Down Syndrome Critical Region 1 Gene, Rcan1, Helps Maintain a More Fused Mitochondrial Network

Abstract

Rationale

The Regulator of Calcineurin 1 (RCAN1) inhibits calcineurin (CN), a Ca²⁺-activated protein phosphatase important in cardiac remodeling. In humans, RCAN1 is located on chromosome 21 in proximity to the “Down syndrome critical region.” The hearts and brains of Rcan1 KO mice are more susceptible to damage from ischemia/reperfusion (I/R), however, the underlying cause is not known.

Objective

Mitochondria are key mediators of I/R damage. The goal of these studies was to determine the impact of RCAN1 on mitochondrial dynamics and function.

Methods and Results

Using both neonatal and isolated adult cardiomyocytes, we show that, when RCAN1 is depleted, the mitochondrial network is more fragmented due to increased CN-dependent activation of the fission protein, Dynamin-1-Like (DRP1). Mitochondria in RCAN1-depleted cardiomyocytes have reduced membrane potential, O₂ consumption, and generation of reactive oxygen species, as well as a reduced capacity for mitochondrial Ca²⁺ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R, however, pharmacological inhibition of CN, DRP1, or calpains (Ca²⁺-activated proteases) restored protection, suggesting that, in the absence of RCAN1, calpain-mediated damage following I/R is greater due to a decrease in the capacity of mitochondria to buffer cytoplasmic Ca²⁺. Increasing RCAN1 levels by adenoviral infection was sufficient to enhance fusion and confer protection from I/R. To examine the impact of more modest, and biologically relevant, increases in RCAN1, we compared the mitochondrial network in induced pluripotent stem cells (iPSC) derived from individuals with Down syndrome to that of isogenic, disomic controls. Mitochondria were more fused and O₂ consumption was greater in the trisomic iPSC, however, coupling efficiency and metabolic flexibility was compromised compared to disomic. Depletion of RCAN1 from trisomic iPSC was sufficient to normalize mitochondrial dynamics and function.

Conclusions

RCAN1 helps maintain a more interconnected mitochondrial network and maintaining appropriate RCAN1 levels is important to human health and disease.

INTRODUCTION

Cardiovascular disease is the leading cause of death worldwide.¹ Restoring blood flow as soon as possible following a myocardial infarction or stroke is essential. However, reperfusion of ischemic tissue (I/R) can itself cause damage through generation of reactive oxygen species (ROS) and Ca²⁺ overload. Mitochondrial function is a critical determinant of the susceptibility of the heart to I/R damage.^2-4 Mitochondria form dynamic networks whose structure is fashioned by the opposing processes of mitochondrial fission and fusion.⁵ Increased fusion tends to increase mitochondrial membrane potential (ΔΨ_m) as well as the ΔΨ_m-dependent processes of ATP generation, ROS generation, and mitochondrial Ca²⁺ uptake. An array of proteins are involved in the fission/fusion process, including the fission protein, Dynamin-1-Like (DNM1L, typically referred to as DRP1). Diverse posttranslational modifications of DRP1 regulate its activity,^6,7 including dephosphorylation by the Ca²⁺-activated protein phosphatase, calcineurin (CN), that promotes DRP1 translocation to mitochondria, thereby initiating fission.⁸ There is a dramatic increase in cytoplasmic Ca²⁺ during ischemia, however, CN remains relatively inactive because of the low pH caused by a build up of lactic acid.⁹ Calpains (CAPN), are Ca²⁺ activated proteases that also play an important role in I/R damage.^10,11 Similar to CN, they remain inactive in the low pH environment of the ischemic heart, but are activated rapidly upon reperfusion.

Sustained activation of CN is sufficient to drive pathological hypertrophy of the myocardium with subsequent heart failure and death.¹² CN activity can be modified by members of the regulator of calcineurin (RCAN) family of proteins that inhibit CN activity through a direct protein-protein interaction.^13,14 The RCAN1 gene encodes two isoforms and was initially designated as Down Syndrome Critical Region 1 (DSCR1) because of its location on human chromosome 21.¹⁵ Altered mitochondrial function and increased oxidative stress have long been associated with Down Syndrome (DS).¹⁶ RCAN1.1 is abundant in striated muscle and brain where its expression is relatively constant, whereas, expression of RCAN1.4 is under the control of CN, thereby acting as a feed-back inhibitor of CN activity.¹⁷ Cardiac-specific over expression of an RCAN1 transgene protects the heart from a variety of pathological stresses including I/R,^18-20 whereas the brains and hearts of mice lacking RCAN1 are more sensitive to I/R.^21-23

Here, we investigate the contribution of RCAN1 to the control of mitochondrial dynamics and function, using neonatal rat ventricular myocytes (NRVM), isolated adult mouse ventricular cardiomyocytes (AMVM), mouse embryonic fibroblasts (MEF), and induced pluripotent stem cells (iPSC) derived from individuals with DS. We show that depletion of RCAN1 increases mitochondrial fission, lowering metabolic function and capacity for Ca²⁺-buffering, thereby increasing CAPN-mediated damage following reperfusion. Conversely, raising RCAN1 levels is sufficient to increase fusion, but may compromise coupling efficiency and respiratory reserve.

METHODS

Full methods are provided in the Online Data Supplement. All data, methods, and study materials are also available upon request by contacting either Dr. Parra (vparra@ciq.uchile.cl) or Dr. Rothermel (beverly.rothermel@utsouthwestern.edu).

