Critical role of insulin‑like growth factor binding protein‑5 in methamphetamine‑induced apoptosis in cardiomyocytes

Abstract

Methamphetamine (MA) is a highly abused amphetamine‑like psychostimulant. At present, the mechanisms underlying MA‑induced cardiotoxicity are poorly understood. The cardiotoxic effects have yet not been clearly elucidated with respect to the apoptotic pathway. Insulin‑like growth factor binding protein‑5 (IGFBP5) is important for cell growth control and the induction of apoptosis. The aim of the present study was to analyze whether IGFBP5 is involved in MA‑induced apoptosis as a novel target. MA‑induced apoptosis was observed in neonatal rat ventricular myocytes (NRVMs) in a concentration‑dependent manner using a terminal deoxyribonucleotide transferase‑mediated dUTP nick end‑labeling assay. Using reverse transcription polymerase chain reaction and western blotting, MA was demonstrated to induce concentration‑dependent increases in the expression of IGFBP5. Silencing IGFBP5 with small interfering RNA significantly reduced apoptosis and suppressed the expression of caspase‑3 in NRVMs following treatment with MA. To the best of our knowledge, the present study provided the first evidence suggesting that IGFBP5 is a potential therapeutic target in MA‑induced apoptosis in vitro, providing a foundation for future in vivo studies.

Figure 1

MA induces apoptosis in NRVMs. (A) Effects of MA treatment in NRVMs with varying concentrations (0, 0.5, 1.0 or 1.5 mM) assessed by TUNEL assay. Nuclei were counterstained with DAPI (blue). Apoptotic cells were stained with TUNEL (green). (B) Quantification of the percentage of apoptotic cells using a standard cell counting method with the TUNEL assay. The apoptotic rate was calculated as follows: (number of apoptotic cells / total number of cells) × 100%. The data are presented as the mean ± standard deviation from three independent experiments (n=5; ^*P<0.01). MA, methamphetamine; TUNEL, terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling; NRVMs, neonatal rat ventricular myocytes; DAPI, 4′,6′-diamidino-2-phenylindole.

Figure 2

The mRNA and protein expression of IGFBP5 increases in a dose-dependent manner in NRVMs treated with MA for 48 h. NRVMs were incubated with MA at varying concentrations (0, 0.5, 1.0 or 1.5 mM). (A) Total RNA was subjected to reverse transcription polymerase chain reaction. (B) Whole cell lysates were analyzed by western blotting. An antibody against β-actin was used as the loading control. The data are presented as the mean ± standard deviation (n=6; ^*P<0.01). MA, methamphetamine; NRVMs, neonatal rat ventricular myocytes; IGFBP5, insulin-like growth factor binding protein-5.

Figure 3

siIGFBP5 silences the protein expression of IGFBP5 in NRVMs. (A) Nonspecific silencing control lane showed no effect of siNC in NRVMs 48 h post-transfection. siIGFBP5-1 and siIGFBP5-2 demonstrated efficient knockdown of IGFBP5 at the protein level. (B) The knockdown efficacy of siIGFBP5 was not affected following treatment with MA. An antibody against β-actin was used as the loading control. The data are presented as the mean ± standard deviation (n=6; ^*P<0.01, ^**P>0.05). MA, methamphetamine; NRVMs, neonatal rat ventricular myocytes; IGFBP5, insulin-like growth factor binding protein-5; si, small interfering.

Figure 4

Effect of siIGFBP5 silencing of the IGFBP5 gene on MA-induced apoptosis evaluated by TUNEL staining. (A) Effects of suppressing the IGFBP5 gene in 1.5 mM MA-treated NRVMs assessed by TUNEL assay. The 0 mM + siNC group was pretreated with nonspecific silencing control without MA as the control group. The 1.5 mM + siIGFBP5-1 group and the 1.5 mM + siIGFBP5-2 group were pretreated with two sequences (siIGFBP5-1 and siIGFBP5-2) that target IGFBP5, respectively. Nuclei were counterstained with DAPI (blue). Apoptotic cells were stained with TUNEL (green). (B) Quantification of the percentage of apoptotic cells using a standard cell counting method with the TUNEL assay. The apoptotic rate was calculated as follows: (number of apoptotic cells / total number of cells) × 100%. The data are presented as the mean ± standard deviation from three independent experiments (n=5; ^*P<0.01, ^**P>0.05). MA, methamphetamine; IGFBP5, insulin-like growth factor binding protein-5; TUNEL, terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling; DAPI, 4′,6′-diamidino-2-phenylindole; NRVMs, neonatal rat ventricular myocytes; si, small interfering.

Figure 5

Expression of caspase-3 was determined by western blot analysis. The intensity of each band was quantified by densitometry and the data were normalized using the β-actin signal. The 0 mM + siNC group (control group) was considered the basal level and the other groups were expressed as fold changes compared with the control. The data are presented as the mean ± standard deviation (n=6; ^*P<0.01, ^**P>0.05). IGFBP5, insulin-like growth factor binding protein-5; si, small interfering.