Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon
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The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.
To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria.
Methods and Results:
Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.
We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.
Meet the First Author, see p 308
The voltage-gated sodium channel (Na-channel) is central to cardiac electrogenesis. Its dysfunction can lead to lethal arrhythmias both in acquired (ischemia1,2; heart failure2) and genetic disorders.3–5 Rare and common genetic variants in SCN5A (encoding NaV1.5), encoding the pore-forming Na-channel subunit NaV1.5, are strongly associated with life-threatening arrhythmias4,6 and with structural heart disease.7–9 Yet, insight into Na-channel function, particularly as it extends beyond electrogenesis, is limited.
Na-channels form macromolecular complexes that cluster at specific subcellular domains (or pools).10–12 An emerging concept is that certain Na-channel partners localize only to one subcellular domain, thus endowing the functional complex with region-specific properties that, if disrupted, facilitate arrhythmias.13–17
Subcellular organelles such as mitochondria also organize in region-specific subdomains. Mitochondria in myocytes are highly organized, spatially separated into at least 3 subpopulations: one just beneath the surface sarcolemma (subsarcolemmal mitochondria [SSM]), a second one (the largest fraction) between myofibrils in longitudinal chains (interfibrillar mitochondria [IFM]), and a third one, occupying the perinuclear subdomain (perinuclear mitochondria [PNM]). Data show that these subpopulations possess distinct biochemical properties, morphology, Ca2+ handling and that they may interact differently with other intracellular structures, causing functional specificity.18,19
The relationship between Na-channel and mitochondrial function is well established in one direction only: mitochondrial function and molecular composition can alter sodium current (INa).20,21 Recently, however, Zhang et al22 demonstrated that reduced levels of Scn5a expression lead to accumulation of myocardial reactive oxygen species (ROS), and Wan et al23 documented that expression of NaV1.5 mutant F1759A leads to mitochondrial injury. The mechanism by which Na-channel expression or activity can affect mitochondrial function or integrity is not known.
We investigated the anatomic and functional relation between mitochondria and NaV1.5 in adult cardiac myocytes. Advanced imaging methods allowed us to observe that a subpopulation of membrane-resident NaV1.5 clusters sits in close apposition to subjacent SSM, and that it is in this mitochondrial subpopulation where the mitochondrial NCLX (mitochondrial Na+/Ca2+ exchanger; encoded by SLC8B1) is primarily located. This anatomic proximity led us to investigate functional changes in mitochondria resulting from Na-channel activity. In particular, exposing adult cardiac myocytes to the Na-channel blocker TTX (tetrodotoxin) caused accumulation of mitochondrial Ca2+ and ROS production, predominantly in SSM. We speculate that proximity to functional NaV1.5 clusters facilitates sodium-dependent activity of NCLX to extrude mitochondrial Ca2+, whereas reduced-sodium entry impairs Ca2+ extrusion. In addition, we observed that the relation between NaV1.5 and NCLX is not only local. Indeed, human transcriptome analysis shows that SCN5A negatively correlates with SLC8B1. Experimental confirmation was obtained from human-induced pluripotent stem cell (hiPSC)–derived cardiomyocytes in which SCN5A was knocked down. We conclude that not only mitochondrial metabolism can affect INa but Na-channels can, in turn, positively affect mitochondrial function. Our data lead us to propose the existence of a Na-channel subpopulation that clusters with subjacent mitochondria to create an anatomic couplon for electro-metabolic coupling.
The authors declare that all supporting data are available within the article and in the Data Supplement.
Expanded Methods available in the Data Supplement.
Wild-type black mice C57BL/6 N strain 027 (Charles River Laboratories) were treated in accordance to the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health. Procedures were approved by the New York University (NYU) Institutional Animal Care and Use (IACUC) Committee, protocol 160726. Both genders were included, and animals were between 3 and 6 months of age.
