, and PKC-epsilon to the particulate fraction at 30 minutes and complete (PKC-alpha and PKC-delta) or 80% (PKC-epsilon) downregulation at 24 hours. PKC-zeta was not affected by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)
Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine.
Authors: Lewis W , Simpson J F , Meyer R R
Published: June 2012

Zidovudine (azidothymidine [AZT]) inhibits human immunodeficiency virus replication and reduces the severity of acquired immunodeficiency syndrome. A limiting side effect of AZT is a mitochondrial cardiac and skeletal myopathy in which the pharmacologically active derivative of AZT (AZT triphosphate) plays a critical role. The present study determined biochemical mechanisms of AZT-induced mitochondrial toxicity and identified AZT triphosphate as an inhibitor of DNA polymerase-gamma in vitro. Inhibition kinetics were defined using purified bovine cardiac mitochondrial DNA polymerase-gamma and AZT triphosphate in vitro. The Km for deoxythymidine triphosphate was 0.8 +/- 0.3 mumol/L. AZT triphosphate incubation with DNA polymerase-gamma in vitro resulted in mixed kinetics with a competitive Ki of 1.8 +/- 0.2 mumol/L and a noncompetitive Ki' of 6.8 +/- 1.7 mumol/L. These Ki and Ki' values were strikingly higher than values for retroviral reverse transcriptase but lower than values for other cellular DNA polymerases. These data support previous molecular and morphological findings in clinical AZT mitochondrial myopathy and in models of AZT myopathy in vivo. Biochemical findings suggest that inhibition of mitochondrial DNA polymerase-gamma may be integral to the pathogenesis of AZT-induced myopathy.
Steady-state mRNA levels of the sarcolemmal Na(+)-Ca2+ exchanger peak near birth in developing rabbit and rat hearts.
Authors: Boerth S R , Zimmer D B , Artman M
Published: June 2012

To functionally compensate for an underdeveloped sarcoplasmic reticulum in immature cardiomyocytes, it has been proposed that the sarcolemmal Na(+)-Ca2+ exchanger may assume a more predominant role for regulating cytosolic Ca2+. Previous studies using sarcolemma prepared from developing rabbit hearts demonstrated that Na(+)-dependent Ca2+ uptake and exchanger protein content were highest at birth and declined postnatally. To further investigate the significance of the Na(+)-Ca2+ exchanger during normal myocardial development, steady-state mRNA levels of the cardiac Na(+)-Ca2+ exchanger were quantitated by Northern blot and slot-blot analyses using poly(A+) RNA isolated from rabbit and rat ventricles at various fetal and postnatal ages. Northern analyses were performed with a 1.35-kb guinea pig cardiac Na(+)-Ca2+ exchanger cDNA probe. Exchanger mRNA levels were quantitated by densitometric scans of the slot blots, and results were normalized by reprobing the same blots with 32P 5'-end-labeled oligo(dT). In both species, exchanger mRNA levels peaked near birth and declined postnatally. Maximal levels were approximately sixfold greater in the late fetal rabbit (gestational day 29) and eightfold greater in the early newborn rat (postnatal day 1) compared with adults of the respective species. The parallel changes in exchanger mRNA and protein levels suggest that developmental regulation of cardiac Na(+)-Ca2+ exchanger expression involves pretranslational control mechanisms. These results support the concept that during normal cardiac development, Na(+)-Ca2+ exchanger expression is maximal near the time of birth and then declines postnatally as Ca2+ regulation by the sarcoplasmic reticulum reaches functional maturity.
The mechanism of termination of reentrant activity in ventricular fibrillation.
Authors: Cha Y M , Birgersdotter-Green U , Wolf P L , Peters B B , Chen P S
Published: June 2012

