Guinea-pig Ventricle Research Articles

Electrophysiological mechanisms by which hypothyroidism delays repolarization in guinea pig hearts.

Thyroid hormone is known to exert important effects on cardiac repolarization, but the underlying mechanisms are poorly understood. We investigated the electrophysiological mechanisms of differences in repolarization between control guinea pigs and hypothyroid animals (thyroidectomy plus 5-propyl-2-thiouracil). Hypothyroidism significantly prolonged the rate-corrected Q-T interval in vivo and action potential duration (APD) of isolated ventricular myocytes. Whole cell voltage-clamp studies showed no change in current density or kinetics of L-type Ca(2+) current, inward rectifier K(+) current, or Na(+) current in hypothyroid hearts. Dofetilide-resistant current (I(Ks)) step current densities were smaller by approximately 65%, and tail current densities were reduced by 80% in myocytes from hypothyroid animals compared with controls. The ratio of delayed rectifier step current at +50 mV to tail current at -40 mV was significantly larger in hypothyroid cells for test pulses from 60- to 4,200-ms duration, reflecting a smaller I(Ks). Dofetilide-sensitive current (I(Kr)) densities were not significantly changed. I(Ks) half-activation voltage shifted to more positive voltages in hypothyroidism (29.5 +/- 2.2 vs. 21.3 +/- 2.7 mV in control, P < 0.01), whereas I(Kr) voltage dependence was unchanged. We conclude that hypothyroidism delays repolarization in the guinea pig ventricle by decreasing I(Ks), a novel and potentially important mechanism for thyroid regulation of cardiac electrophysiology.

Effects of [Na+]o, [Ca2+]o and cyclopiazonic acid on decline of post-extrasystolic potentiation and twitch kinetics in guinea-pig and human myocardial preparations.

The decline of post-extrasystolic potentiation was investigated in atrial and ventricular muscle of guinea-pigs and in human atrial muscle. This decline is described in terms of the recirculation fraction (RF) for calcium in myocardial cells. Under control conditions, the mean values for RF were as follows: 0.61 +/- 0.03 in the guinea-pig atrium, 0.32 +/- 0.05 in the guinea-pig ventricle and 0.51 +/- 0.10 in the human atrium. Upon increasing calcium concentrations within the range of 0.5-10 mM or decreasing the sodium to 70%, the steady-state force and recirculation fraction increased concomitantly in all three types of muscles. Cyclopiazonic acid (CPA), in a 5-20 microM concentration, decreased force in the guinea-pig preparations and decreased RF. The effects of CPA were great in atrial muscle, 20 microM CPA decreasing RF from 0.61 to 0. 39. In ventricular muscle, this effect was small and not statistically significant. When changing calcium or sodium concentrations, increased force was accompanied by slower relaxation in atrial muscle, but an unchanged or slightly faster relaxation in ventricular muscle. In guinea-pig myocardium, CPA (5-20 microM) prolonged the time to peak force (TPF), but it slowed relaxation only in the ventricle. Reducing the calcium outflow by Na/Ca exchange increased RF as expected, and reducing the SR calcium re-uptake decreased RF. These interventions were also expected to reduce the rate of decline of intracellular calcium, but relaxation was not consistently prolonged. This indicates that factors other than the SR calcium pump and sarcolemmal Na/Ca exchange determine, or at least influence, the rate of relaxation.

Isoform-specific regulation of the sodium pump by alpha- and beta-adrenergic agonists in the guinea-pig ventricle.

1. Guinea-pig ventricle was used in the RNase protection assays to determine which alpha-isoforms of the Na+-K+ pumps are present, and ventricular myocytes were used in whole cell patch clamp studies to investigate the actions of alpha- and beta-adrenergic agonists on Na+-K+ pump current. 2. RNase protection assays showed that two isoforms of the alpha-subunit of the Na+-K+-ATPase are present in guinea-pig ventricle. The mRNA for the alpha1-isoform comprises 82 % of the total pump message, the rest being the alpha2-isoform. 3. We have previously shown that beta-adrenergic agonists affect Na+-K+ pump current (Ip) through a protein kinase A (PKA)-dependent pathway. We now show that these beta-effects are targeted to the alpha1-isoform of the Na+-K+ pumps. 4. We have also previously shown that alpha-adrenergic agonists increase Ip through a protein kinase C (PKC)-dependent pathway. We now show that these alpha-isoform effects are targeted to the alpha2-isoform of the Na+-K+ pumps. 5. These results suggest the effects of adrenergic activation on Na+-K+ pump activity in the heart can be regionally specific, depending on which alpha-isoform of the Na+-K+ pump is expressed.

