Complex temporal patterns of spontaneous initiation and termination of reentry in a loop of cardiac tissue.

A two-component model is developed consisting of a discrete loop of cardiac cells that circulates action potentials as well as a pacing mechanism. Physiological properties of cells such as restitutions of refractoriness and of conduction velocity are given via experimentally measured functions. The dynamics of circulating pulses and the pacer's action are regulated by two threshold relations. Patterns of spontaneous initiations and terminations of reentry (SITR) generated by this system are studied through numerical simulations and analytical observations. These patterns can be regular or irregular; causes of irregularities are identified as the threshold bistability (T-bistability) of reentrant circulation and in some cases, also phase-resetting interactions with the pacer.

Regulation of cellular infiltration into tissue engineering scaffolds composed of submicron diameter fibrils produced by electrospinning.

We characterize the infiltration of interstitial cells into tissue engineering scaffolds prepared with electrospun collagen, electrospun gelatin, electrospun poly(glycolic) acid (PGA), electrospun poly(lactic) acid (PLA), and an electrospun PGA/PLA co-polymer. Electrospinning conditions were optimized to produce non-woven tissue engineering scaffolds composed of individual fibrils less than 1000 nm in diameter. Each of these materials was then electrospun into a cylindrical construct with a 2 mm inside diameter with a wall thickness of 200-250 microm. Electrospun scaffolds of collagen were rapidly, and densely, infiltrated by interstitial and endothelial cells when implanted into the interstitial space of the rat vastus lateralis muscle. Functional blood vessels were evident within 7 days. In contrast, implants composed of electrospun gelatin or the bio-resorbable synthetic polymers were not infiltrated to any great extent and induced fibrosis. Our data suggests that topographical features, unique to the electrospun collagen fibril, promote cell migration and capillary formation.

Modulation of cardiac Na(+) current by gadolinium, a blocker of stretch-induced arrhythmias.

Gd(3+) blocks stretch-activated channels and suppresses stretch-induced arrhythmias. We used whole cell voltage clamp to examine whether effects on Na(+) channels might contribute to the antiarrhythmic efficacy of Gd(3+). Gd(3+) inhibited Na(+) current (I(Na)) in rabbit ventricle (IC(50) = 48 microM at -35 mV, holding potential -120 mV), and block increased at more negative test potentials. Gd(3+) made the threshold for I(Na) more positive and reduced the maximum conductance. Gd(3+) (50 microM) shifted the midpoints for activation and inactivation of I(Na) 7.9 and 5.7 mV positive but did not alter the slope factor for either relationship. Activation and inactivation kinetics were slowed in a manner that could not be explained solely by altered surface potential. Paradoxically, Gd(3+) increased I(Na) under certain conditions. With membrane potential held at -75 mV, Gd(3+) still shifted threshold for activation positive, but I(Na) increased positive to -40 mV, causing the current-voltage curves to cross over. When availability initially was low, increased availability induced by Gd(3+) dominated the response at test potentials positive to -40 mV. The results indicate that Gd(3+) has complex effects on cardiac Na(+) channels. Independent of holding potential, Gd(3+) is a potent I(Na) blocker near threshold potential, and inhibition of I(Na) by Gd(3+) is likely to contribute to suppression of stretch-induced arrhythmias.

Cardioplegia-induced cell swelling: prevention by normothermic infusion.

BACKGROUND: Previous work has shown significant swelling of isolated rabbit myocytes exposed to cold hyperkalemic cardioplegia; however, the effect of warm hyperkalemic cardioplegia on myocyte volume is unknown. This study examined the effect of warm hyperkalemic cardioplegia (St. Thomas' solution) on myocyte volume. METHODS: Myocytes were enzymatically isolated and placed on an inverted video microscope. Tyrode's solution (37 degrees C) was infused for 10 minutes to establish baseline cell volumes. Subsequently, either the control Tyrode's or St. Thomas' was infused either at 37 degrees C and 9 degrees C respectively (n = 5 for all groups) for 20 minutes, followed by a 30-minute reperfusion with 37 degrees C Tyrode's. Cell volume was determined from cell images captured every 5 minutes. RESULTS: Myocyte swelling occurred rapidly on exposure to cold St. Thomas' solution to a maximum of 9.8 +/- 2.1% (p < 0.001). In contrast, myocytes exposed to warm cardioplegia did not show any volume changes during exposure to cardioplegia. However, upon reexposure to Tyrode's, these cells showed shrinkage below their baseline volume (p < 0.001). CONCLUSIONS: The cell swelling associated with hypothermic cardioplegia is prevented by normothermic infusion.