The reentrant wave fronts in ventricular fibrillation (VF) have only a limited life span. The mechanisms by which these reentrant wave fronts terminate are unknown. We performed computerized mapping studies in six open-chest dogs before and after right ventricular subendocardial ablation with Lugol's solution. Recordings were made with 56 bipolar electrodes separated by 3 mm. Baseline pacing was performed on the right side of the tissue to create parallel activation wave fronts. A premature 50-V shock of either anodal or cathodal polarity was given to a bar electrode on the upper edge of the tissue. Counterclockwise reentrant wave fronts and VF were induced both before (60 episodes) and after (57 episodes) subendocardial ablation with either anodal or cathodal shocks. Among these reentrant wave fronts, 8 episodes before and 10 episodes after ablation had over 10 rotations (P = NS). The reentrant wave fronts in other episodes terminated with an average of 3.2 +/- 1.9 rotations before and 3.1 +/- 1.8 rotations after the ablation (P = NS). The reentrant wave-front cycle length was 118 +/- 19 milliseconds before and 124 +/- 20 milliseconds after ablation (P = .001). Conduction block occurred when the wave front was traveling across the myocardial fibers. When conduction was blocked in these episodes, the leading edge of the reentrant wave front encountered tissue that had been excited within the past 58 +/- 12 milliseconds (range, 28 to 77 milliseconds), which corresponded to 47 +/- 12% of the preceding VF cycle length. This period was significantly shorter than the recovery period in the same region that had allowed conduction (91 +/- 19 milliseconds; range, 48 to 137 milliseconds), which corresponded to 72 +/- 18% of the preceding VF cycle length (P < .001). In nine episodes, reentrant wave-front activity terminated when wave fronts that had originated from outside the mapped tissue interfered with the reentrant pathways. Conclusions are as follows: (1) The refractory period of fibrillating ventricular muscle ranges from 48 to 77 milliseconds. Because the refractory period is much shorter than the VF cycle length, a large excitable gap is present in the reentrant circuit. The presence of a large excitable gap contributes to reentrant wave-front termination. (2) Myocardial fiber orientation is an important determinant of the site of conduction block. (3) Although subendocardial ablation slowed the wave-front propagation, it did not prevent the generation and the maintenance of reentry and VF.
Immunologic identification of Na+,K(+)-ATPase isoforms in myocardium. Isoform change in deoxycorticosterone acetate-salt hypertension.
Authors: Sweadner K J , Herrera V L , Amato S , Moellmann A , Gibbons D K , Repke K R
Published: June 2012

There are three isoforms of the catalytic (alpha) subunit of the Na+,K(+)-ATPase, each derived from a different gene, that differ in their sensitivity to inhibition by cardiac glycosides. Antibodies specific for the three isoforms were used to study Na+,K(+)-ATPase isoform expression in ventricular myocardium, where an understanding of digitalis receptor diversity is most important. In the rat heart, there is simultaneous expression of two isoforms in adult ventricle, and immunofluorescence studies demonstrated that both isoforms are expressed uniformly in cardiomyocytes. Hypertension and hypertrophy have been reported to selectively depress alpha 2 isoform mRNA levels, and we show in the present study that alpha 2 protein levels were correspondingly depressed in rats made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate and a high-salt diet. In the human heart, where mRNA for all three alpha isoforms has been reported, we detected all three isoform proteins (alpha 1, alpha 2, and alpha 3). Two isoforms (alpha 1 and alpha 3) predominated in the macaque heart; dissection of the heart showed uniformity of isoform expression in different ventricular regions but markedly less alpha 3 in the atrium. Finally, isoform-specific antibodies were used to detect which alpha isoforms were expressed in the ventricles of several commonly used experimental animals to test the correlation of isoform expression with cardiac glycoside-response heterogeneity. Two isoforms (alpha 1 and alpha 3) were found in canine myocardium, whereas only one (alpha 1) was found in sheep and guinea pig. Expression of Na+,K(+)-ATPase isoforms can thus be readily followed and related to the physiology of the digitalis receptor.
Vascular free radical release. Ex vivo and in vivo evidence for a flow-dependent endothelial mechanism.
Authors: Laurindo F R , Pedro M de A , Barbeiro H V , Pileggi F , Carvalho M H , Augusto O , da Luz P L
Published: June 2012

