Muscle fibre mitochondrial [Ca2+] dynamics during Ca2+ waves in RYR1 gain‐of‐function mouse

A fraction of the Ca2+ released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca2+] ([Ca2+]mito). This transient [Ca2+]mito increase may be important in the resynthesis of ATP and other processes. The resynthesis of ATP in the mitochondria generates heat that can lead to hypermetabolic reactions in muscle with ryanodine receptor 1 (RyR1) variants during the cyclic releasing of SR Ca2+ in the presence of a RyR1 agonist. We aimed to analyse whether the mitochondria of RYR1 variant muscle handles Ca2+ differently from healthy muscle.

rapidly to lead to a uniform contraction along the length of the fibre.Voltage sensors on the t-system membrane directly interact with the Ca 2+ release channels/ryanodine receptors (RyRs) on the SR membrane to release Ca 2+ for the rapid activation of contraction. 2To maintain energetic steady state and avoid Ca 2+ release and force decline, it is important that the mitochondria rapidly resynthesize ATP during activity. 3,4n mammalian skeletal muscle, most of the mitochondria in the fibre lie next to the SR, 5 putting the mitochondria close to the SR Ca 2+ release sites and in line with receiving large Ca 2+ transients during SR Ca 2+ release.][8][9][10] It has been shown that a rise in free [Ca 2+ ] mito occurs concomitantly with SR Ca 2+ release, with a lag in the peak of the mitochondrial Ca 2+ transient behind the cytoplasmic transient by the order of 10s of ms. 9 There are a number of potentially important physiological implications of the transient rise in [Ca 2+ ] mito , including modulation of oxidative phosphorylation 11,12 and activation of PGC1α. 13,14n unexplored aspect of Ca 2+ entering the mitochondria during Ca 2+ release is what occurs during pathophysiological situations where Ca 2+ release is activated by direct and uncontrolled stimulation of the RyRs.In malignant hyperthermia-susceptible (MHS) patients, such as those with a gain-of-function RYR1 mutation, the exposure to triggering agents such as volatile anesthetics can cause uncontrolled release of Ca 2+ from the SR and hypermetabolism as all the CaATPases are activated. 15The use and resynthesis of ATP in the muscle cause significant heat generation, raising the body temperature to levels that may cause cardiac arrest or other tissue and organ damage.
The properties of mitochondrial Ca 2+ dynamics under repeatedly uncontrolled SR Ca 2+ release, like what is expected to occur during MH events, are unknown.It is important to know the evolution of the [Ca 2+ ] mito transient ([Ca 2+ ] mito (t)) during these acute pathophysiological events because they could be: (i) modulating the cytoplasmic Ca 2+ transient; (ii) regulating the rate of oxidative phosphorylation, which would promote the generation of heat 3 ; and (iii) may raise the peak of the [Ca 2+ ] mito (t) to levels that cause mitochondrial damage. 16o track the [Ca 2+ ] mito (t) during uncontrolled Ca 2+ release, we developed a technique that is transferable to use in human muscle fibres obtained from needle biopsies, for potential future use in MHS persons or others. 17,18This technique is based on the use of mechanically skinned fibres, which avoids the need for enzymatic dissociation of fibres, where mitochondrial Ca 2+ handling is adversely affected by the enzymes used in the fibre isolation procedure. 19To do this, we used rhod-2/AM, which has been used before to track Ca 2+ transients in mitochondria of muscle fibres. 6,7,20We have previously calibrated the resting [Ca 2+ ] mito ([Ca 2+ ] mito (0)) in wild-type (WT) and RYR1 knock-in (KI) mouse muscle fibres. 20,21Here, we use these values as a base for the calibration of mitochondrialtrapped rhod-2 and [Ca 2+ ] mito (t).We found that the exposure of muscle fibres to caffeine-induced [Ca 2+ ] mito (t) that trailed Ca 2+ waves in the order of seconds and displayed increases in [Ca 2+ ] mito (t) between 50 and 200 nM during cytoplasmic Ca 2+ waves.We also found that the rate of rise and fall of [Ca 2+ ] mito (t) in heterozygous (HET) RYR1 KI fibres exceeded that which occurred in WT for a given cytoplasmic Ca 2+ -wave amplitude.

