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Ca2+ fluxes between adjacent organelles are thought to control many cellular processes, including metabolism and cell survival. In vitro evidence has been presented that constitutive Ca2+ flux from intracellular stores into mitochondria is required for basal cellular metabolism, but these observations have not been made in vivo. We report that controlled in vivo depletion of cardiac RYR2, using a conditional gene knock-out strategy (cRyr2KO mice), is sufficient to reduce mitochondrial Ca2+ and oxidative metabolism, and to establish a pseudohypoxic state with increased autophagy. Dramatic metabolic reprogramming was evident at the transcriptional level via Sirt1/Foxo1/Pgc1α, Atf3, and Klf15 gene networks. Ryr2 loss also induced a non-apoptotic form of programmed cell death associated with increased calpain-10 but not caspase-3 activation or endoplasmic reticulum stress. Remarkably, cRyr2KO mice rapidly exhibited many of the structural, metabolic, and molecular characteristics of heart failure at a time when RYR2 protein was reduced 50%, a similar degree to that which has been reported in heart failure. RYR2-mediated Ca2+ fluxes are therefore proximal controllers of mitochondrial Ca2+, ATP levels, and a cascade of transcription factors controlling metabolism and survival.
Intracellular Ca2+ fluxes regulate an enormous number of processes with the specificity of responses often being ensured by spatial limitation of Ca2+ ions and proximity to targets. Frequently, this takes the form of Ca2+ signals within the nanoscale space between adjacent organelles (1). For example, in vitro studies have shown that Ca2+ flux through channels such as ryanodine receptors and IP3 receptors mediate privileged communication between endoplasmic reticulum/sarcoplasmic reticulum (ER/SR)3 and mitochondria and that this paces cellular metabolism by stimulating oxidative ATP production via interaction with TCA cycle enzymes (2–4). Work from Foskett and colleagues (5) recently demonstrated that knocking out IP3 receptors in DT40 cells causes an energy-deficient state and mTor-independent autophagy. This concept has not been extended to other cell types or to the in vivo situation.
Ryanodine receptor Ca2+ channels (e.g. RYR2) have been observed in close proximity to mitochondria, and beat-to-beat calcium transients have been observed in cardiomyocyte mitochondria (6–8). Furthermore, we and others (9–12) have demonstrated in vitro that RYR2-mediated Ca2+ flux supports ATP levels in other cell types. In pancreatic β-cells, blocking RYR2 also induces calpain-10-dependent, caspase-3-independent, programmed cell death that is associated with presenilin-1-dependent up-regulation of hypoxia-inducible factor 1β (10, 11). To date, analysis of cellular energetics and survival in the context of reduced ER/SR Ca2+ channel expression has not been extended to an in vivo system. Ideally, the hypothesis that intracellular Ca2+ release is required to sustain energy metabolism in vivo would be tested using tissue-specific, inducible deletion of ER/SR Ca2+ channels.
The heart is an ideal model to test this hypothesis because it is a tissue with high metabolic demands. Notably, a single intracellular Ca2+ release channel type, RYR2, is far in excess of other analogous Ca2+ channels, meaning that ER/SR Ca2+ release may be significantly reduced with a single genetic mutation. Examination of cardiomyocytes with specific Ryr2 gene loss-of-function is also of interest because RYR2 protein levels are known to decline as much as 50% with age, in parallel with an increased risk for heart failure (13, 14). RYR2 expression, structural organization, and function are reduced in heart failure, cardiac hypertrophy, and ischemic cardiomyopathy (15–18). It is unclear whether RYR2 loss is sufficient to cause any of the features of heart failure, including contractile dysfunction, hypertrophy, fibrosis, inflammation, transcriptional reprogramming, an energy-starved state of metabolic inflexibility, and cardiomyocyte death (19–21).
In the present study, we demonstrate that acute in vivo deletion of the primary ER/SR Ca2+ release channel reduced ATP, blunted oxidative mitochondrial metabolism, and led to inflexibility of energy substrate utilization. We provide evidence that the reduced energy state associated with loss of RYR2-mediated Ca2+ flux activates hypoxia-inducible factors, and switches off transcriptional control hubs that govern a range of key metabolic pathways. We further observed atypical calpain-10-dependent cell death and cardiac hypertrophy in mice following Ryr2 knock-out. Acute in vivo deletion of cardiomyocyte Ryr2 reproduces many hallmarks of heart failure, suggesting a causal, upstream role for this channel in the pathogenesis of this disease.
