Adult Rabbit Ventricular Myocytes Research Articles

Evidence against Functional Heteromultimerization of theKATP Channel Subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.

Influence of a Na+-H+ exchange inhibitor ethylisopropylamiloride, a Na+-Ca2+ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca2+ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes.

In rabbit, ventricular myocytes loaded with indo-1/AM, angiotensin II (0.1 nM-0.1 microM) exerted a positive inotropic effect with a significant increase in the amplitude of Ca2+ transients. For a given increase in cell shortening, the increase in Ca2+ transients induced by angiotensin II was less than that induced by elevation of extracellular Ca2+ concentration ([Ca2+]0) or isoprenaline, an indication that both the increase in mobilization of intracellular Ca2+ ions and myofibrillar sensitivity to Ca2+ ions contribute to the positive inotropic effect of angiotensin II. The effects of angiotensin II on Ca2+ transients and cell shortening were inhibited by the AT1 receptor antagonist losartan. A Na+ -H+ exchange inhibitor EIPA [5-(N-ethyl-N-isopropyl)amiloride] at 1 and 3 microM did not affect the Ca2+ transients and cell shortening, but it inhibited the angiotensin-II-induced responses in a concentration-dependent manner more effectively than the responses to elevation of [Ca2+]0, indicating that EIPA elicited a selective inhibitory action on the effects of angiotensin II. The observation that EIPA at 10 microM abolished the positive inotropic effect of angiotensin II without a significant depression of the inotropic response to elevation of [Ca2+]0 supports the selective action of EIPA at the high concentration on the response to angiotensin II. A novel selective Na+ -Ca2+ exchange (reverse mode) inhibitor KB-R7943, 2-[2-[4-(-nitrobenzyloxy)phenyl]ethyl] isothiourea methanesulphonate, at 0.3 and 1 microM inhibited also the responses to angiotensin II more effectively than the response to elevation of [Ca2+]0; however, over the same concentration range it suppressed significantly the amplitude of Ca2+ transients and cell shortening. Combination of EIPA (3 microM) and KB-R7943 (0.3 microM), each of which attenuated partially the angiotensin-II-induced responses, abolished the positive inotropic effect and the increase in Ca2+ transients induced by angiotensin II with much less depressant effect on the responses to elevation of [Ca2+]0. Thus, these ion exchange inhibitors exerted selective actions on the respective targets. The results with these selective inhibitors indicate that the activation of Na+ -H+ exchanger and subsequent modulation of the activity of Na+ -Ca2+ exchanger may be responsible for the increase in [Ca2+]i and the myofilament Ca2+ sensitization induced by stimulation of AT1 receptors by angiotensin II in rabbit ventricular myocytes.

Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes.

The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes.

Decreased inward rectifier current in adult rabbit ventricular myocytes maintained in primary culture: a single-channel study.

Regulation of ion channel function in heart has been shown to be affected by changes in the cellular environment. Recently it was shown that rabbit ventricular myocytes kept in primary culture, show a strong reduction in inward rectifier current (IK1). The aim of the present study was to elucidate the mechanism underlying this decrease in IK1, using single-channel measurements. In addition, we studied the effects of primary culture on the ATP-regulated K+ (K.ATP) channel, also a member of the inwardly rectifying K+ channel family. Adult rabbit ventricular myocytes were cultured for up to 3 days in Ham's F-10 medium complemented with 1% rabbit serum and 5% glutamine. IK1 and K.ATP channel activity was studied in the inside-out patch configuration of the patch-clamp technique with equimolar K+ concentrations (140 mM K+) on the intra- and extracellular side. Single channel characteristics were determined at various times during culture and compared to those present in freshly isolated myocytes. IK1 channels in freshly isolated myocytes (day 0) had a single-channel conductance of 56.1 +/- 2.5 pS (mean +/- SEM) and an open probability of 0.64 +/- 0.05 (mean +/- SEM). Neither the single-channel conductance nor the open probability (Po) underwent significant changes during culture. The mean number of channels per patch, however, was drastically reduced from 1.2 +/- 0.3 (mean +/- SEM) at day 0 to 0.17 +/- 0.06 at day three. K.ATP channel density and open probability, on the other hand, were both increased with an optimum at day two. Po increased from 0.27 +/- 0.06 at day 0 to 0.63 +/- 0.06 at day three. The mean number of channels per patch was 2.29 +/- 0.57 and 3.25 +/- 0.48 at days 0 and 3 respectively. The unitary current amplitude at -50 mV remained unchanged, suggesting no change in the K.ATP single-channel conductance. The decrease in IK1 in rabbit ventricular myocytes as has been observed during primary culture is the result of a reduction in the number of active channels and not of altered kinetic or conductive channel properties. The increase in K.ATP channel activity under the same conditions suggests that gene expression of both channel types is differently regulated.

