The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (EGSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline EGSH increase. Importantly, β-adrenergic stimulation resulted in excessive EGSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial EGSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic EGSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial EGSH oxidization in RyR2-R2474S cardiomyocytes.

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca 2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E GSH ). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E GSH increase. Importantly, β-adrenergic stimulation resulted in excessive E GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically β-adrenergic stimulation significantly increased mitochondrial E GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca 2+ leak induce a strong, but dantrolene-inhibited mitochondrial E GSH oxidization in RyR2-R2474S cardiomyocytes.

INTRODUCTION
In the heart, where ATP consumption is highest of any tissue, Ca 2+ homeostasis in the mitochondrial matrix and ATP synthesis in the inner mitochondrial membrane are tightly controlled and interacting in a tissue (muscle)-specific manner regulated by the mitochondrial membrane potential (Wescott et al., 2019;Boyman et al., 2020;Boyman et al., 2021). Pathologically altered intracellular Ca 2+ signaling and impaired bioenergetics due to mitochondrial dysfunction contribute to both ventricular tachyarrhythmia (VT) and heart failure development, at least in part through increased production of reactive oxygen species (ROS) (Zhou and Tian, 2018;Santulli et al., 2021). Under physiological conditions at rest, mitochondrial oxidative phosphorylation and ATP synthesis are accompanied by a low level of cardiac ROS generation mainly through byproducts of the electron transport chain (Murphy, 2009). However, increased stress and enhanced cardiac performance strongly augment mitochondrial respiratory activity significantly increasing ROS production (Bertero and Maack, 2018). Although several enzymatic systems protect cardiomyocyte function through ROS neutralization (Munzel et al., 2017), chronically compromised mitochondrial bioenergetics in heart disease may shift the balance toward increased oxidation of the glutathione (GSH) redox potential (E GSH ) (Bertero and Maack, 2018), especially since an increased energy demand during sustained β-adrenergic stimulation was proposed to weaken mitochondrial antioxidant defense (Bovo et al., 2015). Notably, increased mitochondrial E GSH oxidation itself is a quantitative livecell indicator of a strongly increased ROS production, and hence may identify an early key disease process and mark the beginning of metabolic remodeling in the failing heart (Doenst et al., 2013).
Interestingly, while CPVT has been initially characterized solely as an arrhythmogenic cardiomyopathy (Yano et al., 2009), subsequently seizures originating in the brain (Lehnart et al., 2008;Lieve et al., 2019) and glucose intolerance in the Langerhans cells of the pancreas have been demonstrated in humans and mouse models (Santulli and Marks, 2015;. Although the majority of biophysically investigated RyR2 missense mutations point to a gain-of-function defect following protein kinase A phosphorylation both at the recombinant and native single-channel level (Wehrens et al., 2003;Lehnart et al., 2004;Tester et al., 2007;Lehnart et al., 2008), the role of PKA-dependent RyR2 phosphorylation remains controversial (Leenhardt et al., 2012;Wleklinski et al., 2020) since, e.g., inhibition of cardiac CaMKII by gene therapeutically applied CaMKII inhibitory peptide, but not PKA inhibitory peptide, effectively suppressed ventricular arrhythmias in a murine and an iPSC-CM model of CPTV (Bezzerides et al., 2019). However, a general consensus exists that adult cardiomyocytes and intact hearts from knockin mice expressing different gain-of-function RyR2 mutations are susceptible for significantly increased diastolic Ca 2+ leak and Ca 2+ waves following β-adrenergic stimulation (Lehnart et al., 2008;Fernandez-Velasco et al., 2009;Liu et al., 2009;Uchinoumi et al., 2010;Leenhardt et al., 2012;Borile et al., 2021). Strikingly, consistent with the human phenotype of a normal left-ventricular structure and function at rest, morphological and contractile changes have been excluded in a distinct independent RyR2-R2474S knockin mouse with a clear β-adrenergic driven phenotype of Ca 2+ triggered cardiac VT (Uchinoumi et al., 2010). Given that the altered SR Ca 2+ leak/load balance needs to be acutely compensated at the cost of increased ATP consumption during catecholaminergic stimulation (Eisner et al., 2020), CPVT-affected hearts may face a challenging metabolic state during episodes of transient intracellular Ca 2+ overload and β-adrenergic stimulation, which may lead to increased mitochondrial Ca 2+ load with excessive ROS generation and contribute to electrical and contractile cardiomyocyte dysfunction (Bers, 2014;Wescott et al., 2019;Boyman et al., 2021).
In the failing heart, ROS signaling has been established as a central process of the organic disease progression (Heymes et al., 2003;Yano et al., 2005;Guo et al., 2007;Akki et al., 2009). Increasing evidence suggests that excessive ROS production can lead to chronic cardiac pathologies, such as apoptosis, fibrosis, and hypertrophy jointly promoting the maladaptive ventricular remodeling (von Harsdorf et al., 1999;Cucoranu et al., 2005;Takimoto et al., 2005;Hori and Nishida, 2009;Santulli et al., 2021). Recently, the mechanisms that temporally and spatially link excess ROS and Ca 2+ as two principal detrimental factors have been addressed in the context of diabetic cardiomyopathy. Acute elevation of the extracellular glucose concentration induces cardiomyocyte ROS production through O-GlcNAcylation of CaMKIIδ and activation of NOX2 (NADPH oxidase type 2) resulting in increased ROS production in the cytosol but not in the mitochondria (Lu et al., 2020). On the other hand, RyR2-R2474S +/− knockin mice exhibiting catecholamine-induced intracellular Ca 2+ leak subsequently revealed mitochondrial dysfunction, increased ROS production, increased atrial RyR2 channel oxidation, and arrhythmia susceptibility . Moreover, inhibitory pharmacological treatment selectively targeting the RyR2 Ca 2+ leak or genetic inhibition of mitochondrial ROS production both prevented atrial fibrillation, indicating that RyR2 leakage and mitochondrial ROS generation may indeed constitute a self-enforcing subcellular cycle that can set off arrhythmias . However, the acute mechanism and hence direct but transient link of the catecholamine-stimulated RyR2-R2474S +/− Ca 2+ leak on mitochondrial ROS production have not been investigated previously.
Novel transgenic reporter mouse lines expressing the Grx1-roGFP GSH redox biosensor in a cardiomyocyte-restricted manner targeted to either the cytoplasm or the mitochondrial matrix have been established previously (Swain et al., 2016). Thus, cardiomyocytes from Grx1-roGFP biosensor mice allow the direct live-cell monitoring of changes in compartmentalized -cytosolic vs. mitochondrial -E GSH (Swain et al., 2016), as demonstrated acutely in response to high extracellular glucose concentrations recently (Lu et al., 2020).

