Calcium spike variability in cardiac myocytes results from activation of small cohorts of ryanodine receptor 2 channels

Abstract

In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.

Figure 1. Typical calcium currents, line scan images and calcium spikes

Recordings from the same position were made at 15 s intervals. A, voltage stimuli to 0 mV. B, whole-cell calcium currents. C, x–t line-scan fluorescence images. The numbered arrowheads point to the positions of the release sites generating calcium spikes and their width corresponds to the width of analysed fluorescence profiles. D, fluorescence profiles of the spikes indicated in C. Red lines are the best fits of spikes by eqns (2) (single spikes) or (3) (twin spikes, asterisks). The time coordinate is identical in all panels.

Figure 2. The estimated latencies and amplitudes of all calcium spikes recorded at voltage stimuli to 0 mV

The single, first and second spikes are indicated in white, green and red symbols, respectively.

Figure 3. Statistics of amplitudes of calcium spikes

The same data set as in Fig. 2. Box plots show 25, 50 and 75 percentiles of amplitude distributions of the single, first and second spikes, and of the combined amplitudes of the first and the second spikes. Data points (filled circles) with similar values are laterally offset. Statistically significant differences at P = 0.05 are displayed as horizontal lines.

Figure 4. Analysis of the latencies of calcium spikes

The same data set as in Fig. 2. A, statistics of latencies of the calcium spikes. Box plots show 25, 50 and 75 percentiles of the latency distribution of single, first and second spikes. Statistically significant differences at P = 0.05 are displayed as horizontal lines. Data points (filled circles) with similar values are laterally offset. B, the normalized cumulative latency distribution of single, first and second spikes (continuous, dashed and thin continuous lines, respectively). C, the distribution of intervals between the onsets of the first and of the second spike in twin spikes. The median is 21.0 ms, the minimum 3.3 ms and maximum 62.8 ms.

Figure 5. Kinetic properties of calcium spikes

The same data set as in Fig. 2. A, statistics of the time to peak (TTP) of single, first and second spikes. B, statistics of the full duration at half-maximum (FDHM) of single, first and second spikes. Data points (filled circles) with similar values are laterally offset. Box plots show 25, 50 and 75 percentiles of the distributions

Figure 6. Amplitude distribution of the single, first and second spikes

The same data set as in Fig. 2. A, amplitude histograms of single (open) and first spikes (black). B, amplitude histogram of all primary spikes. Filled black columns show the theoretical distribution for 4 equidistant amplitude levels (see Table 2). C, amplitude histogram of the second spikes (open). Black columns show the theoretical distribution for second spikes with a maximum of 2 open RyR2 channels (see Results).

Figure 7. Testing the quantal character of the single, first and second spikes recorded at voltage stimuli to 0 mV

A, a histogram of the relative occurrence of primary spikes (white) with different quantal contents. The best fit of the data with eqn (7) (n_q = 182, p_A = 0.0060) is shown by black columns. B, the histogram of relative occurrences of second spikes with different quantal contents (white). The best fit of the data with eqn (7) (n_q = 182, p_A = 0.003) is shown as black bars. In B and C, a similar fit could be obtained for n_q = 47–200. C, the goodness of fit, expressed as χ²/df, for description of the amplitude distribution of single/first (filled symbols) and second spikes (open symbols). The lines show the value of n_q at which the fits are accepted by the χ² test at P = 0.05 (continuous and dashed for primary and second spikes, respectively). D, the fraction of primary spikes of the respective quantal content that were followed by a second spike. The best fit with eqn (8) (p_refr = 0.70) is shown by black bars.

Figure 8. Properties of calcium spikes evoked by threshold stimuli

A, the estimated latencies and amplitudes of all calcium spikes evoked by depolarisation to −35 mV. The single, first and second spikes are indicated in white, green and red symbols, respectively. B, white columns represent the amplitude distribution of the primary spikes evoked by depolarization to –35 mV. Black bars show the best theoretical amplitude distribution with 3 equidistant amplitude levels (see Results).