Blockade of L-type Ca2+ channel attenuates doxorubicin-induced cardiomyopathy via suppression of CaMKII-NF-κB pathway

Abstract

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor-kappa B (NF-κB) play crucial roles in pathogenesis of doxorubicin (DOX)-induced cardiomyopathy. Their activities are regulated by intracellular Ca²⁺. We hypothesized that blockade of L-type Ca²⁺ channel (LTCC) could attenuate DOX-induced cardiomyopathy by regulating CaMKII and NF-κB. DOX activated CaMKII and NF-κB through their phosphorylation and increased cleaved caspase 3 in cardiomyocytes. Pharmacological blockade or gene knockdown of LTCC by nifedipine or small interfering RNA, respectively, suppressed DOX-induced phosphorylation of CaMKII and NF-κB and apoptosis in cardiomyocytes, accompanied by decreasing intracellular Ca²⁺ concentration. Autocamtide 2-related inhibitory peptide (AIP), a selective CaMKII inhibitor, inhibited DOX-induced phosphorylation of NF-κB and cardiomyocyte apoptosis. Inhibition of NF-κB activity by ammonium pyrrolidinedithiocarbamate (PDTC) suppressed DOX-induced cardiomyocyte apoptosis. DOX-treatment (18 mg/kg via intravenous 3 injections over 1 week) increased phosphorylation of CaMKII and NF-κB in mouse hearts. Nifedipine (10 mg/kg/day) significantly suppressed DOX-induced phosphorylation of CaMKII and NF-κB and cardiomyocyte injury and apoptosis in mouse hearts. Moreover, it attenuated DOX-induced left ventricular dysfunction and dilatation. Our findings suggest that blockade of LTCC attenuates DOX-induced cardiomyocyte apoptosis via suppressing intracellular Ca²⁺ elevation and activation of CaMKII-NF-κB pathway. LTCC blockers might be potential therapeutic agents against DOX-induced cardiomyopathy.

Figure 1

DOX induced phosphorylation of CaMKII and NF-κB and increased cleaved caspase 3 in cardiomyocytes in a dose-dependent manner. (a) Representative immunoblots of CaMKII, phosphorylated CaMKII, NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in cultured neonatal rat ventricular myocytes (NRVMs) treated with indicated concentrations of DOX. (b–d) Quantitative analysis of phosphorylated CaMKII, phosphorylated NF-κB, and cleaved caspase 3 in NRVMs treated with indicated concentration of DOX (n = 5). The experiment was conducted 2 times. *P < 0.05: post-hoc Tukey’s comparison test.

Figure 2

Blockade of LTCC suppressed DOX-induced phosphorylation of CaMKII and NF-κB and increases in cleaved caspase 3 in cardiomyocytes. (a) Representative immunoblots of CaMKII, phosphorylated CaMKII, NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in NRVMs treated with or without nifedipine (Nif, 1 μM) in the presence or absence of DOX (10 μM) for 24 hour (n = 5). The experiment was conducted 3 times. (b–d) Quantitative analysis of phosphorylated CaMKII, phosphorylated NF-κB, and cleaved caspase 3 in each group (n = 5). (e) Representative immunoblots of CaMKII, phosphorylated CaMKII, NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in NRVMs treated with indicated small interfering RNA (siRNA) in the presence or absence of DOX (10 μM) for 24 hour (n = 5). (f–h) Quantitative analysis of phosphorylated CaMKII, phosphorylated NF-κB, and cleaved caspase 3 in each group (n = 5). The experiment was conducted 3 times. *P < 0.05, **P < 0.01: post-hoc Tukey’s comparison test.

Figure 3

Blockade of LTCC attenuated DOX-induced cardiomyocyte apoptosis. (a,b) Apoptosis evaluated by TUNEL staining in NRVMs treated with nifedipine (Nif, 1 μM) or siRNA for LTCC (1 nM) in the presence or absence of DOX (10 μM) for 24 hours (n = 5–6). The experiment was conducted 3 times. Arrows indicate TUNEL-positive cells. (c) LDH release, a marker of cell death, evaluated by LDH cytotoxicity assay in NRVMs treated with nifedipine (Nif) or siRNA for LTCC in the presence or absence of DOX (10 μM) for 24 hours (n = 5–6). The experiment was conducted 3 times. **P < 0.01: post-hoc Tukey’s comparison test.