Overexpression of a human potassium channel suppresses cardiac hyperexcitability in rabbit ventricular myocytes.

The high incidence of sudden death in heart failure may reflect abnormalities of repolarization and heightened susceptibility to arrhythmogenic early afterdepolarizations (EADs). We hypothesized that overexpression of the human K+ channel HERG (human ether-a-go-go-related gene) could enhance repolarization and suppress EADs. Adult rabbit ventricular myocytes were maintained in primary culture, which suffices to prolong action potentials and predisposes to EADs. To achieve efficient gene transfer, we created AdHERG, a recombinant adenovirus containing the HERG gene driven by a Rous sarcoma virus (RSV) promoter. The virally expressed HERG current exhibited pharmacologic and kinetic properties like those of native IKr. Transient outward currents in AdHERG-infected myocytes were similar in magnitude to those in control cells, while stimulated action potentials (0.2 Hz, 37 degrees C) were abbreviated compared with controls. The occurrence of EADs during a train of action potentials was reduced by more than fourfold, and the relative refractory period was increased in AdHERG-infected myocytes compared with control cells. Gene transfer of delayed rectifier potassium channels represents a novel and effective strategy to suppress arrhythmias caused by unstable repolarization.

Na-Ca Exchange and the Trigger for Sarcoplasmic Reticulum Ca Release: Studies in Adult Rabbit Ventricular Myocytes

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents ( I Ca), Na-Ca exchange currents (I NaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked I Ca by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the “nifedipine-insensitive” component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), I Ca could initiate late-peaking contractions that were ∼70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was ∼10-fold less than that occurring by I Ca. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of I Ca and I NaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.

Effects of Amiodarone and Desethylamiodarone on the Inward Rectifying Potassium Current (IK1) in Rabbit Ventricular Myocytes

We examined the effects of amiodarone (AMI) and desethylamiodarone (DAM) on whole-cell inward rectifying potassium current (IK1) in freshly isolated adult rabbit ventricular myocytes by using the whole-cell voltage-clamp technique, as an index of their effects on resting membrane resistance (Rm). Under control conditions, the current showed a strong inward rectification with a maximal inward current measured at -130 mV of -26.4 +/- 1.3 pA/pF and a maximal outward current measured at -50 mV of 3.5 +/- 0.3 pA/pF The current also exhibit a time-dependent activation, with a time constant of activation (tau(a)) that increased with depolarization. The maximal slope conductance normalized to cell capacitance was 0.509 +/- 0.019 nS/pE After exposure to both DAM (50 microM; n = 8) and AMI (50 microM; n = 7), rapid decrease in inward IK1 was observed. Block was restricted almost exclusively to the inward component. DAM caused a significant reduction of the maximal inward current (-20.0 +/- 2.0 pA/pF; p < 0.05), whereas AMI induced an even greater reduction of the same component (-14.1 +/- 1.2 pA/pF; p < 0.05 with respect to control and to DAM). The outward component of IK1 was not changed by either AMI or DAM (4.0 +/- 0.3 pA/pF and 3.4 +/- 0.4 pA/pF, respectively). AMI and DAM also decreased the maximal slope conductance significantly (0.297 +/- 0.019 nS/pF and 0.421 +/- 0.038 nS/pF, respectively). In addition, AMI but not DAM significantly increased the tau(a). However, the voltage dependence of the acceleration of tau(a) remained unchanged after both AMI and DAM exposure. These results allow us to conclude that AMI may induce a greater increase in the resting Rm than its main metabolite. This effect may counterbalance, at least in part, the conduction slowing due to its sodium channel-blocking properties.