Mechanisms underlying production of vascular free radicals are unclear. We hypothesized that changes in blood flow might serve as a physiological stimulus for endothelial free radical release. Intact isolated aortas from 45 rabbits were perfused with the spin trap alpha-phenyl-N-tert-butylnitrone (PBN, 20 mmol/L) and formed radical adducts detected by electron paramagnetic resonance spectroscopy (EPR). Sequential perfusion at 2, 7.5, and 12 mL/min changed cumulative vascular PBN radical adduct yields, respectively, from 3.2 +/- 0.9 to 4.1 +/- 0.7 (P < .05) and 7.0 +/- 1.5 (P < .005) pmol/mg with endothelium and from 3.6 +/- 1.6 to 3.8 +/- 1.4 and 2.2 +/- 0.8 pmol/mg without endothelium (P = NS). In endothelialized aortas, superoxide dismutase (SOD) completely blocked flow-induced free radical production, whereas inactivated SOD, indomethacin, and the nitric oxide synthetase antagonist nitro-L-arginine methyl ester (L-NAME) had no effect; relaxations to acetylcholine remained unchanged with higher flows. To assess the role of flow on in vivo radical production, femoral arterial plasma levels of the ascorbyl radical, a stable ascorbate oxidation product, were measured by direct EPR in 56 other rabbits. Ascorbyl levels were assessed at baseline (30.2 +/- 0.7 nmol/L) and at peak-induced iliac flow changes. Flow increases from 25% to 100% due to saline injections through an extracorporeal aortic loop induced significant dose-dependent increases in ascorbyl levels (n = 5). In addition, after papaverine bolus injections, flow increased by 114 +/- 8% versus baseline, and ascorbyl levels increased by 5.4 +/- 0.7 nmol/L (n = 31, P < .001); similar results occurred with adenosine, isoproterenol, or hyperemia after 30-second occlusions (P < .05, n = 4 or 5 in each group). Active SOD completely blocked papaverine-induced ascorbyl radical increase, despite preserved flow response (delta ascorbyl = 0.02 +/- 1.6 nmol/L, P = NS); inactivated SOD, catalase, indomethacin, and L-NAME had no effect. Blood flow decreases of 65% to 100% due to phenylephrine or 60-second balloon occlusions were accompanied by an average decrease of 4.4 nmol/L (P < .05) in ascorbyl levels. No change in ascorbyl signal was observed when rabbit blood alone was submitted to in vitro flow increases through a tubing circuit. Thus, increases in blood flow trigger vascular free radical generation; such a response seems to involve endothelium-derived superoxide radicals unrelated to cyclooxygenase or nitric oxide synthetase activities. This mechanism may contribute to explain vascular free radical generation in physiological or pathological circumstances.
8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes.
Authors: Shah A M , Spurgeon H A , Sollott S J , Talo A , Lakatta E G
Published: June 2012

The role of cGMP in myocardial contraction is not established. Recent reports suggest that nitric oxide, released by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. We studied the effects of 8-bromo-cGMP (8bcGMP, 50 mumol/L) on contraction (cell shortening) and simultaneous intracellular Ca2+ transients (indo 1 fluorescence ratio) in intact adult rat ventricular myocytes (0.5 Hz and 25 degrees C) 8bcGMP reduced myocyte twitch amplitude and time to peak shortening (-19.6 +/- 4.2% and -17.6 +/- 1.3%, respectively) and increased steady-state diastolic cell length (+0.6 +/- 0.1 microns, mean +/- SEM, n = 8; all P < .05) but had no effect on shortening velocity, systolic or diastolic fluorescence ratio, or time to peak fluorescence ratio (all P = NS). In 7 of 13 myocytes, this negative inotropic effect was preceded by a transient positive inotropic effect, with small increases in twitch amplitude, shortening velocity, and cytosolic Ca2+ transient. Analysis of 8bcGMP effects on both the dynamic and steady-state relation between cell shortening and intracellular Ca2+ (during twitch contraction and tetanic contraction, respectively) indicated reduction in the myofilament response to Ca2+ in all cases. These 8bcGMP effects were inhibited by KT5823 (1 mumol/L), an inhibitor of cGMP-dependent protein kinase, or by the presence of isoproterenol (3 nmol/L). 8bcGMP had no effect on cytosolic pH in cells (n = 4) loaded with the fluorescent probe carboxyseminaphthorhodafluor-1. These data indicate that cGMP may modulate myocardial relaxation and diastolic tone by reducing the relative myofilament response to Ca2+, probably via cGMP-dependent protein kinase.
Tissue-specific determinants of anisotropic conduction velocity in canine atrial and ventricular myocardium.
Authors: Saffitz J E , Kanter H L , Green K G , Tolley T K , Beyer E C
Published: June 2012