| RESULTS
In this section, a calibration of rhod-2 mito (t) and [Ca 2+ ] mito (t) is used, based on previously measured [Ca 2+ ] mito (0) and k D,Ca of mitochondrial rhod-2. 20With this approach, we describe the Ca 2+ -handling properties of the mitochondria in WT and HET RYR1 KI muscle fibres under conditions that may reflect the repetitive, uncontrolled Ca 2+ -release events that underlie malignant hyperthermia episodes.

| [Ca 2+ ] mito (t) during RyR1 agonist exposure
Figure 1 shows Ca 2+ -dependent fluorescence signals from the cytoplasm and mitochondria in a skinned fibre tracked by xyt imaging during application of caffeine-containing release solution (see also Video S1).xy-images were acquired by line interleaving the 488 and 543 nm laser lines to effectively obtain simultaneous images of cytoplasmic and mitochondrial Ca 2+ -dependent fluorescence from the fibre.The top row of images shows the cytoplasmic fluo-4 fluorescence in the presence of caffeine (Figure 1A).A Ca 2+ transient was initiated as a small local release in the third image marked 268 s, which then propagated in both directions along the fibre. 22The second row of images illustrates the Ca 2+ -dependent mitochondrial rhod-2 fluorescence signal (Figure 1B).The collection of images shows that during the release of Ca 2+ from the SR to the cytoplasm, there is accumulation and then depletion of Ca 2+ inside the mitochondria.
A profile of the cytoplasmic and mitochondrial transients is shown in Figure 1D; each dot represents the spatially averaged fluorescence from within the borders of the fibre in A and B. The initial mitochondria image in the series, prior to the release of SR Ca 2+ , shows inhomogeneity along the length of the fibre in the field of view.To account for rhod-2 signal heterogeneity across the fibre, we subtracted the baseline rhod-2 signal (the initial frame, F mito (0) (x, y)) from each subsequent frame F mito (x, y) (Figure 1C).The manipulated images still show some inhomogeneity, which likely reflects noise between images.Regardless, a clear increase in mitochondrial rhod-2 fluorescence transient that follows the cytoplasmic Ca 2+ transient can be observed.Figure 2 shows an example of repetitive caffeineinduced cytoplasmic and mitochondrial Ca 2+ transients.In both the WT (Figure 2A) and HET (Figure 2B) fibres, the addition of caffeine caused an initial, long-lasting cytosolic Ca 2+ transient that ceased after ~50 s in HET and ~90 s in WT, with Ca 2+ expected to be re-sequestered by the SR.After 10s of seconds of quiescence, SR Ca 2+ release occurred again and propagated through the fibre as Ca 2+ waves, like the images shown in Figure 1 (Video S1).Ca 2+ waves occurred in repetitive fashion and showed a single, abrupt increase in frequency in both Figure 2A,B.][24][25] With each release of Ca 2+ in both WT and HET fibres, the [Ca 2+ ] mito (t) increased and then declined following each cytoplasmic Ca 2+ transients with a delay.We also observed that during the course of the repetitive releases, the cytoplasmic Ca 2+ transients changed their amplitude and duration, which resulted in a change in the associated [Ca 2+ ] mito (t).This is shown in more detail in Figure 2 Aii, Aiii and Bii, Biii, and analysed in Figure 4.
We examine the properties of the [Ca 2+ ] mito (t) in relation to the large initial release and subsequent Ca 2+ waves in Figures 3-6.
Figure 3A,B show the amplitude and duration of the cytoplasmic Ca 2+ transient during the initial, large Ca 2+ release in both genotypes.The duration of the transient was significantly briefer in the HET than WT fibres and the area under the curve (AUC) of the cytoplasmic transient was also smaller in the HET than WT fibres (Figure 3C).Interestingly, for the same cytosolic amplitude, the HET presents a significantly higher amplitude in [Ca 2+ ] mito during the large Ca 2+ release compared to the WT (Figure 3D). Figure 3E reports the lag time between the peaks of the cytoplasmic and mitochondrial Ca 2+ transients were in the order of 10s of seconds for both genotypes and Figure 3F presents the AUC for the mitochondrial transient, which was not different between the genotypes.
The Ca 2+ release properties of the SR and the Ca 2+handling properties of the mitochondria during the brief Ca 2+ waves of the WT and HET fibres are examined in Figure 4.In this figure, the properties of the cytoplasmic waves and the associated mitochondrial Ca 2+ transient are grouped by cytoplasmic Ca 2+ -wave frequency. 18igure 4A shows that the AUC for the cytoplasmic Ca 2+ waves in both genotypes is affected by the wave frequency.We have previously determined that lower wave frequency allows the SR to load more Ca 2+ in the longer period of time between waves and thus provide a greater amount of Ca 2+ release than at the higher wave frequencies. 26During the larger Ca 2+ waves, the AUC of the mitochondrial Ca 2+ transients (Figure 4B) was significantly greater in HET compared to WT.
The lag between the peak of the cytoplasmic and mitochondrial Ca 2+ transients (Figure 4C) during the brief waves was in the order of seconds and declined with the decrease in AUC of the cytoplasmic Ca 2+ transient (Figure 4A).
Figure 4D-F show the rise, rise time, and rate of rise of the [Ca 2+ ] mito at each wave frequency, respectively.The rise (Figure 4D) and rate of rise of the [Ca 2+ ] mito (t) (Figure 4F) were greater in the HET than the WT at the lower wave frequencies, while the rise time itself (Figure 4E) was correlated with the relatively low magnitude of the Ca 2+ release at high wave frequency.These properties are consistent with HET fibre mitochondria being more sensitive to increases in SR Ca 2+ release than WT fibres.
To confirm the difference in relationship between Ca 2+ release and [Ca 2+ ] mito (t) between the genotypes, we plotted the respective amplitudes of the associated Ca 2+ transients against each other (Figure 5).A strong linear relationship for HET fibres (r 2 = 0.85) but not WT (r 2 = 0.06) was observed.It was possible that the strong linear relationship in the HET fibres was observed due to the greater range of cytoplasmic Ca 2+ -release amplitude covered compared to the WT fibres.However, restricting the range of HET cytoplasmic Ca 2+ release amplitudes to that achieved in the WT fibres still provided a strong linear relationship in the HET fibres (r 2 = 0.77) (Figure S1).
Finally, we analysed the mitochondrial Ca 2+ efflux profiles of WT and HET fibres during caffeine-induced Ca 2+ waves (Figure 6).The free Ca 2+ drop in the mitochondria after the cytoplasmic wave passed was greater in HET than WT fibres at low wave frequencies and showed a similar time for this decrease to occur apart from at the lowest wave frequency (Figure 6A,B).Therefore, the rate that the free Ca 2+ dropped in the mitochondria after a cytoplasmic wave passed was greater in HET than WT at the lower wave frequencies (Figure 6C).