Kinetics of rate-dependent shortening of action potential duration in guinea-pig ventricle; effects of IK1 and IKr blockade.

1. The kinetics of shortening of action potential duration (APD) following an increase in pacing rate, from 2 to 3.3 Hz, was characterized in guinea-pig ventricular preparations. Terikalant (RP62719), an inhibitor of the inwardly rectifying K+ current (IK1), and dofetilide, a specific inhibitor of the rapidly activating delayed-rectifier current (IKr), were applied to determine the effect of inhibition of these ion currents on slow APD shortening. 2. Action potentials were recorded from isolated guinea-pig ventricular myocytes using the perforated-patch patch-clamp technique, and monophasic action potentials were recorded from Langendorff-perfused guinea-pig ventricles using a contact epicardial probe. 3. Under control conditions, after an increase in pacing rate, APD immediately decreased, and then shortened slowly with an exponential time course. In ventricular myocytes, the time constant of this exponential shortening was 28+/-4 s and the amount of slow shortening was 21.9+/-0.9 ms (n=8) for an increase in rate from 2 to 3.3 Hz. Similar values were observed in Langendorff-perfused ventricles. 4. Terikalant dose-dependently increased APD and the increase was enhanced by rapid pacing ('positive' rate-dependence). The drug dose-dependently decreased the time constant of shortening and amount of slow APD shortening. In contrast, dofetilide, an inhibitor of IKr, which shows 'reverse' rate-dependent APD widening, had no significant effect on the time constant or amount of slow shortening. 5. These observations suggest that IK1 plays a role in rate-dependent shortening of APD. The results appear to support the hypothesis that 'reverse' rate-dependent effects of IKr blockers are due to these drugs not affecting the ion current(s) mediating intrinsic rate-dependent slow shortening of APD.

Investigating the relaxation rate, following diazo-2 photolysis, of a skinned trabecular preparation from guinea-pig hypertrophied left ventricle

Investigating the relaxation rate, following diazo-2 photolysis, of a skinned trabecular preparation from guinea-pig hypertrophied left ventricle

Inhibition of sodium current by chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in guinea pig cardiac ventricular cells.

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent anion transport blocker, on transmembrane action potentials (APs) and the sodium current (I[Na]) of guinea pig ventricular myocytes were examined by using conventional microelectrode and whole-cell patch-clamp recording techniques. In papillary muscle preparations, DIDS (> or =0.1 mM) suppressed the maximal upstroke velocity (.v[max]) of the AP without significant changes in other AP parameters. Extracellular application of DIDS on single cardiomyocytes isolated from the guinea pig ventricle markedly reduced the peak amplitude of the tetrodotoxin (TTX)-sensitive and voltage-activated sodium current. The concentration-dependent block of DIDS could be expressed by the Hill equation with a Hill coefficient of 0.97 and a dissociation constant of 0.15 mM at a holding potential of (VH) -120 mV. DIDS (0.1 mM) shifted the steady-state inactivation curve for I(Na) toward more negative potentials by 6.0 +/- 0.5 mV and the activation curve to more positive potentials by 5.0 +/- 1.0 mV, although the slope factors were unaffected. With repetitive depolarizing pulses from -120 mV, DIDS produced a use-dependent block on the I(Na). Recovery of I(Na) from inactivation was slowed (time constant = 245 ms, compared with 10 ms of control) in the presence of 0.1 mM DIDS. In the two-pulse experiments, DIDS produced two distinct phases of development of I(Na) block, the rapid phase (tau = 5 ms) caused by an open channel block, and the slower phase (tau = 382 ms) induced by an inactivated channel block. These results suggest that the Cl- transport blocker DIDS has a direct inhibitory effect on the cardiac sodium channel. DIDS-induced use dependence of I(Na) block may result from the interaction of the drug with sodium channels in both the open and inactivated channel states.