Redox Biosensor Measurements and E GSH Calculation in Isolated Cardiomyocytes
Ventricular cardiomyocytes were isolated and quality controlled as described previously (Wagner et al., 2014;Peper et al., 2021). Isolated cardiomyocyte suspensions were plated onto laminincoated glass coverslips (Imaging Dish CG 1.0, Zellkontakt, Nörten-Hardenberg, Germany) at room temperature. Only quiescent brick-shaped cardiomyocytes with cross-striations visualized at high contrast (e.g. see brightfield image below) were used if they contracted regularly in capture to electrical field stimulation (EFS; 4 ms, ± 20 V biphasic, 0.5 Hz, Myopacer, IonOptix, Westwood, MA, United States). Pacing-responsive cardiomyocytes then underwent repeated EFS-trains for 10, 30, and 60 s at 0.1, 1, or 3 Hz, respectively, for up to 20 min (see Supplementary Figure 1). Isolated cardiomyocytes developing sustained spontaneous contractile activity with a frequency above >0.1 Hz after EFS-pacing or during live-cell imaging were excluded from further analysis.
Cardiomyocytes with baseline intensity ratios below 0.15 were paced in series at 0.1, 1, or 3 Hz, respectively, for 10, 30, and 60 s while the redox biosensor signal changes were continuously monitored (for a schematic of the experimental protocol, please refer to Supplementary Figure 1). The non-paced interval between each pacing train lasted 2-3 min to establish steady-state conditions. Only after the baseline redox biosensor signal has reached a stable baseline steady-state, the subsequent EFS pacing period was executed. H 2 O 2 (200 µM) was applied at the end of each experiment to confirm the maximal exogenous increase in intensity ratio (positive control) to capture the maximally oxidized state of the redox biosensor. In some experiments, dithiothreitol (DTT, 1 mM) was applied additionally after H 2 O 2 treatment to obtain the maximal decrease of the redox roGFP2 biosensor signal for calculation of the glutathione redox potential (E GSH ). Isoproterenol (100 nM) and dantrolene (5 µM) were applied acutely for 10 min or incubated for 120 min as indicated, respectively, before the start of the biosensor imaging experiment. All experiments were performed in the HEPES-buffered bath solution (composition in mM: NaCl 140, KCl 5.4, MgCl 2 1.2, CaCl 2 1.2, Na 2 HPO 4 0.3, HEPES 10, glucose 10; pH adjusted to 7.4 with NaOH).
E GSH (glutathione redox potential) calibration was performed by protocols reported previously (Gutscher et al., 2008;Swain et al., 2016). E GSH values were calculated by analysis of the fluorescence intensities at 405 and 488 nm after treatment with H 2 O 2 (maximal oxidation) and DTT (maximal reduction). The redox potential E 0 for roGFP2 was estimated to −280 mV according to protocols published previously (Dooley et al., 2004).
Hearts expressing the roGFP2 sensor were excited using the 385 nm LED and 460 nm LED within the LEDHub and the bandpass filters 385/26 and 475/23 nm (AHF, Tübingen, Germany), respectively. The light was sent to the epicardial surface and the emitted light was captured by the camera using the filter set F46-002XL (AHF, Tübingen, Germany) without the excitation filter. Ratiometric images were acquired at a frame rate of 0.1 Hz using a custom-made software to synchronize both LEDs with the Zyla camera (Andor, Oxford Instruments, Oxford, United Kingdom) through NI 9263 CompactDAQ (National Instruments, Austin, TX, United States). Imaging and cardiac electrogram recording were performed during stable sinus rhythm and after isoproterenol stimulation (33 nM). At the end of each experiment, H 2 O 2 (200 µM) was applied followed by DTT (4 mM) to calibrate and calculate E GSH .

