Down-regulation of OPA1 alters mouse mitochondrial morphology, PTP function, and cardiac adaptation to pressure overload

Abstract

Aims: The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1(+/-) mice, having a 50% reduction in OPA1 protein expression in cardiac tissue.

Methods and results: In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1(+/+). Electron and fluorescence microscopy revealed altered morphology of the Opa1(+/-) mice mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibres, mitochondrial functional properties were maintained, but direct energy channelling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction, Opa1(+/-) hearts demonstrated hypertrophy almost two-fold higher (P< 0.01) than in wild-type mice with altered ejection fraction (decrease in 43 vs. 22% in Opa1(+/+) mice, P< 0.05).

Conclusions: These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.

Conflict of interest statement

Conflict of interest

None

Figures

Figure 1

Anatomical characteristics and cardiac function of 6-month-old Opa1+/+ and Opa1+/− mice. (A) Western blot analysis of OPA1 protein. Upper panel: a representative original recording. Lower panel: mean values of OPA1 protein. ***p<0.001 versus Opa1+/+. (B) Body weight (BW). (C) Heart weight to body weight (HW/BW) ratio. (D) Heart rate. (E) LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV). (F) LV ejection fraction (EF). (G) LV fractional shortening (FS) after dobutamine stimulation.

Figure 2

Morphological description of the mitochondrial network. (A) Representative pictures obtained with Mito-Tracker orange and IMARIS software. Each mitochondrion is identified by a color in accordance with its volume (the smallest mitochondria are blue and the largest are red). (B) Cell volume measured by calcein loading and IMARIS analysis. (C) Total mitochondrial volume quantified by IMARIS. (D) Number of mitochondria per cell. (E) Percentage of mitochondrial size categories. (F) Level of proteins involved in mitochondrial dynamics (Drp1, Mfn1, Mfn2) in Opa1+/+ and Opa1+/− cardiac tissue estimated by Western Blot analysis (normalized to citrate synthase level). *p<0.05; ***p<0.001 versus Opa1+/+.

Figure 3

Electron micrographs of mitochondria in cardiomyocytes from Opa1+/+ and Opa1+/− mice. Upper Panel: Longitudinal (A) and transverse (B) sections of Opa1+/+ cardiomyocytes show individual mitochondria (arrow). Longitudinal (C) and transverse (D) sections of Opa1+/− cardiomyocytes demonstrate mitochondrial clusters (asterisks). Lower Panel: (A) Opa1+/+ cardiomyocytes. Cristae are homogeneously distributed in the mitochondria (arrow). B–D Opa1+/− cardiomyocytes showing enlarged mitochondria and incompletely fused cristae. (B.) Cristae spreading in different directions (arrows); area of disintegrated cristae (asterisks); area of deformation of cristae (arrowhead). (C) Fragmented cristae (arrow); separation between inner and outer mitochondrial membranes (arrowhead). (D) Regions of incomplete mitochondrial fusion (arrow). Note that the peripheral part of the mitochondrion contains normal cristae (arrowhead).

Figure 4

Cardiac energy metabolism of Opa1+/+ and Opa1+/− mice. (A) Oxygen consumption rate of skinned ventricular fibers in the absence (Vo) or presence of 2mM ADP (Vmax) with 10mM glutamate and 4mM malate (complex I, CI), 15mM succinate (both CI and complex II CI+II), and after complex I inhibition with 2mM amytal (CII). (B) Acceptor control ratio. (C) Apparent Km of oxygen consumption for ADP with (+Cr) or without (−Cr) 12 mM creatine. (D) Oxygen consumption rate with 2 mM ADP and 4 mM malate and 0.1mM octanoyl-carnitine. (E) mRNA levels of proteins involved in mitochondrial biogenesis, glucose transport, and lipid utilization. (F) Citrate synthase (CS), complex I and total creatine kinase (CK) activities. (G–H) Efficacy of CK and mitochondria (DANC) to support SERCA (G) or myosin ATPase (H) in permeabilized fibers. *p<0.05 versus OPA1+/+ ***p<0.001 versus OPA1+/+

Figure 5

Kinetics of mitochondrial calcium loading and PTP opening in permeabilized cardiomyocytes and isolated mitochondria of Opa1+/+ and Opa1+/− mice. (A) Upper panel: time course of mitochondrial calcium accumulation following Ca2+ addition (indicated by arrow) measured by Rhod-2 fluorescence. Lower panel: time course of mitochondrial calcein fluorescence following Ca2+ addition (indicated by arrow). (B) Mean values of mitochondrial calcium loading capacity and PTP opening properties. Upper panel, maximal increase of Rhod-2 fluorescence (left panel) and time to the beginning of mitochondrial Ca2+ leak after Ca2+ addition (right panel). Lower panel, time to PTP opening after Ca2+ addition (left panel), and slope of calcein fluorescence decrease after PTP opening (right panel). *p<0.05; **p<0.01; ***p<0.001 versus Opa1+/+. (C) Time course of swelling (left) and depolarization (right) of isolated mitochondria after addition of 25 μM Ca2+.

Figure 6

Anatomical characteristics and echocardiographic data of Opa1+/− mice following transverse aortic constriction (TAC). (A) Heart weight to body weight ratio. (B) Lung weight. (C) Fractional shortening. (D) Left ventricle end-diastolic diameter. (E) Left ventricle end-systolic volume. *p<0.05; **p<0.01; ***p<0.001 versus sham. #p<0.05; ##p<0.01 versus Opa1+/+-TAC.