Remote ischaemic preconditioning ameliorates anthracycline-induced cardiotoxicity and preserves mitochondrial integrity

Abstract

Aims: Anthracycline-induced cardiotoxicity (AIC) is a serious adverse effect among cancer patients. A central mechanism of AIC is irreversible mitochondrial damage. Despite major efforts, there are currently no effective therapies able to prevent AIC.

Methods and results: Forty Large-White pigs were included. In Study 1, 20 pigs were randomized 1:1 to remote ischaemic preconditioning (RIPC, 3 cycles of 5 min leg ischaemia followed by 5 min reperfusion) or no pretreatment. RIPC was performed immediately before each intracoronary doxorubicin injections (0.45 mg/kg) given at Weeks 0, 2, 4, 6, and 8. A group of 10 pigs with no exposure to doxorubicin served as healthy controls. Pigs underwent serial cardiac magnetic resonance (CMR) exams at baseline and at Weeks 6, 8, 12, and 16, being sacrifice after that. In Study 2, 10 new pigs received 3 doxorubicin injections (with/out preceding RIPC) and were sacrificed at week 6. In Study 1, left ventricular ejection fraction (LVEF) depression was blunted animals receiving RIPC before doxorubicin (RIPC-Doxo), which had a significantly higher LVEF at Week 16 than doxorubicin treated pigs that received no pretreatment (Untreated-Doxo) (41.5 ± 9.1% vs. 32.5 ± 8.7%, P = 0.04). It was mainly due to conserved regional contractile function. In Study 2, transmission electron microscopy (TEM) at Week 6 showed fragmented mitochondria with severe morphological abnormalities in Untreated-Doxo pigs, together with upregulation of fission and autophagy proteins. At the end of the 16-week Study 1 protocol, TEM revealed overt mitochondrial fragmentation with structural fragmentation in Untreated-Doxo pigs, whereas interstitial fibrosis was less severe in RIPC+Doxo pigs.

Conclusion: In a translatable large-animal model of AIC, RIPC applied immediately before each doxorubicin injection resulted in preserved cardiac contractility with significantly higher long-term LVEF and less cardiac fibrosis. RIPC prevented mitochondrial fragmentation and dysregulated autophagy from AIC early stages. RIPC is a promising intervention for testing in clinical trials in AIC.

Keywords: Anthracyclines; Cardio-oncology; Cardiotoxicity; Magnetic resonance imaging; Mitochondria; Remote conditioning.

Graphical abstract

Figure 1

Study design. Study 1 consisted of 30 castrated Large-White male pigs; 20 pigs were randomized the AIC protocol with no pretreatment (Untreated-Doxo) or preceded at each cycle by RIPC (RIPC-Doxo). An additional 10 animals were monitored as healthy controls. CMR scans were performed at baseline (Week 0) and at 6, 8, 12, and 16 weeks. Animals were sacrificed, and hearts were excised for ex vivo analysis. Study 2 consisted of 10 animals randomized to Untreated-Doxo or RIPC-Doxo and sacrificed 2 weeks after the third doxorubicin dose. CMR scans were performed at baseline (Week 0) and at 6 weeks.

Figure 2

Functional CMR in Study 1. (A) LVEF over time (left) and at 16 weeks (right). Grouped data are presented as mean ± SE and individual data at the end of the study as a dotplot. N = 10 animals per group. The significance of differences between Untreated-Doxo and RIPC-Doxo groups at 16 weeks was assessed by the Student’s t-test. (B) Wall thickening at 16 weeks in the doxorubicin-infused area (mean of the AHA segments 7 and 8). Grouped data are presented as mean ± SE and individual data at the end of the study as a dotplot. N = 10 animals per group. The significance of differences between Untreated-Doxo and RIPC-Doxo at 16 weeks was assessed by the Student’s t-test.

Figure 3

CMR and histology analysis of cardiac tissue. (A) Native T1 differential at the end of the study (16 weeks – baseline) (right). Grouped data are presented as mean ± SE and individual data at the end of study as a dotplot. N = 10 animals per group. The significance of differences between Untreated-Doxo and RIPC-Doxo groups at 16 weeks was assessed by the Student’s t-test. (B) Collagen fraction area at 16 weeks. Samples were obtained from the medium antero-septal region. Data are presented grouped in a boxplot and ungrouped (individual data) as a dotplot. N = 10 animals per group. Shaded grey lines represented mean and standard deviation of the collagen data from Healthy Control group. The significance of differences between Untreated-Doxo and RIPC-Doxo at 16 weeks was assessed by the Student’s t-test. (C) Representative histology images illustrating myocardial interstitial fibrosis (SR and Masson’s trichrome) at 16 weeks.

Figure 4

TEM analysis of myocardial mitochondria after 16 weeks of AIC (Study 1). (A) Individual mitochondrial area in the infused area in each animal group (dotplot) with its boxplot and violin plot related distribution. (B) Distribution of mitochondrial density vs. mitochondrial area in each animal group. (C) Representative TEM images at 6000× and 40 000× showing cardiac mitochondria replicates for each animal group. N = 5 animals per group. Data were analysed by linear regression nested according to treatment group and animal ID (Untreated-Doxo and RIPC-Doxo).

Figure 5

TEM analysis of myocardial mitochondria after 6 weeks of AIC (Study 2). (A) Individual mitochondrial area in the infused area in each animal group (dotplot) with its boxplot and violin plot related distribution. (B) Distribution of mitochondrial density vs. mitochondrial area in each animal group. (C) Representative TEM images at 6000× and 40 000× showing all cardiac mitochondria replicates for each animal group. N = 5 animals per group. Data were analysed by linear regression nested according to treatment group and animal ID (Untreated-Doxo and RIPC-Doxo). The red arrowhead indicates a fragmented mitochondria and the blue arrowhead a secondary lysosome.

Figure 6

Myocardial mitochondria protein expression in the infused area after 6 weeks of AIC (Study 2). (A) Western blot analysis of the myocardial expression of Beclin1, p62, and DRP1 in all animals. (B) Myocardial mitochondrial DNA content. (C–E) Quantification of protein expression (fold-increased, normalized to GAPDH) for (C) DRP1, (D) Beclin 1, and (E) p62. (n = 4 in Healthy controls, 5 in Untreated-Doxo, and 5 in RIPC-Doxo groups). Significance differences between groups were calculating using one-way ANOVA.