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. 2015 Oct 23;290(43):25907-19.
doi: 10.1074/jbc.M115.665695. Epub 2015 Sep 14.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure

Thomas J Cahill  1 , Vincenzo Leo  2 , Matthew Kelly  1 , Alexander Stockenhuber  1 , Nolan W Kennedy  3 , Leyuan Bao  4 , Grazia M Cereghetti  5 , Andrew R Harper  1 , Gabor Czibik  1 , Chunyan Liao  6 , Mohamed Bellahcene  1 , Violetta Steeples  1 , Safar Ghaffari  1 , Arash Yavari  1 , Alice Mayer  4 , Joanna Poulton  6 , David J P Ferguson  7 , Luca Scorrano  5 , Nishani T Hettiarachchi  8 , Chris Peers  8 , John Boyle  8 , R Blake Hill  3 , Alison Simmons  4 , Hugh Watkins  1 , T Neil Dear  2 , Houman Ashrafian  9
Affiliations

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure

Thomas J Cahill et al. J Biol Chem. .

Erratum in

Abstract

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Keywords: cardiomyopathy; heart failure; mitochondria; mitochondrial metabolism; mitophagy.

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Figures

FIGURE 1.
FIGURE 1.
A, structure of the Drp1 protein. The C452F mutation identified in the Python mouse model of DCM lies in close proximity to the reported human A395D (equivalent to mouse A401D) mutation in the middle domain. VD, variable domain; GED, GTPase effector domain. B, the Drp1 C452F mutation stimulates GTP hydrolysis compared with wild-type Drp1 and the assembly-deficient mutation Drp1 A401D. C, the C452F mutation impairs the NaCl dependence of GTP hydrolysis observed with wild type Drp1. D, assembly was measured via absorbance at 450 nm in reactions containing 20 μm Drp1 WT, 10 μm Drp1 A401D, or 10 μm Drp1 C452F. Reactions were started by the addition of 1 mm GTP. After 20 min of reaction time, buffers that altered the salt concentration of the reaction were added to measure the overall effect on assembly at final [NaCl] of 90 mm, 150 mm, and 300 mm. Representative data at [NaCl]final = 300 mm. E, light-scattering at varying [NaCl], showing a persistently high signal from the C452F mutant compared with the WT and A401D, consistent with failure of disassembly. F, to visualize the distribution of Drp1 C452F, Drp1−/− MEFs were transfected with a YFP-tagged Drp1 WT or Drp1 C45F construct. Drp1 C452F is seen in cytosolic clumps, consistent with increased assembly, in contrast to the diffuse cytosolic pattern of Drp1 WT.
FIGURE 2.
FIGURE 2.
A, representative confocal microscopy images of Drp1 C452F and Drp1 WT MEFs after transfection with mitochondrial red fluorescent protein to show mitochondria. C452F leads to an elongated, tubular, and interconnected mitochondrial morphological phenotype. Cell population analysis of mitochondrial morphology confirms a significant shift to an elongated mitochondrial morphology (50 cells/experiment; n = 3; ***, p < 0.001). B, Western blots of Drp1 C452F and WT lysates for key mitochondrial fusion proteins. Levels of Mfn1 and Mfn2 were unchanged. Levels of Opa1 were increased mildly (three independent experiments). C, Drp1 C452F and WT MEFs were cotransfected with mito-DsRed and mito-PAGFP for imaging the functional connectivity of the mitochondrial network. After activation of GFP in randomly selected regions of interest (circled), the spread of the GFP signal through the highly interconnected mitochondrial network of Drp1 C452F MEFs was evident by 10 min. This was associated with a drop in GFP fluorescence intensity in the original region of interest. In contrast, in Drp1 WT MEFs, the reduction in GFP signal in the region of interest was significantly less, indicating reduced connectivity (n = >10 cells/group, with 1–2 regions of interest/cell; *, p < 0.05). D, autophagosome LC3 and mitochondrial pyruvate dehydrogenase were stained by immunofluorescence and colocalization assessed on an IN Cell Analyzer 1000 automated epifluorescence microscope. Drp1 C452F and Drp1−/− MEFs displayed mitochondrial-autophagosome colocalization after inhibition of autophagosome degradation by bafilomycin (Baf), ***, p < 0.001. E, quantitative PCR analysis of mitochondrial MT-ND1 DNA normalized to nuclear NDUFV1 DNA in MEFs exposed to nutrient deprivation (culture in Hanks' balanced salt solution) for the time shown (n = 3; *, p < 0.05; ns, not significant). In WT but not C452F MEFs, nutrient deprivation results in a drop in mitochondrial DNA consistent with mitochondrial degradation by mitophagy. This decrease is not seen in the Drp1 C452F cells. F, after 4 h of nutrient deprivation, C452F MEFs form fewer mature, spatially separated autophagosomes, as counted by EM (20 cells/group, n = 3). G, large numbers of smaller autophagosomes are present in close apposition to mitochondria in Drp1 C452F cells, possibly consistent with stalled ingestion of mitochondria within autophagosomes (arrows). MV, multiple-membraned vesicles; G, Golgi body; Mi, mitochondrion; N, nucleus. Scale bars = 500 nm. H, representative EM image of MEFs after combined nutrient deprivation and 500 nm bafilomycin A1 treatment for 4 h, showing normal activation of generalized macroautophagy after supramaximal stimulation, with diffuse autophagosome formation in the cytosol. Scale bars = 2 μm. I, ATP concentration measured by firefly luciferase reporter luminescence. **, p < 0.01. J, mitochondrial membrane potential measured by JC1 fluorescence in Drp1 WT compared with C452F MEFs. ***, p < 0.001.