Ryr2 “floxed” mice were generated as described (22). Tamoxifen-inducible, cardiomyocyte-specific Ryr2 knock-out mice were generated by crossing mice harboring Ryr2 alleles containing flanking loxP sites (C57Bl6 Ryr2flox/flox mice) with mice expressing inducible Cre-recombinase under the control of the α-myosin heavy chain promoter (C57Bl6 mer-Cre-mer mice) (23). Tamoxifen was injected into the intraperitoneal cavity of 8–16-week-old Ryr2flox/flox:mer-Cre-mer mice or controls for three consecutive days at 3 mg per 40 g of body weight. Protocols were approved by the University of British Columbia Animal Care Committee.
Myocardial substrate utilization was measured in working hearts as detailed elsewhere (24–26). Briefly, working hearts were perfused with Krebs-Henseleit solution containing 11 mm glucose, 1.5 mm lactate, 0.15 mm pyruvate, 0.6 mm palmitate, and 20 milliunits/ml insulin. Glycolysis, as well as myocardial rates of oxidation of palmitate, glucose, and lactate were determined by the quantitative collection of 3H2O or 14CO2 produced by hearts perfused with either KH solution containing [9,10-3H]palmitate and [U-14C]lactate or [U-14C]glucose, and 5-[3H]glucose.
Paraformaldehyde-fixed ventricular tissue sections were stained with H&E and Masson's trichrome to assess fibrosis. Apoptosis and cell death of adult ventricular myocytes was assessed in heart tissue sections using the TUNEL assay from Roche Applied Science according to the manufacturer's instructions. Images were taken with a Retiga-2000R camera (Q-Imaging). TUNEL-positive cells were normalized to tissue area.
Live cell imaging was conducted on cardiomyocytes isolated from cRyr2KO and control mice using Langendorff reverse perfusion to introduce collagenase in to the cardiac vasculature as detailed elsewhere (27). To assess cytosolic Ca2+ levels, isolated cardiomyocytes were loaded with 5 μm Fura-2-AM (Invitrogen) for 30 min and washed twice for 15 min before imaging. Fura-2-AM-loaded cells were excited at 340 and 380 nm and measured for emission at 510 nm using a imaging system equipped with a 10× air objective (0.3 numerical aperture) on a Zeiss Axiovert-200 M microscope with a CoolSnapHQ2 CCD Camera (Intelligent Imaging Innovations, Denver, CO). Ratiometric images were quantified using Slidebook software (Intelligent Imaging Innovations) to measure relative cytosolic Ca2+ (28). To assess mitochondrial Ca2+ levels, isolated cardiomyocytes were loaded with 5 μm Rhod-2-AM (Invitrogen) for 30 min and washed for 15 min before imaging. In some studies, 5 μm Mitotracker Deep Red (FM) (Invitrogen) was co-loaded. Loaded cells were imaged using the Axiovert-200 M microscope described above and a 40× oil objective (1.3 numerical aperture). Rhod-2 fluorescence was used to measure relative mitochondrial Ca2+ (29), and Mitotracker was used to assess relative mitochondrial content for normalization. The co-loading of these dyes did not alter Ca2+ or cell viability (data not shown). All live-cell microscopy occurred within 8 h of isolation, and only rod-shaped cardiomyocytes with typical cell morphology were considered in analysis.
Total RNA was isolated from heart tissue using TRIzol, followed by cleanup using the RNeasy kit (Qiagen). After reverse transcription (SuperScript III; Invitrogen), TaqMan quantitative RT-PCR was conducted using probes from Applied Biosystems and PerfeCTa quantitative PCR SuperMix (Quanta) on a StepOnePlus thermocycler (Applied Biosystems). SYBR Green quantitative RT-PCR was conducted using PerfeCTa SYBR Green quantitative PCR SuperMix (Quanta). Relative gene expression changes were analyzed by the 2−ΔCt method and plotted in normalized relative units. Hypoxanthine-guanine phosphoribosyltransferase and cyclophilin were used as internal controls after ensuring that they were not altered in cRyr2KO cardiomyocytes. Primer details are available in supplemental Tables S1–S3.