Focused Ion Beam-Scanning Electron Microscopy
Protocol were modified from Wilke et al.24 Tissue was processed, and en bloc lead staining performed to enhance membrane contrast.25 Acquisition was done using Auto Slice and View G3 software.26
For immunolocalization, HL-1 cells were incubated with Mitotracker and NCLX antibody, as described in the Data Supplement.
Adult mouse ventricular myocytes were obtained by enzymatic dissociation, as described in the Data Supplement.
Single-Molecule Localization Microscopy by Stochastic Optical Reconstruction Microscopy
Ventricular cardiomyocytes were incubated with Mitotracker before fixation and immunostained with NCLX, NaV1.5, and α-actinin antibodies. Interaction factor analysis (details in the Data Supplement) provided a measure of the degree to which mitochondria and NaV1.5 proximity can be the result of random (versus deterministic) distribution.
Mitochondrial Ca2+ Dynamics
As described,27 mitochondrial Ca2+ was measured with Rhod-2-AM dye. Analysis and quantification were done using the Leica Application Suite X (LASX) and ImageJ software. To evaluate mitochondrial Ca2+ dynamics in response to drugs, we monitored Rhod-2 fluorescence signal before (F0) and after (F) adding: TTX, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)+oligomycin A and 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157).
Mitochondrial Superoxide Detection
Superoxide production was measured using MitoSOX, per manufacturer’s instructions. To evaluate mitochondrial superoxide changes in response to TTX, we measured MitoSOX fluorescence signal before (F0) and after (F) TTX.
SCN5A Correlation Analysis in Human Left Ventricle
RNA-seq raw data from human left ventricular tissue were downloaded from the Genotype-Tissue Expression v8 consortium (https://www.gtexportal.org/home/datasets). Analysis done using Rstudio.
hiPSC Cardiomyocytes Model of SCN5A Knockout
hiPSCs were maintained as previously described.28 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) was used to generate the SCN5A knockout line. Differentiation into hiPSC cardiomyocytes was modified from a previously described protocol.29,30
Numerical results are given as means±SEM. Statistical tests were used as stated. Differences were considered significant at P<0.05.
Mitochondria Are in Close Apposition to the Sarcolemma
Figure 1A shows an electron microscopy image of mouse left ventricle revealing the alignment of mitochondria along the interfibrillar space. A complete volumetric image of cardiac tissue acquired using focused ion beam-scanning electron microscopy (Figure 1B) showed mitochondria nested immediately under the sarcolemma in the dome-shaped surface spanning the distance between z-disks (the crest31), with both structures (sarcolemma and SSM) reaching points of close apposition (red arrows; small panels show different planes of the same preparation). A zoom-in of Figure 1B-A (top), with detailed description of structures observed in the left end of the image, is presented in Figure I in the Data Supplement. Segmentation analysis of focused ion beam-scanning electron microscopy images facilitated visualization (Figure 1C) and revealed multiple connections of SSM with deeper mitochondria both immediately subjacent and, through mitochondria that bridge across sarcomeres, to other interfibrillar chains in the cell, hence expanding into a complete mitochondrial reticulum31 (Figure 1C and Figure II in the Data Supplement). Approximately 13% of the mitochondrial surface in the subsarcolemmal space was found <20 nm apart from the membrane (Figure III in the Data Supplement).
NaV1.5 Clusters Adjacent to SSM in a Nonrandom Organization
In separate experiments, we used dual-color stochastic optical reconstruction microscopy to localize NaV1.5 in the membrane surface of adult ventricular myocytes. α-Actinin was used to mark sarcomeric edges (z-disks). As shown in Figure 2A, we observed large NaV1.5 clusters between z-disks, in agreement with previous functional data.14 Negative controls are shown in Figure IV in the Data Supplement. NaV1.5 distribution led us to speculate that a subpopulation of Na-channels may localize in close proximity to SSM, creating a macromolecular complex.