The restriction of diffusion of cations at the external surface of cardiac myocytes varies between species

In cardiac muscle sarcolemmal structures such as T-tubules, caveolae and negatively charged protein-polysaccharides may affect the rate of cation exchange on the external surface of the cells. To test this hypothesis, we examined the rate of external cation exchange in adult rabbit and rat ventricular myocytes using a rapid solution switcher to change the bulk external solution within 4 ms. To assess the rate of diffusion of monovalent cations, we increased [K+]o from 4.4 to 6.6 or 8.8 mM and measured the time required to achieve a stable membrane depolarization. In rat myocytes, the mean time to 90% depolarization (t90) was significantly longer than that in rabbit myocytes (137 and 64 ms, respectively) and the difference in t90 was not associated with the cell size. To assess the time course of exchange of external Ca2+, we rapidly exposed the myocytes to 0 Ca2+-2 mM EGTA solution at specific time points before action potentials or voltage clamp steps, and measured the rate of alteration of the normalized peak [Ca2+]i transient (Flux-3) or Ca2+ current. Exposure to 0 Ca2+-2 mM EGTA solution caused a decline in the intracellular calcium transient. In rat myocytes, the rate of decline in the [Ca2+]i transient was much slower (t90 > 1500 ms, the time required for 90% decline) than for the rabbit (t90 = 295 ms). Also, the rate of decline in the Ca2+ current was prolonged in rat myocytes (t90 = 910 ms) compared with rabbit myocytes (t90 = 241 ms). These data indicate that there is a restricted space on the external surface of sarcolemma which limits diffusion of divalent cations more markedly than monovalent cations. The extent of this limitation of cation diffusion varies between species, and may have functional significance.

Sarcoplasmic reticulum and Na+/Ca2+ exchanger function during early and late relaxation in ventricular myocytes.

The relative importance of the Na+/Ca2+ exchanger in the initial and terminal phases of relaxation and the decline in the [Ca2+]i transient was investigated in adult rabbit ventricular myocytes loaded with the Ca2+ indicator fluo 3. For electrically stimulated contractions, the peak intracellular Ca2+ concentration ([Ca2+]i) was 700 +/- 87 nM and end-diastolic [Ca2+]i was 239 +/- 30 nM (0.25 Hz, 37 degrees C, 1.08 mM extracellular Ca2+ concentration; n = 14). Abrupt inhibition of Na+/Ca2+ exchange was produced by removal of extracellular Na+ (KCl substitution) and Ca2+ [2 mM Ca(2+)-free ethylene glycol-bis(beta-aminoethyl either)-N,N,N',N'-tetraacetic acid] by means of a rapid switcher device (SW). Abrupt exposure to high K+ induced an action potential, although sufficient Ca2+ remained adjacent to the sarcolemma to induce a contraction (SW beat) and [Ca2+]i transient that were identical in amplitude to those induced by electrical stimulation (ES beat). The initial relaxation and decline in the [Ca2+]i transient was not significantly prolonged by abrupt elimination of the Na+/Ca2+ exchanger, but the rate and extent of the terminal phase of the decline in the [Ca2+]i transient were significantly reduced. The first derivative of [Ca2+]i with respect to time versus [Ca2+]i during the decline of the [Ca2+]i transient attributable to sarcoplasmic reticulum (SR) function was estimated from the average SW transients, and that attributable to Na+/Ca2+ exchange was estimated from the difference between SW and ES transients. By this analysis, the Na+/Ca2+ exchanger produces 13% of the first half of the decline in [Ca2+]i and 45% of the second half of the decline. We conclude that abrupt inhibition of forward Na+/Ca2+ exchange does not significantly affect the amplitude or the initial rate of decline of the [Ca2+]i transient and relaxation. However, its contribution to the reduction of [Ca2+]i becomes apparent late during the [Ca2+]i transient, when cytosolic [Ca2+]i has been reduced.

