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. 2016 Jan:90:53-8.
doi: 10.1016/j.yjmcc.2015.11.032. Epub 2015 Dec 2.

α-MHC MitoTimer mouse: In vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart

Aleksandr Stotland  1 , Roberta A Gottlieb  2
Affiliations

α-MHC MitoTimer mouse: In vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart

Aleksandr Stotland et al. J Mol Cell Cardiol. 2016 Jan.

Abstract

In order to maintain an efficient, energy-producing network in the heart, dysfunctional mitochondria are cleared through the mechanism of autophagy, which is closely linked with mitochondrial biogenesis; these, together with fusion and fission comprise a crucial process known as mitochondrial turnover. Until recently, the lack of molecular tools and methods available to researchers has impeded in vivo investigations of turnover. To investigate the process at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein characterized by transition from green fluorescence to a more stable red conformation over 48 h, and its rate of maturation is stable under physiological conditions. We fused the Timer cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of a cardiac-restricted promoter. This construct was used to create the alpha-MHC-MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice demonstrated a high degree of heterogeneity in the ratio of red-to-green fluorescence of MitoTimer in cardiac tissue. Further, scattered solitary mitochondria within cardiomyocytes display a much higher red-to-green fluorescence (red-shifted) relative to other mitochondria in the cell, implying a block in import of newly synthesized MitoTimer likely due to lower membrane potential. These red-shifted mitochondria may represent older, senescent mitochondria. Concurrently, the cardiomyocytes also contain a subpopulation of mitochondria that display a lower red-to-green fluorescence (green-shifted) relative to other mitochondria, indicative of germinal mitochondria that are actively engaged in import of newly-synthesized mito-targeted proteins. These mitochondria can be isolated and sorted from the heart by flow cytometry for further analysis. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.

Keywords: Cardiac mitochondria; MitoTimer protein; Mitochondrial biogenesis; Mitochondrial turnover; Mitophagy; Mouse model.

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Figures

Fig. 1
Fig. 1
Constitutive expression of MitoTimer. A) Merged fluorescence microscopy (CFI Plan Apoλ 60xH) images of C2C12 myoblasts expressing MitoTimer. Smaller insets show the green and red channels. Scale bar: 20 μm. B) Fluorescence microscopy (CFI Plan Apoλ 60xH) merged image of MitoTimer C2C12 myoblasts following a 24 h pulse with 10 μM FCCP. Mitochondria engaged in new protein import (green) and mitochondria incapable of importing new protein (red) can be readily observed (white arrows). Scale bar: 5 μm. C) Flow cytometry histogram of derived red-to-green fluorescence of C2C12 MitoTimer cells treated with 10 μM FCCP. D) Flow cytometry histogram of derived red-to-green fluorescence ratio of C2C12 MitoTimer cells treated with 10 μM DECA. Control cells are in gray. Note the red-to-green ratio returning to baseline as mitochondria in cells begin to import newly-synthesized MitoTimer as they regain membrane potential (FCCP) or resume protein import (DECA). E) FAOS dot plots of isolated mitochondria from C2C12 MitoTimer cells 24 h after FCCP addition. The gate denotes newly synthesized green-fluorescent mitochondria. Derived histograms of the data from demonstrate the shift in the fluorescence ratio as the mitochondrial population is depolarized and allowed to recover. Orange number represents the % of mitochondria falling within the normal distribution (95%), while the green number represents the % of mitochondria that are green outliers and the dark red number the % of mitochondria that are red-shifted outliers. At 24 h, the red-shift is apparent, but a substantial increase in the green population is also present. At 48 h the mitochondrial population is returning to equilibrium. F) FAOS dot plot shows mitochondria before (left) or after DAPI staining (right). Fi) Mitochondria that display a lower red:green fluorescence ratio have more DAPI binding (=more mtDNA, right panel, n = 3209, 6.61% of population, MFI PE/FITC = 0.88) than the red mitochondrial population (n = 2589, 4.43% of population, MFI PE/FITC = 5.33). Fii) The black ROI indicates DAPI-stained mitochondria (DAPI Low, n = 44,697, 61.75% of total mitochondria), and the red ROI indicates the brightest population (DAPI High, n = 3170, 6.53% of total mitochondria). (Indo-1 Violet channel used for DAPI) G) Similarly, mitochondria from cells incubated overnight with EdU (right panel) and without EdU (left panel) were subjected to Click Chemistry with Pacific Blue. The black ROI shows the subpopulation of mitochondria that incorporated EdU. Gi) Mitochondria that display a low red:green fluorescence ratio have more EdU incorporation (=more newly synthesized mtDNA, right panel, n = 1099, 2.24% of population, MFI PE/FITC = 0.88) than the mitochondrial population with a high red:green fluorescence (n = 3466, 7.06% of population, MFI PE/FITC = 2.73). Gii) Far-right graph demonstrates the red:green ratio for the Edu + (n = 669, 0.96% of total mitochondria) and EdU-gated subpopulations (n = 44,696), showing that EdU incorporation is higher in mitochondria that are more green-fluorescent. (Pacific Blue channel used for EdU detection) H) Dot plot shows MFN2 immunostaining of mitochondria (no Mfn2 antibody, secondary only, left; MFN2 immunostaining, right). The black ROI indicates Mfn2-positive mitochondria. Hi) MFN2 staining is highest in mitochondria that are mostly green fluorescent (n = 810, 7.52% of population, MFI PE/FITC = 0.60) compared to the mostly-red fluorescent mitochondria ((n = 711, 4.18% of population, MFI PE/FITC = 0.4.87). (Indo-1 Violet channel used to detect Alexa Fluor 350). Hii) MFN2-High (n = 430, 5.83% of the MFN2+ mitochondria) population displays a lower red:green fluorescence compared to MFN2-Low population (n = 6682, 90.8% of MFN2+ mitochondria). All FACS/FAOS experiments presented are plots and statistics from a representative experiment from multiple repeats. Statistical analysis performed from n = 50,000 events (cells or mitochondria). Unpaired t-test used for calculation of significance.
Fig. 2
Fig. 2
α-MHC MitoTimer Mouse. A) Fluorescence microscopy images (CFI Plan Apoλ 4X, image merge) of α-MHC MitoTimer mouse heart transverse and coronal sections. Merge of red, green, and blue (DAPI) channels; smaller insets show the individual channels. Scale bar: 1 mm. B) Cardiac tissue, confocal microscopy merged images of green and red channels is shown. Scale bar: 25 μm. C) Merged fluorescence microscopy images (CFI Plan Apoλ 40X) of green, red and blue (DAPI) channels. Scale bar: 25 μm. Arrows point to solitary red mitochondria in cardiomyocytes. D) Dot plot of FSC (X-axis) vs derived parameter (MitoTimer red:green ratio, PE/FITC) and derived parameter histogram of mitochondria isolated from α-MHC-MitoTimer mouse cardiomyocytes. Subpopulations of mostly-green and mostly-red mitochondria are readily detected. E) Fluorescence microscopy images α-MHC-MitoTimer mouse cardiac tissue immunostained for LC3 (Blue channel, Alexa-Fluor 350 secondary antibody). Arrow points to a solitary red mitochondrion surrounded by LC3. Scale bar: 20 μm. F) Fluorescence microscopy merged images of MitoTimer mouse hearts of littermates (n = 1) at 3 woa (left) and 16 woa (right). G) Derived parameter histogram overlay of the isolated mitochondria from the 3 woa and 16 woa mouse hearts. Statistical analysis performed from n = 50,000 events (mitochondria from each heart). Unpaired t-test used for calculation of significance. Scale bar: 1 mm.
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