Figure 2B presents an image of a single, isolated adult cardiac myocyte labeled for NaV1.5 (green) and Mitotracker (red) to localize mitochondria. At the cell lateral membrane (distinguishable as the edge framing the long axis of the cell), a seemingly preferential proximity of mitochondria and NaV1.5 was noted (white rectangles indicating area enlarged in the bottom of each panel). For some experiments, a third color was added (blue) to mark α-actinin (Figure 2C). Data are consistent with the localization of NaV1.5 and of mitochondria separately detected by focused ion beam-scanning electron microscopy and by stochastic optical reconstruction microscopy (Figures 1 and 2A, respectively). Moreover, the organization of both resembles the shape of the crest, with a NaV1.5 cluster resting on top of the mitochondria and between z-disks. NaV1.5 cluster properties are indicated in Figure V in the Data Supplement.
We developed an analytical tool to address the question of whether proximity is random or deterministic. A line traced over the aligned SSM was used as reference (Figure 3A). Each NaV1.5 cluster was defined as an ellipse. Two parameters were defined: the distance between the centroid of the ellipse and the reference line, and the angle formed between the long axis of the ellipse, and the reference line (details in the Data Supplement). Parameters were correlated for each cluster on an xy plot (Figure 3B). Experimental data were compared with a simulated image where mitochondria were kept as in the experimental data but NaV1.5 clusters were distributed randomly, though constrained by the edge of the cell (Figure 3C and 3D). As shown in Figure 3B, most experimental NaV1.5 clusters assembled in a small window of 10 to 300 nm in distance and a 0° to 60° angle to the reference line. However, in simulated images, green clusters were scattered along the plot (Figure 3D). The method was further validated when comparing the relation between SSM and a line formed by immunostaining of α-actinin, which traces the z-disk perpendicular to the lateral surface of the membrane. In this case, almost all α-actinin clusters were gathered in a very small window, with a 500 to 2000 nm distance and a 70° to 90° angle to the mitochondria long axis, as expected (Figure 3E through 3H). Overall, our results support the concept that the Na-channel complex and mitochondria form a structural macromolecular complex. Whether the association has a functional correlate was investigated next.
Mitochondrial NCLX Is Predominantly Found in SSM Near NaV1.5 Clusters
NCLX plays an important role in maintaining mitochondrial function. Sodium concentration, in turn, is key to the ability of NCLX to extrude Ca2+ from the mitochondrial space. Given the found relation between NaV1.5 and mitochondria, we examined whether NCLX had a preferential localization in the SSM space.
Figure 4 shows that the immunofluorescent signal for NCLX (red) was more abundant in the SSM than in mitochondria localized in the intracellular space; we also show significant colocalization of NCLX with NaV1.5-positive clusters (green). Indeed, ≈80% of NaV1.5-NCLX clusters colocalized (ie, were located within 20 nm of each other) at the lateral membrane versus ≈30% in the cell interior (Figure VI in the Data Supplement). To further validate NCLX antibody specificity for mitochondrial NCLX, we immunolocalized mitochondria with NCLX in HL-1 cells. Figure VII in the Data Supplement shows colocalization of the 2 structures and a round-punctuate pattern, characteristic of mitochondrial structures.
Given the proximity of NCLX to NaV1.5, we postulated that the presence or absence of INa may impact on NCLX function and as such, on the extent of Ca2+ accumulation in the mitochondrial space.
Mitochondrial Ca2+ Dynamics: Differences Between SSM and IFM
To assess the relation between NCLX and Na-channel function, we used a mitochondrial Ca2+ indicator (Rhod2-AM; Figure 5A; see also Figure VIIIA in the Data Supplement, Methods and27). Figure IX in the Data Supplement shows preservation of striated cell morphology after treatment. Rhod2-AM intensity was measured before (F0) and after drug treatment (F), and measurements of the ratio F/F0 in SSM were compared with those obtained in IFM. Measurements in control conditions (no drugs added) are shown as the first 2 bars of the graph in Figure 5B, corresponding to data acquired from SSM (red) and IFM (gray), respectively.