Frequency dependence in the action of the class III antiarrhythmic drug dofetilide is modulated by altering L-type calcium current and digitalis glucoside.

We investigated how modulation of L-type calcium current affects the class II antiarrhythmic effect of dofetilide. Action-potential duration (APD) was determined in guinea pig papillary muscle by microelectrode techniques at different stimulation frequencies (0.5-3 Hz). The APD-prolonging effect (deltaAPD) of 10 nM dofetilide was reversed frequency dependent; it was 51 +/- 6 ms at 0.5 Hz and lower at 3 Hz, 21 +/- 3 ms. Either 10 microM diltiazem or 0.1 microM Bay K 8644 (BayK) was added to decrease or increase L-type calcium currents. In the presence of dofetilide + diltiazem, deltaAPD was reduced to 32 +/- 4 ms at 0.5 Hz but not affected at 3 Hz. Conversely, dofetilide + BayK further prolonged deltaAPD to 78 +/- 10 ms at 0.5 Hz but not at 3 Hz. When 10 microM dihydroouabain, a digitalis glucoside, was added to dofetilide, deltaAPD was more pronounced at 0.5 Hz and reduced at 3 Hz. We conclude that the reversed frequency-dependent effect of dofetilide on APD can be modulated by altering L-type calcium currents. With reduced calcium current, the frequency profile is less reversed and more favorable. The similarity of BayK and dihydroouabain in aggravating the reversed frequency-dependent effect of dofetilide is in line with a contribution of intracellular calcium to this reversed rate-dependent profile in the guinea pig ventricle.

Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: evidence against a cAMP-dependent effect.

In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and 32P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 microM), ACH attenuated PLB phosphorylation stimulated by low concentration (1O-100 microM) but not by high concentrations (> 200 microM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 +/- 6 microM and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.

Actions of cADP-ribose and its antagonists on contraction in guinea pig isolated ventricular myocytes. Influence of temperature.

Although it is becoming widely accepted that cADP-ribose (cADPR) can regulate calcium release from the endoplasmic reticulum in sea urchin eggs and in a variety of mammalian cell types, it remains controversial whether this substance might influence calcium release during excitation-contraction coupling in cardiac muscle. We have investigated possible actions of cADPR in intact cells isolated from guinea pig ventricle, paying particular attention to the possible influence of temperature. At 36 degrees C, myocyte contraction was influenced by cytosolic application of cADPR in a concentration-dependent manner (showing an approximately 30% increase in contraction with 5 mumol/L cADPR applied via a patch pipette in myocytes stimulated to fire action potentials at 1 Hz). Calcium transients measured with fura 2 were also increased by 5 mumol/L cADPR. Antagonists of cADPR reduced contraction at 36 degrees C (by approximately 35% with either 50 mumol/L 8-Br-cADPR or 5 mumol/L 8-amino-cADPR applied via the patch pipette). At room temperature (approximately 20 degrees C to 24 degrees C), no significant effects on contraction were detected with either cADPR or its antagonists. At 36 degrees C, treatment of the cells with a mixture of 2 mumol/L ryanodine and 1 mumol/L thapsigargin to suppress function of the sarcoplasmic reticulum stores of calcium prevented the action of 5 mumol/L cADPR applied via a patch pipette. These observations are consistent with an action of cytosolic cADPR to enhance calcium-induced calcium release from the sarcoplasmic reticulum in guinea pig ventricular myocytes at 36 degrees C. The observed influence of temperature under the conditions of our experiments is one factor that might help to account for failure to detect actions of cADPR and its analogues in some previous studies.

Role of passive electrical properties during action potential restitution in intact heart.