| DISCUSSION
Here, we have designed a simple way to calibrate mitochondrial-trapped rhod-2 and [Ca 2+ ] mito to track the F I G U R E 2 Ca 2+ waves and mitochondrial Ca 2+ -transient properties.WT (Ai) and HET (Bi) fibres both displayed a large Ca 2+ release, followed by Ca 2+ waves that abruptly changed frequency at time point ~600 s and ~450 s, respectively.Aii, Aiii, Bii, and Biii show Ca 2+ transients expanded from A & B, respectively.HET, heterozygous; WT, wild type.
[Ca 2+ ] mito (t) during Ca 2+ release from the SR.This technique relies on (1) previous works that have provided the in situ k D,Ca of rhod-2 trapped in the mitochondria of cardiomyocytes 12 with the assumption that this is similar in skeletal muscle mitochondria, (2) the [Ca 2+ ] mito (0) in these muscle fibres, 20 and (3) the assumption that the [Ca 2+ ] mito (t) changed within the linear range of the Ca 2+ -dependent rhod-2 fluorescence and [Ca 2+ ] mito relationship.Using this method, we quantified the changes in [Ca 2+ ] mito in healthy and MHS mouse muscle fibres during the uncontrolled Ca 2+ -release events induced by the application of low doses of caffeine.We show that [Ca 2+ ] mito typically changes within a relatively small range of 50-200 nM from a [Ca 2+ ] mito (0) of 250-350 nM during caffeine-induced Ca 2+ waves.Additionally, large Ca 2+ waves at low wave frequencies induced a greater increase in [Ca 2+ ] mito in HET than WT fibres; and that the [Ca 2+ ] mito -transient trails that of the cytoplasmic Ca 2+ transients with a typical lag in the order of seconds to 10s of seconds.

