Power Grid Protection of the Muscle Mitochondrial Reticulum

Abstract

Mitochondrial network connectivity enables rapid communication and distribution of potential energy throughout the cell. However, this connectivity puts the energy conversion system at risk, because damaged elements could jeopardize the entire network. Here, we demonstrate the mechanisms for mitochondrial network protection in heart and skeletal muscle (SKM). We find that the cardiac mitochondrial reticulum is segmented into subnetworks comprising many mitochondria linked through abundant contact sites at highly specific intermitochondrial junctions (IMJs). In both cardiac and SKM subnetworks, a rapid electrical and physical separation of malfunctioning mitochondria occurs, consistent with detachment of IMJs and retraction of elongated mitochondria into condensed structures. Regional mitochondrial subnetworks limit the cellular impact of local dysfunction while the dynamic disconnection of damaged mitochondria allows the remaining mitochondria to resume normal function within seconds. Thus, mitochondrial network security is comprised of both proactive and reactive mechanisms in striated muscle cells.

Keywords: 3D electron microscopy; energy distribution; mitochondrial dynamics; mitochondrial retraction; muscle energetics; oxidative phosphorylation.

Figure 1. Heart Mitochondria Form Networks

A) Single FIB-SEM frame. M – mitochondria, C – contractile proteins, L – lipid droplets, yellow arrows - intermitochondrial junctions (IMJ). B) 3D rendering of cardiomyocyte (upper cell in A). C) 3D rendering of paranuclear mitochondria (PNM) and nucleus (N). D) 3D rendering of paravascular mitochondria (PVM) and a capillary (V). E) 3D rendering of fiber parallel mitochondria (FPM) and lipid droplets (L). Represents 5 volumes, 4 animals. See Movie S1 and Dataset S1.

Figure 2. Heart Mitochondrial Morphology and Connectivity

A) Single IMJ (yellow arrows) tomogram slice. Dashed box shown in B. B) Sequential tomograms of membrane to membrane contact site (dotted box). C–E) Mitochondrial morphologies (green) and locations of IMJ (red). C) Compact (upper left) and Elongated (middle and right) PNM. D) Elongated, Nanotube (yellow arrow), and Compact PVM (left to right). E) Connector (left), Compact (right upper), Elongated (right middle), and Non-connected (right lower) FPM. Yellow arrow – sheet-like connecting structure. F) Adjacent FPM (assorted colors) and lipid droplets (white). G) FIB-SEM frame. Mitochondria in the same color are IMJ-coupled. H) 3D rendering of mitochondrial subnetworks. Each color represents a different IMJ-coupled subnetwork. Grey - not network connected. Represents 5 volumes, 4 animals. See Table S1 and Movie S1.

Figure 3. Membrane Potential Conduction

TMRM and MPD loaded cardiomyocyte prior to (A) and after (B) UV irradiation. White dashed lines - UV irradiation zone. Yellow dotted lines - size of FIB-SEM image in Figure 1A. C) Ratio TMRM image before and after irradiation. Black pixels – decreasing signal. White pixels – increasing signal. White dashed lines – irradiation zone for Longitudinal coupling. Represents 9 experiments, 6 mice. D) Ratio TMRM image for Vertical coupling. Represents 8 experiments, 4 mice. E) Ratio TMRM image without MPD (Control). Represents 9 experiments, 5 mice. F) Mitochondrial depolarization extends further along longitudinal axis. See Movie S2 and Figures S1,3.

Figure 4. Mitochondrial Circuit Breaker

TMRM fluorescence before (A), immediately after (B), and 75 seconds after (C) cardiomyocyte regional uncoupling. D) Mitochondrial TMRM timecourse of irradiated (dotted blue) and non-irradiated, depolarized (solid red) regions. Data = mean ± SE. E) Ratio TMRM image before and immediately after irradiation. F) Ratio TMRM image before and 75 sec after UV. Represent 7 experiments, 3 mice. G–I) TMRM fluorescence during muscle fiber regional uncoupling. G) Pre UV. H) Immediately after UV. I) 55 seconds after UV. J) Representative TMRM fast fourier transform (FFT) 0.5 μm⁻¹ frequency amplitude timecourse of irradiated (dotted blue) and non-irradiated (solid red) regions. K) Ratio TMRM image before and immediately after irradiation. L) Ratio TMRM image before and 55 sec after UV. Represents 5 experiments, 5 mice. See Movies S3.

Figure 5. Mitochondrial Network Disconnection

MitoDendra2 fluorescence prior to (A), immediately after (B), and 120 seconds after (C) UV irradiation of muscle fiber w/MPD. D) Timecourse of network disconnectivity. Mean±SE, n=6 fibers, 3 mice. E) 3D rendering after regional uncoupling. F) Timeseries of muscle fiber PVM MitoDendra2 fluorescence after regional uncoupling. G) Number of PVM immediately and 2 minutes after regional uncoupling (n=3). H) Area per PVM immediately and 2 minutes after regional uncoupling (n=3). I,J) 3D schematic of PVM fusion (I) or consolidation (J). K) Timeseries of intrafibrillar MitoDendra2 fluorescence after regional uncoupling. L) Boxed region from K shows retraction of I-band mitochondrial segments (IBS) into fiber parallel segments (FPS). M) Mitochondrial diameters immediately after and 2 minutes after irradiation (n= 16 mitochondria, 3 experiments). N) 3D schematic of mitochondrial retraction. Structures from (Glancy et al., 2015). Represents 6 fibers, 3 mice. *significantly different from 4 sec after UV. See Figure S2 and Movies S4,5.