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. 2023 May 26;132(11):e171-e187.
doi: 10.1161/CIRCRESAHA.122.321833. Epub 2023 Apr 14.

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling

Zuzana Nichtová  1 , Celia Fernandez-Sanz #  2 , Sergio De La Fuente #  2 , Yuexing Yuan  2 , Stephen Hurst  1 , Sebastian Lanvermann  2 , Hui-Ying Tsai  2 , David Weaver  1 , Ariele Baggett  1 , Christopher Thompson  3 , Cedric Bouchet-Marquis  3 , Péter Várnai  4 , Erin L Seifert  1 , Gerald W Dorn 2nd  5 , Shey-Shing Sheu  2 , György Csordás  1
Affiliations

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling

Zuzana Nichtová et al. Circ Res. .

Abstract

Background: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering.

Methods: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype.

Results: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive β-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury.

Conclusions: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.

Keywords: ischemia; mitochondria; muscle cells; myocardium; reperfusion; sarcoplasmic reticulum; transgenes.

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Conflict of interest statement

Disclosures None.

Figures

Figure 1:
Figure 1:. Mitochondria-SR/ER linker expression and basal functional phenotype in the linker-mouse hearts.
A. Schematic of the engineered tether ('linker') between the OMM and ER/SR. Helical spacers (18x, 9x repeats) keep the membrane gap distance <20 nm. B. Immunoblots of mRFP in heart lysates of control, linker- and Doxy-linker-mice. RFP is only expressed in the linker hearts. VDAC serves as loading control. C–L. Basal echocardiography parameters for male (♂) and female (♀) control (Ctr, black), linker (Lnk, red) and Doxy-linker (Lnk/Doxy+, cyan) mice. M. Body weights. Doxycycline administration is indicated under the graphs. Data are normalized to Ctr in each sex. See absolute values in Figure S2. Means±SEM. N is indicated bar wise. Statistics as shown in gray box.
Figure 2:
Figure 2:. Mitochondrial and SR remodeling in the linker myocardium.
A. Representative TEM montages of a control (Ctr) and a linker-expressing (Lnk) cardiomyocyte (yellow shade) in perfusion-fixed papillary muscles. Note the large mitochondrial (cyan shade) clusters in Lnk that can reach from the perinuclear area to the intercalated disc. Nu, nucleus; m, mitochondria; LD, lipid droplet. B. Mitochondrial densities (mitochondria-occupied area of the sarcoplasm). n=14 myocytes each from N=4 Lnk and 4 Ctr hearts; 3-4 myocytes/heart. Means±SEs. P values from mixed-effects regression/Fisher’s LSD. C. Relative expression of matrix protein mtHsp70, mitochondrial biogenesis regulator Pgc1α, jSR-resident calsequestrin (Csq), nSR/ER-resident calreticulin (Crt) and SERCA2a. Pooled data from VCM lysates (N=4 hearts each). Band densities were normalized first to the total protein in the lane (from Ponceau-S gel image), then to the mean of the respective Ctr. Means±SEs. P was calculated for each protein by Mann-Whiney Rank Sum test. See all p values in Online Dataset S1.
Figure 3:
Figure 3:. Mitochondria-jSR contacts are individually enhanced but less frequent in the linker heart.
A-H. TEM analyses of mitochondria-jSR contacts in longitudinal sections of LV papillary muscles. A. Images of typical mitochondrion-jSR contacts occurring at Z-lines from a control (Ctr) and a linker (Lnk) myocyte. Shades: Mitochondrion-yellow, jSR-red, transversal tubule (TT)-blue, nSR-purple. L,T indicate longitudinal and transversal sides of the IFM (T-side faces jSR in the canonical orientation). Zoomed image: T-side traced for the contact length normalization. B. Mitochondria-jSR contact analysis using the MitoCare Tools ImageJ/Fiji plugin; here, instead of surface segments of the whole mitochondrion (Mito), T-side segments are binned. C,D. Gap distance distribution of mitochondrial T-side segments at <50 nm proximity of jSR. C: cumulative (10 nm) binning. D: exclusive, 0-20nm and 20-50 nm distance bins (larger T-side portion in the 0-20nm bin indicate tighter contact in the Lnk). E. Minimum OMM-jSR distances in the individual mitochondria-jSR contacts. F. Mean perimeter of the mitochondria forming jSR contact. G. Lengths of the mitochondrial T-sides involved in contact formation. H. Prevalence of mitochondria-jSR contacts per sarcoplasmic area per cell. For all, n=14 (Ctr) and 17 (Lnk) whole myocyte cross-sectional areas from N=4 Ctr and 4 Lnk hearts. 