Western blot experiments were performed on lysates from mechanically disrupted hearts, homogenized, and sonicated in ice-cold lysis buffer. Samples were quantified, boiled with loading dye, and 15–50 μg of protein was used in SDS-PAGE electrophoresis. Proteins were then transferred to PVDF membranes using standard semi-dry transfer (for protein smaller than 120 kDa) or wet transfer (for proteins larger than 120 kDa) and subsequently treated with targeted primary and horseradish peroxidase-conjugated secondary antibodies. Bands were visualized using an enhanced chemiluminescence detection kit and quantified by densitometry. Antibody details can be found in supplemental Table S4. Cardiac ATP levels were assessed using standardized high-performance liquid chromatography methods (30).
Data are expressed as mean ± S.E. unless otherwise indicated. Results were considered statistically significant when p ≤ 0.05 using the Student's t test. All experiments were repeated on at least three cRyr2KO mice and at least three of their tamoxifen-injected littermate controls.
cRyr2KO mice showed normal Ryr2 expression until they were injected with tamoxifen, a synthetic agonist of the modified estrogen receptor:Cre fusion protein (Mer-Cre-Mer) under the control of a αmhy6 promotor, which allowed temporally controlled targeted gene deletion of Ryr2 in cardiomyocytes. Tamoxifen-injected littermate control mice (Ryr2flox:flox) were used throughout the study; thus, any differences between cRyr2KO and control mice cannot be attributed to tamoxifen. In our hands, this model consistently displays a >90% decrease in whole heart Ryr2 mRNA after only 3 days of tamoxifen injections (Fig. 1A) (22). This leads to a time-dependent reduction in RYR2 protein levels to 51 ± 21% at 4 days post-tamoxifen and 24 ± 22% at 10 days post-tamoxifen in cRyr2KO mouse hearts (22). No effects were seen in the expression of Ryr1, Ryr3, or any of the IP3 receptor Ca2+ release channels (Fig. 1A). As expected for cells lacking their major Ca2+ release channel and without compensatory up-regulation of other SR channels, steady-state cytosolic Ca2+ was reduced in isolated, acutely cultured, non-contracting cRyr2KO cardiomyocytes (Fig. 1B). We predicted that the loss of RYR2 channels juxtaposed with mitochondria would result in lower average Ca2+ levels in mitochondria. Indeed, we observed a significant decrease in steady-state mitochondrial Ca2+ in isolated cRyr2KO cardiomyocytes (Fig. 1C). This result was not dependent on the normalization of the Rhod2 signal to Mitotracker fluorescence (used to control for mitochondrial number). Together, these studies suggest that loss of RYR2 in cardiomyocytes reduces Ca2+ flux from the SR into the mitochondria via the cytosol.
In vitro studies have shown that Ca2+ flux from ER/SR stimulates metabolism in adjacent mitochondria (2, 3, 12). We used cRyr2KO hearts to test the hypothesis that SR-to-mitochondria Ca2+ fluxes are required for ATP production and oxidative metabolism in vivo. Indeed, ATP levels were decreased in flash-frozen cRyr2KO hearts (Fig. 2A). Consistent with this model, metabolic profiling of cRyr2KO working hearts revealed significant reductions in glucose, palmitate, and lactate oxidation (Fig. 2B). These effects were specific to mitochondrial metabolism, as we did not observe a reduction in the rate of glycolysis (Fig. 2C). In fact, glycolysis was increased in cRyr2KO working hearts, suggesting a compensatory mechanism and a switch toward a heart failure-like phenotype (19). These observations were made in hearts with identically oxygenated perfusate and with similar amounts of coronary artery perfusion indicating that the cardiomyocytes had similar access to metabolic substrates (Fig. 2D). Together, these data provide evidence that Ca2+ flux through RYR2 channels is essential for basal mitochondrial metabolism in the heart.