Action potential duration (APD) restitution is classically attributed to membrane ionic currents; however, the role of cell-to-cell coupling in restitution is poorly understood. To test the hypothesis that passive electrical properties of multicellular preparations influence restitution, spatial gradients of transmembrane voltage were measured with high spatial (0.83 mm), voltage (1 mV), and temporal (0.5 ms) resolutions using voltage-sensitive dye in Langendorff-perfused guinea pig ventricle. At short premature coupling intervals, APD failed to shorten in cells located near (< 3 mm) the site of pacing corresponding to the site of earliest repolarization, deviating from classical restitution. In contrast, APD shortened exponentially with increasing stimulus prematurity when pacing was remote from the identical recording site. The mechanism responsible for nonexponential restitution was investigated in a one-dimensional propagation model using the dynamic Luo-Rudy formulation of the ventricular cell and was found to be attributable to depolarizing axial current present in regions of steep repolarization gradients. Moreover, axial current loading attenuated spatial gradients of repolarization that were prominent in the absence of cell-to-cell coupling. These data demonstrate that 1) in contrast to restitution in isolated cells, restitution in multicellular tissue is influenced by axial current from neighboring cells, and 2) in normal myocardium, axial current between cells attenuates dispersion of repolarization during premature stimulation of the heart.

Phosphatase-mediated enhancement of cardiac cAMP-activated Cl-conductance by a Cl- channel blocker, anthracene-9-carboxylate.

An aromatic carboxylate, anthracene-9-carboxylic acid (9-AC), is known as a Cl- channel blocker. However, variable 9-AC effects have hitherto been reported on the cardiac cAMP-activated Cl- conductance, when applied extracellularly. We have reexamined the 9-AC effect on the Cl-conductance activated by isoproterenol or forskolin in guinea pig ventricular myocytes under whole-cell patch-clamp conditions. The inward current was blocked by 9-AC at > or = 0.5 mmol/L, but in contrast, the outward current was enhanced at much lower concentrations (ED50, approximately 13 mumol/L). 9-AC applied by the intracellular perfusion technique increased both the inward and outward currents. In the presence of intracellular 9-AC, deactivation of the conductance after washout of isoproterenol or forskolin was largely prevented. 9-AC produced an enhancing effect, even after inhibiting the deactivation process by okadaic acid (OA), whereas it failed to produce additional-effects in the presence of orthovanadate. Intracellular application of 9-AC together with OA virtually abolished the current deactivation. The 9-AC effects on the Cl-conductance were not dependent on intracellular Ca2+ or pH. Putative inhibitors of alkaline (bromotetramisole) and acid phosphatases (tartrate) were without effect. 9-AC failed to inhibit the activities of purified protein phosphatase (PP)-1, -2A, and -2C. In the extract of guinea pig ventricle, 9-AC (> or = 10 mumol/L for full action) significantly inhibited a fraction of endogenous phosphatase activity that was sensitive to orthovanadate but not to OA, bromotetramisole, and tartrate. It is concluded that 9-AC blocks cardiac cAMP-activated (cystic fibrosis transmembrane conductance regulator) Cl- conductance from the extracellular side but enhances the conductance from the intracellular side by inhibiting an orthovanadate-sensitive phosphatase distinct from PP-1, -2A, -2B, or -2C and alkaline or acid phosphatase.

Angiotensin II inhibits protein kinase A-dependent chloride conductance in heart via pertussis toxin-sensitive G proteins.

Angiotensin II receptors are reported to be abundant in the guinea pig ventricle; their coupling to adenylate cyclase in the heart, however, remains controversial. Therefore, we investigated the effect of angiotensin II on Cl- conductance activated by cAMP-dependent protein kinase. After minimizing the contribution of other ionic currents, exposure of single guinea pig ventricular cells to isoproterenol (40 to 50 nmol/L; 36 degrees C) elicited a typical protein kinase A-dependent Cl- conductance. Subsequent application of angiotensin II reduced the isoproterenol-induced conductance with an IC50 of 0.24 +/- 0.08 nmol/L. Angiotensin II also inhibited the Cl- currents, which were activated through stimulation of adenylate cyclase by forskolin and histamine receptors. CV-11974 (1 mumol/L), an antagonist selective for the angiotensin type 1 receptor, prevented the effect of angiotensin II. Angiotensin II did not inhibit the current that had been persistently activated by intracellular GTP gamma S (100 mumol/L), a nonhydrolyzable guanine nucleotide, plus isoproterenol. In addition, prior incubation of myocytes with pertussis toxin prevented the angiotensin II inhibitory action. Cl- conductance, when activated directly by intracellular dialysis with cAMP (1 mmol/L), was not affected by angiotensin II. Radioimmunologic measurement of cellular cAMP in the dissociated myocytes showed that angiotensin II inhibited the isoproterenol-induced increase of cAMP. Angiotensin II receptors negatively couple to adenylate cyclase via pertussis toxin-sensitive G proteins, thereby inhibiting cardiac protein kinase A-dependent Cl- conductance.