Role of the Sarcoplasmic Reticulum in Contraction and Relaxation of Immature Rabbit Ventricular Myocytes

Previous indirect studies of newborn hearts have suggested a diminished functional role of the SR and a greater dependency upon trans-sarcolemmal Ca2+fluxes to directly elicit contraction and promote relaxation. We tested the hypothesis that the SR in newborn rabbit hearts is functionally incompetent by measuring contraction and relaxation in ventricular myocytes isolated from the hearts of 1–2-day-old (newborn), 10–12-day-old (juvenile) and >150-day-old (adult) rabbits. Electrically stimulated twitch characteristics were compared to those elicited by the rapid application of 10 mmcaffeine in the presence and absence of functional sarcolemmal Na–Ca exchange (disabled using a Na+- and Ca2+-free extracellular solution). During steady state, electrically-induced contractions were lower in amplitude in newborn and juvenile compared to adult myocytes (2.9±0.5 and 3.4±0.3v8.5±0.9% of resting cell length, respectively;n=24–29) and relaxation was slower in immature myocytes (t0.75values: newborn 250±20; juvenile 240±10; adult 130±20 ms,n=14–21). Contrary to our hypothesis, caffeine triggered sufficient SR Ca2+release from immature myocytes to elicit contractions of similar magnitude to adults (newborn 12.8±1.1; juvenile 14.0±0.9; adult 15.0±1.6% of resting cell length,n=25–29). The amplitude of indo-1 Ca2+transients during steady-state twitch was 36±12% of the maximal caffeine-induced Ca2+transient in newborns (n=6) and 59±4% in adults (n=6). Caffeine slightly prolonged relaxation in adult myocytes (t0.75=200±30 ms), but accelerated relaxation in newborn and juvenile myocytes (t0.75=180±20 and 150±30 ms, respectively). When both the SR and Na–Ca exchanger were disabled, the rate of relaxation (attributable to the sarcolemmal Ca2+-ATPase and mitochondrial Ca2+uniporter) of newborn and juvenile myocytes was significantly faster than in the adults (1660±210 and 3030±180v4530±310 ms, respectively;n=14–21). We conclude that neonatal and adult rabbit ventricular myocytes have comparable SR Ca2+load, but neonatal cells exhibit smaller fractional SR Ca2+release during steady-state contractions and greater Ca2+removal by sarcolemmal Na–Ca exchange during relaxation.

Subcellular Distribution of Ankyrin in Developing Rabbit Heart—Relationship to the Na+–Ca2+Exchanger

Ankyrins are a multigene family of proteins that function as adapters between the cytoskeleton and trans-membrane proteins, such as ion channels. Previous studies have shown the linkage between ankyrin and ionic transport proteins such as Na+–K+ATPase, voltage-dependent Na+channels and Ca2+channels. In the present study, we have investigated the subcellular distribution of ankyrin and its relationship to the Na+–Ca2+exchange protein in immature and adult rabbit ventricular myocytes. Isolated single cardiomyocytes from neonatal, juvenile and adult rabbit hearts were examined by immunofluorescence labeling techniques, using antibodies against ankyrin and the Na+–Ca2+exchanger. We found that in neonatal rabbit cardiac myocytes, ankyrin labeling was mainly present at the Z disk, whereas the Na+–Ca2+exchanger was only present on the peripheral sarcolemma. At 2 weeks of age, ankyrin labeling was still predominantly observed at the level of the Z disks as well as in the partially developed T-tubules. In the adult cells, however, ankyrin and the Na+–Ca2+exchanger seem to be co-localized within T-tubules and at the costamere region of the peripheral sarcolemma. Immunogold labeling studies at the higher resolution electron microscopic level using cyrosection tissues of rabbit heart at different ages confirm these findings. These results indicate that the distribution pattern of ankyrin and the Na+–Ca2+exchanger changes with development in rabbit ventricular myocytes.

