Paul Brooksby

The electrophysiological characteristics of hypertrophied ventricular myocytes from the spontaneously hypertensive rat.

Objectives: Previous studies on multicellular preparations have shown that hypertrophied cardiac muscle from the spontaneously hypertensive rat (SHR) has a prolonged action potential. The first aim of the present study was to determine whether the action potential of isolated left ventricular myocytes was similarly prolonged and to study the underlying membrane currents that might be responsible. The second aim was to evaluate the L-type calcium current amplitude of SHR myocytes, as we have recently shown that they have an increased contraction and an increase in the calcium trigger entering via the L-type calcium channel might be one possible mechanism for this. Methods: The electrophysiological characteristics of left ventricular myocytes isolated from the SHR were compared with those from normotensive control rats. Action potentials were recorded with microelectrodes. Cells were voltage-clamped and the membrane currents elicited by steps to different potentials were analysed. Blockers of potassium and calcium currents were used to reveal the contribution made by these currents to net membrane currents. Results: SHR myocytes had prolonged action potentials. The action potential duration of SHR myocytes at 90% repolarization was found to be longer, although at 20% and 50% repolarization no difference was found. There was no difference in the resting membrane potential between SHR and control myocytes. Using a voltage clamp we studied the L-type calcium current and potassium currents. The major change in SHR myocytes was a decrease in the magnitude (normalized to the membrane capacitance) of the inward rectifier potassium current elicited by negative potentials. There was no detectable difference in either the transient outward or delayed rectifier potassium currents. We also found no difference in the magnitude, time course or voltage dependence of L-type calcium current in hypertrophied SHR myocytes. Conclusions: First, the action potential of SHR myocytes was prolonged compared with control myocytes. Secondly, the main change in SHR myocytes was that pulses to negative potentials elicited a lower inward rectifier potassium current. A reduction in the density of inward rectifier channels might play a role in prolonging the SHR action potential, since a lower outward repolarizing current will flow through inward rectifier potassium channels during the SHR action potential repolarization. Thirdly, there was no difference in L-type calcium current density or time course between SHR and control myocytes. Thus, a change in L-type calcium current probably plays no role in causing the prolonged SHR action potential or the increased contraction of hypertrophied SHR ventricular myocytes. Finally, the prolonged action potential in SHR myocytes may itself be one factor responsible for the increased contraction of these cells.

Intracellular calcium transients recorded with Fura-2 in spontaneously active myocytes isolated from the atrioventricular node of the rabbit heart

We have used the fluorescent Ca indicator Fura-2 to assess the changes in intracellular calcium (Ca1) in single spontaneously active myocytes isolated from the rabbit atrioventricular node (avn). Simultaneous recordings of membrane potential and the Fura-2 ratio signal (which reflects Ca1) showed that a transient rise of Ca1 occurred with each spontaneous action potential (ap). The ap upstroke preceded the rise in Ca1 and repolarization of the ap occurred faster than the decline of Ca1. The level of Ca1 remained raised and progressively declined towards a baseline diastolic level during the subsequent pacemaker depolarization. The Fura-2 (Ca1) transient in spontaneously active avn cells had a time-to-peak of 49.2±5.4 ms (mean + s.e.m.; n= 7) and declined with a single exponential time course (time constant = 139.8±23.9 ms; n = 7). Application of 10 µM ryanodine completely and irreversibly abolished the Ca1 transient, identifying the sarcoplasmic reticulum (sr) as the major source of releasable Ca. Both removal of external Ca and block of L-type Ca channels (with 2 µM nifedipine) also abolished Ca1 transients, suggesting that Ca entry via L-type Ca-channels is involved in triggering the sr Ca release underlying the Ca1 transient. Removal of external Na (in the presence of 20 µM nifedipine to block L-type Ca channels) caused a reversible increase in Ca1, showing that Na/Ca exchange is present in avn cells and that it is involved in Ca1 regulation. Spontaneous Ca1 transients were abolished by 1 µM acetylcholine, and this was associated with a hyperpolarization of membrane potential and cessation of action potentials. Isoprenaline (1 µM) increased the rate and amplitude of spontaneous Ca1 transients; this corresponded to an increase in the rate and a change in the shape of spontaneous action potentials observed in patch-clamped avn myocytes.

Contractile properties of ventricular myocytes isolated from spontaneously hypertensive rat.

Objective: To determine whether there are any differences in contractile properties of individual cardiac myocytes isolated from the spontaneously hypertensive rat (SHR) in comparison with its normotensive control — the Wistar—Kyoto (WKY) rat. Design: The effects of cardiac hypertrophy upon individual myocytes from SHR have not been studied previously. Isolated cardiac myocytes do not suffer from a number of problems inherent in experiments on multicellular preparations. Methods: Seven SHR and eight WKY animals were studied. Age-matched animals were compared at 60 and 100 days old. Ventricular myocytes were isolated enzymatically. Myocyte length and width was measured. The cells were stimulated with extracellular electrodes and contraction was measured optically. The effects of altering stimulus rate and extracellular calcium concentration upon contraction were studied. Results: SHR myocytes were found to be significantly wider than WKY myocytes. The contraction (i.e. unloaded cell shortening) of SHR myocytes at stimulation rate of 0.3, 1, 2 and 3 Hz was significantly increased. The time-course of contraction was altered, with SHR myocytes having an increased maximal velocity of shortening and relaxation. The response to changes in bathing calcium was similar in both strains. Conclusions: Individual cardiac myocytes isolated from SHR have an increased contraction. This indicates that cardiac hypertrophy, at least in the early stages, is a protective adaptation allowing the heart to overcome the increased afterload resulting from hypertension.