Confocal Imaging of Cardiomyocytes
Cardiomyocyte endomembrane structures were stained using the lipophilic fluorescent dye di-8-ANEPPS (50 µM, Molecular Probes, Eugene, OR, United States) for 15 min at room temperature followed by two washing steps with buffer solution as reported previously (Wagner et al., 2014). Images were acquired with a confocal laser scanning microscope (LSM710, Carl Zeiss, Oberkochen, Germany) with a Plan-Apochromat × 63/1.4 oil Ph3 objective using 16x line averaging. For combined imaging of roGFP2 expressed in the mitochondrial matrix, the fluorescence excited at 490 nm was detected between 500 and 550 nm, and for di-8-ANEPPS, the fluorescence excited at 480 nm was detected between 550 and 650 nm. Pinhole was set to 0.8 AU.

Chemicals
All chemicals were obtained from Sigma-Aldrich (Merck KgaA, Germany) unless otherwise indicated.

Data Analysis
Data are reported as intensity ratio (I405/I488 in isolated cardiomyocytes and I385/I475 in whole hearts) or as ratio normalized to the baseline obtained at the start of each recording. Data analysis was performed with Prism 7 (GraphPad Software, San Diego, CA, United States). All data sets have passed a normality test (Kolmogorov-Smirnov). Statistical analysis between RyR2-WT control vs. RyR2-R2474S dTG cardiomyocytes was performed using paired or unpaired ANOVA with Dunnett's or Tukey's post-test as appropriate.