Our previous in vitro work on pancreatic β-cells demonstrated that the chemical inhibitor of RyRs, ryanodine, caused a state of ATP depletion and a presenilin-1-dependent induction of hypoxia-inducible factor (11). The hypoxia-inducible factor system is a master sensor of oxidative energy use and may therefore coordinate the response to energy deprivation. Here, we also observed an increase in the levels of presenilin-1 and presenilin-2, as well as Hif1α and Hif1β (Fig. 3, A and B). A dramatic loss of uncoupling protein gene expression suggested that cRyr2KO cardiomyocytes were reprogrammed to conserve and salvage energy (Fig. 3C). Similar to the in vitro observations of others (5), we observed increased autophagy, based on increased levels of LC3-II, even under conditions of nutrient availability (Fig. 3D). Together, these observations suggest that cardiomyocytes display complex signs of energy stress and compensation when ATP levels are reduced following RYR2 reduction.
Given the severity of the metabolic changes, we next tested whether core transcriptional pathways had been affected by the reduction in RYR2-mediated Ca2+ fluxes. We focused our efforts on so-called “master regulator” pathways that modulate the expression of numerous metabolic genes in heart and other tissues. For example, we observed down-regulation of Sirt1 and Foxo1 (Fig. 4A), genes that coordinate cardiac stress responses and metabolism (31–33). Heart failure is associated with metabolic inflexibility and a switch away from fatty acid metabolism (34). Ca2+ signals can act via calcineurin-dependent activation of the Ppargc1a (Pgc1α)/Pparα system to sustain fatty acid oxidation (35, 36). In hearts with reduced RYR2, we observed a striking down-regulation of Ppargc1a and Pparα as well as Pparγ (Fig. 4B). Phosphoenolpyruvate carboxykinase 1 (Pck1), a target of Pgc1α, was virtually eliminated from cRyr2KO hearts (Fig. 4G). The Pparg target gene Cebpa was also robustly inhibited (Fig. 4C). Carbohydrate responsive element-binding protein (Chrebp), a master regulator of complex lipid metabolism, was dramatically decreased in cRyr2KO hearts (Fig. 4C). Accordingly, expression of ATP-citrate lyase (Acly) and acetyl-CoA carboxylase β (Acacb) were decreased significantly; expression of stearoyl-coenzyme A desaturase 1 (Scd1) was virtually eliminated (Fig. 4, D and E). The reduction in lipogenic enzymes was associated with down-regulation of fat transporters (Fig. 4F). At the same time, we observed a dramatic decrease in expression of genes encoding hormone sensitive lipase (Hsl), adiponutrin (Adpn), and adipose triglyceride lipase (Atgl) (Fig. 4H). Atgl is essential for degrading lipid droplets and mice lacking this gene have fatty, hypertrophied, and failing hearts (37). Interesting, we noted reduced expression of the genes encoding the GLUT4 glucose transporter and phosphoenolpyruvate carboxykinase 1 (Fig. 4, G and I). Together, these data suggest that the use of lipids and other substrates by the cRyr2KO heart may be abrogated and provide novel insight into the link between Ca2+ and metabolism.
Heart failure is associated with an increase in cardiomyocyte death, which can be apoptotic, non-apoptotic, and/or associated with ER stress depending on the model (21). cRyr2KO hearts contained numerous TUNEL-positive cells, which were virtually absent in controls (Fig. 5, A and B). This cell death was not caused by ER stress because we observed a decrease in CHOP expression and other ER stress markers (Fig. 5, C and D). In some cell types, RYR2 hyperactivity contributes to classical caspase-3-dependent apoptosis (28). However, we discovered previously that inhibiting RYR2-mediated Ca2+ flux with ryanodine caused caspase-3-independent programmed cell death (10). cRyr2KO hearts did not exhibit a significant increase in caspase-3 cleavage (Fig. 5E), confirming our hypothesis that RYR2 suppresses an atypical form of non-apoptotic cell death. We observed a reduction in the expression of the prosurvival gene Bcl2, perhaps further contributing to cell death (Fig. 5F). We have previously shown that calpain-10 mRNA and small molecular weight isoforms of calpain-10 are up-regulated in cells with depressed energy state resulting from ryanodine treatment (10, 11). Two protein isoforms of calpain-10 were significantly increased in heart tissue from cRyr2KO mice (Fig. 5G). These bands likely represent calpain-10 protein isoforms and cleavage products, as they are not observed in tissue from calpain-10 knock-out mice (10). No effects were observed on the levels of calpain-1 or calpain-2 protein in cRyr2KO hearts. Thus, loss of Ryr2 function activates a conserved form of calpain-10-associated programmed cell death in multiple tissues, including the heart.
