Mitochondrial OPA1, apoptosis, and heart failure

Abstract

Aims: Mitochondrial fusion and fission are essential processes for preservation of normal mitochondrial function. We hypothesized that fusion proteins would be decreased in heart failure (HF), as the mitochondria in HF have been reported to be small and dysfunctional.

Methods and results: Expression of optic atrophy 1 (OPA1), a mitochondrial fusion protein, was decreased in both human and rat HF, as observed by western blotting. OPA1 is important for maintaining normal cristae structure and function, for preserving the inner membrane structure and for protecting cells from apoptosis. Confocal and electron microscopy studies demonstrated that the mitochondria in the failing hearts were small and fragmented, consistent with decreased fusion. OPA1 mRNA levels did not differ between failing and normal hearts, suggesting post-transcriptional control. Simulated ischaemia in the cardiac myogenic cell line H9c2 cells reduced OPA protein levels. Reduction of OPA1 expression with shRNA resulted in increased apoptosis and fragmentation of the mitochondria. Overexpression of OPA1 increased mitochondrial tubularity, but did not protect against simulated ischaemia-induced apoptosis. Cytochrome c release from the mitochondria was increased both with reduction in OPA1 and with overexpression of OPA1.

Conclusion: This is the first report, to our knowledge, of changes in mitochondrial fusion/fission proteins in cardiovascular disease. These changes have implications for mitochondrial function and apoptosis, contributing to the cell loss which is part of the downward progression of the failing heart.

Figure 1

Expression of fission and fusion proteins in heart failure (HF). (A) Westerns from same blot showing relative expression of OPA1, MFN1, MFN2, DRP1, Fis1 in HF rat heart. GAPDH shown as control. (B) Expression of fission and fusion proteins in explanted human hearts compared with normal hearts. Same western shown for all five proteins and GAPDH (loading control). Graphs summarize the relative protein levels normalized to GAPDH. ICM, ischaemic cardiomyopathy; DCM, non-ischaemic cardiomyopathy. n = 3–5/group. *P < 0.05; ***P < 0.001.

Figure 2

(A) Representative EM of sham-operated control and HF rat hearts. (B) Graphs summarize the mitochondria per area (left panel) and average mitochondrial size (right panel). n = 3 hearts/group. (C) (1) Sham-operated control heart showing mitochondrial OPA1 by immuno-EM. (2) Representative image of HF rat heart showing reduced mitochondrial OPA1. (3 and 4) Show enlargement of area outlined in panels (1 and 2). Arrows—5 nM gold particle-antibody bound to OPA1. (D) Upper panel—real-time PCR was done to compare OPA1 mRNA in failing vs. normal human heart samples. Lower panel—real-time PCR for OPA1 mRNA in failing rat hearts vs. sham-operated controls. Results normalized to GAPDH. n = 3–5/group. Bar = 100 nM. *P < 0.05; ***P < 0.001.

Figure 3

(A) Western showing expression of the two main OPA1 variants in normal and post-simulated ischaemia H9c2 cells. GAPDH shown in lower panel. Graph summarizes the relative OPA1 expression normalized to GAPDH. (B) Cells were transfected with an OFP construct. OFP is a fluorescent protein, which localizes to the mitochondria. Normal cells show tubular network, which is lost post-simulated ischaemia (lower panel). (C) Images of cells were collected and analysed for the amount of tubular vs. fragmented mitochondrial network (see Methods for details). (D) Apoptosis post-simulated ischaemia as determined by CDD assay. (E) Graph shows percent of cells that were TUNEL positive. n = 3–12/group. *P < 0.05; ***P < 0.001.

Figure 4

Effect of OPA1 shRNA on the mitochondrial network and cell viability. (A) Western shows OPA1 expression after 48 h of treatment with OPA1 shRNA or to LacZ shRNA (control). Lower panel—GAPDH as control. Right panel shows relative OPA1 expression in control cells (expressing shRNA-LacZ) vs. shRNA-OPA1 cells. Values normalized to GAPDH. (B) Confocal microscopy showing mitochondrial network after OFP expression in cells transfection with shRNA-LacZ or shRNA-OPA1. shRNA constructs also expressed EGFP, so cells with shRNA were clearly identifiable. Left panel shows EGFP, second panel shows OFP expression in the mitochondria, right panel shows enlarged image of mitochondria and far right panel shows merged image of OFP and EGFP. (C) Images of cells were collected and analysed for the amount of tubular vs. fragmented mitochondrial network (see Methods for details). (D) Apoptosis post-simulated ischaemia by CDD assay. (E) Graph shows percent of cells that were TUNEL positive. n = 3–12/group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5

(A) Western demonstrating the changes in OPA1 with overexpression and following ischaemia. Cells expressed either OPA1 and EGFP or EGFP alone. Graph summarizes multiple experiments. (B) Confocal immunocytochemistry of cells overexpressing OPA1 and EGFP or EGFP alone. Mito-OFP was used as a mitochondrial marker. Greater tubularity is visible with OPA1 overexpression (lower panel). (C) Mitochondrial morphology was quantified, as described in Methods. Effect of OPA1 plus EGFP overexpression vs. EGFP alone. (D) Effect of OPA1 overexpression on apoptosis (DNA fragmentation) after simulated ischaemia. (E) Percent of cells undergoing apoptosis (TUNEL) after simulated ischaemia. Comparison of OPA1 overexpressing cells vs. EGFP alone. n = 4–12/group. **P < 0.01; ***P < 0.001.

Figure 6

(A) Effect of changes in OPA1 expression on cytochrome c release with ischaemia—Cytochrome c release was compared with shRNA treatment vs. OPA1 overexpression. Representative western compares release from the treatment groups with and without simulated ischaemia. Graph summarizes multiple experiments. Densitometric values for all were normalized to control with shRNA-LacZ. Reduction in OPA1 with shRNA caused cytochrome c release and this increased with simulated ischaemia. OPA1 overexpression increased the release of cytochrome c. (B) Effect of CsA (500 nM) on OPA1 levels during simulated ischaemia. Upper panels demonstrate protective effect of CsA on OPA1 levels in simulated ischaemia, whereas it has no effect in ischaemia if OPA1 already reduced with shRNA. Lower panels show that even with OPA1 overexpression CsA protects OPA1 during simulated ischaemia. n = 4/group. *P < 0.05; **P < 0.01.