Baseline Mitochondrial Oxidation at Higher Beating Rates in sTG-Cardiomyocytes
Changes of the biosensor signal were initially characterized at increasing durations of pacing-evoked contractions in cardiomyocytes from sTG-mice expressing the redox reporter Grx1-roGFP2 targeted to either the mitochondrial matrix or the cytosol to confirm the imaging workflow as reported previously (Swain et al., 2016;Lu et al., 2020). Mito-Grx1-roGFP2 cardiomyocytes exhibited an apparent increase of the redox I405/I488 ratio after pacing when compared to non-paced cells which was further increased by elongating the 3 Hz EFStrains ( Figure 1A). On average, 10 s of 3 Hz pacing with optically confirmed contractile pacing-capture was already sufficient to significantly increase mitochondrial oxidization ( Figure 1B). Moreover, mitochondrial oxidation was further significantly increased by longer pacing durations both 30 and 60 s, while H 2 O 2 (200 µM) treatment confirmed the maximal level of the biosensor response ( Figure 1B). Likewise, 10 s of 1 Hz pacing considerably increased mitochondrial oxidation which was further enhanced by longer pacing intervals of 30 and 60 s (Figures 2A,B). Of note, the original recording shows that the increase in the redox I405/I488 ratio after 30 s pacing at 1 Hz returned slowly to baseline over several minutes time indicating that the effect of pacing on the biosensor signal is reversible (Figure 2A). In summary, mitochondrial oxidation was significantly increased by pacing at 3 Hz compared to 0.1 Hz beating trains for 10, 30, and 60 s ( Figure 1C).
In contrast, the cytosolic biosensor signals in cyto-Grx1-roGFP2 transgenic cardiomyocytes showed only non-significant smaller oxidation increases at 1 or 3 Hz beating, except when the 3 Hz beating rate lasted 60 s, while H 2 O 2 confirmed the maximal expected response (Figure 3). Taken together, these data indicate that a stronger increase of the beating rate (e.g., from 0.1 to 1 and 3 Hz) leads to significantly increased oxidization of the mitochondrial GSH pool in a time-dependent manner (e.g., from 10 to 30 and 60 s), whereas a significant cytosolic oxidation increase was only observed after the longest (60 s long) 3 Hz beating rate.

Preserved Transverse Tubule and Mitochondrial Network Interface in sTG-Cardiomyocytes
Accounting for ∼30% of the cell volume of adult cardiomyocytes, it is anticipated that the regular subcellular inter-sarcomeric mitochondrial distribution is highly ordered between and along the myofilaments, as well as precisely juxtaposed by the junctional SR and transverse tubule (T-tubule) membrane invaginations at both mitochondria poles, where discontinuous T-tubule/SR contacts function as Ca 2+ release sites containing highly concentrated Ca V 1.2 channels opposed to RyR2 clusters in nanometric proximity (recently reviewed in Rossini and Filadi, 2020). Whereas live-cell confocal mito-roGFP2 imaging showed the expected preserved inter-sarcomeric mitochondrial   density apparent as mature longitudinal chain architecture, the transverse-axial-tubule network furthermore revealed the expected invagination network components mainly composed of T-tubules (Figure 4) as reported previously in living mouse cardiomyocytes (Wagner et al., 2012). Apparently, the high T-tubule density regularly intersects the longitudinal mitochondria strands perpendicularly in register (Figure 4 overlay magnification). These live-cell data indicate that the transgenic mito-roGFP2 expression did not affect the mechano-sensitive electrical conduction conduits in cardiomyocytes, which is an important prerequisite for a preserved excitation-bioenergetic coupling of the mitochondrial matrix reported recently (Wescott et al., 2019;Boyman et al., 2020;Boyman et al., 2021).

Increased Mitochondrial Oxidation in RyR2-R2474S dTG-Cardiomyocytes
Next we extended the post-pacing mitochondrial E GSH imaging to dTG-cardiomyocytes from mouse hearts additionally expressing the heterozygous RyR2-R2474S mutated tetrameric Ca 2+ release channels vs. RyR2-WT control cells. Apparently, relatively large changes in mitochondrial oxidation were readily evidenced in RyR2-R2474S dTG-cardiomyocytes compared to RyR2-WT control ( Figure 5A) particularly following 30 and 60 s of 1 Hz ( Figure 5B) and 3 Hz pacing-evoked beating ( Figure 5C). Together these data suggest that mitochondrial oxidization is significantly increased in heterozygous RyR2-R2474S dTG-cardiomyocytes by the identical 1 and 3 Hz beating rate pacing-trains compared to RyR2-WT control cells.

