Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure

Abstract

Constitutive regulation by PKA has recently been shown to contribute to L-type Ca²⁺ current (I_CaL) at the ventricular t-tubule in heart failure. Conversely, reduction in constitutive regulation by PKA has been proposed to underlie the downregulation of atrial I_CaL in heart failure. The hypothesis that downregulation of atrial I_CaL in heart failure involves reduced channel phosphorylation was examined. Anesthetized adult male Wistar rats underwent surgical coronary artery ligation (CAL, N=10) or equivalent sham-operation (Sham, N=12). Left atrial myocytes were isolated ~18 wk postsurgery and whole cell currents recorded (holding potential=-80 mV). I_CaL activated by depolarizing pulses to voltages from -40 to +50 mV were normalized to cell capacitance and current density-voltage relations plotted. CAL cell capacitances were ~1.67-fold greater than Sham (P ≤ 0.0001). Maximal I_CaL conductance (G_max ) was downregulated more than 2-fold in CAL vs. Sham myocytes (P < 0.0001). Norepinephrine (1 μmol/l) increased G_max >50% more effectively in CAL than in Sham so that differences in I_CaL density were abolished. Differences between CAL and Sham G_max were not abolished by calyculin A (100 nmol/l), suggesting that increased protein dephosphorylation did not account for I_CaL downregulation. Treatment with either H-89 (10 μmol/l) or AIP (5 μmol/l) had no effect on basal currents in Sham or CAL myocytes, indicating that, in contrast to ventricular myocytes, neither PKA nor CaMKII regulated basal I_CaL Expression of the L-type α_1C-subunit, protein phosphatases 1 and 2A, and inhibitor-1 proteins was unchanged. In conclusion, reduction in PKA-dependent regulation did not contribute to downregulation of atrial I_CaL in heart failure.NEW & NOTEWORTHY Whole cell recording of L-type Ca²⁺ currents in atrial myocytes from rat hearts subjected to coronary artery ligation compared with those from sham-operated controls reveals marked reduction in current density in heart failure without change in channel subunit expression and associated with altered phosphorylation independent of protein kinase A.

Keywords: atrial remodeling; coronary artery ligation; voltage-gated Ca2+ channel.

Fig. 1.

Atrial L-type Ca²⁺ current density in heart failure. A: mean tibia length from 12 Sham and 10 CAL rats. *P < 0.05, Mann-Whitney test. B: mean heart weight/tibia length (HW/TL) and lung weight/tibia length (LW/TL) ratios. Sham, filled columns; CAL, open columns. **P < 0.01, ****P < 0.001, Mann-Whitney test.

Fig. 2.

A: whole cell capacitances for Sham (n = 63/12) and CAL (n = 47/10) myocytes. ****P < 0.0001, Mann-Whitney test. B: representative whole cell L-type Ca²⁺ current (I_CaL) traces at +10 mV. Sham, black; CAL, gray. C: mean I_CaL-voltage relations for Sham [filled circles, (n no. of cells/N no. of animals) = 32/12] and CAL (open circles, n/N = 31/10) myocytes. D: mean I_CaL density-voltage relations for Sham (filled circles) and CAL (open circles) myocytes. Same data as shown in C. **P < 0.01, ***P < 0.001, 2-way repeated-measures ANOVA with Bonferroni posttest. Solid lines in C and D represent fits to Eq. 1. E: correlation between I_CaL density at +10 mV in Sham (filled circles, n/N = 32/12) and CAL (open circles, n = 31/10) myocytes. Atrial I_CaL density for the two groups of cells combined was significantly correlated (r = −0.4772, n/N = 63/22, P < 0.0001).

Fig. 3.