Existence of a transient outward K(+) current in guinea pig cardiac myocytes.

A novel transient outward K(+) current that exhibits inward-going rectification (I(to.ir)) was identified in guinea pig atrial and ventricular myocytes. I(to.ir) was insensitive to 4-aminopyridine (4-AP) but was blocked by 200 micromol/l Ba(2+) or removal of external K(+). The zero current potential shifted 51-53 mV/decade change in external K(+). I(to.ir) density was twofold greater in ventricular than in atrial myocytes, and biexponential inactivation occurs in both types of myocytes. At -20 mV, the fast inactivation time constants were 7.7 +/- 1.8 and 6.1 +/- 1.2 ms and the slow inactivation time constants were 85.1 +/- 14.8 and 77.3 +/- 10.4 ms in ventricular and atrial cells, respectively. The midpoints for steady-state inactivation were -36.4 +/- 0.3 and -51.6 +/- 0.4 mV, and recovery from inactivation was rapid near the resting potential (time constants = 7.9 +/- 1.9 and 8.8 +/- 2.1 ms, respectively). I(to.ir) was detected in Na(+)-containing and Na(+)-free solutions and was not blocked by 20 nmol/l saxitoxin. Action potential clamp revealed that I(to.ir) contributed an outward current that activated rapidly on depolarization and inactivated by early phase 2 in both tissues. Although it is well known that 4-AP-sensitive transient outward current is absent in guinea pig, this Ba(2+)-sensitive and 4-AP-insensitive K(+) current has been overlooked.

Activation of mitogen-activated protein kinases is required for alpha1-adrenergic agonist-induced cell scattering in transfected HepG2 cells.

Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.

Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.

Rabbit ventricular myocyte volume changes as a direct result of crystalloid cardioplegia in congestive heart failure induced by aortic regurgitation.

OBJECTIVES: We hypothesized that the cell volume of ventricular myocytes isolated from hearts in volume-overload congestive failure would respond differently to hypothermic cardioplegia than would sham-operated cohorts. METHODS: Adult rabbits underwent either valvotomy and aortic regurgitation-induced heart failure or sham surgery. Congestive failure was confirmed clinically and by means of echocardiography. Cell volumes of isolated myocytes were measured by digital video microscopy. After equilibration in 37 degrees C physiologic solution, cells were suprafused with 9 degrees C standard or low-Cl(-) St Thomas' Hospital solution followed by reperfusion in 37 degrees C physiologic solution. RESULTS: Exposure to cold St Thomas' Hospital solution for 20 minutes caused sham myocytes to swell by 8% (n = 9); cell volumes fully recovered on normothermic reperfusion. In contrast, congestive failure myocytes (n = 9) maintained their cell volume in cold St Thomas' Hospital solution and during reperfusion. Lowering the [K(+)][Cl(-)] product of St Thomas' Hospital solution by partially replacing Cl(-) with an impermeant anion prevented cellular edema in the sham group (n = 8) but caused a 4% swelling in failure myocytes (n = 10) on reperfusion. Osmotically shrinking the failure cells (n = 9) converted their behavior to that of sham cells. CONCLUSIONS: In the absence of ischemia, congestive failure myocytes are less sensitive to cardioplegia-induced edema than sham cells. Low-Cl(-) cardioplegia, which prevents edema and protects the normal heart, induced swelling and may be detrimental in myopathic hearts. Differences in volume regulation in failure and sham myocytes may be due to activation of volume-sensitive channels that are turned off by osmotic shrinkage.

Age-related effects of St Thomas' Hospital cardioplegic solution on isolated cardiomyocyte cell volume.