β-Adrenergic Stimulation Exacerbates Mitochondrial Oxidization in RyR2-R2474S dTG-Cardiomyocytes
An increased sympathetic tone, exercise, and an increased heart rate provoke the characteristic cardiac CPVT phenotype leading to VT episodes in humans (Lehnart et al., 2004;Danielsen et al., 2018;Baltogiannis et al., 2019). Consequently, isolated cardiomyocytes were exposed to selective β-adrenergic stimulation (isoproterenol 100 nM) and pacing ( Figure 6A). First, mitochondrial oxidation was significantly increased by isoproterenol-stimulation in WT control ( Figure 6B) and RyR2-R2474S dTG-cardiomyocytes ( Figure 6C) when compared to the untreated control cardiomyocytes, respectively. Notably, the isoproterenol-stimulation robustly increased mitochondrial oxidation already at post-30 s beating at 3 Hz in RyR2-R2474S dTG-cardiomyocytes compared to control ( Figure 6C). Second, isoproterenol-treatment increased mitochondrial oxidation to a significantly larger extent in RyR2-R2474S dTG-cardiomyocytes compared to RyR2-WT control cells at the 3 Hz beating rate both after 30 and 60 s ( Figure 7A) and even already at the lower 1 Hz beating rate after 60 s ( Figure 7B). Third, mathematical quantification of the E GSH revealed a significant depolarization/oxidation of over 10 mV following isoproterenol-treatment in RyR2-R2474S dTG-cardiomyocytes and a similarly increased oxidation at baseline and isoproterenol-stimulated between RyR2-R2474S dTG-cardiomyocytes and RyR2-WT control cells (Figure 8). Baseline E GSH was not different between RyR2-WT control and RyR2-R2474S dTG-cardiomyocytes. Mean calculated E GSH values ± SEM (in mV) were −290.9 ± 2.2 (n = 8) and −288.8 ± 3.4 (n = 9), respectively. As the level of mitochondrial oxidation was overall robustly increased after isoproterenol-stimulation when compared to non-treated control RyR2-R2474S dTG-cardiomyocytes (Figure 7), β-adrenergic stimulation clearly exacerbated the mitochondrial glutathione oxidization.

Isoproterenol Strongly Increases Mitochondrial Oxidation in Intact RyR2-R2474S dTG-Hearts
To monitor mitochondrial oxidation in the intact beating heart, RyR2-WT control and RyR2-R2474S dTG-hearts were Langendorff-perfused and the ventricular left and right epicardial surface imaged with simultaneous surface field-electrogram rhythm recordings. While color-coded mapping of the RyR2-WT dTG-heart apparently captured no major ventricular mitochondrial E GSH change following acute β-adrenergic stimulation by isoproterenol (33 nM) in RyR2-WT control hearts (Figure 9B), a robust mitochondrial oxidation increase was evident in the beating RyR2-R2474S dTG-heart ( Figure 9A). Meanwhile, both genetic heart groups showed the expected maximally increased oxidation by H 2 O 2 vs. a maximally decreased oxidation after adding DTT (Figures 9A-C).
Comparing the isoproterenol-stimulated E GSH changes confirmed a significant mitochondrial oxidation increase in RyR2-R2474S dTG-hearts, which was, however, not observed in RyR2-WT ventricles ( Figure 9D). Together, these results significantly extend and further confirm the findings mainly documented in isolated RyR2-R2474S dTG-cardiomyocytes, underlining a significantly more oxidized mitochondrial state in beating dTG-hearts following β-adrenergic stimulation evidenced by the pronounced changes of the redox signal I385/I475 ratio monitoring acute E GSH tissue changes.

