Over-expression of a modified bifunctional apoptosis regulator protects against cardiac injury and doxorubicin-induced cardiotoxicity in transgenic mice

Abstract

Aims: Bifunctional apoptosis regulator (BAR) is an endoplasmic reticulum protein that interacts with both the extrinsic and intrinsic apoptosis pathways. We hypothesize that over-expression of BAR Delta RING prevents apoptosis and injury following ischaemia/reperfusion (I/R) and attenuates doxorubicin (DOX)-induced cardiotoxicity.

Methods and results: We generated a line of transgenic mice that carried a human BAR Delta RING transgene under the control of the mouse alpha-myosin heavy chain promoter. The RING domain, which binds ubiquitin conjugating enzymes, was deleted to prevent auto-ubiquitination of BAR and allow accumulation of the BAR protein, which binds apoptosis-regulating proteins. High levels of human BAR Delta RING transcripts and 42 KDa BAR Delta RING protein were expressed in the hearts of transgenic mice. When excised hearts were reperfused ex vivo for 45 min as Langendorff preparations after 45 min of global ischaemia, the functional recovery of the hearts, expressed as left ventricular developed pressure x heart rate, was 23 +/- 1.7% in the non-transgenic hearts compared with 51.5 +/- 4.3% in the transgenic hearts (P < 0.05). For in vivo studies, mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by 4 h of reperfusion. The infarct sizes following I/R injury, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (29 +/- 4 vs. 55 +/- 4%, P < 0.05). In hearts of mice subjected to cardiac I/R injury, BAR transgenic hearts had significantly fewer in situ oligo-ligation-positive cardiac cells (5.0 +/- 0.4 vs. 13.4 +/- 0.5%, P < 0.05). Over-expression of BAR Delta RING also significantly attenuated DOX-induced cardiac dysfunction and apoptosis.

Conclusion: Our results demonstrate that over-expression of BAR Delta RING renders the heart more resistant to I/R injury and DOX-induced cardiotoxicity, and this protection correlates with reduced cardiomyocyte apoptosis.

Figure 1

Bifunctional apoptosis regulator (BAR) is expressed in transgenic heart tissue. (A) Ribonuclease protection assay of human BAR mRNA in mouse tissues. RNA was isolated from the hearts of non-transgenic (NTG) and transgenic (TG) animals and probed with a human BAR riboprobe. (B) Immunoblot analysis of BAR hearts from non-transgenic and transgenic mice. Aliquots of 50 µg of heart homogenates from non-transgenic and transgenic mice were separated on 12% SDS–PAGE and were transferred to nitrocellulose membranes. Immunoblot was carried out with antibodies against BAR, Bcl-2, Bcl-X_L, and caspase-8. α-Actin was used as loading control.

Figure 2

Immunohistochemical staining of BAR and Bcl-X_L in hearts of non-transgenic and transgenic mice. Paraffin-embedded sections of non-transgenic (A) and transgenic heart (B) were immunostained with a rat anti-BAR antibody. (B) BAR immunoreactivity (in red) was demonstrated by diffused cytoplasmic staining in the myocytes. (C and D) Bcl-X_L immunostaining of non-transgenic and transgenic hearts, respectively. (E) Staining pattern of transgenic heart with pre-immune serum. Magnification, ×200.

Figure 3

Interaction of BAR with caspase-8, Bcl-2 and Bcl-X_L. Aliquots of 300 µg of heart homogenates from non-transgenic and transgenic mouse hearts were immunoprecipitated with BAR antibodies, followed by immunoblotting with caspase-8, Bcl-2, or Bcl-X_L antibodies.

Figure 4

Ex vivo studies of BAR-mediated protection from cardiac ischaemia/reperfusion injury. Improvement of left ventricular end-diastolic pressure after 45 min of global ischaemia and 45 min of reperfusion in isolated non-transgenic and transgenic hearts. Values are mean ± SEM of six hearts. a, P < 0.05 vs. reperfusion group in non-transgenics, b, P < 0.05 vs. pre-ischaemic groups.

Figure 5

BAR reduces apoptosis in heart following left anterior descending coronary artery (LAD) ligation. Paraffin-embedded sections of non-transgenic and transgenic hearts subjected to 30 min of LAD ligation followed by 3 h of reperfusion were stained by the in situ oligo-ligation (ISOL) procedure. Immunolabelled nuclei of myocytes were determined by random counting of 10 fields per section. Each bar represents mean ± SEM of six hearts. a, P < 0.05 vs. LAD ligation group in non-transgenic mice, b, P < 0.05 vs. sham groups.

Figure 6

Effect of DOX on apoptosis in hearts from non-transgenic and transgenic mice. ISOL procedure was performed in paraffin-embedded myocardial sections. Immunolabelled nuclei of myocytes were determined by random counting of 10 fields per section. Each bar represents mean ± SEM of six hearts. a, P < 0.05, non-transgenics+DOX vs. other groups.