Since cytoplasmic Na+ concentration is a major driver of NCLX forward movement, we first tested whether blocking INa with TTX would affect mitochondrial Ca2+. Rhod2-AM fluorescence was first recorded in a solution containing 148 mmol/L Na+; then, TTX 10 μmol/L was added. The bar graph (bars with vertical lines) in Figure 5B shows that, upon TTX exposure, SSM accumulated significantly more Ca2+ (detected as increase in Rhod2 fluorescence) than IFM (F/Fo 1.1±0.03 and 0.99±0.03 for SSM and IFM, respectively; P<0.05). This was consistent with the notion that reduced entry of sodium via NaV1.5 limits NCLX activity, and that this effect is more noticeable in the area where the NaV1.5/NCLX complex is abundant (see Figure VIIIB in the Data Supplement). Of note, when low concentrations of TTX (100 nmol/L) were used, we observed a nonstatistically significant tendency for increased Rhod2 intensity in SSM (Figure X in the Data Supplement).
We next compared Ca2+ dynamics in SSM versus IFM in the presence of TTX and after mitochondrial depolarization with 1 μmol/L FCCP +2 μmol/L oligomycin. FCCP alone can induce mitochondrial ATP depletion via the reverse of F0/F1-ATPase, which is prevented by adding the F0/F1-ATPase inhibitor oligomycin. FCCP induces a mitochondrial depolarization of around 100 mV, enough for NCLX to function in reverse mode (Ca2+in/Na+out of the mitochondria).32 Consistent with this notion, FCCP+oligomycin augmented Rhod2 fluorescence exclusively at the SSM (Figure 5B, bars with horizontal lines). Most importantly, adding the NCLX inhibitor CGP-371257 attenuated the mitochondrial Ca2+ accumulation in SSM and no significant difference in Rhod2 fluorescence was observed between SSM and IFM (Figure 5B, bar with dots).
We analyzed Rhod2 fluorescence in cells treated only with 2 μmol/L CGP-37157, a selective NCLX inhibitor. As expected, after adding CGP-37157, Rhod2 fluorescence increased in SSM (ratio 1.10±0.024 versus IFM ratio 0.98±0.01, P<0.001, Figure 5B bars with crosses) indicating that Ca2+ influx through MCU (mitochondrial calcium uniporter) cannot be compensated by extrusion since NCLX is inhibited. Rhod2 fluorescence was practically unchanged after CGP-37157 addition in IFM, suggesting that IFM have another way of extruding Ca2+ that is NCLX- and Na+-independent.
Finally, we measured mitochondrial Ca2+ dynamics at different pacing rates (3 and 5 Hz) alone or in the presence of either TTX or CGP-37157. Rapid pacing led to mitochondrial Ca2+ accumulation in SSM and IFM (versus rest #P<0.05). When TTX was added (10 μmol/L), there was a tendency for SSM to accumulate more Ca2+ in response to pacing (3 versus 3 Hz+ TTX P=0.06; 5 versus 5 Hz+ TTX P=0.07) and when comparing the 2 mitochondrial subpopulations, mitochondrial Ca2+ increased significantly more in SSM than in IFM, *P<0.05). Similar results were obtained with CGP-37157. Details in Figure XI in the Data Supplement. Altogether, the data suggest functional coupling between NaV1.5 and NCLX in the sarcolemmal-subsarcolemmal space in the crest region of adult cardiac myocytes, with functional implications to the adaptation necessary to meet demand at increased rates.
Increased Superoxide Production After TTX Addition
There is a reciprocal interaction between Ca2+ and ROS which, in physiological conditions, plays an important role in regulating cellular signaling networks.33,34 Pathological changes in one can affect the other, leading to more damage. Since TTX increases mitochondrial Ca2+, we wondered if this could lead to increased ROS production.
