Spontaneous, local diastolic subsarcolemmal calcium releases in single, isolated guinea-pig sinoatrial nodal cells

Abstract

Uptake and release calcium from the sarcoplasmic reticulum (SR) (dubbed "calcium clock"), in the form of spontaneous, rhythmic, local diastolic calcium releases (LCRs), together with voltage-sensitive ion channels (membrane clock) form a coupled system that regulates the action potential (AP) firing rate. LCRs activate Sodium/Calcium exchanger (NCX) that accelerates diastolic depolarization and thus participating in regulation of the time at which the next AP will occur. Previous studies in rabbit SA node cells (SANC) demonstrated that the basal AP cycle length (APCL) is tightly coupled to the basal LCR period (time from the prior AP-induced Ca2+ transient to the diastolic LCR occurrence), and that this coupling is further modulated by autonomic receptor stimulation. Although spontaneous LCRs during diastolic depolarization have been reported in SANC of various species (rabbit, cat, mouse, toad), prior studies have failed to detect LCRs in spontaneously beating SANC of guinea-pig, a species that has been traditionally used in studies of cardiac pacemaker cell function. We performed a detailed investigation of whether guinea-pig SANC generate LCRs and whether they play a similar key role in regulation of the AP firing rate. We used two different approaches, 2D high-speed camera and classical line-scan confocal imaging. Positioning the scan-line beneath sarcolemma, parallel to the long axis of the cell, we found that rhythmically beating guinea-pig SANC do, indeed, generate spontaneous, diastolic LCRs beneath the surface membrane. The average key LCR characteristics measured in confocal images in guinea-pig SANC were comparable to rabbit SANC, both in the basal state and in the presence of β-adrenergic receptor stimulation. Moreover, the relationship between the LCR period and APCL was subtended by the same linear function. Thus, LCRs in guinea-pig SANC contribute to the diastolic depolarization and APCL regulation. Our findings indicate that coupled-clock system regulation of APCL is a general, species-independent, mechanism of pacemaker cell normal automaticity. Lack of LCRs in prior studies is likely explained by technical issues, as individual LCRs are small stochastic events occurring mainly near the cell border.

Fig 1. Both GP and R single rhythmically beating SANC generate spontaneous diastolic LCRs beneath sarcolemma under the basal conditions.

Representative examples of confocal line scan images of LCRs (marked with asterisks) and AP-induced Ca²⁺ transient recorded in SANC of GP (A) and R (B). (C) Schematic illustration of the correct scan-line orientation along the cell border. (D) Definition of the LCR period and AP cycle length (APCL).

Fig 2. AP cycle length (APCL) in both GP and R single rhythmically beating SANC is tightly coupled to LCR period under the basal conditions.

(A) Histogram of distributions of all individual APCLs, and (B) Individual LCRs periods in GP (91, LCRs from 13 cells) and R SANC (422 LCRs from 31 cells). Insets in (A) and (B) show the average APCL (interval between AP-induced Ca²⁺ transients) and LCR period (the time between the rapid upstroke of the prior AP-triggered Ca²⁺ transient and the onset of a LCR in diastole) in GP (n = 13) and R (n = 31) SANC. (C) Relationship of the average APCL to the average LCR period is subtended by the same linear function in GP SANC (y = 1.2x + 8.3; R² = 1.0; n = 13) and R SANC (y = 1.1x + 27.8; R² = 1.0; n = 31). (D) Average CV of APCL (measured as SD/Mean) in GP and R SANC.

Fig 3. Distributions and average LCR characteristics measured by confocal microscopy in single spontaneously beating GP and R SANC under the basal conditions.

Histogram distribution of LCR characteristics: (A) amplitude (as normalized Ca²⁺ fluorescence, F/F₀); (B) size (μm); (C) duration (ms); (D) Ca²⁺ signals of individual LCRs (ΔF/F₀*μm*ms*2⁻¹) (see Methods and Panel E) in GP (91, LCRs from 13 cells) and R SANC (422 LCRs from 31 cells). Insets (A-D) show the average data in GP (n = 13 cells) and R SANC (n = 31 cells). *P<0.05 by unpaired t-test. (E) An example of two-dimensional and three-dimensional confocal line-scan images and surface plot of an LCR, demonstrating our measurements of LCR characteristics.

Fig 4. Number of LCR events and magnitude of Ca²⁺ signals of the LCR ensemble in single rhythmically beating GP and R SANC.

(A) The average number of LCR per cycle, normalized to 100 μm of scanning line in each cell. (B) The average number of LCR, normalized per 100 μm of the line-scan image and per 1-s time interval of recording. (C) Average amplitudes of Ca²⁺ signals of the LCR ensemble (as the integrated Ca²⁺ signal produced by each LCR ((ΔF/F₀*μm*ms*#*2⁻¹), see Methods), normalized per 100 μm of the line-scan image during a 1-s time interval in GP (n = 13 cells) and R SANC (n = 31 cells). *P<0.05 by unpaired t-test.

Fig 5. β-AR stimulations (BARs) decreases the AP-induced Ca²⁺ transient cycle lengths (APCL) and LCR periods in single spontaneously beating GP SANC.

Representative examples of confocal line-scan images (upper panels) and AP-induced Ca²⁺ transients (lower panels) of GP SANC prior to (A) and in response to 1 μM of Isoproterenol (ISO) in the same cell (B). LCRs are marked with asterisks. BARs (1 μM ISO) shifts the distributions of APCL (C) and LCR period (D) to the left in GP SANC. (E) The average change of APCL and LCR period following ISO exposure (% of control) (n = 9, *P<0.05 versus control by paired t-test). (F) Relationship of the average APCL (% of control) to the average LCR period change (% of control) in response to ISO in GP SANC (n = 9).

Fig 6. Effect of β-AR stimulations (BARs) on average and distributions of LCR characteristics in spontaneously beating GP SANC.

BARs (ISO, 1 μM) increase (A) the average LCR size (μm) and LCR frequency (#, normalized per 100 μm and per 1 s) in GP SANC and (B) the average amplitude of the Ca²⁺ signal of individual LCRs (ΔF/F₀*μm*ms*2⁻¹) and Ca²⁺ signal of the LCR ensemble (ΔF/F₀*μm*ms*#*2⁻¹). *P<0.05 versus control by paired t-test (n = 9). Distributions of LCR sizes (C) and Ca²⁺ signals of individual LCRs (D) are shifted in GP SANC toward higher values in response to 1 μM of ISO (84 LCRs in control and 168 LCRs with ISO from n = 9 cells).

Fig 7. Spontaneous diastolic LCRs detected by high-speed 2D imaging under the basal conditions and in response to β-AR receptor stimulations (BARs).

(A) Ca²⁺ images under control and (C) during 1 μM of ISO measured by the high-speed 2D camera. The three green rectangles, region of interest (ROI) in (A) and (C) represent arbitrary locations within the cell perimeter to illustrate spontaneous diastolic LCRs. (B) and (D). Respective time series of the of AP-induced Ca²⁺ transients and spontaneous diastolic LCRs (a small bump preceding AP-induced Ca²⁺ transient and indicated by oval), measured within ROI 1–3 in (A) and (C) in control conditions and during superfusion with 1 μM of ISO.