RESULTS

Depletion of RCAN1 increases mitochondrial fragmentation in cardiomyocytes

Transmission electron micrographs comparing wild type (WT) and Rcan1 KO hearts showed evidence of increased mitochondrial fragmentation in the KO (Figure 1A). There was a decrease in the size of individual mitochondria (Figure 1B) and an increase in their number (Figure 1C). Mitochondrial perimeter decreased (Figure 1D), whereas, their circularity index increased (Figure 1E).

Figure 1. Rcan1 KO hearts showed increased mitochondrial fragmentation.

(A) Electron micrographs of the left ventricular wall show disordered and fragmented mitochondria in the KO compared to WT (scale bar: 1 μm). Mitochondrial were quantified for (B) cross-sectional area, (C) density, (D) perimeter, and (E) circularity index. 100 mitochondria were assessed in 3 animals of each genotype (n=3). Mean ± SEM; *P<0.05.

To test whether RCAN1-dependent changes in the mitochondrial network were cell-autonomous, siRNAs were used to deplete the RCAN1.1 and RCAN1.4 isoforms from cultured NRVM, either individually or in combination (dKD) (Online Figure I). Forty-eight hours after siRNA transfection, cells were stained with Mitotracker Green (400 nM) (Figure 2A) and quantified for both the number and size of individual mitochondria using volume-reconstitution of confocal Z-stacks.^24-26 Consistent with the changes observed in the left ventricular wall of KO mice, dKD increased mitochondrial number (Figure 2B) and decreased size (Figure 2C). Depleting RCAN1.1 alone resulted in changes comparable to the dKD, whereas the effect of depleting RCAN1.4, although trending in a similar direction, was not significant. Thus, in this experimental context, the RCAN1.1 isoform had the primary impact on mitochondrial morphology. Electron micrographs of the siRNA-depleted NRVM showed similar changes (Online Figure IIA-E).

Figure 2. Mitochondrial fragmentation increases in RCAN1.1-depleted NRVM.

NRVM were transfected with a nonspecific control siRNA or ones targeting RCAN1.1 and RCAN1.4, individually or combined (dKD). (A) Confocal Z-stack reconstructions of siControl and siRCAN1.1-depleted NRVMs stained with Mitotracker Green (Scale bar: 20 μm) where assessed for (B) the number of mitochondria per cell and (C) volume of individual mitochondria. Data are from 25 cells examined from six separate experiments (n=6). (D–L) FRAP analysis of TMRM stained NRVM was used to assess connectivity of the mitochondrial network. (D) Bleaching of TMRM fluorescence was applied in an ~25-μm² square at randomly chosen regions (scale bar: 10 μm). (E) Fluorescence recovery was tracked over time and normalized to the signal prior to bleaching then quantified for (F) rate of recovery and (G) level of recovery. Data are from 15 cells each condition in five separate experiments (n=5). (H) Mitochondria and cytosol were fractionated from siRNA transfected NRVM then assessed by Western blot for the proteins indicated. (I) DRP1 localized to the mitochondrial fractions in H were quantified by densitometry (n= 4). (J) Total DRP1 protein was immunoprecipitated from total cell extracts of siRNA transfected NRVM then probed with antibody specific for phospho‐Ser⁶³⁷. (K) Signal was normalized to total DRP1 (n=3). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Fluorescence recovery after photobleaching (FRAP) was used to compare the functional connectivity of mitochondria. siRNA-transfected NRVM were labeled with tetramethylrhodamine (TMRM) followed by photo-bleaching of selected regions of the mitochondrial network (Figure 2D). FRAP signal was quantified over time. The rate of recovery and the maximal fluorescence recovered were lower in RCAN1.1-depleted NRVM compared to controls (Figure 2E–G), indicative of decreased mitochondrial connectivity or a lower efficiency for mitochondrial membrane potential recovery. There was no apparent change in total mitochondrial mass, as assessed by either Western blot or flow cytometry (Online Figure IIF-H).

Depletion of RCAN1 increases translocation of DRP1 to mitochondria

We postulated that loss of CN inhibition by RCAN1 promotes mitochondrial fission by increasing CN activation of DRP1. Consistent with this, there was an increase in DRP1 protein in the mitochondrial fraction of RCAN1.1-depleted and dKD NRVM compared to control siRNA or RCAN1.4-depleted (Figure 2H–I). DRP1 phosphorylation at serine 637, the CN substrate site (Ser⁶³⁷ in humans, Ser⁶⁵⁶ in rat), was assessed by immunoprecipitating total DRP1 then probing with a phospho-Ser⁶³⁷ specific antibody. NRVM depleted for RCAN1.1 alone or as the dKD showed a decrease in Ser⁶³⁷ phosphorylation compared to control siRNA and RCAN1.4-depleted cells (Figure 2J–K). Taken together, the changes in phosphorylation and subcellular distribution of DRP1 suggest that RCAN1 helps maintain mitochondrial fusion by suppressing CN-mediated activation of DRP1 and points to the RCAN1.1 isoform playing a primary role in this process. Consistent with RCAN1 acting through CN-dependent control of DRP1, pharmacological inhibition of CN using FK506, or inhibition of DRP1 using Mdivi-1, restored a more fused mitochondrial network to RCAN1.1-depleted NRVM (Online Figure IIIA-F). To verify the impact of RCAN1-depletion on CN activity, we assessed nuclear translocation of endogenous NFAT1, which increased in RCAN1-depleted NRVMs, indicative of an increase in CN activity (Online Figure IIIG-H). Immunocytochemistry likewise showed increased colocalization of DRP1 with mitochondria (Online Figure III-I) along with increased fission (Online Figure IIIJ-K).