OBJECTIVES: We tested the hypothesis that neonatal cells are more sensitive to cardioplegia-induced cell swelling than more mature cells and spontaneous swelling in the absence of ischemia can be prevented by cardioplegia with a physiologic KCl product. METHODS: Cell volumes of isolated ventricular myocytes from neonatal (3-5 days), intermediate (10-13 days), and adult (>6 weeks) rabbits were measured by digital video microscopy. After equilibration in 37 degrees C physiologic solution, cells were suprafused with 37 degrees C or 9 degrees C St Thomas' Hospital solution (standard or low Cl(-)) or 9 degrees C physiologic solution followed by reperfusion with 37 degrees C physiologic solution. RESULTS: Neonatal cells swelled 16.2% +/- 1.8% (P <.01) in 37 degrees C St Thomas' Hospital solution and recovered during reperfusion, whereas more mature cells maintained constant volume. In contrast, 9 degrees C St Thomas' Hospital solution caused significant age-dependent swelling (neonatal, 16.8% +/- 1.5%; intermediate, 8.6% +/- 2.1%; adult, 5.6% +/- 1.1%). In contrast to more mature cells, neonatal cells remained significantly edematous throughout reperfusion (8.1% +/- 1.5%). Swelling was not due to hypothermia because 9 degrees C physiologic solution did not affect volume. Lowering the KCl product of St Thomas' Hospital solution by partially replacing Cl(-) with an impermeant anion prevented cellular edema in all groups. CONCLUSION: In the absence of ischemia, neonatal cells were more sensitive to cardioplegia-induced cellular edema than more mature cells, and edema observed in all groups was avoided by decreasing the KCl product of St Thomas' Hospital solution to the physiologic range. Differences in cell volume regulation may explain the sensitivity of neonatal hearts to hyperkalemic cardioplegic arrest and suggest novel approaches to improving myocardial protection.

Swelling-activated chloride current is persistently activated in ventricular myocytes from dogs with tachycardia-induced congestive heart failure.

The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) anion channels was tested. Digital video microscopy and amphotericin-perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing-induced CHF. In normal myocytes, osmotic swelling in 0.9T to 0.6T solution (T, relative osmolarity; isosmotic solution, 296 mOsmol/L) was required to elicit ICl,swell, an outwardly rectifying swelling-activated Cl- current that reversed near -33 mV and was inhibited by 1 mmol/L 9-anthracene carboxylic acid (9AC), an anion channel blocker. Block of ICl,swell by 9AC simultaneously increased the volume of normal cells in hyposmotic solutions by up to 7%, but 9AC had no effect on volume in isosmotic or hyperosmotic solutions. In contrast, ICl,swell was persistently activated under isosmotic conditions in CHF myocytes, and 9AC increased cell volume by 9%. Osmotic shrinkage in 1.1T to 1.5T solution inhibited both ICl,swell and 9AC-induced cell swelling in CHF cells, whereas osmotic swelling only slightly increased ICl,swell. The current density for fully activated 9AC-sensitive ICl,swell was 40% greater in CHF than normal myocytes. In both groups, 9AC-sensitive current and 9AC-induced cell swelling were proportional with changes in osmolarity and 9AC concentration, and the effects of 9AC on current and volume were blocked by replacing bath Cl- with methanesulfonate. CHF thus altered the set point and magnitude of ICl,swell and resulted in its persistent activation. We previously observed analogous regulation of mechanosensitive cation channels in the same CHF model. Mechanosensitive anion and cation channels may contribute to the electrophysiological and contractile derangements in CHF and may be novel targets for therapy.

Plasmalogen-derived lysolipid induces a depolarizing cation current in rabbit ventricular myocytes.

