Berberine Mediated Positive Inotropic Effects on Rat Hearts via a Ca2+-Dependent Mechanism

Abstract

Previous studies showed that berberine, an alkaloid from Coptis Chinensis Franch, might exert a positive inotropic effect on the heart. However, the underlying mechanisms were unclear. Here, we reported that berberine at 10-20 µM increased the left ventricular (LV) developed pressure and the maximal rate of the pressure rising, and it increased the maximal rate of the pressure descending at 20 µM in Langendorff-perfused isolated rat hearts. These effects diminished with the concentration of berberine increasing to 50 µM. In the concentration range of 50-300 µM, berberine increased the isometric tension of isolated left ventricular muscle (LVM) strips with or without electrical stimulations, and it (30-300 µM) also increased the intracellular Ca²⁺ level in the isolated LV myocytes. The removal of extracellular Ca²⁺ hindered the berberine-induced increases in the tension of LVM strips and the intracellular Ca²⁺ level of LV myocytes. These suggested that berberine might exert its positive inotropic effects via enhancing Ca²⁺ influx. The blockade of L-type Ca²⁺ channels (LTCCs) with nifedipine significantly attenuated 300 μM berberine-induced tension increase in LVM strips but not the increase in the intracellular Ca²⁺ level. Berberine (300 μM) further increased the LVM tension following the treatment with the LTCC opener FPL-64716 (10 μM), indicating an LTCC-independent effect of berberine. Lowering extracellular Na⁺ attenuated the berberine-induced increases in both the tension of LVM strips and the intracellular Ca²⁺ level of LV myocytes. In conclusion, berberine might exert a positive inotropic effect on the isolated rat heart by enhancing the Ca²⁺ influx in LV myocytes; these were extracellular Na⁺-dependent.

Keywords: Ca2+; Na+; berberine; heart; positive inotropic effect.

Figure 1

Berberine increased the capability of Langendorff-perfused isolated rat hearts for contraction. (A) An original recording showing that the effect of berberine on left ventricular pressure (LVP) of an isolated rat heart. (B–D) Summary data showing the effects of berberine on LVDP, +dp/dt_max and |−dp/dt|_max, respectively. The numbers of repeats are as follows: vehicle control group, N = 6; Ber-treated group, N = 6; N is the number of rats. Ber stands for berberine. *p < 0.05, **p < 0.01 vs. vehicle control group; two-way ANOVA with Sidak's multiple comparisons test.

Figure 2

Berberine increased the tension of freshly isolated left ventricular muscle (LVM) strips. (A, B) Original isometric tension recordings showing that berberine (100 µM) increased the tension of LVM strips. (C) Summary data showing that berberine increased the tension of LVM strips in a concentration-dependent manner. The numbers of repeats are as follows: 30 µM Ber, n = 10, N = 10; 50 µM Ber, n = 7, N = 7; 100 µM and 300 µM Ber, n = 11, N = 11; 200 µM Ber, n = 12, N = 12; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. **p < 0.01, ***p < 0.001 vs. vehicle control group; two-way ANOVA with Sidak's multiple comparisons test.

Figure 3

Berberine increased the tension of the freshly isolated left ventricular muscle (LVM) strips pretreated with epinephrine. (A, B) Original isometric tension recordings showing that berberine (100 µM) increased the tension and abolished the epinephrine-evoked phasic contractions in LVM strips. (C) Summary data showing that the effect of berberine on the tension of the LVM strips pretreated with epinephrine. The numbers of repeats are as follows: 30 µM and 50 µM Ber, n = 7, N = 7; 100 µM Ber, n = 8, N = 8; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; two-way ANOVA with Sidak's multiple comparisons test.

Figure 4

Berberine increased the tension of the electrical field stimulation (EFS)-paced left ventricular muscle (LVM) strips. (A, B) Original isometric tension recordings showing that berberine (100 µM) increased the tension of the EFS-paced LVM strips (5 V, 1 Hz). (C) Summary data indicating that berberine increases the tension of the EFS-paced LVM strips in a concentration-dependent manner. The numbers of repeats are as follows: 30 µM Ber, n = 5, N = 5; 50 µM Ber, n = 8, N = 8; 100 µM Ber, n = 6, N = 6; 200 µM Ber and 300 µM Ber, n = 7, N = 7; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; two-way ANOVA with Sidak's multiple comparisons test.