Mitochondrial function is reduced in cardiomyocytes depleted of RCAN1.1

Disruption of the mitochondrial network is often associated with a decrease in mitochondrial function.^5,27,28 Indeed, intracellular ATP (Figure 3A) and ΔΨ_m, (Figure 3B) were significantly reduced in the RCAN1.1-depleted and dKD NRVM. ΔΨ_m is generated by proton pumping through the mitochondrial electron transport chain (ETC) at complexes (I, III, and IV) and then dissipated through complex V to generate ATP (OXPHOS coupling) (Figure 3C). Dissipation of the proton gradient can also occur through other mechanisms, some of which consume ATP. Thus, reductions in ΔΨ_m and ATP levels per se do not necessarily indicate a reduction in mitochondrial activity. The rate of O₂ consumption was used to assess electron flow through the ETC and fidelity of OXPHOS coupling. Baseline O₂ consumption was reduced in RCAN1.1-depleted and dKD NRVM compared to control (Figure 3D). O₂ consumption was lower in RCAN1.1-depleted cells compared to controls, even after the addition of the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating a decrease in maximal ETC capacity (Figure 3E). There was no difference between control and RCAN1.1-depleted cells treated with the complex V inhibitor, oligomycin, demonstrating that loss of RCAN1.1 did not alter OXPHOS coupling. Consequently, oligomycin increased ΔΨ_m in both control and RCAN1.1-depleted cells (Online Figure IVA). ROS production was also lower in the RCAN1.1-depleted and dKD cells compared to control (Figure 3F–G) consistent with a reduction in both their ETC capacity and ΔΨ_m. It is important to note that mitochondria depleted for RCAN1.1 had the same rate of uncoupled proton leak as control cells despite a lower driving force, ergo, they may be intrinsically leakier. If so, this could further contribute to reducing mitochondrial function and ROS production.

Figure 3. Mitochondrial function is decreased in RCAN1.1-depleted NRVM.

Cells were transfected with siRNAs as indicated and analyzed 48 h later. (A) Total cellular ATP levels, were measured by luciferase (n=7). (B) Transfected NRVM were loaded with TMRM and analyzed by flow cytometry to assess Δψ_m. The complex V inhibitor, Oligomycin (Oligo, 10 μM), and the mitochondrial uncoupler CCCP (50 μM) were used as positive and negative controls respectively (n=5). (C) Schematic draws an analogy between mitochondrial electron transport and an electrical circuit. Complexes I, III and IV act in parallel with respect to the proton circuit and in series with respect to the electron flow. The sites of action for Oligo and CCCP are indicated. (D) O₂ consumption is reduced with RCAN1 depletion (n=6). (E) Maximal and proton leak-associated O₂ consumption were assessed by adding CCCP (200 nM) or Oligo (50 nM), respectively (n=4). (F) Total ROS content was measured by flow cytometry using MitoSox® (n=5) or (G) DH123 (n=4). (H) Transcript levels for Hk2, Pfkfb2, Slc2a1, and Atp5b were quantified by qPCR (n=3). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Transcript levels for several genes associated with glycolysis increased, including hexokinase 2 (Hk2), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (Pfkfb2) and solute carrier family 2 member 1 (Slc2a1, also known as Glut1) (Figure 3H), suggesting that the RCAN1.1-depleted and dKD NRVM may compensate for a reduction in mitochondrial function by increasing reliance on glycolysis. The survival rate of RCAN1.1-depleted NRVM following a shift to media lacking glucose was reduced compared to control cells (Online Figure IVB). There was no change in transcript levels of the mitochondrial ATPase synthase beta subunit (Atp5b), consistent with a reduction in mitochondrial efficiency rather than a change in total mitochondrial mass. Treatment with either FK506 or Mdivi-1 was sufficient to prevent the increase in expression of glycolytic genes in RCAN1-depleted NRVM (Online Figure IVC-H).

Increased mitochondrial fragmentation increases susceptibility of RCAN1.1-depleted cardiomyocytes to I/R

Tissues of the Rcan1 KO mice are more susceptible to damage from I/R.^21-23 To examine this in a controlled experimental system, we turned to an in vitro model of simulated cardiac I/R (sI/R). NRVM depleted of RCAN1 were more sensitive to sI/R (Figure 4A), although sI/R itself did not change protein levels of either RCAN1.1 or RCAN1.4 (Online Figure IVI-J). Depleting RCAN1.1 had the greatest impact on I/R sensitivity, whereas, depletion of RCAN1.4 alone was not significant (Figure 4A). Treatment with either FK506 or Mdivi-1 restored resistance to sI/R (Figure 4B–C and Online Figure IVK-L), suggesting that endogenous RCAN1 protects from I/R damage by suppressing CN-dependent activation of DRP1.

Figure 4. siRCAN1.1-depleted NRVM are more sensitive to I/R due to CN-dependent activation of DRP1.

(A) 48 h following transfection NRVM were subjected to simulated I/R (6 h ischemia, 12 h reperfusion) and LDH release was used to assess death (n=4). (B) NRVM were treated with either FK506 (20 nM) to inhibit CN or (C) Mdivi (12.5 μM) to inhibit DRP1, prior to simulated I/R. (n=4). (D–H) NRVM protein extracts were probed for DRP1, HSPA9 (mtHSP70), TUBB, OPA1 (long and short isoforms), PINK1, MFN2, PARK2 and GAPDH by Western blot and quantified by densitometry (n=4). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Abundance of the mitochondrial protein mtHSP70 (HSPA9) was similar in control and RCAN1-depleted NRVM both prior to and following sI/R (Figure 4D) suggesting that, at the time points measured, loss of RCAN1 did not alter total mitochondrial mass. Total DRP1 protein levels also remained constant (Figure 4D, E). In contrast, levels of all isoforms of the fusion protein, mitochondrial dynamin like GTPase (OPA1), were reduced in the RCAN1.1-depleted and dKD NRVM, both under normoxic conditions and following sI/R (Figure 4D, F). There was also an increase in levels of the PTEN induced putative kinase 1 (PINK1)²⁹ in the RCAN1.1-depleted and dKD NRVM, under both normoxic conditions and following I/R (Figure 4D, G). Protein levels for the fusion protein, mitofusin 2 (MFN2), were not different under normoxic conditions, but following sI/R there was a much more pronounced decline in MFN2 in the RCAN1.1-depleted cells compared to controls (Figure 4D, H). These changes in OPA1 and MFN2 levels would favor fission, and may therefore act to further disrupt the mitochondrial network in RCAN1-depleted cells.