| Kinetics of Ca 2+ movements and Ca 2+ -handling properties of the mitochondria
Ca 2+ release under caffeine exposure opens the RyRs via lowering the threshold for activation by [Ca 2+ ] SR . 22,26,27a 2+ release in the presence of caffeine is prolonged, dependent on the [caffeine] and the initial load of SR Ca 2+ .Ca 2+ release terminates due to depletion of [Ca 2+ ] SR .Under low mM levels of caffeine, the RyR shuts allowing the SR to reload Ca 2+ toward the endogenous [Ca 2+ ] SR level.However, the endogenous [Ca 2+ ] SR level is greater than the new threshold for RyR activation by [Ca 2+ ] SR , and Ca 2+ is released again.Oscillatory Ca 2+ release therefore occurs, presenting as Ca 2+ waves along the fibre.26 The initial release of Ca 2+ is large, as the Ca 2+ content of the SR was initially high.The Ca 2+ -buffering power of the SR is reduced by the release of Ca 2+ by caffeine, and the Ca 2+ content of the SR is lower in subsequent releases as the threshold [Ca 2+ ] SR for RyR activation is met.23,28 In A lag is observed in the rise of [Ca 2+ ] mito (t) following the rise of the cytoplasmic Ca 2+ transient when Ca 2+ is released from the SR directly (e.g., following caffeine application) or during EC coupling.During the initial caffeine-induced Ca 2+ release from the SR (Figure 3E), the peak-to-peak lag of the cytoplasmic Ca 2+ transient to the [Ca 2+ ] mito (t) ranged from ~3s to ~60s for WT and ~3s to ~30 for HET, which are much longer than the brief delays of 10s of ms observed following EC coupling in skeletal muscle fibres.9 Thus, the peak to peak of the cytoplasmicto-mitochondrial Ca 2+ transients is determined the SR Ca 2+ flux duration, which will shape the local Ca 2+ transient at the MCU.
It remains difficult to calculate the total amount of Ca 2+ entering the mitochondria during SR Ca 2+ release because the kinetics of Ca 2+ binding and the dynamic nature of the Ca 2+ buffering within the mitochondria are poorly understood. 29We also acknowledge that the mitochondrial Ca 2+ uniporter and mitochondrial Na + -Ca 2+ exchanger may have significant roles in maintaining the [Ca 2+ ] mito (t) within the nM range during Ca 2+ -release events.Additionally, it is important to point out that the

