Intrafibrillar and perinuclear mitochondrial heterogeneity in adult cardiac myocytes

Abstract

Mitochondria are involved in multiple cellular functions, in addition to their core role in energy metabolism. Mitochondria localized in different cellular locations may have different morphology, Ca²⁺ handling and biochemical properties and may interact differently with other intracellular structures, causing functional specificity. However, most prior studies have utilized isolated mitochondria, removed from their intracellular environment. Mitochondria in cardiac ventricular myocytes are highly organized, with a majority squeezed between the myofilaments in longitudinal chains (intrafibrillar mitochondria, IFM). There is another population of perinuclear mitochondria (PNM) around and between the two nuclei typical in myocytes. Here, we take advantage of live myocyte imaging to test for quantitative morphological and functional differences between IFM and PNM with respect to calcium fluxes, membrane potential, sensitivity to oxidative stress, shape and dynamics. Our findings show higher mitochondrial Ca²⁺ uptake and oxidative stress sensitivity for IFM vs. PNM, which may relate to higher local energy demand supporting the contractile machinery. In contrast to IFM which are remarkably static, PNM are relatively mobile, appear to participate readily in fission/fusion dynamics and appear to play a central role in mitochondrial genesis and turnover. We conclude that while IFM may be physiologically tuned to support local myofilament energy demands, PNM may be more critical in mitochondrial turnover and regulation of nuclear function and import/export. Thus, important functional differences are present in intrafibrillar vs. perinuclear mitochondrial subpopulations.

Keywords: Mitochondrial Ca; Mitochondrial dynamic; Mitochondrial heterogeneity.

Figure 1. Perinuclear and intermyofibrillar mitochondrial morphology and Ca²⁺ uptake in intact cardiac myocytes.

(A) Electron microscopy images of sections through left ventricular rabbit heart. Intermyofibrillar mitochondria (IF) are often a rounded brick-shape (left), and perinuclear mitochondria (PN) are more round and loosely arranged (right). (B) Ad-Mitycam indicates mitochondria (and [Ca]_mito), Di-8-ANEPPS indicates T-tubule (red) and Hoechst 33342 indicates the nucleus. Enlarged images from the indicated myocyte regions in B (scale bar 10 μm). (C) Kinetics of [Ca]_mito during 1 Hz pacing frequency in adult rabbit cardiac myocytes, mean initial [Ca]_mito uptake rate (D), Δ[Ca]_mito amplitude (E) and decline rate[Ca]_mito when pacing stopped (F; n=6 cells). (G,H) Mitochondrial membrane potential Δψ_m after 1 Hz pacing, normalized to the maximal value with oligomycin (n=6 cells). Kinetics of [Ca]_mito change during Ca²⁺-clamp in permeablized (and SR disabled) cardiac myoyctes (I), mean Δ[Ca]_mito (K), initial uptake rate (J) and [Ca]_mito decline rate (L; n=6 cells). M,N ψ_m was normalized to the initial value, and mPTP opening time induced by phenylarsine oxide (PAO; n=4 cells) was estimated as the time when a regression line for the first 20 points of sustained ψ_m decay intersect the baseline.

Figure 2. Motility of individual mitochondrion in adult cardiac myocytes.

(A) Right: expression of mitochondrial indicator Ad-mtGFP in adult cardiac myocytes (scale bar: 10 μm), (B) Enlarged images from the indicated myocytes regions in A (IF: lower ROI and PN: upper ROI) at different time points (scale bar: 4 μm). (C) Distance moved by individual mitochondria indicated in B (IFM: mito2 and PNM: mito1) Mito1 (labeled with white *) moved along the nuclear edge indicated by dash lines and away from the neighbor mitochondrion Mito3 labeled with yellow *. (D) Percent of cells (N, left)) and mitochondria (n, right) exhibiting movement and (E) distribution of distances moved by individual mitochondrion moved in PNM and IFM (n= 7 cells).

Figure 3. Visualization of mitochondrial interaction in adult cardiac myocytes.

(A) EM images showed mitochondrial shapes (scale bars: 500 nm). (B) Two mitochondria move towards each other along the path indicated by dash lines. Right: position and distance of mitochondrion center indicated in left image at different time points. (C) mother mitochondrion separates into two daughter mitochondria. Right: position and distance of mitochondrion indicated in left image at different time points. (D) Transient mitochondrial fusion (scale bars: 2μm). (E) Percent of mitochondria exhibiting dynamic interaction.

Figure 4. IMM Δψ_m during mitochondrial separation.

(A) Cardiac myocytes expressing Ad-mitoGFP (green) and mitochondrial membrane potential sensor TMRM (red) (scale bar: 10 μm). Right: enlarged images from the indicated myocytes region in left images at different time points (scale bar: 2 μm). Fluorescence of mitoGFP (B),TMRM (C) and the TMRM/ mitoGFP ratio (D) of mito1 and mito2 (in A) as a function of time.

Figure 5. PNM have faster fission/fusion rates than IFM.

(A, D) Confocal images of cardiomyocytes transfected with Ad-DsRed before and after photobleaching (PB) the ROIs at 540 nm (scale bar: 10 μm, scale bar of enlarged images: 2 μm). (B, C) FRAP time course and rate for PN and IF mitochondria. (D,E) Exemplar ROI where an existing mitochondrion appears to enter the ROI during FRAP period. (F-H) FRAP of DsRed signal after photobleaching with and without Drp1 inhibition by Mdivi-1 (n=7 cells).

Figure 6. Visualization of intermitochondrial communication in adult mouse cardiac myocytes.

(A) Confocal images of cardiomyocytes transfected with Ad-mtPAFGP (Green) and stained with TMRM (Red) indicating individual mitochondrion, at different time points after photoactivation of indicated ROIs. Right: Enlarged PNM region indicated in left images. (B) Enlarged IFM regions indicated in (A) (scale bars: 4 μm). Time courses of mtPA-GFP fluorescence signal of PN and IF mitochondria over time. Individual mitochondria were numbered in A and B as indicated to identify fluorescence changes in each mitochondrion (C).

Figure 7. Perinuclear region is highly active site for starvation-induced mitochondrial turnover.

(A) Confocal images of mitochondrial GFP expression in cardiomyocytes at 15 and 36 hrs after transfecting Ad-mitoGFP. (B) EM image, (C) NBD Golgi staining in live cells indicated that Golgi is enriched around nuclear (indicated by DRAQ5, violet). (D) Confocal images of cardiomyocytes transfected with Ad-mtPA-GFPafter photoactivating ROIs (green) at the cell periphery and stained with LysoTracker red to indicate lysosomes (Red), lower panels are enlarged images from the indicated myocytes region in upper panels. (E) Pearson’s correlation coefficient of mtPA-GFP and LysoTracker signal.

Figure 8. Inhibition of mitochondrial trafficking to the lysosome at the perinuclear region.

Cardiac mycoytes stained with LysoTracker green and expressing mtDsRed before (A) and after (B) applying 100 nM FCCP for 6 hr. (C) Ratio of DsRed signal (PNM/IFM) with and without microtubule disruption (Nocodazole) during FCCP treatment. (D) Pearson’ correlation coefficient of mito-DsRed and LysoTracker green with and without Nocodazole during FCCP treatment (n=6–12 cells).