Phospholamban: a prominent regulator of myocardial contractility.

Our understanding of the role of phospholamban in cardiac physiology has evolved over the past two decades to the point where this protein is now understood to be a critical repressor of myocardial contractility. Phospholamban, through its inhibitory effects on the affinity of the cardiac sarcoplasmic reticulum Ca2+ pump for Ca2+, represses both the rates of relaxation and contraction in the mammalian heart. These inhibitory effects can be relieved through (1) phospholamban phosphorylation, (2) down-regulation of phospholamban gene expression, and (3) disruption of the phospholamban-Ca(2+)-ATPase interaction. Thus, genetic approaches and pharmacological interventions, designed to relieve the phospholamban inhibitory action on the cardiac sarcoplasmic reticulum Ca2+ pump and myocardial relaxation, may prove valuable in reversing the effects of several diseases in the mammalian heart. Such interventions could be designed to inhibit the phospholamban phosphatase, stabilize the phosphorylated state of phospholamban, interrupt the phospholamban-Ca(2+)-ATPase interaction, decrease phospholamban transcription, or disrupt phospholamban mRNA stability. Development of such therapeutic strategies to target phospholamban will be an important future goal for the clinical improvement of contractility in the failing heart.

Effects of class III antiarrhythmic drugs on the Na(+)-activated K+ channels in guinea-pig ventricular cells.

1. Class III antiarrhythmic drugs are known to block the outward currents through voltage-gated K+ channels. However, effects of class III antiarrhythmic drugs on the ligand-gated K+ channels have not been thoroughly examined. In this study effects of amiodarone and newer class III antiarrhythmic drugs, E-4031 and MS-551, on the Na+-activated K+ (KNa) current were examined in inside-out membrane patches and in whole cells isolated from guinea-pig ventricle. 2. The KNa channel current was activated by increasing [Na+]i from 0 mM to 30-100 mM with 150 mM [K+]o in inside-out membrane patches of ventricular myocytes. The channel current showed a larger slope conductance (210 pS), inward-going rectification and subconductance levels of various amplitudes. 3. E-4031 and MS-551 at high concentrations (300 microM) inhibited the K+ current by decreasing the open time (flickering block). On the other hand, amiodarone at relatively low concentrations (0.1-10 microM) inhibited the KNa channel current by decreasing the open probability rather than by decreasing the open time. The IC50 value of amiodarone for inhibiting the KNa channel current was 1.0 microM. 4. These drugs also inhibited the whole-cell outward current activated by intracellular loading of 50 mM [Na+]i and extracellular application of 10 microM ouabain. 5. These results indicate that class III antiarrhythmic drugs inhibit the KNa channel current in cardiac cells. However, there are sharp differences in the effective concentrations and the mode of inhibition between amiodarone and the newer class III antiarrhythmic drugs.

Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin.

Whole cell currents were recorded in single myocytes dissociated from guinea pig ventricles by the patch-clamp technique. The addition of 0.1 mg/ml gramicidin D, a cation-selective ionophore, into the pipette solution induced a gradual spontaneous perforation of the patch membrane under a conventional cell-attached configuration. The access resistance, measured at approximately 12 min after formation of a gigaohm seal, was 9.2 +/- 1.5 M omega (n = 12). The perforated patch membrane exhibited ionic selectivity for various monovalent cations, with a relative order of Cs+ (1.11) > K+ (1.0) > Na+ (0.65) >> tris(hydroxymethyl)aminomethane+ (approximately 0) but was not permeable for Cl-. Under the gramicidin-perforated patch recording configuration, the cells showed the typical electrophysiological properties for ventricular cells reported previously. The intracellular Cl- concentration, estimated from the reversal potential of the catecholamine-induced Cl- current, was 36.3 +/- 2.9 mM (n = 17). We thus conclude that the gramicidin-perforated patch recording mode provides a useful tool for recording the ionic currents while maintaining the intracellular Cl- concentration.