3-5 cells/heart with 8-198 (median 37) contacts/cell). Bars represent Means±SEs. P values were obtained as described in the gray text box. I. Exemplar immunofluorescence image of RyR2 (i, green, 488 nm) and its overlay with mRFP (ii, green/red respective components) in a linker-VCM. RyR2 leaves large ‘empty areas’ (e.g. the white dashed contour), which are filled with tightly packed mRFP-decorated mitochondrial clusters. 2x zoom: packed rings of linker mRFP, contouring the mitochondria and lacking green/RyR2 fluorescence.
Figure 4.
Figure 4.. Assessment of EBC, metabolic fitness and Ca2+ signaling in the linker myocytes.
A-C. NAD(P)H and FAD autofluorescence imaging in VCMs freshly isolated from Ctr and Lnk. Isoproterenol 100 nM was added to the superfusate to provide sympathetic tone. After baseline recording, electric field stimulation (FS) at increasing frequencies (1-2-5 Hz) was applied then stopped (ns), followed by sequential additions of caffeine (Caf 10 mM), NaCN (CN 40 μM), and antimycin A+rotenone (A+R, 10 μM/0.25 μM). (A) NAD(P)H autofluorescence at baseline (basal), after respiratory complex IV (NaCN) and additional complex I&III inhibition (A+R). Means±S.E., N=8 hearts each. B. Representative timecourses of NAD(P)H/FAD ratio from individual Ctrl and Lnk myocytes (n>170 each). C. Cumulated corrected NAD(P)H/FAD ratio levels at the indicated steps of the stimulation protocol, collected from corresponding timecourse trace segments. Before forming the ratio, minimum FAD fluorescence at NaCN or A+R was subtracted to correct for mRFP crosstalk (see details in Expanded Methods). Means±SEs, n=7 independent experiments (one Ctr and Lnk heart each; 4-8 technical replicates/coverslips; 2-8 VCMs/field). D-I. [Ca2+]c (fura-2 ratio) responses of primary VCMs to sequential FS periods at 1Hz, 5Hz, 1Hz, each followed by a short (~30s) pause. After the last pause, caffeine (caff, 10 mM) is added to discharge SR Ca2+. Isoproterenol 100 nM is present throughout to provide sympathetic tone. D. Representative timecourses from a Ctr and Lnk (red) VCM (n(cells)=39 for Ctr and 53 for Lnk). Boxed segments in (i-ii) are overlaid on expanded timescale in (iii). E-I. Cumulated parameters extracted from the timecourses. For each experiment (pair of hearts), individual cells were normalized to the mean of the control (see Figure S5E-G for values without normalization to Ctr). E. Basal Fura-2 ratios. F. Diastolic [Ca2+]c at the indicated FS frequencies. G. [Ca2+]c spike amplitudes (systolic-diastolic difference) during FS. E-G: N=4 pairs of hearts; 2-6 coverslips each (2-7 VCM/field). Means+s.e. P was calculated using mixed random intercept model for E; for F-G, 2-way RM ANOVA on rank-transformed data, Holms-Sidak all-pairwise post hoc comparison. H. Exemplar timecourse of the caffeine response in a linker-VCM (n=10 cells). I. Amplitudes of caffeine responses, (see also Figure S5G). n=15(ctr) and 10(Lnk) myocytes (N=3 pairs of hearts, 3-7 VCMs each. Means ± SEs. P: mixed random intercept model).
Figure 5.
Figure 5.. Altered communication of mitochondria in large clusters with the jSR and each-other.
A. Exemplar TEM image from a linker-mouse LV papillary muscle showing a mitochondrion-jSR contact along the longitudinal (L) side of the mitochondrion at the periphery of a large mitochondrial cluster. B. L-side Mito-jSR contact occurrences as % of all (L+T-side) contacts in Ctr and Lnk myocardium. C. Exemplar TEM images of nanotunnels and connected mitochondria (cyan shade) with different positioning in the large clusters of the linker myocytes (i-iii) and in a row-like IFM cluster of a control myocyte (iv). D. Occurrence of nanotunnel-type intermitochondrial communication in Ctr and Lnk (from 2D TEM analysis) per myocyte (top) and per sarcoplasmic area (bottom). E. FIB-SEM tomographic visualization of nanotunnels (arrows) in a linker LV wall