Figure 1.
Validation of mitochondrial Ca2+ measurements.
(A) Mn2+ (50 µmol/L) quenching of the cytosolic Indo-1 fluorescence ratio 410/490 (top panel), and fluorescence emission at wavelengths of 410 (middle panel), and 490 nm (lower panel). Dashed line under recordings of flourecence emission at individual wavelengths indicates background cell autofluorescence (the fluorescence without Indo-1 loading). (B) Permeabiliziation of the sarcolemma by 5 µmol/L digitonin alone (D5; n = 6) or together with Mn2+ to quench cytosolic Indo-1 (n = 6). Note that Mn2+ plus digitonin decreases the Indo fluorescence to the same level as Mn2+ alone (n = 10). A higher digitonin concentration (25 µmol/L D25; n = 4), permeabilizes the mitochondrial membrane in addition to the sarcolemma, and markedly depletes the cell fluorescence. (C) Application of Mn2+ does not alter the spontaneous AP firing rate. *p<0.05 vs. drug control, **p<0.05 vs. Mn2+.
Figure 2.
The effect of inhibition of mitochondrial fluxes on mitochondrial Ca2+ and spontaneous AP firing rate.
(A) Mean mitochondrial fluorescence intensity in response to specific inhibition of mitochondrial Ca2+ influx or efflux (n = 10 for each drug), (B) AP recordings (before and after 15 min exposure to each of the drugs), and (C) average time-dependent change in the rate of AP induced contractions in the presence of CGP-37157 (n = 12) or Ru360 (n = 11). **p<0.05 vs. Ru360.
Table 1.
Average characteristics of SANC spontaneous APs in response to pharmacological perturbations (n = 9 in each group).
Table 2.
Average characteristics of SANC spontaneous AP induced Ca2+ transients (n = 43).
Table 3.
Average characteristics of SANC spontaneous AP induced Ca2+ transients in response to pharmacological perturbations.
Figure 3.
SR load estimation from rapid caffeine application.
Effects of a rapid application (“spritz”) of caffeine (indicated by the arrow) onto SANC (A) in control, or (B) in the presence of Ru360 or (C) CGP-37157. (D) Average effects of Ru360 or CGP-37157 on peak AP-induced cytosolic Ca2+ prior to a caffeine spritz (left), and the subsequent caffeine-induced cytosolic Ca2+ transient (right) (n = 12, for each group). (The caffeine response can be usually measured only once in a given SANC, because following caffeine application a prolonged period is required for AP firing rate to return to the control AP firing rate. Therefore, the effects of caffeine before (i.e. control) and following application of drugs that affect Ca2+m flux were measured in different cells).
Figure 4.
Specific inhibition of Ca influx into or efflux from mitochondria in intact SANC modifies spatiotemporal characteristics of LCRs.
(A) Confocal line scan Ca2+ images of a representative SANC before and following exposure to 2 µmol/L Ru360 or 1 µmol/L CGP-37157. LCRs are indicated by arrowheads. The LCR period is defined as the time from the peak of the prior AP-induced Ca2+ transient to the LCR onset. Histograms of LCR (B) size (full width at half-maximum amplitude), (C) amplitude (F/F0), and (D) duration (full duration at half-maximum amplitude) in the presence of Ru360 (n = 12; 102 LCRs) and CGP-37157 (n = 12; 92 LCRs).
Figure 5.
Relationships among the Ca2+ release of the LCR ensemble, LCR period, SR Ca2+ load and AP firing rate.
The total LCR Ca2+ ensemble (A) average data and (B) histogram in the presence of Ru360 or CGP-37157. (C) The relationship between caffeine-induced Ca2+ release F/F0 and the total LCR Ca2+ ensemble. Specific inhibition of Ca2+ influx into or efflux from mitochondria in intact SANC shifts the LCR period and AP cycle length. (D) LCR period and (E) AP cycle length in the presence of Ru360 (n = 12; 102 LCRs) or CGP-37157 (n = 12; 92 LCRs). (F) The change in LCR period in response to perturbing mitochondrial Ca2+ flux predicts the concomitant change in AP cycle length. The Ru360-induced decrease in the spontaneous cycle length, and CGP-37157-induced increase in spontaneous cycle length, are both predicted by their effects on the LCR period. The dashed line is the line of identity. *p<0.05 vs. control, **p<0.05 vs. Ru360.
Figure 6.
The effect of CPA on AP cycle length, LCR period and mitochondrial Ca2+.
(A) The effect of CPA on the change in LCR period predicts the concurrent prolongation of AP cycle length. Note that this effect of CPA on LCR period and AP cycle length form a continuum with the effects of Ru360 and CGP-37157 on LCR period and AP cycle length. (B) In the presence of CPA neither Ru360 nor CGP-37157 affect the LCR period or AP cycle length, and [Ca2+]m decreased. The combination of Ru360 plus CGP-37157 does not change [Ca2+]m, AP cycle length or the LCR period.
Figure 7.
Simulations of the coupled clock numerical model, extended to include mitochondrial Ca2+ fluxes.
The simulated effects of specific inhibition of Ca influx into or efflux from mitochondria on (A) membrane potential, (B) cytosolic Ca2+, (C) mitochondrial Ca2+, and (D) sarolemmal Na+-Ca2+ exchanger current in intact SANC.
Figure 8.
The extended coupled clock numerical model simulations of kinetics.
The simulation of the change in (A) AP firing rate, (B) peak systolic cytosolic Ca2+, (C) peak systolic mitochondrial Ca2+, and (D) peak Ca2+ in junctional SR in response to specific inhibition of Ca2+ flux into or flux from mitochondria.