Cell death can be accelerated when growth factor signaling pathways are down-regulated, and hypoxia-like conditions have been linked to insulin resistance (38). cRyr2KO hearts had a significant reduction in the mRNA and protein levels of the insulin receptor (Fig. 5, H and K). A trend toward increased Ptpn1 expression further suggested a suppression of insulin signaling (Fig. 5H), and this was supported by a significant reduction in ERK activity and prosurvival AKT phosphorylation at threonine 308 (Fig. 5K). Cardioprotective adiponectin and leptin signaling systems were down-regulated (Fig. 5, I and J). These data suggest that survival pathways are reduced following Ryr2 reduction.
cRyr2KO mice represented an ideal tool to test the hypothesis that acute cardiomyocyte stress and dysfunction is sufficient to induce cardiac hypertrophy. We observed an increase in gross heart volume and mass (Fig. 6A), and heart fibrosis in cRyr2KO mice (Fig. 6B), already evident 10 days after tamoxifen injection. Many chronic models of cardiac hypertrophy result in a re-expression of fetal genes, including cardioprotective growth factors. In our acute model, brain natriuretic peptide transcription was significantly increased, and adult β-myosin heavy chain gene (Mhy6) was attenuated (Fig. 6, C and D), but we did not find a simultaneous up-regulation of the fetal β-myosin heavy chain gene (Mhy7; Fig. 6D). Interestingly, we observed a reduction of cleaved Notch1 (Fig. 6E), which plays a key role in maintaining cardiac progenitors (39). Heart failure can be associated with an increase in cardiac inflammation and the local release of proinflammatory cytokines (40). We observed a dramatic 20-fold increase in interleukin-6 transcription in cRyr2KO hearts (Fig. 6F). Ryr2 may therefore be upstream of some, but not all, aspects of cardiac inflammation.
Next, we examined pathways that could translate the effects of stress into a hypertrophic pathology. Atf3, a stress gene activated by ischemia/hypoxia (41, 42), was increased ~2.5-fold in cRyr2KO hearts (Fig. 6G). Cardiac Atf3 overexpression is sufficient to induce cardiomyopathy, hypertrophy, and myocyte death (43). We also observed a dramatic loss of several genes negatively regulated by Atf3, including Glut4 (Fig. 4I). cRyr2KO also resulted in a near complete loss of Klf15 (Fig. 6H), which, based on knock-out studies, is sufficient to induce heart failure and fibrosis (44, 45). Klf15 is a positive regulator of Ucp1 and Pparg (46), genes that were decreased in cRyr2KO hearts. Thus, acute Ryr2 knock-out and the concomitant energy-deficient state have profound effects on transcriptional hubs, controlled, at least in part, by hypoxia-inducible factors, Klf15, Atf3, and Foxo1/Sirt1.
Throughout this study, we assessed the survival of cRyr2KO mice. Despite molecular and/or physical compensation, cRyr2KO mice invariably met their humane end point after a period that could range from days to weeks, as we have shown previously (22). Thus, the cardiac expression of the Ryr2 gene is essential for survival in adult mice.
The purpose of this study was to test the hypothesis that Ca2+ flux from intracellular stores, in this case through cardiomyocyte SR RYR2 channels, is required to maintain cellular energy homeostasis and survival in vivo. We used tissue-specific inducible gene ablation to examine the in vivo functions of Ryr2 in the adult mouse heart. This model circumvents the embryonic lethality of Ryr2−/− mice (47), as well as the possible long term compensation from related Ca2+ channels reported in Ryr2+/− mice (48). Our in vivo mouse studies support the concept, previously proposed after in vitro studies (5), that intracellular Ca2+ release paces mitochondrial metabolism and protects cells from atypical programmed cell death caused by energy depletion (Fig. 6I). Our data extend this concept to include the Ca2+-dependent control of key transcription factor networks that modulate metabolic substrate utilization. Our results also demonstrate that partial loss of cardiomyocyte RYR2 protein is sufficient to recapitulate many of the characteristics of human heart failure, providing new mechanistic insight into this devastating condition.