Figure 4

Blockade of LTCC attenuated DOX-induced elevation of intracellular Ca²⁺ levels in cardiomyocytes. (a) The resting levels of intracellular Ca²⁺ concentration in NRVMs were evaluated by fura-2 fluorometry. (b) A representative recording of fura-2 fluorometry in NRVMs treated with nifedipine (Nif) or siRNA for LTCC in the presence or absence of DOX (10 μM) for 24 hours. (c) The resting levels of intracellular Ca²⁺ were expressed as percentages by assigning the levels of intracellular Ca²⁺ obtained with ionomycin in the presence and absence of extracellular Ca²⁺, to be 100% and 0%, respectively. Cap-tipped lines indicate intracellular Ca²⁺ levels (n = 5–8). (d) Representative immunoblots of CaMKII, phosphorylated CaMKII, and GAPDH in NRVMs treated with or without BAPTA (2 μM) in the presence or absence of DOX (10 μM) for 24 hour (n = 5). (e) Quantitative analysis of phosphorylated CaMKII in each group (n = 5). The experiment was conducted 2 times. **P < 0.01: post-hoc Tukey’s comparison test.

Figure 5

CaMKII positively regulated DOX-induced cardiomyocyte apoptosis by activating NF-κB. (a) Representative immunoblots of CaMKII, phosphorylated CaMKII, NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in NRVMs treated with or without AIP (10 μM, 24 h) in the presence or absence of DOX (10 μM) for 24 hours (n = 5–6). (b–d) Quantitative analysis of phosphorylated CaMKII, phosphorylated NF-κB, and cleaved caspase 3 in each group (n = 5–6). The experiment was conducted 3 times. (e) Apoptosis evaluated by TUNEL staining in each group (n = 5). The experiment was conducted 2 times. (f) Representative immunoblots of NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in NRVMs treated with or without PDTC (100 μM, 24 h) in the presence or absence of DOX (10 μM) for 24 hours (n = 5–6). (g–i) Quantitative analysis of phosphorylated NF-κB and cleaved caspase 3 in NRVMs treated with or without PDTC (100 μM, 24 h) in the presence or absence of DOX (10 μM) for 24 hours (n = 5–6). The experiment was conducted 2 times. (j) Apoptosis evaluated by TUNEL staining in each group (n = 5). The experiment was conducted 2 times. *P < 0.05, **P < 0.01: post-hoc Tukey’s comparison test.

Figure 6

Blockade of LTCC suppressed CaMKII-NF-kB pathway in DOX-treated hearts. (a) Representative immunoblots and quantitative analysis of CaMKII, phosphorylated CaMKII, and GAPDH in DOX (3 doses of DOX at 6 mg/kg body weight every third day for 1 week) or control vehicle (phosphate-buffered saline: PBS) treated-C57B/6 J mouse hearts subjected to either nifedipine (Nif, 10 mg/day/day) or saline for 9 days (n = 5). (b) Representative immunoblots and quantitative analysis of NF-κB, phosphorylated NF-κB, cleaved caspase 3, and GAPDH in each group (n = 5). The experiment was conducted 3 times. (c–g) Representative immunoblotsa and quantitative analysis of ERK, phosphorylated ERK, JNK, phosphorylated JNK, Nox4, p53, and GAPDH in each group (n = 5).

Figure 7

Blockade of LTCC ameliorated DOX-induced myocardial injury and apoptosis in mice. (a) HE stained section in DOX (3 doses of DOX at 6 mg/kg body weight every third day for 1 week) or control vehicle (phosphate-buffered saline: PBS) treated-C57B/6 J mouse hearts subjected to either nifedipine (Nif, 10 mg/day/day) or saline for 14 days. Arrows indicate cytoplasmic vacuolization and arrowheads indicate myofibrillar loss. (b) Cardiomyocyte injury as assessed by number of cytoplasmic vaculolization (n = 6). (c) Cardiomyocyte death as assessed by Billingham score in the heart (n = 5). (d) TUNEL stained heart section in each group. Arrow heads indicate TUNEL-positive nuclei. (e) The percentage of TUNEL-positive nuclei in the heart (n = 5). (f) WGA-stained heart section in each group. (g) Cardiomyocyte hypertrophy as assessed by cross-sectional area in each group (n = 5). (h) Masson-trichrome-stained heart section in each group. (i) Interstitial fibrosis as assessed by collagen volume in each group (n = 5). *P < 0.05, **P < 0.01: post-hoc Tukey’s comparison test.

Figure 8

Blockade of LTCC attenuated DOX-induced cardiac dysfunction and loss of heart weight in mice. (a) The representative echocardiographic images of DOX (3 doses of DOX at 6 mg/kg body weight every third day for 1 week) or control vehicle (phosphate-buffered saline: PBS) treated-C57B/6 J mouse hearts subjected to either nifedipine (Nif, 10 mg/day/day) or saline for 14 days. Long two-way arrows and short two-way arrows indicate left ventricular end-diastolic diameter (LVDd) and left ventricular end-systolic diameter (LVDs), respectively. (b–f) Heart rate, LVDd and LVDs, fractional shortening, and LV ejection fraction measured by echocardiography at day14 in each group (n = 6–14). (g,h) Heart weight to tibial length (TL) ratio and LV weight to TL ratio in each group (n = 6–14). *P < 0.05, **P < 0.01: post-hoc Tukey’s comparison test.