Evidence for a Physiological Role of T-Type Ca Channels in Ventricular Cardiomyocytes of Adult Mice

Figure 1. Ca currents in wt and Cav3.2 KO ventricular cardiomyocytes. (A) Original traces of Ca currents of a typical wt and Cav3.2 KO myocyte elicited by the pulse protocol displayed on top. (B) Current density–voltage relationships of wt (n = 20) and Cav3.2 KO (n = 20) myocytes. Data are expressed as means ± SEM. The current density–voltage relations hardly revealed any T-type Ca channel activity (expected to be maximal at −45 mV) but did reveal significant L-type Ca channel activity (peaking at +5 mV), which was similar in wt and Cav3.2 KO myocytes (p = 0.67 at +5 mV, unpaired Student’s t-test). Capacitance values of the examined myocytes were 184 ± 13 pF for wt and 169 ± 11 pF for Cav3.2 KO (p = 0.37). (C) The effect of the external application of 100 nM isoprenaline (ISO) on ICaT in a wt myocyte. From a holding potential of −95 mV, depolarizing voltage steps to −45 mV were applied every 3 s to elicit the currents. The arrows indicate the beginning and end of ISO application. Current peaks were finally plotted against time. (D) ICaT peaks before, during application after the steady-state was reached, and after the washout of ISO in wt (top) and Cav3.2 KO (bottom) myocytes. There was no…

Figure 2. Immunostaining of T-type Ca channels in ventricular cardiomyocytes isolated from adult wt mice. Cav3.1 (top) channel expression and localization was detected using a selective anti-Cav3.1 antibody (anti-CACNA1G, #ACC-021, source: rabbit, Alomone Labs, 1:500). Cav3.2 (bottom) was detected with the selective anti-Cav3.2 antibodies (anti-CACNA1H, #ACC-025, source: rabbit, Alomone Labs, 1:1000; left and right cell; or anti-Cav3.2 polyclonal, #PA5-106771, source: rabbit, Invitrogen, 1:200; middle cell). Secondary antibody: Alexa Fluor 488, #A11008, goat anti-rabbit, Invitrogen, 1:500.

Figure 3. Ca current density–voltage relationships recorded from ventricular cardiomyocytes isolated from wt BL/10 mouse hearts under different experimental conditions. (A) Left: standard solution (see Materials and Methods) at holding potential −95 mV; T/L-type current amplitude: 9%; Right: standard solution at holding potential −135 mV; T/L amplitude: 19%; a significant difference existed between different ICaT amplitudes at −50 mV (p = 0.006, paired Student’s t-test). Data represent means ± SEM (n = 11). (B) Left: solution set 2 (see Materials and Methods) at holding potential: −95 mV; T/L amplitude: 0%; Right: solution set 2 at holding potential: −135 mV; T/L amplitude: 5% (n = 14).

Figure 4. Electrically induced and caffeine-induced intracellular Ca transients in wt and Cav3.2 KO ventricular cardiomyocytes. (A) Left: Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca concentration during electrical field stimulation at 0.1 Hz frequency in a single wt and Cav3.2 KO myocyte. The orange lines represent fits of the data with a single exponential function. Right: Comparison of mean Ca peak fluorescence relative to baseline (F/F0) between wt and Cav3.2 KO myocytes. Each data point represents a single cell, and values are expressed as means ± SEM (n= 68 for wt and 59 for Cav3.2 KO myocytes). * p < 0.05 (0.02); unpaired Student’s t-test. The Ca transients decayed with time constants of 0.30 ± 0.02 s in wt and 0.25 ± 0.01 s in Cav3.2 KO myocytes, which were significantly different (p = 0.04). (B) Comparison of mean Ca peak fluorescence (F/F0) before and after the application of 100 nM ISO in wt (n = 20) and Cav3.2 KO (n = 21) myocytes. **** p < 0.0001 (paired Student’s t-test). There was no significant difference between wt and Cav3.2 KO in the presence of ISO (p = 0.37; unpaired Student’s t-test). The Ca transients under ISO decayed with time…