Direct, differential effects of tamoxifen, 4-hydroxytamoxifen, and raloxifene on cardiac myocyte contractility and calcium handling

Abstract

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.

Figure 1. Experimental Timelines.

A) For sarcomere length measurements, cells were covered in Tyrode’s containing Tam, 4OHT or Ral, allowed to reach 36 ± 1°C, and then stimulated at 0.2 Hz. Data was collected from 15-45 minutes. B) For cell contractility measurements, the number of contracting cells were counted at baseline, and 5, 15, 30 and 45 minutes after administration of the treatment. C) For Ca²⁺ transient measurements, cells were stimulated at 0.5 Hz for 3 minutes and then data was collected for 20 minutes.

Figure 2. Sarcomere Length and Calcium Transient Measurements in Tamoxifen-Treated Rat Cardiac Myocytes.

A) Representative traces of sarcomere shortening normalized to baseline. B-D) Sarcomere length data was collected from myocytes treated with Tam and paced at 0.2 Hz and 36 ± 1°C. Data are from four rat cardiac myocyte preparations, N = 46-52 cells/treatment. E) Representative traces of Ca²⁺ transients normalized to baseline. F-H) Myocytes were treated with Fura-2AM, and Ca²⁺ transient data was collected at 0.5 Hz and 36 ± 1°C after Tam treatment. Data are from three rat cardiac myocyte preparations, N = 11-30 cells/treatment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 µM.

Figure 3. Stimulation-Induced Contractility with Tamoxifen, 4-Hydroxytamoxifen, and Raloxifene.

Contracting cells in a field of view containing 30-50 cells treated with A) Tam, B) 4OHT, or C) Ral were counted over 45 minutes at 0.2 Hz. The percentage change from baseline in contracting cells at each time point was calculated, and data are presented as mean ± SEM, N = 3-4 counting experiments/treatment. **P < 0.01, ***P < 0.001 for 10 µM compared to 0 µM.

Figure 4. Sarcomere Length and Calcium Transient Measurements in 4-Hydroxytamoxifen-Treated Rat Cardiac Myocytes.

A) Representative traces of sarcomere shortening normalized to baseline. B-D) Sarcomere length data was collected from myocytes treated with 4OHT and paced at 0.2 Hz and 36 ± 1°C. Data are from four rat cardiac myocyte preparations, N = 45-50 cells/treatment. E) Representative traces of calcium transients normalized to baseline. F-H) Myocytes were treated with Fura-2AM, and calcium transient data was collected at 0.5 Hz and 36 ± 1°C after 4OHT treatment. Data are from three rat cardiac myocyte preparations, N = 38-42 cells/treatment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 µM.

Figure 5. Sarcomere Length and Calcium Transient Measurements in Raloxifene-Treated Rat Cardiac Myocytes.

A) Representative traces of sarcomere shortening normalized to baseline. B-D) Sarcomere length data was collected from myocytes treated with Ral and paced at 0.2 Hz and 36 ± 1°C. Data are from four rat cardiac myocyte preparations, N = 39-47 cells/treatment. E) Representative traces of Ca²⁺ transients normalized to baseline. F-H) Myocytes were treated with Fura-2AM, and Ca²⁺ transient data was collected at 0.5 Hz and 36 ± 1°C after Ral treatment. Data are from three rat cardiac myocyte preparations, N = 12-26 cells/treatment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared to 0 µM.

Figure 6. Phosphorylated cTnI with Raloxifene and Isoproterenol.

Cells were treated with vehicle (Ctl), 10 nM isoprenaline (Iso), 10 µM raloxifene (Ral), or isoprenaline and raloxifene (Iso + Ral) for 15 min, after which cells were collected and lysed, protein concentration determined, and subjected to SDS-PAGE. Protein was transferred to PVDF membrane and probed for phospo-cTnI (Ser 23/24) and actin. The data are presented as mean ± SEM for the ratio of phospho-cTnI to actin. N = 14-18 coverslips of cells/group, ***P < 0.001.

Figure 7. Sarcomere Length Measurements in Tamoxifen and Raloxifene-Treated Mouse Cardiac Myocytes.

Sarcomere length data was collected from myocytes treated with 10 µM Tam or Ral and paced at 0.2 Hz and 36 ± 1°C. Data are from two mouse cardiac myocyte preparations, N = 21-25 cells/treatment. A) Peak height of contraction; B) Time from peak to 50% relaxation. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 compared to 0 µM.