Effects of norepinephrine on atrial L-type Ca²⁺ current (I_CaL) density. A: representative Ca²⁺ current traces at +10 mV in an atrial myocyte from a Sham-operated rat in control conditions and in the presence of 1 μmol/l norepinephrine. B: representative Ca²⁺ current traces in an atrial myocyte from a CAL rat in control conditions and in the presence of 1 μmol/l norepinephrine. Arrows indicate zero current level. C: mean I_CaL density-voltage relations under control conditions from Sham-operated (n/N = 5/3, filled circles) and CAL (n/N = 9/4, open circles). *P < 0.05, **P < 0.01, ***P < 0.001, Bonferroni posttest following 2-way repeated-measures ANOVA. D: mean I_CaL density-voltage relations in the presence of 1 μmol/l norepinephrine from Sham-operated (n/N = 5/3, open circles) and CAL (n/N = 9/4, filled circles). Data correspond to the control data shown in C. Note the difference in current density scale between C and D. Solid lines in C and D represent fits to Eq. 1.

Fig. 4.

Atrial L-type Ca²⁺ currents following phosphatase inhibition. A, i: representative current traces recorded at +10 mV from a Sham myocyte before and after superfusion with 100 nmol/l calyculin A. A, ii: I_CaL density-voltage relations from Sham myocytes (n/N = 6/3) in control (filled circles) and in the presence of 100 nmol/l calyculin A (open circles). *P < 0.05, ***P < 0.001, 2-way repeated-measures ANOVA and Bonferroni posttest. B, i: representative current traces recorded at +10 mV from a CAL myocyte before and after superfusion with 100 nmol/l calyculin A. B, ii: I_CaL density-voltage relations from CAL myocytes (n/N = 9/3) in control (filled squares) and in the presence of 100 nmol/l calyculin A (open squares). ***P < 0.001, 2-way ANOVA and Bonferroni posttest.

Fig. 5.

Effect of the protein kinase A inhibitor, H-89. A, i: representative Ca²⁺ current traces at +10 mV in an atrial myocyte from a Sham-operated rat in control Tyrode’s, in the presence of 10 μmol/l H-89, and in the presence of 10 μmol/l H-89 and 1 μmol/l norepinephrine (NE). A, ii: mean I_CaL density-voltage relations from Sham myocytes (n/N = 8/4) in control (black-filled circles), in the presence of 10 μmol/l H-89 (gray-filled circles) and in the presence of 10 μmol/l H-89 and 1 μmol/l NE (open circles). B, i: representative Ca²⁺ current traces in an atrial myocyte from a CAL rat in control Tyrode’s, in the presence of 10 μmol/l H-89, and in the presence of 10 μmol/l H-89 and 1 μmol/l NE. Right-pointing arrows indicate zero current level. B, ii: mean I_CaL density-voltage relations from CAL myocytes (n/N = 13/4) in control (black filled squares), in the presence of 10 μmol/l H-89 (gray-filled squares) and in the presence of 10 μmol/l H-89 and 1 μmol/l NE (open squares).

Fig. 6.

Effect of the Ca²⁺-calmodulin-dependent protein kinase II inhibitor, AIP. A: mean I_CaL density-voltage relations for Sham myocytes with AIP included in the pipette solution (n/N = 4/1). Black-filled circles, AIP-dialyzed cells in control Tyrode’s; open circles, AIP-dialyzed cells in the presence of 100 nmol/l calyculin A. B: mean I_CaL density-voltage relations for CAL myocytes with AIP included in the pipette solution (n/N = 5/1). Black-filled squares, AIP-dialyzed cells in control Tyrode’s; open squares, AIP-dialyzed cells in the presence of 100 nmol/l calyculin A. Data from Sham (A) and CAL (B) myocytes under control conditions (respectively, n/N = 33/12 and 31/10) are shown as, respectively, gray-filled circles and gray-filled squares, for comparison. ***P < 0.001, 2-way repeated-measures ANOVA and Bonferroni posttest vs. corresponding AIP control.

Fig. 7.

Left atrial expression of the Ca_v1.2 LTCC α_1c subunit and various protein phosphatases in heart failure. A: representative Western blots of LTCC, protein phosphatases, and GAPDH from Sham and CAL rats. For each protein, the bands are taken from the same gel and have not been manipulated for contrast, color-balance, brightness, or background. Solid lines demarcate the bands for the target protein and for the GAPDH loading control, which were taken from the same gel. For each protein, the molecular weights of the bands are indicated. B: mean band intensity expressed relative to Sham as 100%. Data are means ± SE from 3 Sham and 3 CAL hearts.