Impact of Overnight Storage of Human Atrial Myocytes on Intracellular Calcium Homeostasis and Electrophysiological Utility

Abstract

Human atrial myocytes afford an attractive experimental model to investigate mechanisms underlying electrophysiological alterations in cardiovascular disease. However, this model presents limitations, such as the availability of human atrial tissue and a variable yield of myocytes isolation. Therefore, we aimed to determine whether overnight storage can increase the time window where the electrophysiological properties of human atrial myocytes can be determined. To address this issue, human atrial myocytes isolated from patients undergoing cardiac surgery were used for patch-clamp experiments on the day of cell isolation (Day 1) and the following day (Day 2). The shape of the current-voltage (I-V) relationship for the calcium current (I_Ca) depended on the access resistance and the cell capacitance, with large cells (>75 pF) requiring a lower access resistance (<15 MΩ) than small cells (<40 pF) to avoid distortion of the I-V curve. Importantly, overnight storage did not significantly affect (1) the I_Ca amplitude or properties, (2) sarcoplasmic reticulum calcium homeostasis or (3) the frequency-dependency of the beat-to-beat response. In conclusion, overnight storage of isolated human atrial myocytes at 4 °C does not affect essential features of intracellular calcium homeostasis and, therefore, affords a simple protocol to extend the experimental lifetime of human atrial myocytes.

Keywords: beat-to-beat response; calcium currents; calcium homeostasis; human atrial myocyte; patch-clamp technique; sarcoplasmic reticulum.

Figure 1

Impact of cell size and access resistance on the shape of the current–voltage relationship. (A) Representative I_Ca recordings at voltages between −20 and +20 mV at access resistances (R_s) < 15 mΩ, between 25 and 40 MΩ and >60 MΩ. Recordings were classified according to the cell capacitance (C_S) as small (left), medium-sized (center), or big myocytes (right). (B) Current–voltage relationship determined in 51 cells from 15 patients with different R_s given on the right. Values were normalized to the current density at 0 mV. (C) I–V relationship determined with R_s < 15 mΩ in small, medium-sized, and big myocytes. Statistical significance in panels (B,C) was determined using a two-way ANOVA followed by a Tukey’s HSD post hoc test. (D) Fraction of myocytes with fast I_Ca inactivation (tau-1) at different R_s given below bars. Statistical significance in panel (D) was determined using a multivariate logistic regression model. (E) Tau-1 measured at different R_s (given below bars) in small, medium-sized, and big myocytes. Statistical significance in panel E was determined using two-way ANOVA followed by Tukey’s HSD post hoc test. Groups significantly different from the R_s < 15 MΩ group are indicated with *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 2

Impact of overnight storage on L-type calcium current properties. (A) Representative I_Ca traces recorded at different test potentials (given above traces) on Day 1 and Day 2 in myocytes from the same patient. The I–V relationship is shown for all myocytes. (B) Conductance–voltage relationship for the same myocytes. Solid lines represent average values. (C) Steady-state I_Ca recordings in myocytes paced at 0.5 Hz. The bar graph shows the I_Ca density for Day 1 and Day 2. (D) Fast and slow time constants for steady-state I_Ca inactivation. p-values given above bars were obtained using a paired t-test comparing data from the same patients recorded on Day 1 and Day 2.

Figure 3

Impact of overnight storage on SR calcium loading. (A) Experimental protocol used to estimate SR calcium load by exposing myocytes transiently to 10 mM caffeine (Caf-1). Subsequently, 20 stimulation pulses (p1–p20) were used to reload the SR, and a second caffeine application (Caf-2) was used to determine the amount of calcium re-accumulated in the SR. Representative current traces elicited by Caf-1 (B) and Caf-2 (C) with their respective time integrals are shown on the top and mean values of the time integrals are shown below, illustrating changes on the left (paired values are connected with bars) and confidence intervals on the right. Number of cells/patients is given below bars. Statistical significance in panels (B,C) was determined using a paired t-test to compare data from the same patients recorded on Day 1 and Day 2.

Figure 4

Impact of overnight storage on SR calcium re-loading. (A) Superimposed I_Ca traces elicited by stimulation pulses #1, 2, 5, 10, and 20 after Caf-1 on Day 1 and Day 2. (B) The time constant for fast I_Ca decay (tau-1) was normalized to its maximum and plotted as a function of the number of stimulation pulses used to reload the SR. Tau-1 values were fit with an exponential decaying function. (C) Number of pulses required to achieve half-maximal recovery of the SR calcium content on Day 1 and Day 2, assuming that it is inversely proportional to tau-1. Statistical significance in panel (C) was determined using a paired t-test to compare data from the same patients recorded on Day 1 and Day 2.

Figure 5

Impact of overnight storage on the incidence of transient inward currents. (A) Representative current traces recorded in myocytes from the same patient isolated on Day 1 (blue) and Day 2 (yellow). (B) Mean I_TI frequency recorded on Day 1 and Day 2. Error bars represent 95% confidence intervals. The p-value given above bars was determined using the Wilcoxon signed-rank test to compare data from the same patients recorded on Day 1 and Day 2.

Figure 6

Impact of overnight storage on the beat-to-beat response. (A) Representative current traces recorded over a 6 s period in a myocyte paced at increasingly shorter stimulation intervals (given on the right). Horizontal scale bars indicate 200 ms for the traces. The peak I_Ca is indicated with asterisks at stimulation intervals of 1 s or shorter. (B) Fraction of 46 myocytes from 15 patients showing a uniform I_Ca amplitude for each stimulation frequency, recorded on Day 1 (blue bars) or Day 2 (yellow bars).