We used the fluorescent dye MitoSOX Red, which detects mitochondrial superoxide, to measure ROS in live myocytes before and after TTX. When in mitochondria, MitoSOX exhibits red fluorescence. As seen in Figure 6, at resting conditions (F0) ventricular myocytes showed low MitoSOX fluorescence, higher in SSM, as previously reported.35 Co-staining with MitoTracker confirmed MitoSox mitochondrial localization (Figure XII in the Data Supplement).
After exposure to TTX 10 μmol/L (pacing at 0.5 Hz), MitoSOX fluorescence increased in both SSM and IFM, the latter being less pronounced (Paired Student t test: P<0.001 +TTX versus rest in SSM; P<0.05 +TTX versus rest in IFM). This result indicates that blocking Na+ entry with TTX leads to increased mitochondrial ROS production, though in this case there was no predominant effect in the SSM, suggesting that the role of INa on mitochondrial function may go beyond its local interaction with NCLX.
Correlation Analysis of Human Left Ventricle Transcriptome in Relation to SCN5A Expression
We used the Genotype-Tissue Expression repository (v8) of 386 samples of left ventricle tissue from deceased human donors to compare the abundance of SCN5A transcript against SLC8B1 and SLC8A1, which encode the mitochondrial NCLX and the plasma membrane NCX, respectively. Resulting plots are shown in Figure 7A and 7B. Data show that SLC8A1 is positively correlated with SCN5A (regression coefficient β=0.62, log P value=4×10−58) whereas SLC8B1 is negatively correlated with SCN5A (regression coefficient β=−0.17, log P value=1.66×10−30). This observation is only correlational. We, therefore, tested whether reduced abundance of one transcript would affect the abundance of the other. To this aim, we used an hiPSC cardiomyocytes line deficient in SCN5A (see Methods and Figures XIII and XIV in the Data Supplement). As shown in Figure 7C the abundance of SLC8B1 was significantly increased in cells where SCN5A was knocked down, supporting the notion of a transcriptional relation between the 2 gene products.
Our data show that SSM and a subpopulation of NaV1.5 at the lateral membrane are in close apposition and that in ventricular cardiomyocytes, not only mitochondria can regulate INa,20,21 but also INa can regulate NCLX activity. NCLX preferentially localized at SSM near NaV1.5 clusters. We speculate that such distribution creates a high-Na+ microdomain that is energy-efficient, comparable to the high-Ca2+ microdomain formed between MCU and the sarcoplasmic reticulum. Many diseases have been linked to changes in intracellular Na+ concentration36 which, in turn, could affect NCLX function, therefore affecting ATP production and ROS generation. Overall, we show the presence of a Na-channel-mitochondria complex and suggest that NaV1.5-NCLX proximity may have functional consequences to metabolic homeostasis via crosstalk with the mitochondrial reticulum.
Cardiomyocytes are rich in mitochondria, which occupy a third of total cell volume.18 Although mitochondria form a network reaching throughout the cell,37,38 they are divided into at least 3 subpopulations with distinct morphology, composition, biochemical properties and function, contributing to their capacity for adaptation.18,19,35 Here, we focus on SSM. As others,39 we found SSM nested in-between z-disks. A nanometric-resolution cell surface scan shows a pattern of crests and valleys (or grooves) with ≈2 μm periodicity, that is, the length of a sarcomere.31 In this pattern, grooves match the position of the z-disk (prompting the name z-grooves), with crests being dome-shaped surfaces spanning the distance between 2 z-grooves.31 Previously, using scanning patch clamp, we measured larger INa in the crest compared with grooves.14 This matches our data showing NaV1.5 clusters preferentially aligned with SSM at the crest. We also show that the cell membrane in the crest region reaches close proximity with the outer membrane of the mitochondria, in a manner reminiscent of the T-tubule-junctional sarcoplasmic reticulum dyad (see eg,40,41) or between cell membranes in the perinexus.42 In these structures, the narrow intermembrane space allows functional coupling between channels residing in opposing membranes. The molecular mechanisms of formation of this NaV1.5-mitochondrial complex remain undefined. Yet, we show that the relation between mitochondria and NaV1.5 is deterministic and of a particular topology.