Cultured adult rabbit myocytes: effect of adding supplements to the medium, and response to isoprenaline.

The aims of this study were to investigate: (1) the effect of supplementing the culture medium on preservation of L-type calcium channel current (1Ca,L) in adult rabbit ventricular myocytes cultured for 4 days; and (2) preservation of the ICa,L response in cultured myocytes to the beta-adrenergic agonist isoprenaline. Adult rabbit myocytes were cultured on laminin-pretreated glass coverslips. The basic, serum-free culture medium was supplemented with 2 mM L-carnitine, 5 mM creatine, and 5 mM taurine. Myocytes were whole cell patch-clamped, and the L-type Ca channel current was recorded selectively as Ba flux (IBa,L) via the channels. IBa,L density (i.e., IBa,L amplitude normalized to membrane capacitance) in myocytes maintained in supplemented medium did not change significantly during culture (P > 0.1). By comparison, IBa,L density in myocytes cultured in nonsupplemented medium declined by 36% after 24 hours in culture (day 1) and then recovered by the fourth day (day 4). There was no significant difference in the response to isoprenaline of acutely isolated myocytes and 4-day cultured myocytes. Isoprenaline 100 nM increased peak IBa,L by 149% +/- 32% (mean +/- SEM) in acutely isolated myocytes (n = 4 cells), and by 224% +/- 60% (n = 6 cells) and 159% +/- 24% (n = 8 cells) in day 1 and 4 cultured myocytes, respectively. Supplemented medium improved IBa,L density in cultured myocytes. beta-Adrenergic receptors and intracellular messenger pathways appear to remain intact in adult rabbit myocytes cultured for up to 4 days.

Negative inotropic effect of adrenomedullin in isolated adult rabbit cardiac ventricular myocytes.

Adrenomedullin (AM) is a potent vasodilator peptide. AM-induced vasodilatation is mediated by an increase of NO as well as cAMP. Both AM and binding sites for this peptide have been found in cardiac tissue, indicating the possible existence of an autocrine or paracrine system of AM in the heart. Myocytes were isolated by use of retrograde coronary perfusion with physiological solution containing collagenase and hyaluronidase from adult rabbit ventricles. Contraction of cardiac myocytes was traced with a video motion detector, and [Ca2+]i was measured with indo 1 at 37 degrees C. The Ica was measured with a whole-cell patch clamp at 23 degrees C. AM and calcitonin gene-related peptide (CGRP), another member of the same peptide family, showed a concentration-dependent negative inotropic effect (10(-7) mol/L AM: contraction amplitude, 64 +/- 7% of control; [Ca2+]i, 52 +/- 5% of control; n = 10; 10(-6) mol/L CGRP: contraction amplitude, 64 +/- 25%; [Ca2+]i, 70 +/- 3%; n = 5; mean +/- SD). Ica was decreased to 60 +/- 39% by superfusion with AM after the cessation of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor. Pretreatment with L-NMMA (10 mumol/L) abolished the negative inotropic effect of AM, whereas switching from AM+L-NMMA to AM+L-arginine (1 mmol/L) restored it. Superfusion with 8-bromo-cGMP also showed a negative inotropic effect. AM significantly increased the intracellular content of cGMP, a second messenger of NO, but not that of cAMP. AM (10 nmol/L) blunted the effect of 1 mumol/L forskolin. AM has a negative inotropic effect and decreased both [Ca2+]i and Ica, with these effects being at least party mediated via the L-arginine-NO pathway in adult rabbit ventricular myocytes.

Effects of cGMP on L-type calcium current of adult and newborn rabbit ventricular cells.