Electrical conduction is very rapid and highly anisotropic in atrial fiber bundles, such as the crista terminalis. In contrast to left ventricular myocardium in which the ratio of longitudinal to transverse conduction velocities is approximately 3, propagation velocity in the crista terminalis is approximately 10 times greater in the longitudinal than in the transverse direction. To elucidate potential determinants of these distinct conduction properties, we characterized structural and molecular features of intercellular coupling in the crista terminalis and left ventricular myocardium of the canine heart. Analysis of the number and spatial orientation of myocyte interconnections at gap junctions revealed that a typical left ventricular myocyte was connected to 11.3 +/- 2.2 other myocytes. Approximately equal numbers of connections occurred between ventricular myocytes juxtaposed in side-to-side and end-to-end orientation. In contrast, a typical myocyte of the crista terminalis was connected to only 6.4 +/- 1.7 other cells (P < .05), but nearly 80% of these connections occurred between cells oriented in an end-to-end configuration. In comparison with the ventricular pattern, this spatial distribution of connections would limit intercellular current transfer between laterally apposed cells and thereby enhance anisotropy of conduction velocity in the longitudinal and transverse directions. Ultrastructural analysis showed that crista terminalis myocytes were connected by numerous small gap junctions that occurred in relatively simple, straight intercalated disks. Northern blot analysis showed approximately equivalent amounts of mRNAs encoding the gap junction channel proteins connexin43 and connexin45 but approximately four times more connexin40 mRNA in crista terminalis than in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)
A dynamic model of the cardiac ventricular action potential. II. Afterdepolarizations, triggered activity, and potentiation.
Authors: Luo C H , Rudy Y
Published: June 2012

The action potential model presented in our accompanying article in this journal is used to investigate phenomena that involve dynamic changes of [Ca2+]i, as described below. Delayed afterdepolarizations (DADs) are induced by spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), which, in turn, activates both the Na(+)-Ca2+ exchanger (INaCa) and a nonspecific Ca(2+)-activated current (Ins(Ca)). The relative contributions of INaCa and of Ins(Ca) to the generation of DADs are different under different degrees of Ca2+ overload. Early afterdepolarizations (EADs) can be categorized into two types: (1) plateau EADs, resulting from a secondary activation of the L-type Ca2+ current during the plateau of an action potential, and (2) phase-3 EADs, resulting from activation of INaCa and Ins(Ca) by increased [Ca2+]i due to spontaneous Ca2+ release from the SR during the late repolarization phase. Spontaneous rhythmic activity and triggered activity are caused by spontaneous Ca2+ release from the SR under conditions of Ca2+ overload. Postextrasystolic potentiation reflects the time delay associated with translocation of Ca2+ from network SR to junctional SR. The cell is paced at high frequencies to investigate the long-term effects on the intracellular ionic concentrations.
Triamterene inhibits the delayed rectifier potassium current (IK) in guinea pig ventricular myocytes.
Authors: Daleau P , Turgeon J
Published: June 2012

