Simon M. Harrison

Different regional effects of voluntary exercise on the mechanical and electrical properties of rat ventricular myocytes

Short‐term (6 weeks) voluntary wheel running exercise in young female rats that were in an active growth phase resulted in whole‐heart hypertrophy and myocyte concentric hypertrophy, when compared to sedentary controls. The cross‐sectional area of ventricular myocytes from trained rats was significantly greater than for those isolated from sedentary rats, with the greatest change in morphology seen in sub‐endocardial cells. There was no statistically significant effect of training on cell shortening in the absence of external mechanical loading, in [Ca2+]i transients, or in myofilament Ca2+ sensitivity (assessed during re‐lengthening following tetanic stimulation). Under the external mechanical load of carbon fibres, absolute force developed in myocytes from trained rats was significantly greater than in those from sedentary rats. This suggests that increased myocyte cross‐sectional area is a major contractile adaptation to exercise in this model. Training did not alter the passive mechanical properties of myocytes or the relative distribution of titin isomers, which was exclusively of the short, N2B form. However, training did increase the steepness of the active tension‐sarcomere length relationship, suggesting an exercise‐induced modulation of the Frank‐Starling mechanism. This effect would be expected to enhance cardiac contractility. Training lengthened the action potential duration of sub‐epicardial myocytes, reducing the transmural gradient in action potential duration. This observation may be important in understanding the cellular causes of T‐wave abnormalities found in the electrocardiograms of some athletes. Our study shows that voluntary exercise modulates the morphological, mechanical and electrical properties of cardiac myocytes, and that this modulation is dependent upon the regional origin of the myocytes.

TNF-α and IL-1β increase Ca2+ leak from the sarcoplasmic reticulum and susceptibility to arrhythmia in rat ventricular myocytes

Sepsis is associated with ventricular dysfunction and increased incidence of atrial and ventricular arrhythmia however the underlying pro-arrhythmic mechanisms are unknown. Serum levels of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are elevated during sepsis and affect Ca2+ regulation. We investigated whether pro-inflammatory cytokines disrupt cellular Ca2+ cycling leading to reduced contractility, but also increase the probability of pro-arrhythmic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). Isolated rat ventricular myocytes were exposed to TNF-α (0.05 ng ml−1) and IL-1β (2 ng ml−1) for 3 hr and then loaded with fura-2 or fluo-3 to record the intracellular Ca2+ concentration ([Ca2+]i). Cytokine treatment decreased the amplitude of the spatially averaged Ca2+ transient and the associated contraction, induced asynchronous Ca2+ release during electrical stimulation, increased the frequency of localized Ca2+ release events, decreased the SR Ca2+ content and increased the frequency of spontaneous Ca2+ waves at any given cytoplasmic Ca2+. These data suggest that TNF-α and IL-1β increase the SR Ca2+ leak from the SR, which contributes to the depressed Ca2+ transient and contractility. Increased susceptibility to spontaneous SR Ca2+ release may contribute to arrhythmias in sepsis as the resulting Ca2+ extrusion via NCX is electrogenic, leading to cell depolarisation.

Effects of Isoflurane, Sevoflurane, and Halothane on Myofilament Ca2+Sensitivity and Sarcoplasmic Reticulum Ca2+Release in Rat Ventricular Myocytes

BackgroundThe aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes. MethodsSingle rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine. ResultsIsoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane. ConclusionsDepressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).

Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium–calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.

Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes.

