Mitochondrial transporter ATP binding cassette mitochondrial erythroid is a novel gene required for cardiac recovery after ischemia/reperfusion

Abstract

Background: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart.

Methods and results: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion.

Conclusions: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Figure 1. ABC-me +/- hearts have normal structure and function

A) Left ventricular pressure–volume relationships in isolated Langendorff perfused wild type (black symbols) and ABC-me +/- hearts (open symbols) (n=7). Upper traces represent systolic pressure and lower traces represent end diastolic pressure (EDP). B) Representative tracings of mitral inflow and tissue Doppler analysis of wild type and ABC-me +/- hearts. For values see table 1.

Figure 2. ABC-me +/- hearts have normal mitochondrial function and oxidative status under basal conditions

A) & B) Oxygen consumption rates (OCR) of isolated mitochondria (20μg) from wild type and ABC-me +/- hearts after Langendorff (n=4). State III was induced by incubating mitochondria with 0.25 mM ADP, State IV by 2 μM Oligomycin and uncoupled respiration by 100 μM 2,4-dinitrophenol (DNP). Respiration was driven by A) complex I (5mM pyruvate and 5 mM malate) or B) complex II (5 mM succinate and 2 μM rotenone). C) Representative Western blot analysis from total lysates of wild type (WT) and ABC-me +/- (+/-) hearts (n=7) to detect carbonylated proteins (the comassie blue staining of the transferred gel was used as a loading control). D) Mitochondrial superoxide production rates were addressed by monitoring the linear increase on Mitosox fluorescence in 96-well plate with 5-10 μg of mitochondria from wild type and ABC-me +/- hearts. Antimycin A was used as a positive control, as it increases superoxide production (black bars, wild type, open bars, ABC-me +/-, n=6). E) Representative Western blot analysis from total lysates of wild type (WT) and ABC-me +/- (+/-) hearts (n=7) to detect mitochondrial superoxide dismutase (sod2) and catalase. The comassie blue staining of the transferred gels and Porin were used as loading controls. Data are shown as mean with ± SEM.

Figure 3. ABC-me +/- hearts have increased oxidative damage and impaired recovery after ischemia-reperfusion, which is specifically rescued by the superoxide dismutase / catalase mimetic, EUK-207

Continuous recording of left ventricular pressure (baseline and during ischemia-reperfusion) was performed in isolated Langendorff perfused hearts. A) Developed pressure (difference between systolic and diastolic pressures, mmHg) B) End diastolic pressure and C) Systolic pressure of wild type (black symbols) and ABC-me +/- hearts (open symbols) (n=7) pre-treated (20 minutes before ischemia) with vehicle (continuous line) or 50 μM EUK-207 (dashed line). D) Percentage of recovery of contractile function (ratio of Developed Pressure at the end of reperfusion vs. baseline) of wild type and ABC-me +/- hearts (n=7) pre-treated with vehicle or 50 μM EUK-207. *, p=0.02, wild type vs. ABC-me +/-; #, p=0.002, vehicle ABC-me +/- vs. EUK-207 ABC-me +/-. †, p=0.03, wild type vs. wild type EUK-207. E) Quantification of lipid oxidation by TBARS in isolated mitochondria from wild type and ABC-me +/- hearts after ischemia-reperfusion, or ABC-me +/- hearts pre-treated with EUK-207 or with vehicle after ischemia-reperfusion (n=5). All data are shown as mean with SEM. *, p= 0.0079, wild type vs. ABC-me +/-; #, p= 0.03, vehicle +/- vs. EUK-207. F) Representative images of heart sections immunostained with an antibody detecting sulfonylated SERCA on cysteine 674 from wild-type (WT) and ABC-me +/- hearts under basal conditions, after ischemia reperfusion (I-R) and pre-treated with EUK-207 or vehicle after ischemia-reperfusion. Scale bar, 25 μm.

Figure 4. EUK-207 restores mitochondrial function and ATP levels in ABC-me +/- hearts after ischemia-reperfusion

A) Oxygen consumption rates (OCR) driven by complex II after ischemia-reperfusion (5 mM succinate + 2 μM rotenone) of isolated mitochondria (40μg) from vehicle and EUK-207 (50 μM) wild type (black, vehicle; dark grey, EUK-207) and ABC-me +/- pre-treated hearts (white, vehicle; light grey, EUK-207) (n=3). Data are shown as mean with ± SEM. State III was induced by incubating mitochondria with 0.25 mM ADP, State IV by 2 μM Oligomycin and uncoupled respiration by 100 μM 2,4-dinitrophenol (DNP). For detailed respirometry procedure see supplementary material. Data are shown as mean with ± SEM. *, p≤0.01, vehicle wild type vs. vehicle- ABC-me +/- hearts; #, p≤0.04, vehicle vs. EUK-207 ABC-me +/- hearts. B) Measurement of ATP synthesis rates in isolated mitochondria (nmols synthesized per minute and milligram of protein) from wild type (WT) and ABC-me +/- hearts after ischemia-reperfusion (n=5) or ABC-me +/- vehicle or EUK-207 (50 μM) after ischemia-reperfusion (n=5). Data are shown as mean with ± SEM. *, p=0.016, wild type vs. ABC-me +/-; #, p=0.03, vehicle vs. EUK-207 pre-treated ABC-me +/- hearts. C) Measurement of total ATP content after ischemia-reperfusion (μmols of nucleotide per mg of total protein) in freeze-clamped ABC-me +/- pre-treated with EUK-207 or with vehicle (n=4). Data are shown as mean with ± SEM. #, p=0.03.