In humans, proarrhythmia during therapy with action potential-prolonging drugs can be associated with hypokalemia often provoked by concomitant administration of diuretic agents. Consequently, therapy with class III antiarrhythmics and K(+)-sparing diuretics, such as triamterene, may be indicated. Triamterene, along with its K(+)-sparing properties, exhibits other pharmacological effects. In the heart, it can increase action potential duration (guinea pig atria and papillary muscles), protect against reperfusion-induced arrhythmias (rat), and increase the QT interval (humans). Therefore, studies were undertaken to assess effects of triamterene on cardiac K+ repolarizing currents. Guinea pig ventricular myocytes were superfused at 30 degrees C with Cd(2+)-containing solution to block Isi and held at -40 mV to inactivate INa. Currents were measured in the whole-cell configuration of the patch-clamp technique. The delayed rectifier outward current (IK) was elicited by short (250-millisecond) and long (5000-millisecond) depolarizing pulses, and time-independent currents were assessed by a rapid ramp test protocol. After high-voltage long pulses (+50 mV; 5000 milliseconds), tail current amplitude of the slow component of IK (IKs) was decreased 36 +/- 6% (n = 6) and 51 +/- 8% (n = 6) by triamterene 10(-5) and 10(-4) mol/L, respectively. After low-voltage short pulses (-20 mV; 250 milliseconds), tail current amplitude corresponding essentially to the rapid component of IK (IKr) was decreased only 14 +/- 11% (n = 9) and 19 +/- 10% (n = 10) by triamterene 10(-5) and 10(-4) mol/L, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
In vivo myocardial kinetics of air-filled albumin microbubbles during myocardial contrast echocardiography. Comparison with radiolabeled red blood cells.
Authors: Jayaweera A R , Edwards N , Glasheen W P , Villanueva F S , Abbott R D , Kaul S
Published: June 2012

Myocardial contrast echocardiography (MCE) is a new technique for assessing myocardial perfusion that uses intracoronary injections of microbubbles of air. Because these microbubbles have a mean diameter of 4.3 +/- 0.3 microns and an intravascular rheology similar to that of red blood cells (RBCs), we hypothesized that their mean myocardial transit rates recorded on echocardiography would provide an estimation of regional myocardial blood flow in the in vivo beating heart. Accordingly, blood flow to the left anterior descending coronary artery (LAD) of 12 open-chest anesthetized dogs (group I) was adjusted to 4 to 6 flows (total of 60 flows), and microbubbles and radiolabeled RBCs were injected into the LAD in a random order at each stage. The mean myocardial RBC transit rates were measured by fitting a gamma-variate function to time-activity plots generated by placing a miniature CsI2 probe over the anterior surface of the heart, and the mean myocardial microbubble transit rates were measured from time-intensity plots derived from off-line analysis of MCE images obtained during the injection of microbubbles. An excellent correlation was noted between flow (measured with an extracorporeal electromagnetic flow probe) and mean myocardial RBC transit rate (y = 2.83 x 10(-3)x + 0.01, r = .96, SEE = 0.02, P < .001). A close correlation was also noted between mean RBC and microbubble myocardial transit rates (y = 1.01x + 0.01, r = .89, SEE = 0.02, P < .001). Despite its theoretical advantages, a lagged normal density function did not provide a better fit to the MCE data than the gamma-variate function.(ABSTRACT TRUNCATED AT 250 WORDS)
DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions.
Authors: Wu M L , Tsai M L , Tseng Y Z
Published: June 2012

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP acidified the pHi in both normal and SHR myocytes (by 0.21 +/- 0.03 and 0.33 +/- 0.03 pH units, respectively); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pHi by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardiac myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropyl amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only partially inhibited the pHi recovery from internal acidosis under nominally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pHi recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pHi regulators can be significantly activated under resting or acidified pHi in HEPES-buffered medium, probably because of the cellular production of CO2. The contribution of these bicarbonate-dependent pHi regulators, ie, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger, cannot therefore be ignored even under nominally HCO3-free conditions.
Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B.
Authors: Au Y P , Dobrowolska G , Morris D R , Clowes A W
Published: June 2012