L-type Ca2+ current, iCa, has been recorded in guinea-pig ventricular myocytes at 36° C using the whole cell patch clamp technique. Intracellular Ca2+ was buffered with ethylenebis(oxonitrilo)tetraacetate (EGTA). An increase in the rate of stimulation from 0.5 to 3 Hz resulted in an abrupt decrease in iCa in the first beat at the high rate, followed by a progressive decrease (τ approx. 7 s) over the next 30 s. The changes were not the result of Ca2+-dependent inactivation, because similar changes occurred with either Ba2+ or Na+ as the charge carrier. During 20-s voltage clamp pulses there was an ultra-slow phase of inactivation of Ba2+ or Na+ current through the Ca2+ channel (τ approx. 6 s at 0 mV). This was confirmed by applying test pulses after conditioning pulses of different duration: the Ba2+ current during the test pulse decreased progressively when the duration of the conditioning pulse was increased progressively to 20 s. Ultra-slow inactivation of Ba2+ current was voltage dependent and increased monotonically at more positive potentials. Recovery of Ba2+ current from ultra-slow inactivation occurred with a time constant of 3.7 s at −40 mV and 0.7 s at −80 mV. The gradual decrease in iCa on increasing the rate to 3 Hz may have been the result of the development of ultra-slow voltage-dependent inactivation.

The role of inward Na(+)‐Ca2+ exchange current in the ferret ventricular action potential.

1. Inward Na(+)‐Ca2+ exchange current (iNaCa) was either blocked in ferret ventricular cells by replacing extracellular Na+ with Li+ or substantially reduced by the almost complete elimination of the Ca2+ transient by buffering intracellular Ca2+ with the acetoxymethyl ester form of BAPTA (BAPTA AM). 2. During square wave voltage clamp pulses to 0 mV, replacing extracellular Na+ with Li+ or buffering intracellular Ca2+ with BAPTA AM resulted in the loss of a transient inward current. This current was increased by the application of isoprenaline (expected to increase the underlying Ca2+ transient) and displayed the voltage‐dependent characteristics of inward iNaCa. 3. Replacing extracellular Na+ with Li+ or buffering intracellular Ca2+ caused a significant shortening of the action potential (at ‐65 mV, 44 +/‐ 2% with Li+ and 20 +/‐ 2% with BAPTA AM). The shortening can be explained by changes in iNaCa. 4. The action potential clamp technique was used to measure the BAPTA‐sensitive current (putative iNaCa) and the Ca2+ current (ica; measured using nifedipine) during the action potential. Under control conditions, the inward BAPTA‐sensitive current makes approximately the same contribution as iCa during much of the action potential plateau. These results suggest an important role for inward iNaCa in the ferret ventricular action potential.

The role of Na(+)-Ca2+ exchange current in electrical restitution in ferret ventricular cells.

1 The mechanisms underlying electrical restitution (recovery of action potential duration after a preceding beat) were investigated in ferret ventricular cells. The time to 80% recovery (t80) of action potential duration was ∼204 ms. 2 At a holding potential of −80 mV, the Ca2+ current (ICa) reactivated and the delayed rectifier K+ current (IK) deactivated very rapidly (t80: ∼32 and ∼93 ms, respectively). The kinetics of both currents are too fast to account for electrical restitution alone. 3 The putative inward Na+−Ca2+ exchange current (INa‐Ca) produced by the Na+−Ca2+ exchanger in response to the intracellular Ca2+ transient reprimed (t80: 189 ms) with the same time course as mechanical restitution (recovery of contraction) and with a similar time course to electrical restitution. 4 Substantial reduction of inward INa‐Ca, by buffering intracellular Ca2+ with the acetyl methyl ester form of BAPTA, shortened the action potential and greatly altered the electrical restitution curve. Subsequent addition of nifedipine (to block ICa) or 4–aminopyridine (4–AP) (to block the transient outward current, ITO) further altered the electrical restitution curve. 5 Any time‐dependent current that contributes to the action potential is likely to affect the time course of electrical restitution. Although ICa:, IK and ITO were previously thought to be the only currents involved in electrical restitution, we conclude that inward INa‐Ca also plays an important role.