The capacity for mitochondrial Ca²⁺ uptake is reduced in cardiomyocytes depleted of RCAN1.1

Uptake through the mitochondrial Ca²⁺ uniporter (MCU) is dependent upon ΔΨ_m, thus, a more connected mitochondrial network is a more efficient buffer of cytosolic Ca²⁺.^30,31 Close proximity of mitochondria to sites of Ca²⁺ release also facilitates mitochondrial Ca²⁺ uptake. In RCAN1.1-depleted NRVM there was a general redistribution of mitochondria away from the perinuclear zone (Figure 5A and Online Figure V), a region of close interaction between mitochondria and the endoplasmic reticulum (ER). To assess capacity for mitochondrial Ca²⁺ uptake, NRVM were loaded with RhodFF and then treated with histamine to evoke Ca²⁺ release from the ER. Mitochondrial Ca²⁺ uptake was significantly reduced in RCAN1.1-depleted cells compared to siRNA controls (Figure 5B–C). To test the impact on cytosolic Ca²⁺ transients in the setting of calcium-induced calcium release (CICR), NRVM were loaded with Fura2 then stimulated with 50 mM KCl to depolarize the plasma membrane and trigger CICR. Cytoplasmic Ca²⁺ levels were similar in RCAN1.1-depleted and control cells at rest. Following KCl stimulation, a rapid increase in cytosolic Ca²⁺ was observed in both cell types. However, cytosolic Ca²⁺ returned to base line levels in the control NRVM, whereas, in the RCAN1.1-depleted cells cytosolic Ca²⁺ levels remained elevated (Figure 5D–E). The increases in cytosolic Ca²⁺ following histamine treatment were also much higher in RCAN1.1-depleted NRVM compared to controls (Online Figure VIA-B).

Figure 5. Depletion of RCAN1.1 reduces mitochondrial Ca⁺² buffering capacity, and increases CAPN-mediated damage following I/R.

(A) siRNA transfected NRVM stained with Mitotracker Green have been pseudo-colored to indicate the relative density of mitochondrial signal. Yellow indicates lower density, whereas red-violet indicates higher density (scale bar: 20 μm). (B&C) Mitochondria Ca⁺² uptake was assessed by loading cells with Rhod-FF prior to the addition of histamine (100 μM) to release Ca⁺² from ER stores (n=4). (D) Cytosolic Ca⁺² was assessed by loading cells with Fura2 prior to the addition of KCl 50 mM to trigger Ca⁺²-induced Ca⁺² release. (E) Signal from D was quantified as the maximal fluorescence ratio reached during the first 150 s. Data are from 50 cells examined in five separate experiments. (F) α-spectrin cleavage products were assessed by Western blot. Ionomycin (Iono) was added as a control for CAPN activation. Quantification in lower panel (n=4). (G–I) RCAN1-depleted NRVM where treated with the CAPN inhibitors E-64D (10 μM), MDL (10 μM), or PD 150606 (10 μM) prior to sI/R (n=5). (J) A mixture of siRNA’s depleting CAPN 1 and 2 (50 nM) also conferred protection to the RCAN1.1-depleted NRVM (n=5). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Depletion of RCAN1.1 increases calpain-dependent cardiomyocyte damage following I/R

During ischemia, acidification of the cytosol by lactic acid increases cytosolic Ca²⁺ through the combined compensatory actions of the Na⁺/H⁺ exchanger, the Na⁺/K⁺-ATPase, and the Na⁺/Ca²⁺ exchanger. CAPNs are activated rapidly upon reperfusion^32,33 and contribute to the degradation of several structural proteins in cardiomyocytes following I/R, including α-spectrin (SPTAN1).^34,35 An increase in the levels of SPTAN1 cleavage products following sI/R verified activation of CAPNs in our model (Figure 5F). The accumulation of cleavage products following sI/R was greater in the RCAN1.1-depleted cells than in control siRNA-treated NRVM suggesting increased activation of CAPN. Under normoxic conditions SPTAN1 cleavage products were not significantly increased in RCAN1-depleted cells compared to controls, suggesting that depletion of RCAN1 does not elevate CAPN activity under homeostatic, resting conditions. Treatment with Mdivi-1 reduced the accumulation of SPTAN1 cleavage products under all conditions (Online Figure VIC-D). Treatment with any one of three different CAPN inhibitors (E-64D, MDL, or PD 150606) restored protection from I/R to the RCAN1.1-depleted and dKD cells (Figure 5G–I and Online Figure VIE-G). E-64D and MDL also inhibit cathepsins.³⁶ PD 150606 is more specific, inhibiting both CAPN 1 and 2 (μ‐CAPN and m-CAPN), which are the primary forms found in heart.³⁴ Targeted siRNAs were used to knock down both Capn 1 and 2. This conferred significant protection to the RCAN1.1-depleted NRVM (Figure 5J and Online Figure VIH-J). Preincubation of NRVM with Ruthenium Red (RuRed), an inhibitor of MCU, increased cell death under normoxia and following I/R in RCAN1.1-depleted cells, whereas, chelating intracellular Ca⁺² with BAPTA-AM prior sI/R reduced death (Online Figure VIK-O). Taken together, these results suggest that NRVM deficient for RCAN1.1 are more susceptible to damage from I/R due to elevated activation of CAPN following reperfusion and that this may be due to a decrease in mitochondrial Ca²⁺ buffering capacity, the result of enhanced fission.