In the physiological state, the cardiomyocyte is always working. Our analysis of metabolism and survival were conducted in vivo and in ex vivo working heart models. The single-cell Ca2+ measurements were the only experiments in this study not conducted in working cardiomyocytes. Nevertheless, these measurements provide some interesting information and suggest a role for RYR2 channels in cardiomyocyte Ca2+ homeostasis. Taken at face value, these data suggest that basal Ca2+ flux through RYR2 channels normally supports a significant component of resting Ca2+ levels in both the cytosolic and mitochondrial compartments of isolated cardiomyocytes. Indeed, microscopic and submicroscopy RYR2-mediated Ca2+ release events have previously been implicated in the diastolic SR Ca2+ leak into the cytosol (49), and subthreshold RYR2 release events are known to increase Ca2+ in nearby mitochondria (50). Future studies will be required to assess Ca2+ in these compartments in beating cardiomyocytes, ideally within the intact heart.
One of the key findings of our study is that cRyr2KO mouse hearts had decreased ATP levels, as well as lower substrate utilization when compared with control hearts. This can be attributed to defects in the utilization of metabolic substrates via oxidative ATP generation. The Ca2+ dependence of the TCA cycle was proposed many years ago based on the finding that Ca2+ in the matrix of isolated mitochondria stimulates several enzymes in the pathway, including pyruvate dehydrogenase, which controls entry of glucose carbon into the TCA cycle (4, 51, 52). Similarly, emerging evidence suggests that the F1F0 ATP synthase is also directly regulated by mitochondrial Ca2+ such that the maximal rate of oxidative ATP production at given mitochondrial membrane potential is dependent on Ca2+ concentration (12). As such, the rate and efficacy of many energy-producing substrates via the TCA cycle could be affected by mitochondrial Ca2+ dynamics. Collectively, these enzymes are thought to rely on Ca2+ signals from across nanoscale spaces between ER/SR and mitochondria. Local microdomains of concentrated Ca2+ are required to access the mitochondria and the duration of the mitochondrial Ca2+ transients is limited by rapid export mechanisms (2, 3, 6). Our results suggest that ablation of RYR2 in vivo disrupts this signaling pathway, leading to a decrease in TCA cycle enzymatic activity and, consequently, a diminished ability to sustain cellular ATP levels. Inability to employ the TCA cycle likely promotes the metabolic inflexibility observed in heart failure, as the utilization of fat as fuel requires oxidative metabolism (19). Indeed, decreased RYR2 expression or function is associated with several models of metabolic cardiomyopathy (15–18). Our data support the concept that decreased RYR2 function in cardiac pathology may directly contribute to metabolic inflexibility seen in heart failure.
Our data also provide evidence for a role for RYR2 and Ca2+ in regulating lipid utilization and ATP production by transcriptional reprogramming. It is well established that failing hearts switch from metabolizing free fatty acids toward carbohydrate utilization (19). Knock-out and overexpression studies show that changes in transcriptional regulators and enzymes involved in lipid metabolism are sufficient to cause cardiac hypertrophy and steatosis (35, 37, 53). Our data suggest a role of the cytoprotective and metabolism-controlling Sirt1/Foxo1/Pgc1α axis in hearts lacking Ryr2. RYR2 deficiency is sufficient to flip a transcriptional metabolic “master switch,” which down-regulates lipid utilization pathways and effectors. We also found reduced insulin signaling and dramatic loss of Glut4, which might be expected to have deleterious effects given the cardioprotective effect of glucose uptake (54). Therefore, our study suggests that acute Ryr2 deletion disrupts both lipid and carbohydrate metabolism indirectly via transcriptional programming, as well as directly at the mitochondria.
In our previous studies of other cell types, loss of metabolism-pacing Ca2+ communication between RyR channels and mitochondria caused cellular ATP levels to decline and induced the presenilin-dependent expression of key hypoxia response genes (3, 11). Similarly, cRyr2KO mice had significantly increased proteins levels of presenilin-1, presenilin-2, HIF1α, and HIF1β, and they displayed numerous signs of energy starvation with an associated increase in compensatory mechanisms for energy conservation and optimization. This included decreased expression of uncoupling proteins and down-regulated carbohydrate and lipid storage pathways. Thus, cRyr2KO hearts exist in a pseudohypoxic stress state. Hypoxia-inducible factors are known to increase Atf3 and decrease Klf15 expression, a combination of events that is sufficient to drive cardiomyopathy, hypertrophy, fibrosis, and cell death (41, 43, 45, 55). Although we cannot rule out other parallel mechanisms of cardiac dysfunction in other models of heart failure, our evidence does demonstrate that disruption of RYR2-dependent metabolism is sufficient to cause heart failure.