Dantrolene Prevents Mitochondrial Oxidation in RyR2-R2474S dTG-Cardiomyocytes
Dantrolene is used clinically as acute RyR1 channel blocker targeted to skeletal muscles and other tissues as emergency intervention to treat patients who develop life-threatening malignant hyperthermia episodes induced by certain anesthetic drug compounds during surgical interventions. Recent studies demonstrated that dantrolene may also cause beneficial antagonistic effects against the chronic cardiac RyR2 channel Ca 2+ leak in human diseased cardiomyocytes isolated from failing hearts (Hartmann et al., 2017). Therefore, the acute potential of the large-conductance channel-inhibitory efficacy of dantrolene against mitochondrial oxidation was investigated in RyR2-R2474S dTG-cardiomyocytes following β-adrenergic stimulation (isoproterenol 100 nM). Strikingly, dantrolene pre-treatment (5 µM for 2 h) significantly decreased the large mitochondrial oxidation after 3 Hz 60 s-beating and non-significantly after 3 Hz 30 s-pacing (Figure 10). In summary, these data suggest that a pathologically increased mitochondrial oxidation in isoproterenol-stimulated RyR2-R2474S cardiomyocytes can be partly prevented by pharmacological dantrolene treatment.

DISCUSSION
This work has examined how mitochondrial E GSH oxidation is increased by essential factors in cardiomyocytes with important physiological and pathophysiological consequences that converge at the dynamic interface between RyR2 channel function, sympathetic catecholaminergic stimulation, and contractile  Numbers indicate the number of cells used (five cells from two mice, each). Statistical analysis was performed by a paired (control vs. isoproterenol) or unpaired (RyR2-WT control vs. RyR2-R2474S dTG) Student's t-test. **P = 0.001; *P = 0.02; * # P = 0.01; n.s., non-significant. The baseline heart rate for RyR2-WT and RyR2-R2474S was 340 bpm and 275 bpm, respectively. In-between, we performed electrical pacing with 600 bpm and 900 bpm (red lines). Red arrows refer to time points for representative images shown in (A,B). (D) Paired dot-line plot showing E GSH calculated before and after application of isoproterenol each for RyR2-WT and RyR2-R2474S dTG-hearts. Each dot represents one heart/experiment. Paired Student's t-test; *P = 0.013; n.s., non-significant.
activity. Overall, we find that a significant increase following β-adrenergic stimulation significantly changes mitochondrial E GSH in dTG-cardiomyocytes and hearts, which was further dependent on both the presence of the RyR2-R2474S missense mutation and faster beating rates. Importantly, dantrolene treatment of RyR2-R2474S dTG-cardiomyocytes results in a pronounced reduction of this increased mitochondrial oxidation. We discuss our findings on these interrelated RyR2-R2474S regulatory and E GSH driven mitochondrial mechanisms in the context of previously published reports.
Recent quantitative imaging studies established that an increase of mitochondrial Ca 2+ uptake was necessary to drive inner mitochondrial membrane depolarization and enhanced ATP synthesis in a heart-specific and previously unknown voltage-dependent fashion (Wescott et al., 2019;Boyman et al., 2020;Boyman et al., 2021). This contrasts with heart disease where alterations of mitochondrial E GSH may profoundly shift organelle metabolism and thus cardiac bioenergetics (Bertero and Maack, 2018). Beyond bioenergetics, increased mitochondrial E GSH oxidation revealed a strongly increased ROS production, which might be of immediate relevance for heart disease progression and metabolic remodeling (Doenst et al., 2013). In analogy, increased mitochondrial oxidation and E GSH depolarization in RyR2-R2474S cardiomyocytes and hearts indicate a strongly boosted ROS generation during faster beating rates concurrent with β-adrenergic stimulation.
Interestingly, increased SR Ca 2+ leak and mitochondrial dysfunction have been demonstrated to enhance ROS production in heart disease previously (Zhou and Tian, 2018;Santulli et al., 2021). Remarkably, mitochondrial ROS scavenging attenuated the effects of augmented RyR2 activity leading to proarrhythmic Ca 2+ waves in Calsequestrin2-null mice serving as a model for CPVT (Hamilton et al., 2020). In line with this view, ROS scavenging by mito-TEMPO was shown FIGURE 10 | Dantrolene diminishes mitochondrial oxidation in β-adrenergic stimulated RyR2-R2474S dTG cardiomyocytes. Dot plot showing the individual and mean ± SEM values of the normalized I405/I488 nm ratio recorded after 3 Hz beating for 10, 30, and 60 s in the presence of isoproterenol (100 nM) either with or without dantrolene pre-treatment (5 µM for 2 h). Triangles and squares represent individual experiments ± dantrolene as given by the legend. Cell/heart numbers are indicated. Unpaired one-way ANOVA with Tukey's post-test. ***P < 0.0002; n.s., non-significant.