Plasmalogen rather than diacyl phospholipids are the preferred substrate for the cardiac phospholipase A2 (PLA2) isoform activated during ischemia. The diacyl metabolite, lysophosphatidylcholine, is arrhythmogenic, but the effects of the plasmalogen metabolite, lysoplasmenylcholine (LPLC), are essentially unknown. We found that 2.5 and 5 micromol/L LPLC induced spontaneous contractions of intact isolated rabbit ventricular myocytes (median times, 27.4 and 16.4 minutes, respectively) significantly faster than lysophosphatidylcholine (>60 and 37.8 minutes, respectively). Whole-cell recordings revealed that LPLC depolarized the resting membrane potential from -83.5+/-0.2 to -21.5+/-1.0 mV. Depolarization was due to a guanidinium toxin-insensitive Na+ influx. The LPLC-induced current reversed at -18.5+/-0.9 mV and was shifted 26.7+/-4.2 mV negative by a 10-fold reduction of bath Na+ (Na+/K+ permeability ratio, approximately 0.12+/-0.06). In contrast, block of Ca2+ channels with Cd2+ and reducing bath Cl failed to affect the current. The actions of LPLC were opposed by lanthanides. Gd3+ and La3+ were equally effective inhibitors of the LPLC-induced current and equally delayed the onset of spontaneous contractions. However, the characteristics of lanthanide block imply that Gd3+-sensitive, poorly selective, stretch-activated channels were not involved. Instead, the data are consistent with the view that lanthanides increase phospholipid ordering and may thereby oppose membrane perturbations caused by LPLC. Plasmalogens constitute a significant fraction of cardiac sarcolemmal choline phospholipids. In light of their subclass-specific catabolism by phospholipase A2 and the present results, it is suggested that LPLC accumulation may contribute to ventricular dysrhythmias during ischemia.

Persistent activation of a swelling-activated cation current in ventricular myocytes from dogs with tachycardia-induced congestive heart failure.

The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) channels was tested. Digital video microscopy and amphotericin-perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing-induced CHF. In normal myocytes, osmotic swelling in 0.9x to 0.6x isosmotic solution (296 mOsm/L) was required to elicit an inwardly rectifying swelling-activated cation current (I(Cir,swell)) that reversed near -60 mV and was inhibited by 10 micromol/L Gd3+, a mechanosensitive channel blocker. Block of I(Cir,swell) by Gd3+ simultaneously reduced the volume of normal cells in hyposmotic solutions by up to approximately 10%, but Gd3+ had no effect on volume in isosmotic solution. In contrast, I(Cir,swell) was persistently activated under isosmotic conditions in CHF myocytes, and Gd3+ decreased cell volume by approximately 8%. Osmotic shrinkage in 1.1x to 1.5x isosmotic solution inhibited both I(Cir,swell) and Gd3+-induced cell shrinkage in CHF cells, whereas osmotic swelling only slightly increased I(Cir,swell). The K0.5 and Hill coefficient for Gd3+ block of I(Cir,swell) and Gd3+-induced cell shrinkage were estimated as approximately 2.0 micromol/L and approximately 1.9, respectively, for both normal and CHF cells. In both groups, the effects of Gd3+ on current and volume were blocked by replacing bath Na+ and K+ and were linearly related with varying Gd3+ concentration and the degree of cell swelling. CHF thus altered the set point for and caused persistent activation of I(Cir,swell). This current may contribute to dysrhythmias, hypertrophy, and altered contractile function in CHF and may be a novel target for therapy.

Using gadolinium to identify stretch-activated channels: technical considerations.

Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+ insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.

Flanking proline residues identify the L-type Ca2+ channel binding site of calciseptine and FS2.

Calciseptine and FS2 are 60-amino acid polypeptides, isolated from venom of the black mamba (Dendroaspis polylepis polylepis), that block voltage-dependent L-type Ca2+ channels. We predicted that these polypeptides contain an identical functional site between residues 43 and 46 by searching for proline residues that mark the flanks of protein-protein interaction sites [Kini, R. M., and Evans, H. J. (1966) FEBS Lett. 385, 81-86]. The predicted Ca2+ channel binding site also occurs in closely related toxins, C10S2C2 and S4C8. Therefore, it is likely that these toxins also will block L-type Ca2+ channels. To test the proposed binding site on calciseptine and FS2, an eight-residue peptide, named L-calchin (L-type calcium channel inhibitor), was synthesized and examined for biological activity. As expected for an L-type Ca2+ channel blocker, L-calchin reduced peak systolic and developed pressure in isolated rat heart Langendorff preparations without affecting diastolic pressure or heart rate. Furthermore, L-calchin caused a voltage-independent block of L-type Ca2+ channel currents in whole-cell patch-clamped rabbit ventricular myocytes. Thus the synthetic peptide exhibits the L-type Ca2+ channel blocking properties of the parent molecules, calciseptine and FS2, but with a lower potency. These results strongly support the identification of a site in calciseptine and FS2 that is important for binding to L-type Ca2+ channels and reinforce the importance of proline brackets flanking protein-protein interaction sites.