THE ROLE OF THE NA+-CA2+ EXCHANGER IN THE RATE-DEPENDENT INCREASE IN CONTRACTION IN GUINEA-PIG VENTRICULAR MYOCYTES

1. The intracellular sodium activity (alpha Na1), contraction and membrane current were recorded simultaneously in voltage‐clamped guinea‐pig ventricular myocytes. 2. Increasing the frequency (from 0.5 to 3 Hz) of voltage clamp pulses to 0 mV from a holding potential of ‐80 mV led to an increase in both alpha Na1 and contraction. The rate‐dependent increase in contraction was reduced by 25 microM tetrodotoxin (TTX) and abolished with a holding potential of ‐40 mV. There was no rate‐dependent rise in alpha Na1 with a holding potential of ‐40 mV. These results suggest an important role for alpha Na1 and in particular Na+ influx via Na+ channels during rate‐dependent changes in contraction. 3. After an increase in frequency from 0.5 to 3 Hz, membrane current at the end of voltage clamp pulses became progressively more outward and the tail current upon at repolarization became progressively more inward compared with those recorded at 0.5 Hz. TTX reduced the magnitude of both the outward and inward rate‐dependent shifts of current. 4. The addition of extracellular CsCl blocked the inward rectifier potassium current (IK.1) and the delayed rectifier (IK), but did not change the rate‐dependent shift in current. 5. The difference between current‐voltage relationships at 0.5 and 3 Hz showed that the rate‐dependent outward shift of current at the end of voltage clamp pulses was small at potentials negative to ‐20 mV, was larger at more positive potentials and was reduced by TTX at most potentials. The TTX‐sensitive component reversed at ‐47 mV. 6. These results are consistent with a net increase in outward Na(+)‐Ca2+ exchange current during a voltage clamp pulse in response to the rise of alpha Na1. The increase in outward current (resulting from either enhanced Ca2+ influx or reduced Ca2+ efflux) will augment the Ca2+ load of the cell and contribute to the rate‐dependent increase in contraction.

Negative inotropic effects of tumour necrosis factor-α and interleukin-1β are ameliorated by alfentanil in rat ventricular myocytes

Serum levels of tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) increase during an inflammatory response and have been reported to induce a negative inotropic effect on the myocardium. Alfentanil, an opioid analgesic often used in the critical care of patients with sepsis, has been shown to enhance ventricular contractility. This study characterised the effects of TNF‐α and IL‐1β on contraction and the Ca2+ transient and investigated whether depressed ventricular function was ameliorated by alfentanil.

Effects of halothane on the transient outward K(+) current in rat ventricular myocytes.

Halothane has been shown to affect several membrane currents in cardiac tissue including the L‐type calcium current (ICa), sodium current and a variety of potassium currents. However, little is known about the effects of halothane on the transient outward K+ current (Ito). Single ventricular myocytes from rat hearts were voltage clamped using the whole cell patch configuration and an EGTA‐containing pipette solution to record the Ca2+‐independent, 4‐aminopyridine sensitive component of Ito. 300 μM Cd2+ or 10 μM nifedipine was used to block ICa. At +80 mV, Ito (peak current minus current at the end of the pulse) was 1.8±0.2 nA under control conditions which was reduced to 1.3±0.2 nA by 1 mM halothane (P<0.001, mean±s.e.mean, n=9). The inhibition of Ito by halothane was concentration‐dependent (K0.5, 1.1±0.2 mM). One mM halothane led to a 16 mV shift in the steady‐state inactivation curve towards negative membrane potentials (P=0.005, n=8) but had no significant effect on the activation‐voltage relationship (P=0.724). One mM halothane also increased the rate of inactivation of Ito; the dominant time constant of inactivation was reduced from 14±1 to 9±1 ms (P=0.017, mean±s.e.mean, n=6). These data show that halothane reduced Ito; 0.3 mM, close to the MAC50 value for halothane, inhibited the current by 15% and as such, the inhibition of Ito will be relevant to the clinical situation. Halothane induced a shift in the steady‐state inactivation curve and accelerated the inactivation process of Ito which could be responsible for its inhibitory effect. Due to the differential transmural expression of Ito in ventricular tissue, inhibition of Ito would reduce the transmural dispersion of refractoriness which could contribute to the arrhythmogenic properties of halothane.