Figure 4. Effects of ryanodine receptor (RyR) dispersion on intracellular Ca²⁺ homeostasis are reversed by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) or protein kinase A (PKA) inhibition.

(a, b) Representative examples of Ca²⁺ sparks of control and isoproterenol (60 min)-treated cardiomyocytes obtained by confocal line-scan imaging. The time course for each spark is shown on right, with indicated time to peak (TTP) and full duration at half maximum (FDHM) measurements. (c–h) Experimental data indicate that increasing spark frequency, geometry, and slowing of kinetics during isoproterenol were all reversed by CaMKII or PKA inhibition (control: n_sparks = 47, n_cells = 38, n_hearts = 3; Iso 10: n_sparks = 166, n_cells = 44, n_hearts = 3; Iso 30: n_sparks = 194, n_cells = 44, n_hearts = 3; Iso 60: n_sparks = 222, n_cells = 47, n_hearts = 3; Iso 60 + AIP: n_sparks = 147, n_cells = 69, n_hearts = 5; Iso 60 + H89: n_sparks = 135, n_cells = 55, n_hearts = 4). (i) Representative line-scan images illustrating the last in a series of electrically paced Ca²⁺ transients, followed by a pause to examine Ca²⁺ wave generation. Enlargements of the Ca²⁺ transients are shown on right to highlight differences in Ca²⁺ release synchrony. (j–l) Data showing initial increases in Ca²⁺ transient amplitude and synchrony were reversed with continued exposure to isoproterenol, while overall release kinetics slowed (control: n_cells = 38, n_hearts = 3; Iso 10: n_cells = 35, n_hearts = 3; Iso 30: n_cells = 36, n_hearts = 3; Iso 60: n_cells = 41, n_hearts = 3). (m) Ca²⁺ wave incidence increased during early time points following isoproterenol treatment, but then reversed (control: n_cells = 43, n_hearts = 3; Iso 10: n_cells = 44, n_hearts = 3; Iso 30: n_cells = 46, n_hearts = 3; Iso 60: n_cells = 44, n_hearts = 3). The bar charts present mean measurements ± SEM, with superimposed data points representing averaged values from each cardiomyocyte. Statistical significance (p<0.05) between groups is indicated by a comparison bar.

Figure 4—figure supplement 1. Isoproterenol elicits a biphasic increase in sarcoplasmic reticulum (SR) Ca²⁺ content.

SR Ca²⁺ content was estimated by rapidly changing the superfusate to one containing 10 mmol/L caffeine, and measuring the amplitude of the elicited Ca²⁺ release. Representative recordings (a) and mean data (b) show that a marked increase in SR content during early isoproterenol treatment was reversed with continued exposure. SERCA and NCX activity (c and d, respectively) were estimated based on fits of the decays of caffeine- and electrically elicited Ca²⁺ transients (see ‘Materials and methods’) (control: n_cells = 16, n_hearts = 4; Iso 10: n_cells = 13, n_hearts = 4; Iso 60: n_cells = 15, n_hearts = 4).