We identify NCLX, a molecule key for Na+ and Ca2+ homeostasis, as resident of the NaV1.5-mitochondria hub. NCLX (encoded by SLC8B1) is proposed to be the main pathway for mitochondrial Ca2+ extrusion in excitable cells, while MCU is responsible for Ca2+ transport into mitochondria.43,44 NCLX transport is voltage-dependent and electrogenic, with stoichiometry of 3 to 4 Na+ for 1 Ca2+.45 Unlike plasma membrane NCX, unique residues confer NCLX Li+ selectivity.46 The Ca2+ extrusion rate of NCLX is ≈100× slower than MCU-Ca2+ influx rate. Therefore, NCLX activity is the rate-limiting step of mitochondrial Ca2+ cycling.44,47,48
NCLX is fundamental to life. A conditional knockout of NCLX quickly leads to heart failure associated with mitochondrial Ca2+ overload and consequent increase in ROS generation.43 In contrast, NCLX overexpression in mouse heart increases mitochondrial Ca2+ extrusion preventing pathogenic Ca2+ accumulation and ROS generation.43 Whether loss or over-abundance of NCLX affects INa or subcellular localization of NaV1.5 remains unclear.
NCLX is very sensitive to changes in cytoplasmic [Na+],32,48,49 given that the Km of association is similar to resting [Na+]i (Km ≈7–10 mmol/L). Moreover, studies from Skogestad et al show that [Na+]i in an active, excitable myocyte, is not homogeneous. Rather, it is highest in the subsarcolemma and distributes in pools, likely, corresponding to the location of Na+-channel clusters.50 We propose that NCLX is close to NaV1.5 channels to rapidly sense Na+ signals and properly extrude Ca2+ out of mitochondria, in a way similar to how MCU’s low affinity for Ca2+ is compensated by proximity to the sarcoplasmic reticulum (diagram in Figure VIII in the Data Supplement).51,52 Several studies describe an interaction of NCLX with plasma membrane transporters: (1) in pancreatic β-cells, glucose-dependent depolarization leads to cytosolic Na+ influx via voltage-gated Na+ channels, which promotes NCLX activity, maintaining ATP production and controlling insulin secretion.53,54 (2) TRPV1 (the nociceptive noxious heat-activated transient receptor potential vanilloid 1) triggers a Na+-dependent depolarization that controls NCLX Ca2+ extrusion, which at the same time regulates TRPV1 conductance.55,56 (3) In macrophages and melanoma cells, activation of a splice variant of NaV1.6 causes release of Na+ from vesicular compartments and Na+-uptake-Ca2+ release by NCLX.57
Through human transcriptome analysis, we found SLC8B1 negatively correlating with SCN5A. Though the common denominator of this relation remains unknown, its presence may have important functional consequences. Indeed, reduced expression of SCN5A may be compensated by increased expression of SCL8B1 to increase the ability to capture Na+ entering the cell. The reverse would also have functional effects: an excess Na+ entry could be compensated by reduced expression of the exchanger, maintaining mitochondrial homeostasis. This is consistent with the notion that NCLX activity is strongly regulated by even slight changes in cytosolic Na+.48,58–63 Their physical proximity (shown here) and transcriptional inverse relation would facilitate maintenance of this delicate equilibrium.