A specific cyclic ADP-ribose antagonist inhibits cardiac excitation–contraction coupling

Background Cyclic ADP-ribose (cADPR) has been shown to act as a potent cytosolic mediator in a variety of tissues, regulating the release of Ca2+ from intracellular stores by a mechanism that involves ryanodine receptors. There is controversy over the effects of cADPR in cardiac muscle, although one possibility is that endogenous cADPR increases the Ca2+ sensitivity of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum. We investigated this possibility using 8-amino-cADPR, which has been found to antagonize the Ca2+-releasing effects of cADPR on sea urchin egg microsomes and in mammalian cells (Purkinje neurons, Jurkat T cells, smooth muscle and PC12 cells).Results In intact cardiac myocytes isolated from guinea-pig ventricle, cytosolic injection of 8-amino-cADPR substantially reduced contractions and Ca2+ transients accompanying action potentials (stimulated at 1Hertz). These reductions were not seen with injection of HEPES buffer, with heat-inactivated 8-amino-cADPR, or in cells pretreated with ryanodine (2μM) to suppress sarcoplasmic reticulum function before injection of the 8-amino-cADPR. L-type Ca2+ currents and the extent of Ca2+ loading of the sarcoplasmic reticulum were not reduced by 8-amino-cADPR.Conclusions These observations are consistent with the hypothesis that endogenous cADPR plays an important role during normal contraction of cardiac myocytes. One possibility is that cADPR sensitizes the CICR mechanism to Ca2+, an action antagonized by 8-amino-cADPR (leading to reduced Ca2+ transients and contractions). A direct effect of 8-amino-cADPR on CICR cannot be excluded, but observations with caffeine are not consistent with a non-selective block of release channels.

Cyclic AMP levels in ventricular myocytes from noradrenaline-treated guinea-pigs

Chronic activation of the sympathetic nervous system in human heart failure is believed to cause cardiac β-adrenoceptor desensitisation. We have investigated the relationship between β-adrenoceptor desensitisation and cyclic AMP levels in cardiac myocytes isolated from the ventricle of guinea-pigs chronically infused with noradrenaline hydrochloride for 7 days. Functional β-adrenoceptor desensitisation was confirmed by a significant decrease in the maximum isoprenaline-stimulated contraction amplitude and an increased EC 50 for isoprenaline. In the absence of β-adrenoceptor stimulation, basal cyclic AMP levels were significantly depressed in populations of myocytes from noradrenaline-treated animals compared to sham-operated controls, and this was not accounted for by myocyte hypertrophy or necrosis. Similarly, there was a significant decrease in cyclic AMP levels at maximally inotropic isoprenaline concentrations. Threshold and maximum inotropic concentrations of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), restored isoprenaline-stimulated cyclic AMP levels in noradrenaline-treated guinea-pig cardiac myocytes, although we have previously reported no increase in maximum inotropic effect of isoprenaline with these compounds.

Angiotensin II and contraction of isolated myocytes from human, guinea pig, and infarcted rat hearts.