Pharmacological studies have shown that Ca2+ released through RYRs or IP3 receptors into adjacent mitochondria modulates apoptosis (9, 50). When in excess, Ca2+ transfer from SR/ER to mitochondria triggers caspase-3-dependent apoptosis (28). We and others (9, 10, 56) have shown that constitutive intracellular Ca2+ signals are required to stimulate cellular respiration and prevent a hypoxia-like state with calpain-dependent, caspase-3-independent cell death. Interestingly, calpain-10 is localized to mitochondria (57) where it is poised to sense metabolic state. Our data indicate that disruption of RYR2 function, via its effects on mitochondrial calcium signaling and cellular metabolism, is also sufficient to cause cell death in vivo. Additional work will be required to further elucidate the mechanisms involved in this atypical cell death mode. We found evidence of autophagy in cRyr2KO hearts similar to the data on cells lacking IP3 receptors (5), suggesting that the control of metabolism and autophagy by SR/ER-to-mitochondria Ca2+ shuttling, both the barely perceptible Ca2+ microsignaling from stochastic channel opening as well as the larger global Ca2+ signals found in excitable cells such as cardiomyocytes, may be a ubiquitous metabolic control system.
Our inducible knock-out approach was designed to mitigate chronic compensatory gene expression induced by lifelong genetic alterations. Accordingly, our data were not confounded by compensation from other ER/SR Ca2+ release channels. We speculate that this may be the reason for the discrepancy between our observations of cardiac hypertrophy and the results obtained in mice with chronic Ryr2 haploinsufficiency, which were protected from pressure overload-induced hypertrophy (48). Interestingly, increased levels of cell death were reported in Ryr2+/− mice. The majority of in vivo studies of Ryr2 have used point mutation knock-in mice, typically with gain-of-function mutations causing “leaky channels” (58, 59). Studies have focused on the susceptibility of these mice to tachycardic arrhythmia. Recently, a loss-of-function knock-in model was created (Ryr2ADA/ADA mice) and reported to undergo calcineurin-independent hypertrophy (60). Additional studies are required to determine whether the hypertrophy observed in our cRyr2KO hearts is mediated through a similar mechanism. Given the fact that basal cytosolic Ca2+ appears to be lower in cRyr2KO cardiomyocytes, one might speculate that a calcineurin-independent pathway is involved.
Although ours is the first in vivo study to target an intracellular Ca2+ channel and examine metabolism, our conclusions are well supported by more reductionist studies on the roles of RYR2 in cardiac metabolism (2–5, 12), cell survival (10, 11, 48), and cardiac dysfunction (15–17). In our model, deletion of Ryr2 rapidly led to many alterations in cardiac phenotype. Because our only manipulation was Ryr2 deletion, all of the observed changes must ultimately be downstream of RYR2 action in cardiomyocytes, either immediately downstream or indirectly downstream. Future studies are required to elucidate the specific mechanisms involved and the temporal order in which these events occur.
In summary, our results illustrate that Ca2+ flux through the ER/SR RYR2 Ca2+ channels is required to maintain mitochondrial Ca2+, oxidative metabolism, metabolic transcriptional pathways, and cellular survival in the mouse heart in vivo. These observations, which support concepts advanced from in vitro studies, demonstrate a paramount role for RYR2 in cardiomyocyte energetics. This creates a paradigm in which RYR2 Ca2+ fluxes have simultaneous roles in the regulation of heart rate and rhythmicity (22), as well as effects on metabolic mitochondrial pacing, and transcriptional programming in cardiomyocytes. This comprehensive model may explain how functional and metabolic demands of cardiomyocytes can be so exquisitely coupled to ensure energy needs are met.
We thank Edwin Moore for helpful comments throughout this project. We thank Dr. Tatyana B. Kalynyak, Xiaoke Hu, Farnaz Taghizadeh, and Saba Marzara for technical assistance. We thank Kenneth Boheler (University of Hong Kong) for helpful comments throughout this project and for providing the original line of Ryr2flox/flox mice.
*This work was supported by a Canadian Institutes for Health Research operating grant to J. D. J.
This article contains supplemental Tables S1–S4.
3The abbreviations used are: