Mitochondrial Calcium Increase Induced by RyR1 and IP3R Channel Activation After Membrane Depolarization Regulates Skeletal Muscle Metabolism

Abstract

Aim: We hypothesize that both type-1 ryanodine receptor (RyR1) and IP₃-receptor (IP₃R) calcium channels are necessary for the mitochondrial Ca²⁺ increase caused by membrane depolarization induced by potassium (or by electrical stimulation) of single skeletal muscle fibers; this calcium increase would couple muscle fiber excitation to an increase in metabolic output from mitochondria (excitation-metabolism coupling). Methods: Mitochondria matrix and cytoplasmic Ca²⁺ levels were evaluated in fibers isolated from flexor digitorium brevis muscle using plasmids for the expression of a mitochondrial Ca²⁺ sensor (CEPIA3mt) or a cytoplasmic Ca²⁺ sensor (RCaMP). The role of intracellular Ca²⁺ channels was evaluated using both specific pharmacological inhibitors (xestospongin B for IP₃R and Dantrolene for RyR1) and a genetic approach (shIP₃R1-RFP). O₂ consumption was detected using Seahorse Extracellular Flux Analyzer. Results: In isolated muscle fibers cell membrane depolarization increased both cytoplasmic and mitochondrial Ca²⁺ levels. Mitochondrial Ca²⁺ uptake required functional inositol IP₃R and RyR1 channels. Inhibition of either channel decreased basal O₂ consumption rate but only RyR1 inhibition decreased ATP-linked O₂ consumption. Cell membrane depolarization-induced Ca²⁺ signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondrial membrane potential; Ca²⁺ signals propagated toward intermyofibrillar mitochondria, which displayed increased membrane potential. These results are compatible with slow, Ca²⁺-dependent propagation of mitochondrial membrane potential from the surface toward the center of the fiber. Conclusion: Ca²⁺-dependent changes in mitochondrial membrane potential have different kinetics in the surface vs. the center of the fiber; these differences are likely to play a critical role in the control of mitochondrial metabolism, both at rest and after membrane depolarization as part of an "excitation-metabolism" coupling process in skeletal muscle fibers.

Keywords: energy distribution; inositol 1,4,5-trisphosphate receptor; mitochondria heterogeneity; mitochondrial network; ryanodine receptors.

Figure 1

Membrane depolarization promotes oxygen consumption ratio in skeletal muscle fibers. Muscle fibers were isolated from FDB muscle and cultured overnight; fibers were depolarized using 65 mM K⁺ during 5 min before measurement started. (A) Representative kinetics of OCR in control or depolarized muscle fibers. (B) Quantification of the basal, ATP-linked, reserve capacity, proton leak and non-mitochondrial OCR were determined as described in section Materials and Methods. (C) Percent distribution of the basal, ATP-linked, reserve capacity, proton leak and non-mitochondrial OCR were calculated. OCR after FCCP administration was considered as 100% (maximal OCR). N = 5 different animals ^**p < 0.01; ^***p < 0.001 compared with the control condition.

Figure 2

Mitochondrial Ca²⁺ levels visualized with CEPIA3mt. Adult FDB muscles were electroporated with plasmids encoding the molecular Ca²⁺ sensor CEPIA3mt and/or RCaMPs directed to mitochondria and cytoplasmic compartment, respectively. (A) Muscle fibers were co-electroporated with plasmids encoding CEPIA3mt (Left top panel) mtDsRed (left middle panel). High co-localization was observed between both sensors (Left bottom panel). Mander's coefficient were evaluated (right panels), scale bar is 15 μm. (B) Standard deviation of fluorescence, representative ROI (ROI1 and ROI2) selected by fiber and representative kinetics of 5 different ROIs are shown. Scale bar is 2 μm. (C) The fibers expressing CEPIA3mt (green line) plus RCaMPs (red line) were stimulated with 65 mM K⁺; FCCP (1 μM) was added at the end of experiments to uncouple mitochondria. Mean of 5 different animals is shown. C) FDB fibers electroporated with plasmids encoding CEPIA3mt (green line) and RCamPs (red line) were exposed to electrical stimulation; representative kinetics and the maximal fluorescence obtained is shown (D,E). (F) Intramitochondrial calcium wave propagation in fibers expressing CEPIA3mt stimulated with 65 mM K⁺. N = 4 different animals and 25 fibers were evaluated in each case. ^**p < 0.01 vs. control.

Figure 3

Activation of IP₃R and RyR1 participate in the mitochondrial Ca²⁺ increase after membrane depolarization. Adult FDB muscle was electroporated with plasmids encoding the molecular Ca²⁺ sensor CEPIA3mt. Fibers were maintained in Krebs Ringer buffer with BTS (10 μM) and pre-incubated for 1 h with xestospongin B (10 μM), dantrolene (50 μM) or both. The representative kinetics and maximal fluorescence was calculated in each condition (A,B). sh-IP₃R1-RFP partially reduced the calcium increases in response to high K⁺ (C,D), scale bar is 15 μm. (E) Muscle fibers electroporated with plasmids encoding CEPIA3mt and pre-incubated with caged IP₃. The photolysis increased the mitochondrial calcium level. Color bar represents the relative change in fluorescence. (F) Percent of response to different number of UV flashes is shown. N = 6 experiments were performed and 25 fibers were evaluated each time. †Means difference vs. control stimulated with K⁺. ^*Difference vs. resting; ^*p < 0.05; ^**/††p < 0.01.