Prevention of cellular edema directly caused by hypothermic cardioplegia: studies in isolated human and rabbit atrial myocytes.

OBJECTIVES: This study tested the hypothesis that edema during hypothermic cardioplegia is caused by the hypotonicity of the perfusate at cold temperatures. METHODS: The volume of isolated human and rabbit atrial myocytes was measured by video microscopy under nonischemic conditions. Each cell served as its own control. RESULTS: After equilibration in 37 degrees C physiologic buffer (Tyrode's solution), exposure to 9 degrees C St. Thomas' Hospital solution for 20 minutes caused human atrial cells to swell by 20% and rabbit atrial cells to swell by 10%. Cell volume fully recovered on rewarming in 37 degrees C physiologic solution. Cell swelling was due to the composition of St. Thomas' Hospital solution rather than hypothermia alone. Exposure to 9 degrees C physiologic solution did not significantly affect cell volume. Swelling of myocytes was largely prevented by replacing most of the Cl- in St. Thomas' Hospital solution with an impermeant anion so that the product of the concentrations of K+ and Cl- were the same as in the physiologic solution. CONCLUSIONS: This study suggests that cell swelling during hypothermic cardioplegia is caused in part by the composition of the cardioplegic solution. The volume of cardiac myocytes appears to follow a Donnan equilibrium in the cold, and the perfusate KCl product determines water movement. Thus, the tonicity of hyperkalemic cardioplegic solutions can be adjusted to a physiologic value by replacing most Cl- by an impermeant anion. Following this simple principle, a reformulation of cardioplegic solutions may be able to minimize iatrogenic myocardial edema.

Prevention of cell swelling with low chloride St. Thomas' Hospital solution improves postischemic myocardial recovery.

OBJECTIVE: In isolated myocytes cardioplegia-induced cell swelling can be prevented by lowering the KCl product by replacing Cl- with an impermeant ion. This study tested the hypothesis that Cl- substitution in St. Thomas' Hospital cardioplegic solution would result in superior myocardial protection in the intact, blood-perfused heart. METHODS: Using a parabiotic, isolated rabbit heart Langendorff model, hearts were exposed to 1 hour of hypothermic (10 degrees to 12 degrees C), global ischemia followed by 30 minutes of reperfusion. Isosmotic cardioplegia was administered as a single 50 ml bolus of either standard St. Thomas' Hospital solution ([K+]o x [Cl-]o = 2566.4 (mmol/L)2) or low Cl- St. Thomas' Hospital solution ([K+]o x [CI-]o = 700 (mmol/L)2). Chloride was replaced by a large, impermeant ion, methanesulfonate. Postreperfusion systolic function and atrioventricular conduction times were measured before ischemia and after reperfusion. RESULTS: Hearts receiving low Cl- St. Thomas' Hospital cardioplegia demonstrated significantly better postischemic functional recovery (74% +/- 3%) compared with those treated with standard high Cl- St. Thomas' Hospital solution (55% +/- 4%, p = 0.003). In addition, atrioventricular conduction times remained normal in the low Cl- group but were significantly prolonged in the St. Thomas' Hospital group. CONCLUSIONS: Lowering the KCl product of St. Thomas' Hospital solution makes it isotonic with plasma and prevents cellular edema. This ameliorates the detrimental functional and electrophysiologic sequelae of hypothermic, hyperkalemic cardioplegia.

Human tryptase fibrinogenolysis is optimal at acidic pH and generates anticoagulant fragments in the presence of the anti-tryptase monoclonal antibody B12.

Human tryptase is uniquely regulated by its association with heparin and resists inhibition by biological protease inhibitors. The effects of pH and B12, an IgG anti-tryptase mAb, on cleavage of the synthetic substrate tosyl-Gly-Pro-Lys-p-nitroanilide and of the biological substrate fibrinogen by tryptase were examined. Tosyl-Gly-Pro-Lys-pnitroanilide cleavage was optimal at neutral pH and was inhibited by the B12 mAb at acidic and neutral pH values. At pH 7.5, inhibition was reversible and noncompetitive. In contrast, the optimal pH for tryptase to cleave fibrinogen was acidic. B12 dramatically enhanced the rate and extent that tryptase cleaved all three fibrinogen subunits at pH 6.0 to 6.5, but inhibited these activities at neutral pH. Major fibrinogen cleavage fragments generated at acidic pH by the B12:tryptase complex were identical with those made by plasmin. Thus, at acid pH, tryptase alone destroyed the ability of fibrinogen to clot, while the B12:tryptase complex increased the rate of fibrinogenolysis and also generated the anticoagulant, fragment D. The acidic pH optimum for tryptase fibrinogenolysis may direct this activity to tissue sites of inflammation. A putative biological equivalent to B12 would limit tryptase fibrinogenolytic activity at sites of neutral pH, such as blood, but would augment activity at acidic sites.