to reduce the number of Ca 2+ waves also in cardiomyocytes from rabbit hearts (Bovo et al., 2012;Cooper et al., 2013). As isoproterenol-stimulated RyR2-R2474S cardiomyocytes exhibit an over threefold increased Ca 2+ spark frequency that additionally sustains regenerative intracellular Ca 2+ waves, here the observed robust increase in mitochondrial oxidation will depend both on acute catecholamine stimulation and the beating rate-dependent extent and complex spatiotemporal dynamics of the cell-wide diastolic Ca 2+ leak reported previously (Lehnart et al., 2008;Xie et al., 2015;Sadredini et al., 2021). However, even under physiological conditions, an increased mitochondrial oxidation was evidenced at higher beating rates in sTG-cardiomyocytes albeit much more pronounced in RyR2-R2474S dTG-cardiomyocytes. For the latter, the faster beating rate clearly led to mitochondrial E GSH oxidation and this was most strongly increased after β-adrenergic stimulation.
Tight control of nanomolar diastolic Ca 2+ levels is essential for normal cardiac function depending on the precise balance between cytosolic Ca 2+ influx and efflux (Eisner et al., 2020). If the local control mechanisms become compromised, cardiomyocytes will develop arrhythmia and hypercontraction from cytosolic Ca 2+ overload. This balance is disturbed when the diastolic Ca 2+ spark frequency is significantly increased, a phenomenon linked molecularly to RyR2 channel gainof-function defects (Cheng and Lederer, 2008;Bers, 2014). Interestingly, more than a dozen RyR2 mutations originally identified in CPVT patients have been shown to increase Ca 2+ leak (Jiang et al., 2002;Wehrens et al., 2003;Lehnart et al., 2004;Kontula et al., 2005;Tester et al., 2007;Lehnart et al., 2008;Marjamaa et al., 2011;Shan et al., 2012). However, only two knockin models were confirmed to exhibit exercisetriggered VTs, RyR2-R2474S +/− (Lehnart et al., 2008) and RyR2-R4496C +/− (Priori and Napolitano, 2005;Liu et al., 2006), and are hence reminiscent of the human arrhythmia phenotype diagnosed by exercise stress testing. In line with the in vivo pathology, RyR2-R2474S +/− cardiomyocytes exhibited an overall normal diastolic Ca 2+ spark behavior at rest, but a strong spark frequency increase occurred after isoproterenol treatment (Lehnart et al., 2008). In this context, increased diastolic Ca 2+ extrusion mainly by SERCA2a and NCX will necessarily consume additional ATP at rest at the cost of concomitantly increased ROS generation (Bers, 2002(Bers, , 2014. Thus, our data further link increased catecholaminergic RyR2 channel dysfunction in a murine CPVT model to acute mitochondrial oxidation. In the present study, we monitored the effect of increased contractile activity on E GSH in the mitochondrial matrix, while the former process has been long known to correlate with mitochondrial metabolic activity and ROS generation (Gudbjarnason et al., 1962). Since a similar extent of E GSH change was excluded in the cytoplasm, NOX2 seems unlikely as ROS source here (Santos et al., 2011). In another context, high extracellular glucose concentrations increased E GSH in the cytosol through NOX2 but not in the mitochondrial matrix (Lu et al., 2020). In contrast, we found that mitochondrial oxidization depended on the duration and frequency of dTG-cardiomyocyte beating. This finding is in line with reported changes of NADH in response to a stepwise increase of the beating frequency from 0.1 to 3.3 Hz in guinea-pig cardiomyocytes (Jo et al., 2006). As the increase in the mitochondrial E GSH following beating was observed both in RyR2-WT and RyR2-R2474S dTG-cardiomyocytes, a relation with increased SR Ca 2+ leak seems plausible after pacing-evoked contractile activity reported previously (Satoh et al., 1997). Finally, blocking RyR2 Ca 2+ leak with dantrolene diminished E GSH oxidation as hypothesized previously (Hartmann et al., 2017).