Heparin is a potent inhibitor of the proliferation and migration of vascular smooth muscle cells. This agent selectively inhibits the transcription of tissue-type plasminogen activator and interstitial collagenase, probably by decreasing the binding of activator protein-1 (AP-1) to phorbol ester-responsive elements in the promoters of these genes. Decreased AP-1 binding is not due to a direct inhibition by heparin, since heparinase digestion of nuclear extracts prepared from heparin-treated smooth muscle cells does not restore AP-1 binding activity. Treatment of cells with heparin suppresses the expression of Jun B, one of the components of AP-1. The major effect of heparin is at the level of posttranslational modification of Jun B. Results from pulse-chase labeling experiments show that the newly synthesized Jun B is rapidly converted to a higher-molecular-weight form and that conversion is suppressed by heparin. Evidence is presented suggesting that the heparin-inhibited event is phosphorylation of Jun B.
Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents.
Authors: Maltsev V A , Wobus A M , Rohwedel J , Bader M , Hescheler J
Published: June 2012

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.
Rat dilated cardiomyopathy after autoimmune giant cell myocarditis.
Authors: Kodama M , Hanawa H , Saeki M , Hosono H , Inomata T , Suzuki K , Shibata A
Published: June 2012

One of the possible causes of dilated cardiomyopathy is considered to be a sequel to myocarditis. Two mechanisms have been proposed in the process of progression of myocarditis into dilated cardiomyopathy: one is a persistent viral infection, and the other is an autoimmune myocardial injury. To clarify the possible part played by the autoimmune mechanism in the process, using an animal model, we investigated whether autoimmune myocarditis, exclusively not related to viral infection, might develop into dilated cardiomyopathy. Experimental autoimmune myocarditis was elicited in Lewis rats by immunization with cardiac myosin fraction. Rats of the control group were immunized with ovalbumin. The clinical course was observed over 4 months. Six rats from the myosin-immunized group died during the acute phase and the healing phase, and all those rats had severe myocarditis. All rats that survived until the end of the study showed enlarged and discolored hearts. Aneurysmal changes were observed in the right ventricle during thoracotomy. The ratio of heart weight to body weight of the myosin-immunized group was significantly higher than that of the control group (3.36 +/- 0.49 versus 2.69 +/- 0.06 g/kg, respectively; P < .005). The lengths of the anterior interventricular fissure and the posterior interventricular fissure of the hearts of the myosin-immunized group were significantly longer than those of the control group. The external diameter of the left ventricle of the myosin-immunized group was also significantly larger than that of the control group. Diffuse myocardial muscle loss and replacement fibrosis were the prominent histological findings of the rats of the myosin-immunized group.(ABSTRACT TRUNCATED AT 250 WORDS)
Relation between myocardial function and expression of sarcoplasmic reticulum Ca(2+)-ATPase in failing and nonfailing human myocardium.
Authors: Hasenfuss G , Reinecke H , Studer R , Meyer M , Pieske B , Holtz J , Holubarsch C , Posival H , Just H , Drexler H
Published: June 2012

Expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase was shown to be reduced in failing human myocardium. The functional relevance of this finding, however, is not known. We investigated the relation between myocardial function and protein levels of SR Ca(2+)-ATPase in nonfailing human myocardium (8 muscle strips from 4 hearts) and in myocardium from end-stage failing hearts with dilated (10 muscle strips from 9 hearts) or ischemic (7 muscle strips from 5 hearts) cardiomyopathy. Myocardial function was evaluated by the force-frequency relation in isometrically contracting muscle strip preparations (37 degrees C, 30 to 180 min-1). In nonfailing myocardium, twitch tension rose with increasing rates of stimulation and was 76% higher at 120 min-1 compared with 30 min-1 (P < .02). In failing myocardium, there was no significant increase in average tension at stimulation rates above 30 min-1. At 120 min-1, twitch tension was decreased by 59% (P < .05) in dilated cardiomyopathy and 76% (P < .05) in ischemic cardiomyopathy compared with nonfailing myocardium. Protein levels of SR Ca(2+)-ATPase, normalized per total protein or per myosin, were reduced by 36% (P < .02) or 32% (P < .05), respectively, in failing compared with nonfailing myocardium. SR Ca(2+)-ATPase protein levels were closely related to SR Ca2+ uptake, measured in homogenates from the same hearts (r = .70, n = 16, and P < .005).(ABSTRACT TRUNCATED AT 250 WORDS)
Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin.
Authors: Wettwer E , Amos G J , Posival H , Ravens U
Published: June 2012

In various mammalian species, shapes of action potentials vary within the cardiac wall because of differences in transient outward current (Ito). A prominent Ito exists in human ventricular myocytes, but cells have not been separated according to their original localization. Human ventricular myocytes were isolated from separated subepicardial and subendocardial tissue, and regional variations in Ito were studied. Ito was larger in subepicardial than subendocardial cells. Current density at +60 mV was 7.9 +/- 0.7 pA/pF (n = 28) in subepicardial cells and 2.3 +/- 0.3 pA/pF (n = 16) in subendocardial cells. When cells from explanted failing and nonfailing donor hearts were compared, Ito was not different in subepicardial cells; however, it was larger in subendocardial cells from nonfailing hearts. The potential of half-maximal activation (V0.5) was more positive in subendocardial cells (+25.6 +/- 3.5 mV, n = 15) than in subepicardial cells (+9.2 +/- 1.8 mV, n = 28). There was no difference in V0.5 between cells from failing and nonfailing hearts. Ito inactivation was similar in all cell types and independent of membrane depolarization (time constant [tau] = approximately 60 milliseconds at 22 degrees C). The potential of half-maximal steady-state inactivation was similar in all cell types. Recovery from inactivation of Ito was fast in subepicardial cells at -100 mV (tau = 24 +/- 4 milliseconds, n = 6), exceeding control values transiently (overshoot), and slow at -40 mV without overshoot (tau = 638 +/- 91 milliseconds, n = 6). In subendocardial cells, Ito recovered at -100 mV with a fast phase (tau = 25 milliseconds) and a slow phase (tau = 328 milliseconds), and recovery was not complete after 6 seconds at -100 mV. In conclusion, regional differences in Ito between subepicardial and subendocardial cells may have clinical implications with respect to rhythmic disturbance during heart failure.
Anisotropic conduction in monolayers of neonatal rat heart cells cultured on collagen substrate.
Authors: Fast V G , Kléber A G
Published: June 2012

Anisotropic impulse conduction was studied in neonatal rat heart cell monolayers produced by culturing cells on a growth-directing substrate of collagen. Monolayers consisting of parallel-oriented cells without visible intercellular clefts were selected for experiments; cell lengths and widths were 65.8 +/- 12.5 and 12.2 +/- 3.2 microns (n = 49), respectively. Action potential upstrokes were measured by using 12 photodiodes selected within a 10x10 diode array and a voltage-sensitive dye (RH-237). The size of the area sensed by a single diode was 14 x 14 microns. High-density multiple recordings (resolution, up to 15 microns) demonstrated the variability of local activation delays and of the maximal rate of rise of the action potential upstroke (Vmax), which are presumably related to the microscopic cellular architecture. Mean macroscopic conduction velocities measured over distances of 135 microns were 34.6 +/- 4.5 and 19.0 +/- 4.3 cm/s (mean +/- SD, n = 13, P < .0001) in longitudinal and transverse directions, respectively. The anisotropic velocity ratio was 1.89 +/- 0.38 (n = 13). Mean Vmax was not significantly different in two directions (122.0 +/- 17.4 V/s longitudinally versus 125.2 +/- 15.6 V/s transversely, n = 13, P = NS). In conclusion, we developed an anisotropic cell culture model suitable for studying impulse conduction with cellular resolution. The anisotropic velocity ratio was close to values measured in vivo. By contrast, Vmax was not dependent on the direction of propagation.
Multiple phenotypically distinct smooth muscle cell populations exist in the adult and developing bovine pulmonary arterial media in vivo.
Authors: Frid M G , Moiseeva E P , Stenmark K R
Published: June 2012

Different smooth muscle cell (SMC) functions may require different cell phenotypes. Because the main pulmonary artery performs diverse functions, we hypothesized that it would contain heterogeneous SMC populations. If the hypothesis were confirmed, we wished to determine the developmental origin of the different populations. Using specific antibodies, we analyzed the expression of smooth muscle (SM) contractile and cytoskeletal proteins (alpha-SM-actin, SM myosin, calponin, desmin, and meta-vinculin) in the main pulmonary artery of fetal (60 to 270 days of gestation), neonatal, and adult animals. We demonstrated the existence of a complex, site-specific heterogeneity in the structure and cellular composition of the pulmonary arterial wall. We found that at least four cell/SMC phenotypes, based on immunobiochemical characteristics, cell morphology, and elastic lamellae arrangement pattern, were simultaneously expressed within the mature arterial media. Further, we were able to assess phenotypic alterations in each of the four identified cell populations during development. We found that each cell population within the arterial media expressed alpha-SM-actin at least at certain stages of development, thus demonstrating its smooth muscle identity. However, each cell population progressed along different developmental pathways, suggesting the existence of multiple and distinct cell lineages. A novel anti-metavinculin antibody described in this study reliably distinguished one SMC population from the others during all the developmental stages analyzed. We conclude that the pulmonary arterial media is indeed composed of multiple phenotypically distinct cell/SMC populations with unique lineages. We speculate that these distinct cell populations may serve different functions within the arterial media and may also respond in unique ways to pathophysiological stimuli.
Physiological concentrations of nitric oxide do not elicit an acute negative inotropic effect in unstimulated cardiac muscle.
Authors: Weyrich A S , Ma X L , Buerke M , Murohara T , Armstead V E , Lefer A M , Nicolas J M , Thomas A P , Lefer D J , Vinten-Johansen J
Published: June 2012

We examined the effect of several nitric oxide (NO) donors, authentic NO gas, and L-arginine in isolated cat and rat papillary muscles. We did not observe significant inotropic effects in response to any NO donor (ie, SPM-5185, C87-3754, and S-nitroso-N-acetylpenicillamine [SNAP]) from 1 nmol/L to 100 mumol/L. Similarly, authentic NO, at concentrations far in excess of those that maximally dilate the coronary vasculature (ie, 500 nmol/L), also failed to exert a detectable inotropic effect in these preparations. However, in the presence of 5 mumol/L norepinephrine, 500 nmol/L NO exerted a 12 +/- 3% decrease in isolated rat papillary muscle contractility (P < .05). Addition of L-arginine up to 25 mmol/L exerted no inotropic effects in isolated rat papillary muscles. However, at 50 mmol/L, L-arginine decreased contractile force by 21 +/- 4% (P < .01). On further examination, the negative inotropic effect of 50 mmol/L L-arginine appeared to be nonspecific, since the inactive stereoisomer, D-arginine, at 50 mmol/L exerted the same effect. Further studies in isolated adult rat cardiac myocytes elicited similar results, in that 50 mmol/L of L- and D-arginine equally decreased contraction amplitude and the underlying cytosolic calcium transient. Moreover, 500 nmol/L of the NO donor SPM-5185 only modestly decreased contraction amplitude or intracellular calcium in isolated rat cardiac myocytes. These results indicate that administration of physiological concentrations of exogenous NO does not acutely depress the inotropic state of the rat or cat heart to a physiologically significant extent.(ABSTRACT TRUNCATED AT 250 WORDS)