In resting Na+ and Ca2+ conditions and with an inner mitochondrial membrane potential of ≈−180 mV, NCLX operates in forward mode, exporting Ca2+ out of the mitochondrial matrix in exchange for cytosolic Na+. However, in depolarized mitochondria, NCLX functions in reverse mode, transporting Ca2+ into the mitochondria and extruding Na+.64 Studies have shown that increasing cytosolic Na+ accelerates mitochondrial Ca2+ decay due to increased NCLX forward mode activity,63,64 but these studies did not compare SSM to IFM. Our mitochondrial Ca2+ dynamic experiments show that NCLX is predominantly located at SSM, confirming our single-molecule localization microscopy experiments, and TTX leads to mitochondrial Ca2+ accumulation more prominently in SSM, connecting NaV1.5, and NCLX function. The Ca2+ accumulation after TTX+FCCP+Oligomycin exposure in SSM seen in our experiments could be explained by (1) active uptake of calcium by the reverse NCLX or (2) increased mitochondrial uptake via MCU. However, MCU open probability decreases with mitochondrial depolarization (ie, with FCCP).64 In fact, IFM show a slight decrease in Rhod2 fluorescence after TTX+FCCP+oligomycin which could be accounted for an inhibited MCU and lack of reverse mode NCLX-Ca2+ uptake in IFM. We used the benzothiazepine compound CGP-37157 to selectively block NCLX (IC50=0.36 μmol/L).59,62 At least a 10-fold higher concentration of CGP-37157 is needed to affect plasma membrane NCX, SERCA, and RyR2.59 It is known that cytosolic Na+ is required for powering NCLX to activate mitochondrial Ca2+ efflux. Therefore, our mitochondrial Ca2+ dynamics data suggest that a healthy INa is needed to maintain mitochondrial Ca2+ homeostasis. However, and compared with Ca2+ imaging, studying cellular Na+ dynamics is challenging, particularly due to the relatively small changes of intracellular Na+ concentration.53,58
There is mutual interaction between ROS and Ca2+, where ROS can regulate Ca2+ signaling and Ca2+ signaling is essential for ROS production.33,43 Physiologically, ROS are generated as by-product of mitochondrial respiratory chain activity or by specialized ROS-generating enzymes to regulate signaling functions such as cell proliferation, migration, and vascular tone; but toxic levels associate with pathology. Mitochondrial Ca2+ accumulation leads to ROS production. Our results show increased ROS production after TTX exposure in both SSM and (less pronounced) IFM, even though only SSM showed increased mitochondrial Ca2+ after TTX. It is worth noting that, even though they are part of distinct mitochondrial subpopulations, SSM and IFM are connected, forming a mitochondrial reticulum in which ROS can propagate.37,38,65 Most importantly, ROS production leads to decreased INa.66 ROS can alter the oxidative state of protein kinase A (PKA), protein kinase C (PKC), CaMKII (calcium/calmodulin-dependent protein kinase II), and NAD(H), which in turn regulates INa.33,67 Genetically engineered Scn5a heterozygous mice show increased ROS accumulation.68 This, together with our results, suggests that there could be an amplification loop: increased ROS decreases INa, leading to less Na+ entering the cytoplasm, which in turn affects NLCX activity which leads to mitochondrial Ca2+ accumulation, leading to more ROS production.
One question that arises is how do IFM extrude calcium if NCLX is mainly found in SSM? Even though NCLX is the major transporter for Ca2+ extrusion, mitochondria contain both Na+-dependent and Na+-independent mechanisms for Ca2+ extrusion.45,64 This may include LETM1 (leucine zipper EF-hand-containing transmembrane protein 145,64,69). Somehow controversial, it has been proposed that transient mitochondrial permeability transition pore openings could also help discharge mitochondrial Ca2+ load.45,47,70 Other studies show Cx43 hemichannels in mitochondria71 and particularly in SSM72 which could act as mitochondrial Ca2+ gateway. Overall, our data are consistent with previous descriptions of proteins that distribute and work differently in various subpopulations of mitochondria (see eg 73,74).