A recent study demonstrated that Ca 2+ leak increased mitochondrial ROS production in cultured WT rat cardiomyocytes transfected for 48 h with the OMM-HyPer mito-ROS biosensor (Hamilton et al., 2020). An increased ROS production has also been demonstrated during increased beating in healthy cardiomyocytes with the fluorescent probe dichlorodihydrofluorescein, which is confirmed by our sTGcardiomyocyte data (Heinzel et al., 2006). In rat trabeculae, NADH oxidation was observed at 0.25-2 Hz after 60 s with pronounced ATP hydrolysis (Brandes and Bers, 2002). A similar time frame in response to contractile activity was observed in the present study, where the maximal change of the biosensor signal occurred within 2-3 min after beating. This further agrees with the reported change of the redox signal observed in cardiomyocytes in the same biosensor cardiomyocytes after a high glucose or pH challenge (Lu et al., 2020;Kreitmeier et al., 2021). Since the observed redox changes occurred only after completion of the given beating period, it is suggested to correlate further with the aftercontraction related metabolic activity observed frequently in isoproterenol-stimulated RyR2-R2474S dTG-cardiomyocytes.
Importantly, our report provides the first evidence that E GSH changes happen under quasi physiological conditions in the intact beating heart as well. Using the genetically encoded reporter targeted to mitochondria allowed to monitor the average E GSH from the whole ventricular, anterior heart surface ( Supplementary Videos 1, 2) and to perform a ratiometric imaging excluding any major interference due to cardiac contractions. In comparison to the first description of the glutathione redox sensor mouse lines, we were able to increase the ratio differences from maximal oxidization to reduction to comparable signal-to-noise values to single-cell measurements (see Figures 5, 9). This increase in signal amplitude was the key for the detection of the comparable relatively small steps in oxidization during β-adrenergic stimulation, which we could only detect in the intact hearts of dTG mice expressing the RyR2-R2474S mutated channels. However, in contrast to isolated cardiomyocytes, we could not detect a change in oxidation by additional faster pacing in the spontaneously beating Langendorff hearts. One possible explanation may be that these changes are in comparison subtle and thus still not detectable in this setting. The other is that we focused on the effects of isoproterenol and such changes would occur only after prolonged phases of pacing which we could not implement in these experiments.
In summary, we have shown that contractile activity induced by EFS-pacing in isolated cardiomyocytes increased the mitochondrial E GSH to more oxidized levels in a time-and frequency-dependent manner. The increase was most pronounced after β-adrenergic stimulation and in cardiomyocytes expressing the human RyR2-R2474S mutated tetrameric channel. The catecholaminergic E GSH increase, however, was significantly attenuated by the pharmacological RyR2 channel antagonist dantrolene. Likewise, we observe a larger increase in the mitochondrial E GSH toward more oxidized levels in spontaneously beating hearts expressing the RyR2-R2474S mutated channels after β-adrenergic stimulation as compared to control hearts. Hence, we suggest that the RyR2-R2474S mutation induces a metabolic burden in cardiomyocytes through excess ROS generation, especially after β-adrenergic stimulation, tipping the balance from a compensated to a high energetic cost and acutely increased diastolic Ca 2+ leak, a multifactorial process, which may acutely contribute to the development of the arrhythmogenic CPVT phenotype as proposed for calsequestrin2-null mice previously (Hamilton et al., 2020).

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional IACUC. This study has not used in vivo examination of mice but ex vivo investigations, which are not subject to an ethics protocol at our institution.

AUTHOR CONTRIBUTIONS
JW and AW performed the research and analyzed the data. JW, TB, DK, and SL wrote the manuscript. DK contributed to the biosensor mouse models and analyzed the data. JW, SL, TB, and GH designed the study. All authors approved the manuscript.

FUNDING
This work was supported by grants from Deutsche Forschungsgemeinschaft SFB1002 to SL (projects A09 and S02), TB (A14), and GH (project D01), Germany's Excellence Strategy (EXC 2067/1-390729940) to SL, and SPP1926 to TB and SL. JW and AW were supported by DZHK (German Centre for Cardiovascular Research) Partner Site GÃűttingen. SL is a principal investigator of DZHK.

ACKNOWLEDGMENTS
Excellent technical assistance by Birgit Schumann is gratefully acknowledged.