Increasing RCAN1.1 levels is sufficient to promote cardiomyocyte mitochondrial fusion and increase O₂ consumption

An adenovirus encoding full length human RCAN1.1 (Ad-hRCAN1.1) was used to test whether increasing RCAN1.1 levels is sufficient to increase mitochondrial fusion (Online Figure VIIA-D). Twenty-four hours after infection, Ad-hRCAN1.1 restored mitochondrial fusion to the RCAN1.1-depleted cells in a dose-dependent fashion (Figure 6A–C). In contrast, infection with an adenovirus encoding the RCAN1.4 isoform (Ad-hRCAN1.4) was not sufficient to restore the mitochondrial network to RCAN1.1-depleted NRVM (Online Figure VIIIA-G). Ad-hRCAN1.1 also increased fusion in control siRNA cells, demonstrating that increasing RCAN1 levels is sufficient to promote fusion. Consistent with the changes seen in mitochondrial morphology, Ad-hRCAN1.1 increased O₂ consumption (Figure 6D), ROS generation (Figure 6E), and conferred protection from sI/R (Figure 6F). In contrast, Ad-hRCAN1.4 did not confer significant protection (Online Figure VIIIH). Although Ad-hRCAN1.1 infection of control cells increased O₂ consumption, this was at least in part due to an increase in uncoupling (+ oligomycin in Figure 6G) rather than a change in the capacity for maximal electron flow through the ETC (+ CCCP in Figure 6G).

Figure 6. Exogenous expression of human RCAN1.1 restores mitochondrial parameters and protection from I/R to RCAN1.1-depleted NRVM.

(A) Cells were transfected with indicated siRNAs followed by adenoviral infection to express human RCAN1.1 (Ad-RCAN1.1) or β-galactosidase (Ad β-gal). 48 h later, cells where loaded with Mitotracker Green and imaged by confocal (n=4) (scale bar: 10 μm) and quantified for (B) the number of mitochondria per cell and (C) individual mitochondrial volume. (D) Infection with Ad‐hRCAN1.1 increased O₂ consumption in both siControl and siRCAN1.1-depleted NRVM (n=4). (E) Infection with Ad-hRCAN1.1 increased ROS production in both siControl and siRCAN1.1-depleted NRVM (n=4). (F) Infection with Ad-hRCAN1.1 restored protection from sI/R to siRCAN1.1-depleted NRVM (n=4). (G) Maximal and proton leak-associated O₂ consumption were assessed by adding CCCP (200 nM) or oligomycin (50 nM), respectively to control cells infected with either Ad-β-gal or Ad-hRCAN1.1 (n=4). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Loss of RCAN1 in vivo decreases mitochondrial metabolism and the capacity for mitochondrial Ca²⁺ uptake and function in adult cardiomyocytes

To determine whether our findings in NRVMs translated to the adult myocardium, AMVM were isolated from adult WT and KO mice and evaluated to compare mitochondrial morphology, capacity for Ca²⁺ uptake, and metabolic activity. Despite the compact organization of the mitochondrial network, 3D reconstruction revealed a more fragmented network in KO myocytes compared to WT, with an increase in the total number of mitochondria per cell and a decrease in mitochondrial size (Figure 7A–C). Interestingly, separate analysis of interior regions of interest (ROI) verses peripheral ROI suggested that increased fission in the KO localized primarily to mitochondrial populations located in the interior of cardiomyocytes, whereas, peripheral populations were not significantly different between KO and WT (Figure 7A, D–E). Directly relevant to the increase in I/R sensitivity, the capacity for mitochondrial calcium uptake was significantly reduced in KO AMVM compared to WT (Figure 7F–H). The localization of the Ca²⁺ signal was consistent with mitochondrial morphology. Uptake by MCU was validated using the inhibitor RuRed (Online Figure IX). O₂ consumption was measured in permeabilized AMVM. State III respiration was significantly lower in AMVM from the Rcan1 KO compared to those from WT using either pyruvate (Figure 7I) or glutamate (Figure 7J) as a substrate. Taken together these findings indicate that loss of RCAN1 in adult cardiomyocytes replicates many of the key features observed in NRVMs.

Figure 7. Rcan1 KO adult cardimyocytes show increased fission, decreased mitochondrial Ca⁺² buffering capacity, and decreased mitochondrial function. (A).

