Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients

Figures

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  FIGURE 1. The effect of muscarinic receptor stimulation on cytoplasmic Ca2+ transients depends on the level of β-adrenergic receptor stimulation. (A) Representative fluorescence profiles obtained from the line-scan images recorded in mouse ventricular myocytes electrically stimulated at 1 Hz in the presence of isoproterenol (Iso) alone or the presence of Iso plus 10 μM carbachol (CCh). Bar graphs with individual data points illustrate the effect of 10 μM CCh on the amplitude (B) and the decay rate (C) of Ca2+ transients recorded in the presence of various concentrations of Iso.

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  FIGURE 2. Formamide treatment disrupts t-tubule organization and amplifies the inhibitory effect of CCh. Representative images of control (A) and formamide-treated (B) ventricular myocytes stained with membrane dye Di-4-AN(F)EPPTEA. Lower panels represent scaled-up regions indicated by white line rectangles and illustrate morphological skeletons of t-tubules (red lines). White scale bars are 20 μm. (C) Summary data along with the individual data points are shown for the density of t-tubules in control (n = 13) and formamide-treated (n = 13) ventricular myocytes, respectively. (D) Representative fluorescence profiles were obtained from the line-scan images recorded in formamide-treated ventricular myocytes electrically stimulated at 1 Hz in the presence of Iso alone or the presence of Iso plus 10 μM CCh. Bar graphs with individual data points illustrate the effect of 10 μM CCh on the amplitude (E) and the decay rate (F) of Ca2+ transients recorded in the presence of various concentrations of Iso in formamide-treated myocytes. CCh–induced changes in the amplitude (G) and the decay rate (H) of Ca2+ transients recorded in control and formamide-treated myocytes are plotted against the…

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  FIGURE 3. Formamide treatment delays Ca2+ release activation. (A,B) Linescan images of fluo-4 fluorescence (left panels) and corresponding images illustrating the rate of fluorescence change (right panels) recorded in control (A) and formamide-treated (B) ventricular myocytes in the presence of Iso (100 nM). Ca2+ transients were induced by electrical field stimulation at 1 Hz. White lines indicate the maximal rate of fluorescence increase [(dF/dt)max] that was calculated by averaging data from at least five consecutive Ca2+ transients. (C) Average time distribution of (dF/dt)max obtained from the line-scan images of control (n = 12) and formamide-treated (n = 33) myocytes. Time distribution in each myocyte was adjusted to the timing of fastest (dF/dt)max. The time distribution bin is 2.1 ms. Formamide treatment significantly changed the relative frequency of occurrence of (dF/dt)max at each time interval except where indicated by NS (not significant). Blue and red lines indicate the time intervals used for the classification of image pixels into the “early” [blue rectangles in panel (A,B)] and “delayed” [red rectangles in panel (A,B)] regions, respectively.

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  FIGURE 4. Downregulation of t-tubules augments the anti-adrenergic effect of CCh on the amplitude of Ca2+ transients. (A,B) Ca2+ transients were recorded in formamide-treated ventricular myocyte in the presence of 100 nM Iso alone (A, n = 32) and the presence of 100 nM Iso plus 10 μM CCh (B, n = 39). The inset on the left illustrates spatially averaged fluorescence profiles recorded from the cells displaying moderate (a, corresponding to the line-scan image in panel a) and pronounced (b, corresponding to the line-scan image in panel b) levels of detubulation, respectively. (A,a,b,B,a,b) Linescan images of fluo-4 fluorescence (left) and corresponding images illustrating the rate of fluorescence change (right). White lines indicate the timing of (dF/dt)max, and red lines indicate the time point used to differentiate delayed regions (red rectangles). Early and intermediate regions are indicated by blue and gray rectangles, respectively. (C,D) Regression analysis of the relationship between the detubulation level and the amplitude of Ca2+ transients showed the lack of correlation for the transients recorded in a presence of Iso alone (C) and the inverse correlation for the transients recorded…

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  FIGURE 5. Muscarinic receptor stimulation does not affect the sarcoplasmic reticulum (SR) Ca2+ content in control and formamide-treated ventricular myocytes. (A,B) spatially averaged fluo-4FF fluorescence profiles illustrating field-stimulated (1 Hz) and caffeine-induced Ca2+ transients recorded in control (A) and formamide-treated (B) ventricular myocytes in the presence of 100 nM Iso alone, and in the presence of 100 nM Iso plus 10 μM CCh. Bar graphs with data points demonstrate the effect of CCh on the amplitude caffeine-induced Ca2+ transients (C) and fractional release (D) in control and formamide-treated (F) myocytes. The fractional release was calculated as the ratio of amplitudes of electrically-stimulated to caffeine-induced Ca2+ transients, respectively. Multiple pairwise comparisons were performed with Tukey’s test. The number of control myocytes studied was 14 for both Iso and Iso plus CCh groups. Number of formamide-treated myocytes studied was 10 for the Iso group and 6 for the Iso plus CCh group.