Figure 5

Berberine concentration-dependently increased the intracellular Ca²⁺ level of freshly isolated left ventricular (LV) myocytes. (A, B) Original Ca²⁺ imaging recordings showing that berberine (100 µM) increased the intracellular Ca²⁺ level of freshly isolated LV myocytes. (C) Summary data showing that berberine concentration-dependently increased the intracellular Ca²⁺ level. The numbers of repeats are as follows: 30 µM Ber, n = 54, N = 3; 50 µM Ber, n = 65, N = 3; 100 µM Ber, n = 35, N = 3; 300 µM Ber, n = 36, N = 3; n and N are the numbers of LV myocytes and rats, respectively. Ber stands for berberine. *p < 0.05, ***p < 0.001 vs. vehicle control group; two-way ANOVA with Sidak's multiple comparisons test.

Figure 6

The removal of extracellular Ca²⁺ hindered the berberine-induced tension increase in left ventricular muscle (LVM) strips. (A–D) Original isometric tension recordings of the LVM strips treated with vehicle (A), 300 μM Ber (B); vehicle (C) and 300 μM Ber (D) under the Ca²⁺-free condition, respectively. (E) Summary data showing that the removal of extracellular Ca²⁺ significantly reduced the ability of berberine to increase the tension of LVM strips. The numbers of repeats are as follows: vehicle control of 300 µM Ber, n = 6, N = 6; 300 µM Ber, n = 6, N = 6; vehicle control of 300 µM Ber in the Ca²⁺-free Krebs-Hensseleit (K-H) buffer, n = 6, N = 6; 300 µM Ber in the Ca²⁺-free K-H buffer, n = 7, N = 7; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; ^###p < 0.001 vs. Ber-treated group in the normal K-H buffer; two-way ANOVA with Tukey's multiple comparisons test.

Figure 7

Nifedipine attenuated the berberine-induced tension increase in left ventricular muscle (LVM) strips. (A–D) Original isometric tension recordings of the LVM strips treated with vehicle (A), 300 μM Ber (B), 10 μM nifedipine (C), and 300 μM Ber in the presence of 10 μM nifedipine (D), respectively. (E) Summary data showing nifedipine hindered the 300 μM berberine-induced tension increase in LVM strips. The numbers of repeats are as follows: vehicle control of 300 µM Ber, n = 6, N = 6; 300 µM Ber, n = 6, N = 6; 10 μM nifedipine, n = 7, N = 7; 300 µM Ber plus 10 μM nifedipine, n = 5, N = 5; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; ^#p < 0.05, vs. 300 μM Ber-treated group; two-way ANOVA with Tukey's multiple comparisons test.

Figure 8

Berberine increased the tension of the freshly isolated left ventricular muscle (LVM) strips pretreated with FPL-64716. (A, B) Original isometric tension recordings of the LVM strips treated with 10 µM FPL-64716 (A) and 300 μM berberine in the presence of 10 µM FPL-64716 (B). (C) Summary data showing berberine induced an additional increase in the tension of the LVM strips pretreated with FPL-64716. The numbers of repeats are as follows: 10 µM FPL-64716, n = 4, N = 3; 300 µM Ber plus 10 μM FPL-64716, n = 3, N = 3; n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. *p < 0.05 vs. 10 µM FPL-64716-treated group; two-tailed unpaired Student's t-test.