Figure 4

Activation of RyR1 but not of IP₃R is necessary to increase the mitochondrial O₂ consumption after membrane depolarization. Adult FDB muscle fibers maintained in Seahorse medium with BTS (10 μM) were pre-incubated for 1 h with xestospongin B (10 μM) or dantrolene (50 μM). The fibers were depolarized 5 min before the measurement. (A,D) Representative kinetics of OCR in fibers pre-incubated with xestospongin B or dantrolene respectively. (B,E) Quantification of basal and ATP-linked OCR in muscle fibers pre-incubated with xestospongin B or dantrolene respectively. (C,F) Percent distribution of the basal, ATP-linked, reserve capacity, proton leak and non-mitochondrial OCR were calculated. OCR after FCCP administration was considered as 100% (maximal OCR). N = 5 independents experiments. ^*p < 0.05; ^**p < 0.01 compared with the control condition.

Figure 5

Heterogeneity in intramitochondrial proteins and calcium handling between SSM and IMFM in FDB fibers. (A) Immunostaining of complex IV and TOM20 in 1 μm confocal slices of adult muscle fibers, scale bar is 15 μm. (B) 3D reconstruction of immunostaining of MCU and ATP5a in adult muscle fibers, scale bar is 15 μm. (C) Immunostaining of Cytochrome C (CytC) and TOM20 in 1 μm confocal slices (upper panel). The Z-projection reconstruction of the whole fiber is shown (bottom panel), scale bar is 15 μm. (D) Inmunostaining of MCU plus CytC. The longitudinal or z-projection reconstruction is shown in the upper and bottom panel, respectively, scale bar is 15 μm. (E) Inmunostaining of MICU1 plus CytC. The longitudinal or z-projection reconstruction is shown in the upper and bottom panel respectively, scale bar is 15 μm. (F) Mander's coefficient for Cyt C/MICU1 and Cyt C/MCU. (G) Muscle fibers isolated from FDB muscle were incubated during 30 min with TMRE⁺ plus mitotracker green (MTG). One optical slice at 2 μm from the center of the fiber is shown. The ratio of TMRE⁺/MTG fluorescence quantification is shown. (H) Adult FDB muscle expressing the molecular Ca²⁺ sensor CEPIA3mt were maintained in Krebs Ringer buffer with BTS (10 μM). The change of fluorescence in the x,z,t axis was evaluated. Representative kinetics from the subsarcolemmal area (first 5 μm) or the intermyofibrillar area was determined (Left panel) and maximal fluorescence was calculated in the subsarcolemmal or intermyofibrillar area (right panel). (I) Maximal fluorescence of SSM or IMFM was adjusted as 100% in order to evaluate the kinetic of fluorescence increase. There is no difference in the slope of calcium increase between SSM and IMFM. N = 6 different animals and 25 fibers were evaluated in each case. ^**p < 0.01 vs. IMFM. ^***p < 0.001 difference vs Cyt/MICU1.

Figure 6

ΔΨm appearsto propagate away from the surface regions toward the central zone. (A) Muscle fibers electroporated with plasmids encoding CEPIA3mt were incubated with TMRE+ (20 nM) during 30 min. The subsarcolemmal mitochondrial fluorescence was measured after depolarization with high K⁺ medium. Representative kinetics is shown. (B) Muscle fibers were incubated with TMRE⁺ (20 nM) and the fluorescence in the subsarcolemmal region was evaluated after electrical stimulation or high K⁺ medium. At the end of the experiment 0.5 μM FCCP was added to depolarize the mitochondria (left panel). The quantification of the fluorescence is shown in the right panel. (C) Muscle fibers incubated with TMRE⁺ (20 nM) were stimulated with ES in presence of different inhibitors. Slope of mitochondrial depolarization is shown (botton panel). (D) Muscle fibers incubated with TMRE⁺ (20 nM) were stimulated with high K⁺ medium in presence of different inhibitors. Slope of mitochondrial depolarization is shown (botton panel). (E) Line scan of the subsarcolemmal and intermyofibrillar region was performed after K⁺-induced depolarization; the representative image of ΔΨm change and calcium handling (upper panel) and kinetics (bottom panel) are shown. Vertical scale bar represents 10% fluorescence change of. Horizontal scale bar corresponds to 25 s. (F) Muscle fibers were incubated with TMRE+ (20 nM) and the images were acquired before and 60 s after potassium depolarization. Red arrowhead shows reduction of fluorescence near the surface of the fiber and green arrowhead shows areas of increase in fluorescence toward the center of the fiber. Scale bar 5 μm. N = 6 different animals and 20–25 fibers were analyzed in each condition. ^*p < 0.05.