HET WT
SR is the dominant Ca 2+ buffer in the fibre.The SR holds ~90% of the fibre Ca 2+ content at steady state.The mitochondria hold <5% of the fibre Ca 2+ content in healthy muscle 20 and it is not expected to sequester significant amounts of Ca 2+ during SR Ca 2+ release.The rate of Ca 2+ entry into the mitochondria is correlated with the Ca 2+ release rate during the Ca 2+ waves in this study (Figure 4).We can compare this to what has been found in fibres during depolarization-induced Ca 2+ release.The Ríos lab 9 determined the rate that Ca 2+ entered the mitochondria during voltage-clamp depolarizations.This rate was some 5 orders of magnitude greater than during Ca 2+ waves (~4.1 mM s −1 vs. ~20 nM s −1 , respectively).It is possible to gain some understanding of the properties of the skeletal muscle MCU by comparing these mitochondria Ca 2+ -uptake rates with the respective SR Ca 2+ -release rates that provide the increased driving force for Ca 2+ entry into the mitochondria.The rate that Ca 2+ is released by voltage and caffeine is about 2 orders of magnitude different, where depolarization-induced Ca 2+ release moves at about 20 mM s −1 and the rate that Ca 2+ is released from the SR by caffeine is 0.1-1 mM s −1 .The ratio of mitochondrial Ca 2+ -uptake rate to SR Ca 2+ -release flux rate for the two different types of Ca 2+ release therefore increases significantly under depolarization compared to caffeine agonism (4.1/20 > 0.00002/0.1-1).This suggests that the uptake of mitochondrial Ca 2+ is a non-linear function of the SR Ca 2+ -release flux.
The large difference in rates that the mitochondria take up Ca 2+ as a function of SR Ca 2+ release rate suggests that during slower SR Ca 2+ -release fluxes, such as in the presence of low-dose caffeine, that the rate of Ca 2+ making it to the MCU pore is limiting the uptake flux of the mitochondria.There are factors that favour and potentially temper the uptake of Ca 2+ by mitochondria during SR Ca 2+ release.These factors are related to the proximity of the SR Ca 2+ -handling proteins to the mitochondria.Favouring mitochondrial Ca 2+ uptake is the proximity of the RyRs and MCU, at some 100-150 nM apart.Potentially tempering mitochondrial Ca 2+ uptake is the action of the SR Ca 2+ pumps that outcompete the MCUs for Ca 2+ .The SR Ca 2+ pumps are located on the longitudinal SR and are much closer to the MCUs than the RyRs. 5,30he correlation between Ca 2+ -release amplitude and [Ca 2+ ] mito amplitude was strong in the HET and weak in the WT (Figure 5).The greater uptake of mitochondrial Ca 2+ in HET during similar Ca 2+ -release amplitudes in WT and HET muscle suggests a difference in the properties of the MCUs in responding to spikes in cytoplasmic Ca 2+ .The MCUs of WT and HET mouse muscles are chronically exposed to different resting levels of bulk [Ca 2+ ] cyto , 21 which not only set the steady-state mitochondrial Ca 2+ content 20,31 but may regulate post-translational modifications of the MCUs that affect its response to SR Ca 2+ release.
The increases in [Ca 2+ ] mito during the uncontrolled release of Ca 2+ in the presence of an RyR agonist may help stimulate the rate of oxidative phosphorylation. 11,12Given the similarity of caffeine-and halothane-induced Ca 2+ waves, 18,26 we can consider this type of caffeine-induced Ca 2+ release comparable to what happens in the muscles during a MH event.For an MH event to continue, or indeed increase in severity, the ATP hydrolysis rate at the SR Ca 2+ pump cannot outcompete the rate of oxidative phosphorylation or Ca 2+ release will fail regardless of the normal Ca 2+ content of the SR being maintained. 4If it is the case that oxidative phosphorylation rate increases with the [Ca 2+ ] mito (t), then this process may support the malignant nature of the hyperthermic event under a general anesthetic, as net heat is likely generated by the process of ATP resynthesis inside the mitochondria. 3However, direct evidence for this possibility is still required.We observed no signs of mPTP opening or dysfunction or death of mitochondria in our experiments.The repetitive, low amplitude of the [Ca 2+ ] mito (t) may not activate the mPTP but it is not possible to say from our results whether the Ca 2+ -buffering properties or any restriction on Ca 2+ entering mitochondria are responsible for "protecting" the organelle from Ca 2+ -induced damage.
In this study we have presented a simple way to calibrate mitochondrial-trapped rhod-2 fluorescence and [Ca 2+ ] mito .We observed the changes in [Ca 2+ ] mito (t) during Ca 2+ waves in muscle fibres, which presented significant lags behind the cytoplasmic Ca 2+ transients in the order of many seconds and differences in Ca 2+ -uptake capacity between WT and HET fibres.Our data provide a platform for understanding what may be occurring in the muscle during an MH event and help us understand the handling of Ca 2+ by the mitochondria, in conjunction with other studies that have focused on mitochondrial Ca 2+ handling during EC coupling. 6,7,9,10,19 4 |MATERIALS AND METHODS

| Ethics and muscle preparation
All experiments performed were approved by The University of Queensland Animal Ethics Committee.Male C56Bl/6 J mice WT and HET for the p.G2435R variant of RYR1 21 were euthanized by CO 2 asphyxiation.The EDL muscles were quickly excised and pinned to Sylgard set in a Petri dish under a layer of paraffin oil.Single fibres were isolated, and the sarcolemma was mechanically removed by microdissection along the fibre using a pair of jeweler's fine forceps. 32

| Solutions
Skinned fibres were positioned on a custom-built well that used a coverslip as a base and bathed in a K + -based internal solution containing HEPES 90 mM, EGTA 0.1 mM, ATP 8 mM, creatine phosphate 10 mM, BTS 50 μM, Mg 2+ 1 mM, Na + 36 mM, K + 126 mM, and Ca 2+ 300 nM.The solution pH was set to 7.1 and osmolality was 300 ± 10 mOsmol/kg.To image Ca 2+ in the mitochondria, fibres were loaded with rhod-2/AM.To do this, fibres were incubated in a K + -based solution with 2.5 μM rhod-2/AM and 0.005% Pluronic F-127 detergent at 4°C for 10 minutes.Then, the rhod-2/AM containing solution was substituted for a standard K + -based internal solution without rhod-2/AM to wash the excess dye away.To track cytoplasmic Ca 2+ , the K + -based internal solution contained 10 μM of fluo-4 salt.To induce RyR1 Ca 2+ release, caffeine (1-3 mM for RYR1 WT/KI and 5-7 mM for WT) was added to the standard K + -based solution containing fluo-4.