Cyclic GMP has been shown to be in some respects an inhibitory modulator of heart function. Various studies on the modulation of cardiac L-type calcium current (ICa) by cGMP in different species show inconsistency and the role of cGMP remains unclear and controversial. The present study was focused on the differences in the modulation of basal ICa by cGMP in adult and newborn rabbit ventricular cells. Enzymatically isolated adult and newborn (1-4-day-old) rabbit ventricular myocytes were used to measure ICa under whole-cell voltage clamp conditions with internal perfusion of isolated cells. We have shown that in adult ventricular cells, the intracellular perfusion of 8BrcGMP did not produce any effect on basal ICa, while intracellular perfusion of 8BrcGMP or 8CPT-cGMP in newborn ventricular cells significantly and reversibly increased basal ICa without changing the voltage dependence for activation of ICa. Both methylene blue and LY-83583 (which inhibit guanylyl cyclase and thus lower cGMP levels), in adult ventricular cells, failed to produce any significant effect on basal ICa, while in newborn ventricular cells the application of methylene blue or LY-83583 produced irreversible inhibition of basal ICa. Similarly, KT-5823, an inhibitor of cGMP-dependent protein kinase, also inhibited basal ICa in newborn ventricular cells but not in adult ventricular cells. However, extracellular application of methylene blue during the intracellular perfusion of 8BrcGMP was unable to inhibit ICa. Extracellular application of nitrosoglutathione which releases nitric oxide produced a significant increase in ICa in newborn but not in adult ventricular cells. Intracellular application of a cAMP-dependent protein kinase inhibitor peptide blocked the stimulatory effect of cAMP but not of 8CPT-cGMP, while the stimulatory effect of nitrosoglutathione on ICa was not blocked by the presence of a phosphodiesterase inhibitor (isobutylmethyl-xanthine). We propose that, for newborn rabbit ventricular cells, cGMP plays a crucial role in maintaining basal ICa by a mechanism mediated via protein-kinase-G-dependent phosphorylation of calcium channels or some associated protein.

Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.

Our laboratory has been testing the hypothesis that genetic modulation of the beta-adrenergic signaling cascade can enhance cardiac function. We have previously shown that transgenic mice with cardiac overexpression of either the human beta2-adrenergic receptor (beta2AR) or an inhibitor of the beta-adrenergic receptor kinase (betaARK), an enzyme that phosphorylates and uncouples agonist-bound receptors, have increased myocardial inotropy. We now have created recombinant adenoviruses encoding either the beta2AR (Adeno-beta2AR) or a peptide betaARK inhibitor (consisting of the carboxyl terminus of betaARK1, Adeno-betaARKct) and tested their ability to potentiate beta-adrenergic signaling in cultured adult rabbit ventricular myocytes. As assessed by radioligand binding, Adeno-beta2AR infection led to approximately 20-fold overexpression of beta-adrenergic receptors. Protein immunoblots demonstrated the presence of the Adeno-betaARKct transgene. Both transgenes significantly increased isoproterenol-stimulated cAMP as compared to myocytes infected with an adenovirus encoding beta-galactosidase (Adeno-betaGal) but did not affect the sarcolemmal adenylyl cyclase response to Forskolin or NaF. beta-Adrenergic agonist-induced desensitization was significantly inhibited in Adeno-betaARKct-infected myocytes (16+/-2%) as compared to Adeno-betaGal-infected myocytes (37+/-1%, P < 0.001). We conclude that recombinant adenoviral gene transfer of the beta2AR or an inhibitor of betaARK-mediated desensitization can potentiate beta-adrenergic signaling.

Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture

Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture

Effects of TNF-alpha on [Ca2+]i and contractility in isolated adult rabbit ventricular myocytes.