Confocal Z-stack reconstructions of WT and KO AMVMs stained with TMRM (Scale bar: 20 μm) where assessed for (B) the number of mitochondria per cell and (C) volume of individual mitochondria. Data are from 25 cells examined from three separate experiments. (D–E) Interior (Inter) or peripheral (Peri) regions of interests from the images in A were assessed independently. (F) Mitochondrial Ca²⁺ uptake in permeabilized AMVM stained with Rhod2. (G) Quantification of F (H) area under the curve during 300 s. Data are from 50 cells examined in three separate experiments. Oxygen consumption of permeabilized AMVMs using (I) Pyruvate and (J) glutamate as substrates. n=4 from three different mice in each group. Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Mitochondrial fusion is increased in iPSC derived from individuals with down syndrome

The preceding studies provide evidence that RCAN1 helps to maintain the mitochondrial network in a more fused configuration and that this affects fundamental metabolic parameters in cardiomyocytes. To determine whether these findings extend to other cell types, we first isolated mouse embryonic fibroblasts (MEF) from the Rcan1 KO and compared the mitochondrial network to that of WT MEF. Consistent with the studies in RCAN1-depeleted NRVM, the mitochondrial network in Rcan1 KO MEF showed evidence of increased fission compared to that of WT MEF (Online Figure X), thus, RCAN1’s impact on mitochondrial dynamics was not specific to cardiomyocytes. Our studies over expressing RCAN1.1 in NRVM, also suggest that increasing RCAN1 levels beyond their normal homeostatic controls is not necessarily beneficial, and can have detrimental consequences with regard to increased uncoupling and ROS generation (Figure 6).

To examine this in the context of human health and disease, we turned to individuals with DS who are trisomic for chromosome 21. RCAN1 protein levels are elevated in the brain human fetuses with DS^15,37 and studies have linked RCAN1 with oxidative stress respnses.^38-40 We obtained human trisomy 21 induced pluripotent stem cells (T21-iPSC) and isogenic disomic controls (D21-iPSC) through the PCBC Disease Lines resource (https://progenitorcells.org/).⁴¹ Mitochondrial morphology was assessed and showed a reduction in the number of mitochondrial per cell and an increase in the mean volume of individual mitochondria in trisomic T21-iPSC when compared to disomic D21-iPSC, indicative of a more interconnected mitochondrial network (Figure 8A–C). The rate of O₂ consumption was also substantially elevated in the T21-iPSC (Figure 8D). O₂ consumption did not further increase after addition of CCCP, suggesting that the mitochondria in these cells are already working at maximal capacity. Importantly, there was also an increase in uncoupled proton leak in the T21-iPSC similar to that observed in control NRVM infected with Ad-hRCAN1.1 (Figure 6), suggesting increased dosage of RCAN1 may have detrimental effects on mitochondrial function.

Figure 8. Trisomic iPSC from individuals with Down syndrome have a more fused mitochondrial network and increased O₂ consumption.

(A) Disomic D21-iPSC (2S) and Trisomic T21-iPSC (3S) were stained with Mitotracker-green and then analyzed by confocal microscopy to reconstruct the mitochondrial network. Representative images are provided from the D21-iPSC line #409 (2S) and the T21-iPSC line #416 (3S). Identical results where obtained from independent lines #406 (2S) and #419 (3S) (scale bar: 10 μm). (B) Trisomic T21-iPSC contained fewer (C) and larger mitochondria compared to disomic D21-iPSC. Data are from 25 cells in three independent experiments. (D) O₂ consumption rate was higher in T21-iPSC than in D21-iPSC. Maximal capacity and proton leak were assessed by adding Oligo, (50 nM) or CCCP (200 nM) respectively. (n=5). (E) Transcript levels for RCAN1.1 were higher in T21-iPSC than in D21-iPSC. Human-specific siRNAs targeting each of the RCAN1 isoforms were used to deplete endogenous RCAN1 from both lines and changes in endogenous RCAN1.1 transcripts quantified by qPCR (n=4). (F) The siRNA-depleted iPSC were analyzed by Western blot for RCAN1, SOX2, POU5F1 and TUBB proteins. HEK293 cells were used as controls. (G) Densitometry was used to quantify RCAN1.1 protein levels in F normalized to TUBB (n=4). (H) Trisomic T21-iPSC were depleted of RCAN1.1 and RCAN1.4, individually and in combination (dKD) then stained for analysis of the mitochondrial network (scale bar: 10 μm). (I) Depletion of RCAN1.1 alone or in combination with RCAN1.4 (dKD) increased mitochondrial number, (J) reduced average mitochondrial size, and (K) reduced basal ROS generation to levels (n=5). Mean ± SEM; *P<0.05, **P<0.01, ***P<0.001.

Although chromosome 21 is the smallest of the human somatic chromosomes, it contains over 200 genes along with an array of noncoding RNAs, any one, or combination of, could contribute to this change in mitochondrial morphology and function. To test whether increased dosage of the RCAN1 gene underlies the changes in mitochondrial form and function, human-specific siRNAs were used to deplete each of the RCAN1 isoforms individually and in combination from the trisomic T21-iPSC. At baseline, transcript levels for RCAN1.1 were elevated in the T21-iPSC compared to the disomic D21-iPSC (Figure 8E). The disomic and trisomic iPSC were transfected with siRNAs targeting RCAN1.1 and RCAN1.4 alone or in combination. siRNA targeting RCAN1.1 was effective at reducing RCAN1.1 transcript levels in both genotypes (Figure 8E). In the T21-iPSC transcript and protein levels for RCAN1.1 were reduced to at, or below, those found in D21-iPSC (Figure 8E–G). This occurred without impacting markers of pluripotency as assessed by western blot (Figure 8F) or qRT-PCR (Online Figure XI). RCAN1.4 protein was below the level of detection in these cells (Figure 8F). Remarkably, siRNA depletion of RCAN1.1 was sufficient to restore the mitochondrial network in the T21-iPSC to levels comparable to control siRNA transfected D21-iPSC (Figure 8H–J). The rate of O₂ consumption in T21-iPDC was likewise restored to that measured in D21-iPSC (Figure 8K). Taken together, these data suggest that increased dosage of RCAN1 may underlie a fundamental increase in mitochondrial fusion in the setting of trisomy 21, but may come at the cost of compromised coupling efficiency and reduced respiratory reserve.