| Confocal imaging
Fibres were imaged on an Olympus FV1000 confocal microscope using a 40× objective and continuous xyt scanning of the fibre with the scanning lasers positioned perpendicular to the fibre axis.Images were built by line interleaving of the excitation lines 488 nm and 543 nm.Images had an aspect ratio of 256 × 512 pixels with the long axis of the image parallel with that of the fibre.Our protocol captured images at 1.8 or 3.6 s per frame, which we considered appropriate to capture the dynamics of Ca 2+ movements from the cytoplasm to mitochondria and back from the transients.Therefore, we maintained these imaging protocols throughout the study and moved to calibration of mitochondrial rhod-2 fluorescence and [Ca 2+ ] mito .

| Calibration of mitochondrial rhod-2 fluorescence and [Ca 2+ ] mito
We have previously calibrated mitochondrial rhod-2 fluorescence and [Ca 2+ ] mito in resting muscle fibres isolated from RYR1 KI mice using a technique where the Ca-phosphate network is disrupted following the depolarization of the mitochondria. 20This maneuver caused [Ca 2+ ] mito to surge over several seconds, close to saturating the mitochondrial-trapped rhod-2, allowing the maximum fluorescence signal to be determined (F max ).Secondarily to this, the following extrusion of Ca 2+ from the mitochondria in the absence of the electrical potential (the driving force for Ca 2+ entry into the organelle) allowed [Ca 2+ ] mito to drop to low levels determining the minimum fluorescence signal for the preparation to be evidenced (F min ).An example of the mitochondrial rhod-2 fluorescence transient in the presence of depolarizing FCCP is shown in Figure 7.As we expect that FCCP depolarization causes a near saturation and depletion of rhod-2 with Ca 2+ , we can estimate the dynamic range (DR) of mitochondria rhod-2.A DR value of 2.6 was calculated from similar experiments as shown in Figure 7 with nine fibres.
To calibrate mitochondrial rhod-2 transients (rhod-2 mito (t)), we used a similar set of assumptions as described by Royer et al. 28 for the calibration of nonratiometric dyes in the absence of individually obtained Fmax and Fmin values for each preparation.We used the known initial resting [Ca 2+ ] mito for WT and HET fibres ([Ca 2+ ] mito (0)) 20 and the in situ k D,Ca for rhod-2 in mitochondria (rhod-2 mito (t)) of 1.74 μM 12 and assumed that the rhod-2 mito (t) remained on the linear part of the rhod-2 fluorescence-[Ca 2+ ] mito relationship.This assumption is reasonable given the DR of rhod-2 mito (t) in Figure 2 is <<2, which is much less than the DR of rhod-2 mito (Figure 7).Additionally, [Ca 2+ ] mito (0) is below the k D,Ca with the [Ca 2+ ] mito (t) displaying values that typically increase above the [Ca 2+ ] mito (0) (Figure 2).Under these conditions, we used the expression: where [Ca 2+ ] mito (0) is the reference resting [Ca 2+ ] mito for WT and HET RYR1 KI mice (0.25 and 0.36 μM, respectively 20 ); F(t) is rhod-2 mito (t); and k d * d(F(t)/d(t))/F 0 is the time required for Ca 2+ -dye equilibration.The sampling rate used here is considered significantly slower than the rate that Ca 2+ -dye equilibration and thus this component of the relationship can be ignored.It follows that the simplified equation can be used: The advantage of using this approach is the rhod-2 mito (t) calibration of every fibre recorded without the need for subsequent exposure to FCCP following caffeine exposure.

| Wave property measurements
The AUC was calculated using GraphPad Prism 9.5.1 as described in detail: https:// www.graph pad.com/ guides/ prism/ latest/ stati stics/ stat_ area_ under_ the_ curve.htm.Amplitude of the wave was determined as the difference from baseline immediately before the start of the wave to the peak of the wave (Amplitude = Peak value -Wave baseline).Duration of the wave was determined as the full duration calculated from the baseline (initial) of the wave to return to baseline (Final) (Duration = Time final -Time initial ).

| Statistical analysis
Statistical data analysis was performed on GraphPad Prism 9.5.1.Unpaired t-test, non-parametric test was performed to compare the cytosolic Ca 2+ -to-mitochondria Ca 2+ transients on the initial caffeine-induced Ca 2+ release.Two-way ANOVA with Sidak's multiple comparisons was used to analyse the mitochondria Ca 2+ handling at multiple cytosolic Ca 2+ -wave frequencies.A p-value < 0.05 was considered statistically significant.Data are plotted as mean ± standard error mean.