Our data suggest the need of healthy INa for (proper) mitochondrial Ca2+ balance. Many diseases associate with cytoplasmic unbalanced Na+ concentration. A pathogenic increase in cytoplasmic Na+ accelerates mitochondrial Ca2+ efflux trough NCLX, which inhibits mitochondrial metabolic rate and ATP production, as seen in heart failure, ischemia, and seizure-like events.48,59,63,75,76 In melanoma cells, an increase in Na+ mediated by vesicular NaV1.6 activates NCLX, which accelerates invasiveness of these tumor cells.57 Conversely, hypoxia is known to reverse NCLX mode, which leads to mitochondrial Ca2+ overload and ROS production, in turn accelerating cytoplasmic Na+ accumulation.77 Future studies need to be done to analyze NCLX activity in pathologies where INa is reduced (eg, Brugada syndrome).
human-induced pluripotent stem cell
leucine zipper EF-hand-containing transmembrane protein 1
mitochondrial calcium uniporter
mitochondrial Na+/Ca2+/Li+ exchanger
reactive oxygen species
We thank Chris Petzold for EM sample preparation and image acquisition. The GTEx Project was supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by the National Cancer Institute (NCI), National Human Genome Research Institute (NHGRI), National Heart, Lung, and Blood Institute (NHLBI), National Institute on Drug Abuse (NIDA), National Institute of Mental Health (NIMH), and National Institute of Neurological Disorders and Stroke (NINDS).
Sources of Funding
Supported by National Institutes of Health (NIH) RO1-HL134328, RO1-HL136179, 5R01HL148609-02, and RO1-HL145911 (M. Delmar), the Wilton W. Webster Fellowship in Pediatric Electrophysiology from the Heart Rhythm Society (M. Pérez-Hernández), a Leducq Foundation Transatlantic network of Excellence (M. Delmar and P. Burridge) and the Swiss National Science Foundation P1BEP3_172237 (S. Vermij). Microscopy Laboratory is supported by NIH/NCI P30CA016087 and S10 ODO019974-01A1.
Expanded Materials and Methods
Online Figures I–XIV
References 78 and 79
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Novelty and Significance
What Is Known?
The voltage-gated sodium channels, formed by the protein NaV1.5, carry the excitatory inward current (the sodium current) that is responsible for the action potential upstroke in adult cardiac ventricular myocytes.
NaV1.5 channels localize to different subdomains within the myocyte, associating with other molecules in a domain-specific manner.
Mitochondrial function can alter the magnitude and properties of the sodium current.
What New Information Does This Article Contribute?
NaV1.5 clusters that localize to the membrane region between two z-lines (the crest of the sarcomere) are in close apposition to subsarcolemmal mitochondria and to NCLX (mitochondrial Na+/Ca2+ exchanger).
This anatomic proximity leads to a functional relation between NaV1.5 and the mitochondrial NCLX.
NaV1.5 channel malfunction leads to unbalanced mitochondrial calcium dynamics, potentially impacting cell metabolism.
Na-channels form macromolecular complexes that, in adult ventricular myocytes, cluster at specific subdomains. Dysfunction of the channel itself, or of its partners, can lead to arrhythmias that cause sudden cardiac death. The relation between Na-channels and mitochondrial function has been described in one direction only: mitochondrial byproducts (such as reactive oxygen species [ROS]) affect sodium current. Here we show that this relation is reciprocal, namely, Na-channel activity influences mitochondrial function. Using imaging techniques, we describe the deterministic localization of Na-channels and subsarcolemmal mitochondria, where we primarily locate the mitochondrial NCLX. Functionally, the blockade of Na-channels leads to a local mitochondrial Ca2+ accumulation and ROS production. We propose that NCLX is powered by the entry of Na+ through Na-channels to properly extrude Ca2+. This proposed model for electro-metabolic coupling redefines NaV1.5 as a key regulator of cell metabolism and opens a new window to understand electrical and structural disorders of the heart.