The effects of angiotensin II on myocardial contractility were assessed in isolated cardiac myocyte preparations, using video microscopy with a computerized edge-detection system. Angiotensin II (1 nM-10 microM) did not affect the contraction of rat (n = 10), guinea pig (n = 11), or human ventricular myocytes (n = 8) or of human atrial myocytes (n = 12). Isoproterenol or raised extracellular calcium increased the contraction amplitude of the cardiac myocytes to a maximum of between 150 and 560% above basal, and there were corresponding increases in the velocities of contraction and relaxation. In rat and guinea pig ventricular myocytes 1 microM angiotensin II did not affect the inotropic response to isoproterenol. Seven days after left coronary artery ligation in seven rats, the basal contraction amplitude was reduced in myocytes from the infarcted region (4.0 +/- 1.9%) compared with the noninfarcted region (5.0 +/- 2.8%, P = 0.03) and with myocytes from six sham-operated hearts (5.0 +/- 2.8%, P = 0.03). There was a switch in myosin isoform expression from the V1 to the V3 isoform in myocytes from both the infarcted and noninfarcted regions. Angiotensin II (1 nM-10 microM) had no significant effect on the contraction characteristics of myocytes from the infarcted rat hearts. In conclusion, angiotensin II had no significant inotropic effect on isolated cardiac myocyte preparations from guinea pig ventricle, normal and infarcted rat ventricle, human ventricle, and human atrium.

Evidence for the Presence of M Cells in the Guinea Pig Ventricle

Recent studies have described the presence of M cells in the deep layers of the canine and human ventricle displaying electrophysiologic and pharmacologic features different from those of epicardial (EPI) and endocardial (ENDO) cells. The M cell is distinguished electrophysiologically by the ability of its action potential to prolong disproportionately to that of other myocardial cells with slowing of the stimulation rate and pharmacologically by its unique sensitivity to Class III antiarrhythmic agents. The present study was designed to test the hypothesis that similar cells are present in the guinea pig ventricle. We used a dermatome to obtain-thin strips of left ventricular free wall from the hearts of guinea pigs (8 to 14 weeks old) and standard microelectrode techniques to record transmembrane activity. Action potential duration measured at 90% repolarization (APD90) was significantly longer in mid-myocardial (MID) cells than in surface EPI or ENDO cells at all basic cycle lengths (BCLs) tested. At a BCL of 300 msec, APD90 was 102 +/- 21,136 +/- 9, and 95 +/- 15 msec in EPI, MID, and ENDO cells (mean +/- SD; n = 12). At a BCL of 5000 msec, APD90 was 133 +/- 14, 185 +/- 24, and 135 +/- 13 msec in EPI, MID, and ENDO cells ([K+]o = 4 mM). Thus, APD-rate relations were more pronounced in the MID cells. MID cells were also more sensitive to agents with Class III actions (e.g., d,I-sotalol: 10 to 100 microM), exhibiting a greater APD prolongation than EPI or ENDO. d,I-Sotalol also induced early afterdepolarizations in MID cells but not in EPI or ENDO cells. The rate of rise of the action potential upstroke (Vmax) was significantly greater in MID cells: 129 +/- 13, 240 +/- 42, and 192 +/- 28 V/sec in EPI, MID, and ENDO cells (n = 10 to 18). Our results demonstrate the existence of important transmural electrical heterogeneity in guinea pig ventricular myocardium. The study provides data in support of the existence of M cells in the mid-myocardial layers of the guinea pig ventricle exhibiting longer APDs and a greater sensitivity to agents with Class III antiarrhythmic action.

Putative cardiotoxicity of the venoms of three mamba species.

The venoms of all mamba species are known to contain potent neurotoxins. Some authors suggest that cardiotoxins from mamba venom are responsible for heart failure in envenomated patients. This study was undertaken to come to a better understanding of the mechanisms and possible effects of mamba venom on the hearts of animals. The venoms of three mamba species namely Dendroaspis polylepis, Dendroaspis angusticeps, and Dendroaspis jamesoni were screened for cardiotoxicity by the cardiomyocyte viability test on cardiomyocytes isolated enzymatically from guinea pig ventricle muscle. This analysis was followed by an electrophysiological evaluation of the effects of venoms (from the Dendroaspis species) on cardiac ion channels by employing the whole-cell clamp procedure. In this study the cardiomyocyte viability test indicates differences among the venoms of the three mamba species. The venom of D. jamesoni seems to be the most potent followed by D. angusticeps and then D. polylepis. The whole-cell clamp results indicate that the venoms have no affinity for cardiac potassium channels but have an inhibitory influence on cardiac L-calcium channels. Although this study provides evidence that mamba venoms have a specific effect on isolated myocytes of guinea pig, it is doubtful that it will have a profound influence on a human heart in case of envenomation.