F I G U R E 7
The dynamic range of mitochondrial-trapped rhod-2.A mitochondrial rhod-2 transient during addition of FCCP shows the depolarization of the mitochondria and the dissipation of the driving force for Ca 2+ entry into mitochondria result in a surge of free Ca 2+ inside the mitochondria (due to the disruption of the poly-phosphate network).This process is followed by the slow extrusion of Ca 2+ from the mitochondria and the net loss of Ca 2+ from the mitochondria.The Fmax and Fmin of mitochondrialtrapped rhod-2 can be estimated from this transient.The "top" and "bottom" of the fluorescence signals here represent the dynamic range of the dye in the mitochondria.

F I G U R E 1
Caffeine-induced Ca 2+ release.Images obtained from simultaneous recording of cytosolic-wave fluo-4 fluorescence (A) and mitochondrial Ca 2+ -uptake rhod-2 fluorescence (B) in a mechanically skinned fibre of WT EDL.The cytosolic wave originates at 268 s and propagates in both directions along the fibre.The cytosolic wave reaches its maximum fluorescence at 275 s.The mitochondrial rhod-2 fluorescence starts to increase at 271 s and reaches the maximum fluorescence at 285 s, indicating a delay between the peak Ca 2+ release and the mitochondrial Ca 2+ uptake.(C) Subtraction of noise in mitochondrial images.The frame 260 s in (B) was defined as reference and subtracted from the subsequent frames.The results are presented below the corresponding images in (C).(D) The spatially average profile of cytoplasmic fluo-4 fluorescence (blue line) and mitochondrial rhod-2 fluorescence (red line).WT, wild type.

F I G U R E 3
Mitochondrial Ca 2+ handling during the initial, large caffeine-induced Ca 2+ release in WT and HET fibres.(A) cytoplasmic amplitude of the Ca 2+ release.(B) Duration of initial Ca 2+ release.(C) AUC of cytoplasmic Ca 2+ release.(D) Increase in [Ca 2+ ] mito during Ca 2+ release.(E) Lag in peak of Ca 2+ transients in cytoplasm and mitochondria.(F) AUC of mitochondrial Ca 2+ transient.t-Tests: (B) *p = 0.0317.(C) *p = 0.0318.(D) *p = 0.0317.AUC, area under the curve; HET, heterozygous; WT, wild type.allcases, the rise of the cytoplasmic Ca 2+ transient occurs over seconds, which is a much longer duration than the rise of the Ca 2+ transient during normal EC coupling.These types of agonist-induced Ca 2+ release thus provide Ca 2+ release of different durations and release fluxes to examine the kinetics of [Ca 2+ ] mito changes across RYR1 genotypes and compare to normal EC coupling.

F I G U R E 4 5
Ca 2+ handling by mitochondria at various frequencies of caffeine-induced Ca 2+ waves in WT and HET fibres.(A) AUC of cytoplasmic Ca 2+ waves.(B) AUC of mitochondrial Ca 2+ transient.(C) Time between the peaks of the cytoplasmic and mitochondrial Ca 2+ transients.(D) Increase in [Ca 2+ ] mito per wave.(E) Rise time of [Ca 2+ ] mito per wave.(F) Rate of increase in [Ca 2+ ] mito per wave.n WT = 5 fibres, n HET = 5 fibres, n WT waves = 126, and n HET waves = 61.*p < 0.05, †p < 0.01, and ‡p < 0.001.AUC, area under the curve; HET, heterozygous; WT, wild type.Mitochondrial Ca 2+ uptake amplitude as a function of cytoplasmic Ca 2+ -wave amplitude.The delta increase in [Ca 2+ ] mito for its associated cytoplasmic Ca 2+ -wave amplitude is plotted in WT and HET fibres.The shades of blue and red represent data from individual fibres for WT and HET, respectively.Linear regression is plotted as solid lines and 95% CI is plotted as broken lines.n WT = 5 fibres, n HET = 5 fibres, n waves WT = 126, n waves HET = 61.HET, heterozygous; WT, wild type.