Hsp70 regulates the doxorubicin-mediated heart failure in Hsp70-transgenic mice

Abstract

The aim of this study was to investigate the potential protective effect of the Hsp70 protein in the cardiac dysfunction induced by doxorubicin (DOX) and the mechanisms of its action. For this purpose, we used both wild-type mice (F1/F1) and Hsp70-transgenic mice (Tg/Tg) overexpressing human HSP70. Both types were subjected to chronic DOX administration (3 mg/kg intraperitoneally every week for 10 weeks, with an interval from weeks 4 to 6). Primary cell cultures isolated from embryos of these mice were also studied. During DOX administration, the mortality rate as well as weight reduction were lower in Tg/Tg compared to F1/F1 mice (P < 0.05). In vivo cardiac function assessment by transthoracic echocardiography showed that the reduction in left ventricular systolic function observed after DOX administration was lower in Tg/Tg mice (P < 0.05). The study in primary embryonic cell lines showed that the apoptosis after incubation with DOX was reduced in cells overexpressing Hsp70 (Tg/Tg), while the apoptotic pathway that was activated by DOX administration involved activated protein factors such as p53, Bax, caspase-9, caspase-3, and PARP-1. In myocardial protein extracts from identical mice with DOX-induced heart failure, the particular activated apoptotic pathway was confirmed, while the presence of Hsp70 appeared to inhibit the apoptotic pathway upstream of the p53 activation. Our results, in this DOX-induced heart failure model, indicate that Hsp70 overexpression in Tg/Tg transgenic mice provides protection from myocardial damage via an Hsp70-block in p53 activation, thus reducing the subsequent apoptotic mechanism.

Fig. 1

Diagram showing the timeline of the study

Fig. 2

The Hsp70 overexpression is followed by decreased apoptotic activity in primary embryonic cells. a NIH-3T3, F1/F1 and Tg/Tg cells were exposed or not to heat shock (90 min at 43 °C and 90-min recovery at 37 °C) as indicated. Protein lysates (10 μg per sample) were analyzed to SDS-PAGE and subjected to Western blot using a MAb specific for the inducible Hsp70 (C92). bThe same primary embryonic cells were used for flow cytometric analysis of intracellular Hsp70, using the same anti-Hsp70 antibody as in a. Control unstained sample, IgG incubation only with secondary anti-mouse-FITC, FITC-Mock incubation with anti-Hsp70 and anti-mouse-FITC, FITC-heat shock incubation with anti-Hsp70 and anti-mouse-FITC, after 90-min heat shock at 42.5 °C and 3-h recovery to 37 °C. c, d The same cells were exposed to a range of doxorubicin (DOX) concentrations for 24 h (c) or 48 h (d) and analyzed for PI/Annexin V FITC by flow cytometry. In c and d, each point represents the mean ± SD of three measurements in three separate experiments. In all points, except for treatment with 0.1 μM of DOX (P = NS), P values were <0.01 and <0.005 for positive pI/Annexin V cells %: apoptosis and necrosis %, respectively

Fig. 3

Hsp70 blocks the doxorubicin-induced apoptotic cascade upstream of p53 phosphorylation. F1/F1 and Tg/Tg cells were exposed to 1, 5, 10, or 25 μM DOX for 24 h. Simultaneously, the same cells were exposed to heat shock (60 min at 42.5 °C and 90-min recovery at 37 °C) or staurosporine (3 h at 1 μM) that were used as markers of Hsp70 expression or as markers of apoptosis activation. Ten micrograms of each cellular fraction was analyzed by Western blot with anti-Hsp70 (A1, A2), anti-PARP-1 (B1, B2) anti-phospho-p53-[ser15] (C1, C2), anti-Bax (D1, D2), anti-cleaved caspase-9 (E1, E2), anti-active caspase-3 (F1, F2), and anti-tubulin antibodies as indicated

Fig. 4

The Hsp70 expression in F1/F1 and Tg/Tg mice during their exposure to DOX treatment. a Real picture of wild-type control (left) and Hsp70-transgenic mice (right). b Determination of Hsp70-transgenic mice genotype: PCR analysis of DNAs isolated from control (F1/F1 mice), heterozygous (F1/Tg mice), and homozygous (Tg/Tg mice) Hsp70-transgenic mice. The amplification of the 18S mouse rRNA was used as internal marker of equal loading (241 bp). c Cardiac tissue protein extracts (20 μg/sample) were analyzed in SDS-PAGE and subjected to Western blotting using specific antibodies. The α-tubulin was used as marker for equal sample loading

Fig. 5

DOX-induced cardiac dysfunction is attenuated in Tg/Tg transgenic mice overexpressing Hsp70. a Echocardiographic M-mode images of murine hearts. Upper row wild-type animals, control that served as baseline (left) and 10 weeks after DOX initiation (right). Lower row transgenic mice overexpressing Hsp70, control that served as baseline (left), and 10 weeks after DOX initiation (right). FS represents fractional shortening, an index of left ventricular systolic function. b Fractional shortening, an index of systolic function in wild-type (WT) and transgenic mice overexpressing Hsp70. *P < 0.05 compared to control wild-type (WT) mice group (without DOX administration), ^§ P < 0.05 compared to control transgenic mice group (without DOX administration)

Fig. 6

Inhibition of caspace-9 and caspace-3 in cardiac tissues of Hsp70-transgenic mice. F1/F1 and Tg/Tg mice were exposed to DOX (3 mg/kg) for 4, 6, or 10 weeks. Cardiac tissue protein extract (20 μg/sample) obtained from F1/F1 and Tg/Tg were analyzed in SDS-PAGE and subjected to Western blotting using specific antibodies against cleaved caspase-9 (a) and active caspase-3 (b). The α-tubulin was used as marker for equal loading of samples. C2 cells were exposed to heat shock (60 min at 42.5 °C and 90 min recovery at 37°C) or staurosporine (3 h at 1 μM) to be used as markers of Hsp70 expression or as markers of apoptosis activation

Fig. 7

Activation via phosphorylation of p53 in mouse myocardial tissue after doxorubicin administration. a Confocal microscopy analysis of phospho-p53 positive cells (red stained cells) derived from F1/F1 and Tg/Tg myocardial sections 10 weeks after DOX (3 mg/kg) initiation. Tissues were fixed for immunofluorescence staining of phosphorylated-p53, followed by co-staining with Alexa-Fluor 568 and SYBR Green I. b Percentages of p53 activation were determined by cell counting. Data are expressed as mean ± SD (n = 5), P = 0.002. c The same amounts of nuclear and cytoplasmic proteins derived from cells of fresh myocardial tissues of F1/F1 and Tg/Tg mice were analyzed by SDS-PAGE and in turn subjected to Western blotting using specific antibodies against the phospho-p53

Fig. 8

Hypothetical model of the Hsp70 function on the response to doxorubicin treatment, in the cytoplasm and nucleus. Doxorubicin translocates to the nucleus and produces DNA damages that promote the activated p53-apoptotic pathway via p53, Bax, caspase-9, and caspase-3