The mechanism of the acute negative inotropic effect of tumor necrosis factor-alpha (TNF-alpha) was studied in enzymatically isolated adult rabbit ventricular myocytes. In cells loaded with fura 2 acetoxymethyl ester (AM) and paced intermittently at 0.2 Hz, TNF-alpha at doses < or = 10,000 U/ml caused a significant reduction in active cell shortening at 20 min, without reducing the amplitude of the accompanying intracellular Ca2+ concentration ([Ca2+]i) transient. Similar results were obtained in cells loaded with indo 1-AM and paced continuously at 0.2 Hz during exposure to TNF-alpha (10,000 U/ml). The effect of TNF-alpha on cell shortening could be prevented by the nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME) but not its inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). The NO scavenger hemoglobin also attenuated the effects of TNF-alpha. TNF-alpha also caused a significant increase in diastolic cell length without any change in diastolic [Ca2+]i. The effect on cell length was prevented by L-NAME but not D-NAME. In cells loaded with the pH indicator seminaphthorhodafluor-AM, TNF-alpha did not alter pH sufficiently to account for the negative inotropic effect. These data suggest that high doses of TNF-alpha can acutely induce NO synthesis in isolated myocytes and reduce contractility by decreasing myofilament [Ca2+]i responsiveness. The mechanism of this altered myofilament [Ca2+]i response is unknown but does not appear to be pH mediated.

Survival of metabolically inhibited ventricular myocytes is enhanced by inhibition of rigor and SR Ca2+ cycling.

During severe ATP depletion, sarcolemmal rupture resulting from rigor- and/or Ca(2+)-induced myofilament force development is considered to be an important cause of irreversible cell injury. Recent experiments in our laboratory demonstrated that during prolonged metabolic inhibition (MI) in adult rabbit ventricular myocytes, in which rigor was prevented by exposure to 30 mM 2,3-butanedione monoxime (BDM), cyclic uptake and release of cystolic Ca2+ occurred and was associated with strong phasic contractions. To investigate the relative contribution of this sarcoplasmic reticulum Ca2+ cycling and associated force development to energy depletion injury, the effects of BDM together with 7 mM caffeine were examined in isolated rabbit ventricular myocytes subjected to MI with 2 mM NaCN and 20 mM 2-deoxyglucose (2-DG). During 90 min of MI with CN and 2-DG, no cells retained a rod shape in the absence of BDM or caffeine. In the presence of both 30 mM BDM and 7 mM caffeine during MI, preservation of rod morphology was enhanced, and 52 +/- 6.2% of cells retained a rod shape 48 h after metabolic inhibition and had normal ATP content and resting membrane potential. Both systolic and diastolic functions of cells that survived MI, however, were impaired. We conclude that exposure to caffeine together with BDM markedly enhances survival of myocytes during severe prolonged ATP depletion. After recovery, these isolated myocytes show some characteristics of stunning.

Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture.

Adult rabbit ventricular myocytes were cultured in a basic medium (Medium 199) for up to 6 days to assess preservation of morphology and ion channel currents. In culture, cells remained rod shaped and striated but their ends became progressively rounded. Cell cross-sectional area declined slightly (by 14%) over the first 24 h, in contrast, whole-cell capacitance (which reflects external surface membrane plus membrane infoldings) decreased by 42% over the same time. Using whole-cell patch-clamp, we observed that the typical "N" shape steady-state current-voltage (I-V) relation became flattened after 24 h in culture. L-type Ca channel density was assessed as barium flux (IBa,L) via the channel. IBa,L (normalised to cell capacitance) declined by 50% after 24 h and recovered partially by days 4 and 6. The density of inward rectifier K current declined by 54% after 24 h and showed no recovery subsequently. In contrast, there was no significant decline in the density of transient outward K current after 24 h, but it declined subsequently by 65% after 6 days. We speculate that the time course of change in each ion channel density may reflect a change in pattern of ion channel expression, or differential membrane loss since the density of transverse tubules decreased by 57% after 6 days in culture. These results suggest that even by 24 h in culture, ion channel density in myocytes has changed substantially from the acutely isolated state.

Stimulation and binding of myocardial phospholipase C by phosphatidic acid

Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.