Mechanisms underlying the frequency dependence of contraction and [Ca(2+)](i) transients in mouse ventricular myocytes

Abstract

In most mammalian species force of contraction of cardiac muscle increases with increasing rate of stimulation, i.e. a positive force-frequency relationship. In single mouse ventricular cells, both positive and negative relationships have been described and little is known about the underlying mechanisms. We studied enzymatically isolated single ventricular mouse myocytes, at 30 degrees C. During field stimulation, amplitude of unloaded cell shortening increased with increasing frequency of stimulation (0.04 +/- 0.01 Delta L/L(0) at 1 Hz to 0.07 +/- 0.01 Delta L/L(0) at 4 Hz, n = 12, P < 0.05). During whole cell voltage clamp with 50 microM [K5-fluo-3](pip), both peak and baseline [Ca(2+)](i) increased at higher stimulation frequencies, but the net Delta[Ca(2+)](i) increased only modestly from 1.59 +/- 0.08 Delta F/F(0) at 1 Hz, to 1.71 +/- 0.11 Delta F/F(0) at 4 Hz (n = 17, P < 0.05). When a 1 s pause was interposed during stimulation at 2 and 4 Hz, [Ca(2+)](i) transients were significantly larger (at 4 Hz, peak F/F(0) increased by 78 +/- 2 %, n = 5). SR Ca(2+) content assessed during caffeine application, significantly increased from 91 +/- 24 micromol l(-1) at 1 Hz to 173 +/- 20 micromol l(-1) at 4 Hz (n = 5, P < 0.05). Peak I(Ca,L) decreased at higher frequencies (by 28 +/- 6 % at 2 Hz, and 45 +/- 8 % at 4 Hz), due to slow recovery from inactivation. This loss of I(Ca,L) resulted in reduced fractional release. Thus, in mouse ventricular myocytes the [Ca(2+)](i)-frequency response depends on a balance between the increase in SR content and the loss of trigger I(Ca,L). Small changes in this balance may contribute to variability in frequency-dependent behaviour. In addition, there may be a regulation of the contractile response downstream of [Ca(2+)](i).

Figure 1. Frequency dependence of cell shortening in mouse myocytes

A, example of cell shortening measurements in an unloaded cell, externally stimulated at 1, 2 and 4 Hz, showing a positive frequency response. B, frequency response of amplitude of cell shortening (ΔL), normalized to resting cell length (L₀) of individual cells, represented by thin lines, one cell showed a negative response. Average data show an increase in amplitude with increasing stimulation frequency (mean ± s.e.m., n = 12, *P < 0.05 vs. 1 Hz).

Figure 2. Frequency dependence of [Ca²⁺]_i transients

A, original current traces, I, and [Ca²⁺]_i transients, F/F₀, recorded in a voltage clamped cell during steady-state stimulation with 25 ms depolarizing steps from -70 to +20 mV at the indicated frequencies. B, individual data for frequency dependence of [Ca²⁺]_i transients, F/F₀, showing peak values (continuous lines) and baseline values (dashed lines) of 17 cells. Pooled data show an increase in peak [Ca²⁺]_i (□) and baseline [Ca²⁺]_i (▪) at higher stimulation frequencies (mean ± s.e.m., *P < 0.05 vs. 1 Hz). C, amplitude of the [Ca²⁺]_i transients, ΔF/F₀, with increasing stimulation rate (mean ± s.e.m., *P < 0.05 vs. 1 Hz).

Figure 3. [Ca²⁺]_i transients following 1 s pause after different stimulation frequencies

A, typical example of [Ca²⁺]_i transients, F/F₀, elicited by a 25 ms depolarizing pulse from -70 to +20 mV following 1 s interval (test pulse, t) after stimulation with 10 conditioning pulses (conditioning, c) at different frequencies of 1, 2 and 4 Hz. B, pooled data of peak [Ca²⁺]_i, F/F₀, of the test pulse (□) and of the last conditioning pulse (▪) as a function of increasing stimulation frequency (# P < 0.05 for t vs. c, *P < 0.05 for 2 and 4 Hz vs. 1 Hz, mean ± s.e.m., n = 5).

Figure 4. Frequency dependence of SR Ca²⁺ content

A, typical example of integrated Na⁺-Ca²⁺ exchange current, shown as the running integral, and [Ca²⁺]_i transients, F/F₀, induced by 8 s application of 10 mm caffeine following a 1 s pause after stimulation with 10 conditioning pulses at the indicated frequency (1, 2 and 4 Hz). B, peak of caffeine-induced transients, F/F₀, with increasing frequency of stimulation (mean ± s.e.m., n = 5, P < 0.05). C, SR Ca²⁺ content, calculated from integrating I_Na,Ca, and normalized to accessible cell volume, as described in the Methods section (mean ± s.e.m., n = 5, P < 0.05).

Figure 5. Reduced ‘fractional’ release at higher frequencies

A, the ratio of the amplitude of the [Ca²⁺]_i transients of the conditioning pulses, ΔF/F_c, and the [Ca²⁺]_i transient of caffeine-induced transient ΔF/F_caff, from experiments as shown in the inset, as an estimate of fractional release (mean ± s.e.m., n = 5, P < 0.05). B, for comparison we also calculated the ratio between the amplitude of the [Ca²⁺]_i transients of the conditioning pulses, ΔF/F_c, relative to the [Ca²⁺]_i transient of the test pulse after a 1 s pause, ΔF/F_t, at different frequencies (mean ± s.e.m., n = 5, P < 0.05). Data from the same experiments as shown in Fig. 3.

Figure 6. Frequency dependence of L-type Ca²⁺ current

A, time course of a typical experiment measuring I_Ca,L during stimulation at 1, 2 and 4 Hz with a 10 s interval between the different frequencies, showing absolute peak values of I_Ca,L at each frequency. I_Ca,L was measured with 25 ms depolarizing steps from -70 to 0 mV in K⁺-free internal and external solutions. B, peak of steady-state current, expressed as percentage of I₀ (mean ± s.e.m., n = 6). C, recovery from inactivation of I_Ca,L; a 25 ms depolarizing step from -70 to +20 mV was followed by a test step after a 125 ms interval, and this interval was increased by 25 ms steps, interpulse duration was 10 s.

Figure 7. Simultaneous measurement of cell shortening and [Ca²⁺]_i transients during field stimulation

Pooled data of eight cells loaded with fluo-3AM, and calibrated [Ca²⁺]_i signals. A, amplitude of fractional cell shortening. B, peak (□) and basal (▪) [Ca²⁺]_i of transients. C, relative amplitude of cell shortening (○), calculated as percentage of the amplitude at 1 Hz, compared to the relative increase in the amplitude of the [Ca²⁺]_i transients (•).