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. 2015 Nov 23;211(4):795-805.
doi: 10.1083/jcb.201507035.

Titration of mitochondrial fusion rescues Mff-deficient cardiomyopathy

Hsiuchen Chen  1 , Shuxun Ren  2 , Clary Clish  3 , Mohit Jain  4 , Vamsi Mootha  5 , J Michael McCaffery  6 , David C Chan  7
Affiliations

Titration of mitochondrial fusion rescues Mff-deficient cardiomyopathy

Hsiuchen Chen et al. J Cell Biol. .

Abstract

Defects in mitochondrial fusion or fission are associated with many pathologies, raising the hope that pharmacological manipulation of mitochondrial dynamics may have therapeutic benefit. This approach assumes that organ physiology can be restored by rebalancing mitochondrial dynamics, but this concept remains to be validated. We addressed this issue by analyzing mice deficient in Mff, a protein important for mitochondrial fission. Mff mutant mice die at 13 wk as a result of severe dilated cardiomyopathy leading to heart failure. Mutant tissue showed reduced mitochondrial density and respiratory chain activity along with increased mitophagy. Remarkably, concomitant deletion of the mitochondrial fusion gene Mfn1 completely rescued heart dysfunction, life span, and respiratory chain function. Our results show for the first time that retuning the balance of mitochondrial fusion and fission can restore tissue integrity and mitochondrial physiology at the whole-organ level. Examination of liver, testis, and cerebellum suggest, however, that the precise balance point of fusion and fission is cell type specific.

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Figures

Figure 1.
Figure 1.
Cardiomyopathy in Mffgt mice. (A) Survival curve. Mffgt mutant (mut) mice (n = 22) die by 13 wk on average, as opposed to wild-type controls (n = 14). (B) Increased size, dilation of chambers, and thinning of walls in mutant hearts. (C) Heart mass normalized to tibia length. (D) Masson’s trichrome stain of heart sections. Arrowheads indicate fibroblasts. Blue strands (arrows) denote collagen deposition. (E) TUNEL staining of heart sections. Arrows indicate apoptotic nuclei. (F) Quantification of apoptotic nuclei per field (n ≥ 11). (G) Heart rate (n ≥ 6). (H) M-mode echocardiogram of 13–14-wk-old mice. Electrocardiogram is superimposed. (I and J) Longitudinal echocardiography measuring fractional shortening (I) and ejection fraction (J). Study conducted on 5 wild-type and 10 Mffgt mice (five mutants died during the course of the study). Error bars = SEM. *, P ≤ 0.01; **, P ≤ 0.001. Bars, 50 µm. WT, wild type.
Figure 2.
Figure 2.
Mitochondrial function and structure in Mffgt hearts. (A) Respiratory control ratio (ADP stimulated/endogenous) of isolated heart mitochondria. A significant decrease is observed at 6 wk (n = 2 samples; two mice/sample) and ≥10 wk (three mutants; five wild types), but not at 4 wk (n = 3 samples; two mice/sample). (B) ATP levels in heart (n = 6). (C) Histochemical stains of mitochondrial respiration complex activities in hearts. Bar, 50 µm. (D) EM of cardiomyocytes. Bars, 10 µm. (E) Mean mitochondrial length. Error bars = SEM. ^, P ≤ 0.05; *, P ≤ 0.01. WT, wild type.
Figure 3.
Figure 3.
Rescue of Mffgt phenotypes by deletion of Mfn1. (A) Survival curve demonstrating extension of life with loss of Mfn alleles (n ≥ 14). (B) Echocardiography of different genotypes (n = 3–8). (C) Masson's trichrome stained heart. Bar, 50 µm. (D) Normalized oxygen consumption rates (OCR) of isolated heart mitochondria (≥10 wk; n = 3–5). (E) Respiratory control ratios (n = 3–5). (F) ATP levels in heart (n = 6). (G) Normalized oxidized glutathione levels of 13–14-wk-old hearts (n = 5). (H) Normalized oxidized/reduced glutathione ratio. Increased level in mutants (n = 8) over the wild type (n = 7) is abolished in dm hearts (n = 3). (I and J) Ubiquitin protein blots of 4- (I) and 13–14- (J) wk-old heart mitochondria. Error bars = SEM. P-values reflect comparisons to the wild type unless otherwise noted: ^, P ≤ 0.05; *, P ≤ 0.01; **, P ≤ 0.001. Ant A, complex III inhibitor; CCCP, uncoupler; EF, ejection fraction; Endo, endogenous; FS, fractional shortening; Oligo, ATPase inhibitor; WT, wild type.
Figure 4.
Figure 4.
Loss of mitochondria in Mffgt myocardium. (A) Low-magnification EM showing differences in mitochondrial mass. Bars, 5 µm. (B) Quantification of mitochondrial area in EM images. (C) HSP60/actin protein ratio, normalized to wild type (n ≥ 7). (D) mtDNA per cell, normalized to wild-type content (n ≥ 7). (E) EM of mutant myocardium, showing examples of defective mitochondria. Arrow indicates mitophagic structure. Bars, 0.5 µm. (F) Heart sections immunostained with LC3 or p62 (red) and HSP60 (green). Arrows indicate positive puncta. Mutant inset is enlarged. Bars: 10 µm; (mutant inset) 50 µm. Error bars = SEM. P-values as compared with wild type: ^, P ≤ 0.05; *, P ≤ 0.01; **, P ≤ 0.001. nDNA, nuclear DNA; WT, wild type.
Figure 5.
Figure 5.
Mffgt and dm phenotypes in liver, testis, and cerebellum. (A) Comparison of liver to body mass ratio (n ≥ 10). (B) ALT levels in serum (n = 5–13). (C and E) Testis to body mass ratio in Mffgt and dm (C; n ≥ 5) and Stra8-Cre lines (E; n ≥ 6). (D and F) Hematoxylin and eosin stain of testicular cross sections in 8–9-wk-old Mffgt and dm (D) and Stra8-Cre lines (F). Note that only control and Mffgt sections contain spermatozoa (arrows). (G) Anticalbindin immunofluorescence illuminating cerebellar PC. Error bars = SEM. P-values as compared with the wild type: ^, P ≤ 0.05; *, P ≤ 0.01; **, P ≤ 0.001. Bars, 50 µm. WT, wild type.
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