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  FIGURE 6. Similar regulation of sarcolemmal Ca2+ influx by muscarinic receptor stimulation in control and detubulated myocytes. (A,B) Representative traces of Ca2+ currents and corresponding time-courses of changes in the amplitude of Ca2+ currents observed at the baseline (Base), in response to application of 100 nM Iso alone followed by application of 100 nM Iso plus 10 μM CCh. Currents were recorded in control (A, n = 6–10) and formamide-treated (B, n = 4) ventricular myocytes. (C) Bar graphs with data points show the peak Ca2+ current density recorded at the baseline (Base), in the presence of Iso alone, and in the presence of Iso plus CCh in control (C) and formamide-treated (F) ventricular myocytes, respectively. Summary data along with individual data points are shown for myocyte capacitance (D), peak Ca2+ current response to Iso, normalized to the baseline (E), and peak Ca2+ current response to CCh, normalized to the peak Ca2+ current recorded in the presence of Iso (F).

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  FIGURE 7. Detubulation reduces the fidelity of excitation-contraction (EC) coupling. (A,B) Representative line-scan images of fluo-4 fluorescence with the corresponding spatially averaged fluorescence profiles and ICa traces recorded in control (A) and formamide-treated (B) ventricular myocytes in a presence of 100 nM Iso. Depolarization levels used to evoke ICa are indicated above each line scan image. The upper panel in A illustrates the voltage protocol used to evoke Ca2+ currents. (C) The relationship between EC coupling gain and membrane voltage is shown for control (n = 5) and formamide-treated (n = 6) myocytes, respectively *p < 0.05; **p < 0.01 (control vs. formamide). (D) Voltage-dependent reduction in ICa decreased the amplitude of Ca2+ transients in formamide-treated but not in control myocytes, respectively. Data shown in panel C were transformed so that for each myocyte both peak Ca2+ transients and peak ICa were normalized to the corresponding values recorded at 0 mV.

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  FIGURE 8. Muscarinic receptor stimulation reduces CaMKII activation in formamide-treated myocytes. (A) Representative images of formamide-treated myocytes immunostained for activated CaMKII (phospho T286). Right panels are the scaled-up parts of the corresponding left panel images. AB” images illustrate myocyte labeling with secondary antibody only. (B) Summary data along with individual data points illustrate average myocyte fluorescence observed in formamide-treated ventricular myocytes incubated with 100 nM Iso alone (n = 41), 100 nM Iso plus 10 μM CCh (n = 26), and 100 nM Iso and 2 μM KN93, a CaMKII inhibitor (n = 20). In all groups, myocytes were field-stimulated for 1 min at 2 Hz followed by 1 min at 1 Hz. Multiple pairwise comparisons were performed with Tukey’s test.

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  FIGURE 9. CaMKII inhibitor KN93 mimics the effect of CCh on Ca2+ transients in formamide-treated myocytes. Ca2+ transients were recorded in formamide-treated ventricular myocytes in the presence of 100 nM Iso and KN93 (2 μM). (A) Inset on the left shows spatially averaged fluorescence profiles recorded from the cell displaying moderate (a, corresponding to the line-scan image in panel a) and pronounced (b, corresponding to the line-scan image in panel b) levels of detubulation, respectively. (A,a,b) Linescan images of fluo-4 fluorescence (left) and corresponding images illustrating the rate of fluorescence change (right). The white line indicates the timing of (dF/dt)max, and the red line indicates the time point used to differentiate delayed regions (red rectangles). Early and intermediate regions are indicated by blue and gray rectangles, respectively. (B) The amplitude of Ca2+ transients is inversely correlated with the fraction of the delayed regions. (C) The relationship between (dF/dt)max at the early Ca2+ release regions and the delayed region fraction recorded in the presence of Iso plus KN93 is shown along with a linear fit to the data. The slope of the regression line (–1.2 ±…