DISCUSSION

Mitochondrial metabolism and function play an important role in human health, disease, and aging. This is particularly true in highly oxidative organs such as the heart and brain where mitochondrial dysfunction is often a prominent feature of disease related decline. In the setting of reperfusion injury following acute MI or stroke, mitochondria are the source of fundamental signals mediating cellular survival and death. In the context of RCAN1 deficiency, cardiac and neuronal damage following I/R is greater in Rcan1-KO mice and forced expression in either cardiomyocytes or neurons can confer protection.^21-23,42 Here, we provide evidence that altering the levels of RCAN1 has a direct impact on mitochondrial dynamics. Based on these findings, we propose that the following cascade of events underlies the increase in sensitivity to reperfusion damage: In the absence of inhibition by RCAN1, increased CN activity promotes DRP1-mediated mitochondrial fission. The increase in fission decreases ΔΨ_m and metabolic activity, lowering the capacity for mitochondrial Ca²⁺ uptake. Our studies suggest that, as a consequence, buffering of cytosolic Ca²⁺ is reduced, thereby increasing CAPN-mediated damage following reperfusion. Although both cytosolic and mitochondrial localized CAPN can cause cellular damage, in the context of RCAN1 deficiency, the relevant pool, or pools, of CAPN are most likely cytosolic (Online Figure XII).

The reduction in OPA1 protein levels seen in RCAN1-depleted NRVM is consistent with lower ΔΨ_m, as decreased membrane potential promotes OPA1 cleavage and degradation. Because of OPA1’s role in mitochondrial fusion, the decline in OPA1 abundance may further shift the mitochondrial population toward fission in RCAN1-deficient cells. An increase in PINK1 protein is likewise consistent with reduced ΔΨ_m as membrane potential is required for translocation of PINK1 into the mitochondrial matrix for subsequent degradation. Activation of PINK1 can initiate tagging of mitochondria for degradation via mitophagy. However, we saw no reduction in total mitochondrial mass in RCAN1-depleted cells, suggesting that the increase in PINK1 was either not sufficient to increase mitochondrial degradation or that an increase in mitochondrial biogenesis compensated for increased catabolism.

ROS generation during reperfusion is a well-documented mediator of I/R damage. However, the decrease in basal ROS activity and decline in maximal ETC capacity in the RCAN1-depleted NRVM (Figure 3) would suggest that ROS-mediated damage may not be the primary mechanism mediating increased I/R sensitivity in culture. That does not preclude a mechanism involving elevated ROS in the context of the working heart and neurons where energetic demands might drive an increase in mitochondrial content to compensate for the decline in mitochondrial efficiency.

Our studies suggest that RCAN1.1 is the primary isoform impacting mitochondrial dynamics and function. However, in our experiments the protein levels following Ad‐hRCAN1.1 infection were an order of magnitude higher than for Ad-hRCAN1.4 (Online Figure VIIC-D and VIIIC-D), thus limiting the ability to draw definitive conclusions regarding isoform-specificity based on the adenoviral complementation studies. The two isoforms are comparable in their ability to inhibit CN in biochemical assays, although, some isoform specific properties have been reported in vivo. For instance, we recently demonstrated an isoform-specific role for RCAN1.4 in the process of VEGF receptor internalization.⁴³

In our studies, forced expression of RCAN1.1 was sufficient to increase mitochondrial fusion and confer protection from sI/R to both RCAN1.1-depleted and control siRNA transfected NRVM (Figure 6). Although over expression using adenoviruses increased RCAN1.1 levels well over those of the endogenous protein, the studies comparing trisomic T21-iPSC to disomic D21-iPSC demonstrate that modest changes in RCAN1 levels can have a meaningful impact on mitochondrial dynamics and function. Altered mitochondrial function and increased oxidative stress have long been associated with DS.¹⁶ Although there is no universal agreement regarding morphological changes to the mitochondrial network in trisomic tissues, an increase in mitochondrial ROS generation is widely reported and would be consistent with the increased fusion observed in the T21-iPSC. In addition, increased mitochondrial Ca²⁺ levels are documented in both heart and cultured fibroblasts from DS fetuses.⁴⁴ Our findings point toward a mechanism through which increased RCAN1 dosage could underlie these changes.

In the context of I/R damage, these studies suggest that increased fusion protects cardiomyocytes by reducing the extent of CAPN activation following reperfusion. However, a sustained increase in fusion may also have negative consequences. Indeed, although an increase in ΔΨ_m increased the capacity for ATP generation, it also increased the capacity for ROS generation, thereby increasing the potential for cellular damage. Sustained fusion could also slow the process of mitochondrial turnover and repair. Over time, these two mechanisms would act to fuel the generation and accumulation of damaged mitochondrial components. This may be the underlying cause of the increase in uncoupling observed in both trisomic T21-iPSC and NRVM infected with Ad-hRCAN1.1. Alternative explanations for the decline in coupling efficiency are activation of protective compensatory mechanisms or a direct impact of RCAN1 on mitochondrial coupling. Along these lines it is relevant to note that RCAN has been reported to interact both directly⁴⁵ and indirectly⁴⁶ with the adenine nucleotide translocase, a protein which can act to uncouple the inner mitochondrial membrane proton gradient.⁴⁷

Transgenic over expression of RCAN1 is reported to increase mitochondrial ROS in both neurons and pancreatic islets.^48,49 Under normal glucose conditions ΔΨ_m is elevated in islets over expressing RCAN1, but is depressed in response to elevated glucose.⁵⁰ Thus, changes mediated by RCAN1 may be context dependent. As an example, we recently showed that brain-specific over expression of RCAN1.1 caused age-dependent cognitive impairments similar to early on-set dementia seen in individuals with Down syndrome⁵¹. Mitochondrial ROS and the number of large, globular mitochondria increased in the brains of these animals; consistent with the mechanism we report here, however, there was also an accompanying, age-dependent decline in DRP1 phosphorylation, not predicted by our model. We postulate that this may be the combined outcome of primary effects, cumulative damage, and compensatory mechanisms. The mitochondrial network of trisomic Ts21-iPSC may more closely represent primary fundamental differences in mitochondria dynamics because iPSC are predominately glycolytic and therefore not yet impacted by functional selection as might occur in the setting of an intact animal or even differentiated cell types in culture.