Swelling-activated Gd3+-sensitive cation current and cell volume regulation in rabbit ventricular myocytes.

The role of swelling-activated currents in cell volume regulation is unclear. Currents elicited by swelling rabbit ventricular myocytes in solutions with 0.6-0.9x normal osmolarity were studied using amphotericin perforated patch clamp techniques, and cell volume was examined concurrently by digital video microscopy. Graded swelling caused graded activation of an inwardly rectifying, time-independent cation current (ICir,swell) that was reversibly blocked by Gd3+, but ICir,swell was not detected in isotonic or hypertonic media. This current was not related to IK1 because it was insensitive to Ba2+. The PK/PNa ratio for ICir,swell was 5.9 +/- 0.3, implying that inward current is largely Na+ under physiological conditions. Increasing bath K+ increased gCir,swell but decreased rectification. Gd3+ block was fitted with a K0.5 of 1.7 +/- 0.3 microM and Hill coefficient, n, of 1.7 +/- 0.4. Exposure to Gd3+ also reduced hypotonic swelling by up to approximately 30%, and block of current preceded the volume change by approximately 1 min. Gd3+-induced cell shrinkage was proportional to ICir,swell when ICir,swell was varied by graded swelling or Gd3+ concentration and was voltage dependent, reflecting the voltage dependence of ICir,swell. Integrating the blocked ion flux and calculating the resulting change in osmolarity suggested that ICir,swell was sufficient to explain the majority of the volume change at -80 mV. In addition, swelling activated an outwardly rectifying Cl- current, ICl,swell. This current was absent after Cl- replacement, reversed at ECl, and was blocked by 1 mM 9-anthracene carboxylic acid. Block of ICl,swell provoked a 28% increase in swelling in hypotonic media. Thus, both cation and anion swelling-activated currents modulated the volume of ventricular myocytes. Besides its effects on cell volume, ICir,swell is expected to cause diastolic depolarization. Activation of ICir, swell also is likely to affect contraction and other physiological processes in myocytes.

Modulation of atrial repolarization by site of pacing in the isolated rabbit heart.

BACKGROUND: Single-site or multisite atrial pacing may reduce the incidence of atrial fibrillation in humans. The therapeutic mechanisms may include synchronization of atrial repolarization (repolarization "memory") and/or decreased dispersion of atrial repolarization. These responses have not been well documented in intact atria. METHODS AND RESULTS: Monophasic action potential recordings were made from six atrial epicardial sites in 39 isolated perfused rabbit heart preparations during 3 hours of continuous right atrial, left atrial, or biatrial pacing. Action potential recordings obtained at times 0, 45, 90, 135, and 180 minutes were computer analyzed for activation time (AT) and 90% action potential duration (APD) at each site. No consistent relationship could be demonstrated between APD and AT at any time during atrial pacing (all P > .05). On average, left atrial APDs were longer than right atrial APDs by up to 6.3 ms at all times, regardless of the site of pacing (P < or = .05). At all times, dispersion of atrial repolarization was minimized by left atrial pacing compared with right atrial pacing (21.6 +/- 9.1 versus 32.4 +/- 15.1 ms, respectively, at time 0; P < .05). Biatrial pacing provided no further reduction in dispersion of repolarization compared with left atrial pacing (all P > .05). CONCLUSIONS: No relationship can be demonstrated between atrial AT and APD in the isolated rabbit heart preparation. This differs from ventricular repolarization "memory," which is demonstrable under the same conditions. Left atrial APD is, on average, longer than right atrial APD, suggesting spatial heterogeneity in repolarization. Dispersion of atrial repolarization is minimized by left atrial pacing in this preparation with no further advantage to biatrial pacing.

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes.

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein-free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.