Neuronal nitric oxide synthase regulation of calcium cycling in ventricular cardiomyocytes is independent of Cav1.2 channel modulation under basal conditions

Figures

• 

  Fig. 1. External application of the nNOS inhibitor L-VNIO increases electrically induced cytosolic Ca2+ transients in mouse (a) and rat (b) ventricular cardiomyocytes. Left: representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca2+ concentration during electrical field stimulation, at 0.1 and 0.2 Hz frequency, in single mouse (a) and rat (b) ventricular cardiomyocytes, respectively. Following a time period under control conditions, the cells were superfused with bath solution containing 100 μM L-VNIO for the respective indicated durations (see black bars). Right: mean Ca2+ peak fluorescence relative to baseline (F/F0) elicited by electrical field stimulation for mouse (a) and rat (b) myocytes in the absence (ctl) and the presence (L-VNIO) of 100 μM of the drug. Each data point represents a single cell (n = 11 and 59 for mouse and rat cardiomyocytes, respectively), and values are expressed as means (calculated from these total cell numbers) ± SD. Cells originated from four individual mouse and nine rat hearts in total. Triple asterisks indicate a significant difference between the drug-free control condition and the presence of L-VNIO (p < 0.001, paired…

• 

  Fig. 2. Whole cell currents through Ca2+ channels recorded from mouse ventricular cardiomyocytes. a The left figure part shows typical original traces of Ca2+ currents elicited by the pulse protocol displayed on top. On the right, the respective inward current peaks were plotted against the applied membrane potentials to obtain the current–voltage relationship. The solid line represents a fit of the data points with a function described in the “Materials and methods” section. b Original Ba2+ current traces (left) and the respective current–voltage relationship (right). c, d Left: examples of continuous Ba2+ current recordings, in the conventional whole cell patch clamp mode, showing permanent rundown over 500 s. The currents were elicited by 50-ms pulses to + 20 mV every 3 s from a holding potential of − 80 mV, and their peaks were plotted against time. The insets display the respective currents at the beginning (time point, 10 s) and the end (time point, 480 s) of the experiments. The solid lines (green) represent fits of the data points with a single exponential function. Right: rundown-corrected current peaks, expressed in percentage relative to the respective value at experiment…

• 

  Fig. 3. External application of the nNOS inhibitor L-VNIO does not affect currents through L-type Ca2+ channels in ventricular cardiomyocytes. a Left: original trace of a Ca2+ current, elicited by the pulse protocol displayed on top, of a mouse ventricular cardiomyocyte under control (drug-free) condition. Right: the rundown-corrected peaks of the currents, elicited by pulses every 3 s, before drug application, during superfusion with 100 μM L-VNIO, and after drug washout were plotted over time. b Evaluation summary of a series of experiments with mouse cardiomyocytes as described in a. Ca2+ current amplitudes (left) and decay kinetics (right) in the absence (ctl, wash) and presence of L-VNIO are compared. The control (ctl) amplitude values were detected immediately before drug application, and the experimental values (in the presence of the drug) were taken 180 s after the start of superfusion with L-VNIO. The “wash” amplitude values were detected 180 s after the start of drug washout. The current amplitudes are expressed in percentage relative to the respective value at experiment start (= 100%). Each data point represents a single cell, and data variation is expressed as SD. Decay…

• 

  Fig. 4. Internal application of nNOS inhibitors via the pipette solution does not affect currents through L-type Ca2+ channels in mouse ventricular cardiomyocytes. a Left: Ca2+ current density–voltage relationships (top) and decay kinetics (bottom) of myocytes recorded under control conditions (standard pipette solution, ctl), or in the presence of 100 μM L-VNIO in the pipette. The currents were elicited by 400-ms pulses to various depolarizing potentials from a holding potential of − 80 mV. Data points are given as means ± SD (n = 7 for ctl and 6 for drug-treated cells). Cells originated from three mouse hearts. No significant difference (p always > 0.23, unpaired Student’s t test) existed between ctl and drug-treated cells over the whole voltage range studied. Right: Ba2+ current density–voltage relationships (top) and decay kinetics (bottom; given as time constant tau derived from a single exponential fit of the current decay) of myocytes recorded under control conditions, or in the presence of L-VNIO in the pipette. There was no significant difference (p always > 0.10, unpaired Student’s t test) between ctl and drug-treated cells at all voltages (n = 6 for ctl and 5 for…

• 

  Fig. 5. External application of the nNOS inhibitor L-VNIO does not affect Ca2+ currents in mouse ventricular cardiomyocytes recorded with the perforated patch clamp technique. a Left: voltage-clamp protocol and corresponding original Ca2+ current trace under control conditions. Right: maximal inward current amplitude before drug application and during superfusion with 100 μM L-VNIO was plotted over time. b Evaluation summary of a series of experiments with mouse cardiomyocytes as described in a. Ca2+ current amplitudes (left) and decay kinetics (right) in the absence (ctl, wash) and presence of L-VNIO are compared. Drug application did not generate any significant difference (p always > 0.36, paired Student’s t test; n = 8 for ctl, 8 for drug-treated, and 5 for wash). Cells originated from four mouse hearts

• 

  Fig. 6. Application of NO donors does not affect single-channel open probability of the long N-terminal isoform of human Cav1.2 channel. a Left: representative single-channel currents recorded at + 150 mV in the absence (ctl) or presence of 150 μM NO donor GSNO. Right: relative mean ± SD channel open probability for currents recorded in control solution (ctl) and in the presence of GSNO (n = 4, number of liposome patches).b NO donor SNP had no effect on single-channel current of the long N-terminal isoform of Cav1.2. Left: representative single-channel currents recorded at + 150 mV in control conditions (ctl), and in the presence of 100 μM SNP. Right: relative mean ± SD channel open probability for currents recorded in control condition, in the presence of SNP (n = 6). c PKA increases open probability of the single-channel current of the human long N-terminal isoform of Cav1.2. Left: representative single-channel currents recorded at + 150 mV in the absence (ctl) or presence of 0.5 μM PKA (modified after [9]). Right: relative mean ± SD channel open probability for currents recorded in control solution and during PKA treatment (n = 7). Single asterisk indicates a significant difference…