FIGURE 3.
FIGURE 3.
A, mitochondria were analyzed from the hearts of Python C57BL/6J mice after exposure to different conditions. Pyruvate, glutamate, and malate (Pyr/Mal/Glu) were added to initiate respiration. ADP and succinate are substrates of ATP synthase and complex II, respectively. FCCP uncouples the OXPHOS system, representing the maximum capacity of the electron transport chain. Rotenone and antimycin A are inhibitors of complexes I and III, respectively. General respiration begins to decline at 4 months of age. ATP synthase activity gradually declines from 3 months of age until onset of heart failure. Complexes I and II both show no alteration of OXPHOS activity until 5 months of age, when a dramatic reduction becomes evident for both complexes and remains the same until the onset of overt heart failure at 6 months. The maximum capacity of the electron transport chain also follows a dramatic decline from 5 months. Inhibition with antimycin A displays a gradual reduction from 3 months of age (*, p < 0.05, **, p < 0.01). B, mitochondrial OXPHOS activity in wild-type hearts. No significant decreases in O2 flux were observed between any of the time points measured.
FIGURE 4.
FIGURE 4.
A, representative traces showing [Ca2+]i in WT (top panel) and C452F (bottom panel) MEFs following emptying of ER stores with the reversible ER Ca2+ ATPase inhibitor CPA, followed by emptying of mitochondrial Ca2+ stores using FCCP in the presence of oligomycin (Oligo). r.u., relative units. B, results (mean ± S.E.) for the peak response to CPA and the peak response to FCCP and oligomycin in WT and C452F MEFs (n = 10/group). Ab units, absorbance units. *, p < 0.05; ns, not significant. C, representative traces and area under the curve (AUC) of mitochondrial Ca2+ uptake in MEFs (measured by mitochondrially targeted aequorin) after ATP-evoked ER Ca2+ release (n = 2/group). *, p < 0.05. D, representative traces of [Ca2+]i during ATP-evoked Ca2+ release from ER stores, followed by capacitative calcium entry in WT and C452F MEFs. 100 μm ATP was applied for the duration of the black bar to evoke Ca2+ release from the ER prior to reintroducing Ca2+ (2.5 mm) in the perfusate to determine the capacitative calcium entry. E, results (mean ± S.E.) for resting Ca2+-containing baseline, the peak response to ATP, the integral of the response (AUC), and the t½ in WT and C452F MEFs (n = 12 recordings for WT and 11 for C452F). ***, p < 0.01.
FIGURE 5.
FIGURE 5.
A, quantitative PCR of inflammatory cytokines in young (age, 50 days) Python mice prior to the development of left ventricular dysfunction (n = 10/group). B, inflammatory cytokines in old (age, 150 days) Python mice, which display severe left ventricular dysfunction, compared with age-matched littermate WT mice and age-matched mice with left ventricular failure induced by transverse aortic constriction (TAC) mice (n = 9–11/group). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.
FIGURE 6.
FIGURE 6.
A, Western blot analysis for LC3 in Python cardiac samples demonstrating activation of macroautophagy by an isocaloric LPD. B and C, overall body weight and ratio of heart weight to tibia length was significantly lower in the Python group on LPD (n = >5/group) compared with Python mice on a normal chow diet. ***, p < 0.01 Python (Py)/LPD versus Py/chow. D, representative M mode echocardiography images performed at an age of 150 days, showing improved cardiac function. E, LPD decreased left ventricular end-diastolic dimension (LVEDD). ***, p < 0.01 Py/LPD versus Py/chow. F, improved ejection fraction (EF). ***, p < 0.01 Py/LPD versus Py/chow. G, the overall mitochondrial:nuclear DNA ratio was unchanged by LPD, suggesting that intact mitochondrial DNA is not the primary driver of cardiomyopathy.
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References

    1. Cahill T. J., Ashrafian H., and Watkins H. (2013) Genetic cardiomyopathies causing heart failure. Circ. Res. 113, 660–675 - PubMed
    1. Neubauer S. (2007) The failing heart: an engine out of fuel. N. Engl. J. Med. 356, 1140–1151 - PubMed
    1. Ashrafian H., Docherty L., Leo V., Towlson C., Neilan M., Steeples V., Lygate C. A., Hough T., Townsend S., Williams D., Wells S., Norris D., Glyn-Jones S., Land J., Barbaric I., Lalanne Z., Denny P., Szumska D., Bhattacharya S., Griffin J. L., Hargreaves I., Fernandez-Fuentes N., Cheeseman M., Watkins H., and Dear T. N. (2010) A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy. PLoS Genet. 6, e1001000. - PMC - PubMed
    1. Youle R. J., and van der Bliek A. M. (2012) Mitochondrial fission, fusion, and stress. Science 337, 1062–1065 - PMC - PubMed
    1. Otera H., Ishihara N., and Mihara K. (2013) New insights into the function and regulation of mitochondrial fission. Biochim. Biophys. Acta 1833, 1256–1268 - PubMed

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