sample. The high 3D-resolution (5 nm voxels) gives high confidence in membrane continuities. Individual mitochondria are distinctly colored to follow continuities. Note the multiple nanotunnels connecting the green mitochondrial ‘bodies’ and the blunt-ended nanotunnel-like processes indicative of additional (dynamic) future or past continuities. See also Supplemental Movie 1. F. Schematic for grouping the nanotunnels by their relation to the cluster periphery/border (myofibrils). Periphery⥃inside (Periph⥃inside) peripheral nanotunnel(s) contact(s) inner mitochondrion (i) or peripheral/outer mitochondrion forms nanotunnel ‘inward’ (ii). Inside⥃inside: nanotunnel and connected mitochondrion are both ‘deep’ in the clusters (iii). Periph⥃Periph nanotunnel and connected mitochondrion are both adjacent to myofibril (iv). Each group is exemplified by the TEM images in C, with matching labels. Bar graph: Distribution of the three groups expressed as % of all nanotunnels in Lnk myocytes. TEM analysis: N=4 hearts/condition. n=3-5 cell/heart, Means ± SEs. P determined by mixed random intercept model (B,D) and Chi square test (F, against even distribution; with nanotunnel counts 142, 79 and 15 in the respective groups). G. Right: Exemplar volume rendering of nanotunnel-connected mitochondria (yellow) and nearby dyads from a FIB-SEM (Thermo/FEI Helios 4) tomography volume. Segmentation of the T-tubule (cyan) and SR (red) networks is partial; focused on the dyad junction areas (see also Supplemental Movie 2). Scheme and table: 3D analysis of the prevalence of nanotunnel-forming mitochondria that also contact dyad(s) (jSR). Mitochondrial cross sections from the top and bottom slices of FIB-SEM tomography stacks (from 2 linker hearts) were followed inside the whole volume. Limitation: mitochondria with a cross-sectional surface at the top/bottom slice will have a part outside the volume. Even so, almost all nanotunnel-forming mitochondria have jSR contact (Table).
Figure 6.
Figure 6.. Improved resistance to acute massive β-adrenergic stress in the linker-mice.
Mice were injected intraperitoneally with a single 300 mg/kg isoproterenol bolus. Echocardiography was performed before (Basal), then 30s, 30min and 24h post injection. A-F. Cumulated time-points for the indicated parameters for male (♂) and female (♀) mice. Means are shown as pseudo-timecourses (Ctr, black; Lnk, red). Individual animals are shown as vertical scatters. Means±SEs. N=13 Ctr♀, 10 Ctr♂, 11 Lnk♀, 15 Lnk♂. Gray box: statistical analysis information. G. Individual SV vs. HR fold changes from basal to the 30min point. To maintain CO (blue dashed ‘iso-CO’ line), an HR drop would be compensated by a proportional increase in SV. Mice with HR<1x under the iso-CO line (pink-shaded area) are not fully compensating (those with HR<1x & SV<1x are decompensating). Most control females are in the pink-shaded area. ✞, animal died in the first hour. H. CI distribution at 30 minute post-isoproterenol. Bins contain %portions of all Ctr or Lnk mice (actual males/female distribution is indicated by the symbols on top of the bins). Note that 5 Ctr and only one linker female have CI<1.2 ml/min/body-surface*. I. Survival traces (as labeled) at the indicated time points. Only control females died in 1 hour post-injection. See Doxy-linker mice in Figure S6.
Figure 7.
Figure 7.. Linker-mediated protection from ex vivo I/R injury.
A Exemplar LabChart record of LV pressure (LVP) and perfusion pressure (PP). Three phases of the experiment are indicated: equilibration/stabilization (Stabil, 20 minutes), no-flow ischemia (Isch, 40 min), reperfusion (60 min). B Timecourses of LV contractility (dP/dtmax) from control (black/gray) and linker (red/pink) hearts without (black/red) or with (gray/pink) CSA (2 μM) in the buffer. Inset i, the initial period of reperfusion on expanded timescale. Inset ii, bar graph representation of +dP/dtmax values at the end of reperfusion. C Infarct sizes (weighted average of triphenyltertazolium chloride-positive %areas of LV slices). Means±SE., N=6 animals/group/condition. Gray boxes: statistical analysis information.
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