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. 2011 Dec 9;109(12):1327-31.
doi: 10.1161/CIRCRESAHA.111.258723. Epub 2011 Nov 3.

Mitochondrial fusion is essential for organelle function and cardiac homeostasis

Yun Chen  1 , Yingqiu Liu, Gerald W Dorn 2nd
Affiliations

Mitochondrial fusion is essential for organelle function and cardiac homeostasis

Yun Chen et al. Circ Res. .

Abstract

Rationale: Mitochondria constitute 30% of myocardial mass. Mitochondrial fusion and fission appear essential for health of most tissues. Mitochondrial fission occurs in neonatal cardiomycyte and is implicated in cardiomyocyte death. Mitochondrial fusion has not been observed in postmitotic myocytes of adult hearts, and its occurrence and function in this context are controversial.

Objective: Determine the consequences on organelle and organ function of disrupting cardiomyocyte mitochondrial fusion in vivo.

Methods and results: The murine mfn1 and mfn2 genes, encoding mitofusins (Mfn) 1 and 2 that mediate mitochondrial tethering and outer mitochondrial membrane fusion, were interrupted by Cre-mediated excision of essential exons in neonatal (Nkx2.5-Cre) and adult (MYH6 modified estrogen receptor-Cre-modified estrogen receptor plus tamoxifen or Raloxifene) hearts. Embryonic combined Mfn1/Mfn2 ablation was lethal after e9.5. Conditional combined Mfn1/Mfn2 ablation in adult hearts induced mitochondrial fragmentation, cardiomyocyte and mitochondrial respiratory dysfunction, and rapidly progressive and lethal dilated cardiomyopathy. Before heart failure developed, cardiomyocyte shortening and calcium cycling were unaffected by absence of Mfn1 and Mfn2. Based on the time course over which fusion-defective mitochondrial size decreases, a mitochondrial fusion/fission cycle in adult mouse hearts occurs approximately every 16 days.

Conclusions: Mitochondrial fusion in adult cardiac myocytes is necessary to maintain normal mitochondrial morphology and is essential for normal cardiac respiratory and contractile function. Interruption of mitochondrial fusion causes lethal cardiac failure at a time corresponding to 3 or 4 cycles of unopposed mitochondrial fission.

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Figures

Figure 1
Figure 1. Combined ablation of Mfn1 and Mfn2 in the early embryonic heart is lethal
(A) Schematic representation of Cre-Lox strategy for combined cardiomyocyte-specific deletion of mfn1 exon 4 and mfn2 exon 6 using Nkx2.5-Cre knockin. (B) Cardiomyocyte gene recombination by Nkx-2.5 Cre knock-in crossed to ROSA-26 LacZ reporter line and assayed for recombination (blue staining) at 13.5 days p.c. (E13.5), the second day post birth (P2) and three weeks of age (P21). (C) Yield of Nkx2.5-Cre driven cardiac Mfn1, Mfn2, and Mfn1/Mfn2 double cardiac knockout (DCKO) mice.
Figure 2
Figure 2. Conditonal combined ablation of Mfn1 and Mfn2 in adult hearts induces mitochondrial fragmentation and cardiomyocyte respiratory dysfunction
(A) Schematic representations of conditional Cre-Lox strategy for combined cardiomyocyte-specific deletion of mfn1 and mfn2 using MYH6-driven modified estrogen receptor-Cre and tamoxifen or Raloxifene. (B) Immunoblot analysis of mitochondrial fusion and fission proteins 3 weeks after tamoxifen administration. Control (Ctrl) is mfn1fl/fl + mfn2fl/fl without Cre treated with tamoxifen. Each column is a separate mouse heart. (C) Time course of gene recombination induced by tamoxifen, assayed by LacZ staining of ROSA-26 crosses as in Figure 1b. (D) Flow cytometric analysis of isolated cardiac mitochondria size (forward scatter; FSC) and sphericity (side scatter) in Mfn1/Mfn2 double cardiac KO mice 3 weeks after tamoxifen. Left graph shows representative mitochondrial size (Forward scatter) distribution from identically treated ctrl (black) and Mfn1/Mfn2-DKO (red) hearts. Group mean data for forward and side scatter are to the right (n=3; *=P<0.05 vs ctrl). (E) Mitochondrial mass measured as protein content indexed to heart weight (n=4 ctrl and 3 DKO; * is P=0.0026 vs ctrl). (F) Transmission electron microscopic examination of cardiomyocyte mitochondria (5,000x). (G) Time-dependent oxygen consumption of digitonin-permeabilized cardiomyocytes from ctrl (black) and Mfn1/Mfn2 DKO (red) mice. Right panel shows group data; white is ctrl, black is DKO. n=3. (H) Isolated cardiac mitochondrial respiration studies. Data are plotted as in (G). The respiratory control index (state 3 ADP-stimulated/state 2 ADP-limited) is shown immediately to the right, and maximal uncoupled respiration in response to FCCP at the extreme right (n=3 each).
Figure 3
Figure 3. Cardiomyocyte contraction and calcium cycling are normal 1 week after combined cardiac Mfn1 and Mfn2 ablation
(A) Unloaded cardiomyocyte shortening with field stimulation at 1Hz. Representative tracings show time-dependent change in cell length. Left tracing is tamoxifen-treated mfn1fl/fl, mfn2fl/fl without Cre; right tracing is identically treated mfn1fl/fl, mfn2fl/fl Cre+. Bar graphs show group quantitative data (ctrl is white, DKO is black). (B) Phasic calcium transients in Fura-2 loaded cardiomyocytes as above. Group quantitative data for transient amplitude and time for 50% normalization of the transient (t50) are shown to the right. Data are mean ± SEM of n= 3 paired hearts, with each experimenting averaging 8–12 cardiomyocytes myocytes/heart. There were no differences between DKO and ctrl.
Figure 4
Figure 4. Combined Mfn1 and Mfn2 ablation in adult hearts induces rapidly progressive dilated cardiomyopathy
(A) Representative M-mode echocardiograms of un-anesthetized mouse left ventricles before (Pre) and at intervals after conditional ablation of Mfn1 and Mfn2 with tamoxifen. Group data for fractional shortening (top) and LV end diastolic dimension (LVEDD, bottom) are to the right (n = 4–5 per group). (B) Same as in (A), but using Raloxifene to activate MER-Cre-MER (n=4 per group).
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