Taken together, our studies indicate a dose-dependent response of the mitochondrial network to changes in the availability of RCAN1. RCAN1 insufficiency favors fission, thereby reducing mitochondrial metabolic activity and capacity for mitochondrial Ca²⁺ uptake. In the case of RCAN1-depleted NRVM, this increases vulnerability to sI/R damage, due to increased CAPN-mediated damage upon reperfusion. Although forced RCAN1.1 expression is sufficient to promote fusion and increase metabolic activity, in the setting of trisomy 21, this may occur at the cost of compromising coupling efficiency and loss of metabolic flexibility. Our findings highlight the need for maintaining appropriate RCAN1 levels, and suggest a mechanism through which increased dosage of the RCAN1 locus in DS may impact human health at the fundamental level of mitochondrial dynamics.

NOVELTY AND SIGNIFICANCE.

What Is Known?

• The Regulator of Calcineurin 1 gene (RCAN1), located on human chromosome 21, encodes a protein that inhibits the protein phosphatase calcineurin.

• Excessive activation of calcineurin in the heart contributes to pathological hypertrophy and the progression to failure.

• Calcineurin promotes mitochondrial fission by dephosphorylating the pro-fission protein DRP1.

• The hearts and brains of mice lacking RCAN1 are more susceptible to damage from ischemia/reperfusion.

• Down Syndrome, a condition caused by triplication of chromosome 21, has been associated with altered mitochondrial function and increased oxidative stress.

What New Information Does This Article Contribute?

• The mitochondrial network is more fragmented in cells deficient for RCAN1, due to a calcineurin-dependent increase in DRP1 activation and mitochondrial fission.

• Cardiomyocytes deficient for RCAN1 have decreased mitochondrial membrane potential, O₂ consumption, generation of reactive oxygen species (ROS), and capacity for mitochondrial Ca²⁺ uptake.

• This reduces the capacity of mitochondria to buffer cytosolic Ca²⁺, thereby increasing damage from Ca²⁺-activated calpain proteases upon reperfusion.

• Over expression of the RCAN1.1 isoform is sufficient to generate a more fused network and increase oxygen consumption.

• Mitochondria in trisomic iPSCs derived from individuals with Down syndrome are larger and more metabolically active than isogenic, disomic controls. Furthermore, siRNA depletion of RCAN1.1 is sufficient to restore the mitochondrial network and function.

Mitochondria carry out critical functions beyond making ATP, such as acting as sites for Ca²⁺ uptake and mediating cell death or survival. Here we show that RCAN1 helps maintain a functional mitochondrial network in cardiomyocytes and other cell types both in vitro and in vivo. Loss of RCAN1 increases mitochondrial fission while decreasing the capacity for Ca²⁺ uptake. This leaves tissues more susceptible to calpain-mediated damage following ischemia/reperfusion, such as occurs with heart attack or stroke. Conversely, excess RCAN1, is sufficient to promote fusion and increase mitochondrial metabolism. Although this is beneficial in the heart, we postulate, that in the context of Down syndrome, where RCAN1 levels are chronically elevated, enhanced fusion may both increase generation of reactive oxygen species and interfere with the process of mitochondrial repair.

Nonstandard Abbreviations and Acronyms

Atp5b

Mitochondrial ATPase Synthase, beta subunit

CAPN

calpain

CCCP

carbonyl cyanide m-chlorophenylhydrazone

CICR

calcium-induced calcium release

CN

calcineurin

dKD

double knockdown of RCAN1.1 and RCAN1.4

DRP1

Dynamin-1-Like

DSCR1

Down Syndrome Critical Region 1

E-64D

calpain inhibitor

ER

endoplasmic reticulum

ETC

electron transport chain

FRAP

Fluorescence recovery after photobleaching

Hk2

hexokinase 2

HSPA9

Heat Shock Protein Family A (Hsp70) Member 9

iPSC

induced pluripotent stem cells

T21-iPSC

iPSC derived from individuals with Down syndrome

D21-iPSC

disomic derivative of T21-iPSC

I/R

ischemia/reperfusion

MDL

calpain inhibitor

MEF

mouse embryonic fibroblasts

MFN2

mitofusin 2

MOI

multiplicity of infection

NANOG

Nanog homeobox

NRVM

neonatal rat ventricular myocytes

Oligo

oligomycin

OPA1

mitochondrial dynamin like GTPase

PD 150606

calpain inhibitor

PINK1

PTEN induced putative kinase 1

Pfkfb2

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2

POU5F1

POU class 5 homeobox

RCAN1

Regulator of Calcineurin 1

RCAN1.1

exon 1 isoform of RCAN1

RCAN1.4

exon 4 isoform of RCAN1

ROS

reactive oxygen species

Slc2a1

solute carrier family 2 member 1

SPTAN1

α-spectrin

SOX

SRY-box 2

TMRM

tetramethylrhodamine

TUBB

beta-tubulin

WT

wild type

ΔΨ_m

mitochondrial membrane potential