Figure 9

The removal of extracellular Ca²⁺ hindered the berberine-induced increase in the intracellular Ca²⁺ level of freshly isolated LV myocytes. (A–D) Original Ca²⁺ imaging recordings of the LV myocytes treated with vehicle (A) and 300 μM berberine (B); vehicle (C) and 300 μM berberine (D) in the Ca²⁺-free modified Krebs-Hensseleit (K-H) buffer, respectively. (E) Summary data showing that the removal of extracellular Ca²⁺ significantly hindered the 300 μM berberine-induced increase in the intracellular Ca²⁺ level of LV myocytes. The numbers of repeats are as follows: vehicle control of 300 µM Ber, n = 57, N = 3; 300 µM Ber, n = 83, N = 3; vehicle control of 300 µM Ber in the Ca²⁺-free modified K-H buffer, n = 27, N = 3; 300 µM Ber in the Ca²⁺-free modified K-H buffer, n = 47, N = 3; n and N are the numbers of LV myocytes and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; ^###p < 0.001 vs. Ber-treated group in the normal modified K-H buffer; two-way ANOVA with Tukey's multiple comparisons test.

Figure 10

Nifedipine did not influence the 300 μM berberine-induced increase in the intracellular Ca²⁺ level of freshly isolated left ventricular (LV) myocytes. (A–D) Original Ca²⁺ imaging recordings of the LV myocytes treated with vehicle (A), 300 μM Ber (B), 10 μM nifedipine (C) and 300 μM Ber in the presence of 10 μM nifedipine (D). (E) Summary data showing nifedipine did not affect the 300 μM berberine-induced increase in the intracellular Ca²⁺ level of LV myocytes. The numbers of repeats are as follows: vehicle control of 300 µM Ber, n = 57, N = 4; 300 µM Ber, n = 83, N = 4; 10 μM nifedipine, n = 37, N = 4; 300 µM Ber plus 10 μM nifedipine, n = 46, N = 4; n and N are the numbers of LV myocytes and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; two-way ANOVA with Tukey's multiple comparisons test.

Figure 11

The berberine-induced tension increase in left ventricular muscle (LVM) strips was extracellular Na⁺-dependent. (A, B, D) Original isometric tension recordings of the LVM strips treated with 300 μM berberine (A), and 300 μM berberine under the low Na⁺ condition where NaCl of the Krebs-Hensseleit (K-H) buffer was replaced with CsCl (B) or with NMDG (D). (C, E) Summary data indicating that the 300 µM berberine-induced tension increase has a positive correlation with the concentrations of extracellular NaCl. The numbers of repeats are as follows: experiments with the normal K-H buffer, 300 µM Ber, n = 6, N = 6. Experiments with the K-H buffer in which NaCl was replaced with CsCl: 300 µM Ber in the buffer containing 77 or 10 mM NaCl, n = 5, N = 5; 300 µM Ber in the buffer containing 0 mM NaCl, n = 6, N = 6. Experiments with the K-H buffer in which NaCl was replaced with NMDG: 300 µM Ber in the buffer containing 77 or 0 mM NaCl, n = 6, N = 6; 300 µM Ber in the buffer containing 10 mM NaCl, n = 5, N = 5. n and N are the numbers of LVM strips and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Ber-treated group in the normal K-H buffer; two-way ANOVA with Tukey's multiple comparisons test.

Figure 12

The berberine-induced increase in the intracellular Ca²⁺ level of left ventricular (LV) myocytes was extracellular Na⁺-dependent. (A–D) Original Ca²⁺ imaging recordings of the LV myocytes treated with vehicle (A) and 300 μM Ber (B); vehicle (C) and 300 μM Ber (D) in the modified Krebs-Hensseleit (K-H) buffer containing 62.5 mM NaCl and 62.5 mM NMDG, respectively. (B) Summary data indicating that the berberine-induced increase in the intracellular Ca²⁺ level in a buffer containing 62.5 mM NaCl was smaller than that in a buffer containing 125 mM NaCl. The numbers of repeats are as follows: vehicle control of 300 µM Ber, n = 42, N = 3; 300 µM Ber, n = 58, N = 3; vehicle control of 300 µM Ber in the K-H buffer containing 62.5 mM NaCl, n = 24, N = 3; 300 µM Ber in the K-H buffer containing 62.5 mM NaCl, n = 21, N = 3; n and N are the numbers of LV myocytes and rats, respectively. Ber stands for berberine. ***p < 0.001 vs. vehicle control group; ^###p < 0.001 vs. Ber-treated group in the modified K-H buffer containing 125 mM NaCl; two-way ANOVA with Tukey's multiple comparisons test.