You are on page 1of 11

Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Contents lists available at ScienceDirect

Journal of Molecular and Cellular Cardiology


journal homepage: www.elsevier.com/locate/yjmcc

Mitochondrial cardiomyopathies feature increased uptake and diminished MARK


eux of mitochondrial calcium
Salah Sommakiaa,1, Patrick R. Houlihanb,1,2, Sadiki S. Deanea, Judith A. Simcoxc,
Natalia S. Torresa, Mi-Young Jeongc,d, Dennis R. Wingec,d, Claudio J. Villanuevac,
Dipayan Chaudhuria,
a
Nora Eccles Harrison Cardiovascular Research and Training Institute, Cardiology Division, Department of Internal Medicine, University of Utah, Salt Lake City, UT,
United States
b
Department of Cardiology, Boston Children's Hospital, Boston, MA, United States
c
Department of Biochemistry, University of Utah, Salt Lake City, UT, United States
d
Department of Internal Medicine, University of Utah, Salt Lake City, UT, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Calcium (Ca2 +) inux into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether
Mitochondrial calcium uniporter mitochondrial Ca2 + transport pathways are altered in the setting of decient mitochondrial energy synthesis, as
Mitochondrial sodium-calcium exchanger increased matrix Ca2 + may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which
Whole-mitoplast electrophysiology feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription
Cardiac metabolism
factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in
Respiratory-chain decient cardiomyopathy
cardiomyocytes. By the second postnatal week (1015 day old mice), these mice have developed a dilated
OXPHOS decient cardiomyopathy
cardiomyopathy associated with impaired oxidative phosphorylation. We nd evidence of increased mi-
tochondrial Ca2 + during this period using imaging, electrophysiology, and biochemistry. The mitochondrial
Ca2 + uniporter, the main portal for Ca2 + entry, displays enhanced activity, whereas the mitochondrial sodium-
calcium (Na+-Ca2 +) exchanger, the main portal for Ca2 + eux, is inhibited. These changes in activity reect
changes in protein expression of the corresponding transporter subunits. While decreased transcription of Nclx,
the gene encoding the Na+-Ca2 + exchanger, explains diminished Na+-Ca2 + exchange, the mechanism for
enhanced uniporter expression appears to be post-transcriptional. Notably, such changes allow cardiac mi-
tochondria from Tfam knockout animals to be far more sensitive to Ca2 +-induced increases in respiration. In the
absence of Ca2 +, oxygen consumption declines to less than half of control values in these animals, but rebounds
to control levels when incubated with Ca2 +. Thus, we demonstrate a phenotype of enhanced mitochondrial
Ca2 + in a mitochondrial cardiomyopathy model, and show that such Ca2 + accumulation is capable of rescuing
decits in energy synthesis capacity in vitro.

1. Introduction Upon entering the mitochondrial matrix, Ca2 + can stimulate ATP
production [3], but excess Ca2 + entry disrupts mitochondrial function.
Heart failure leads to a profound reprogramming of energetic pro- Because Ca2 + has bimodal eects, deciphering its contribution to en-
duction in cardiomyocytes [1]. As heart failure progresses, energetic ergetic homeostasis during heart failure has remained dicult. Perhaps
supply fails to meet demand, and individuals with more severe supply- such diculties arise from the relatively late occurrence of clinically-
demand mismatch have reduced survival [2]. Amongst the pathways evident energetic depletion during heart failure [2], with mechanisms
altered in heart failure, our interest focuses on calcium (Ca2 +), which is regulating mitochondrial Ca2 + becoming obscured by other homeo-
central for excitation-contraction coupling, and is increasingly re- static pathways triggered during heart failure progression. To identify
cognized as both an enhancer and inhibitor of mitochondrial function. clear examples of altered mitochondrial Ca2 + regulation, we sought a

Abbreviations: AA, antimycin A; CSA, cyclosporin A; ETC, Electron transport chain; IMAC, Inner membrane anion channel; OXPHOS, Oxidative phosphorylation

Corresponding author at: 95 South 2000 East, Building 500 (CVRTI), Salt Lake City, UT 84108, United States.
E-mail address: d.chaudhuri@utah.edu (D. Chaudhuri).
1
Contributed equally.
2
Current address: Howard Hughes Medical Institute, Ashburn, VA, United States.

http://dx.doi.org/10.1016/j.yjmcc.2017.09.009
Received 27 April 2017; Received in revised form 7 September 2017; Accepted 25 September 2017
Available online 28 September 2017
0022-2828/ 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

disease model that features energetic failure as an early phenotype. We 2. Methods


expected that early onset of energetic dysfunction during heart failure
would allow us to examine mitochondrial Ca2 + regulation in a rela- Full description of methods is available in the Supplementary ma-
tively isolated manner. terials.
A spectrum of diseases featuring energetic imbalance arise from a
heterogeneous group of mutations in proteins encoded in either the 2.1. Animal use
nuclear or mitochondrial genome (mtDNA) that are involved in oxi-
dative phosphorylation (OXPHOS) [4]. These disorders often feature The oxed Tfam (a kind gift of Dr. Nils-Gran Larsson) and -
cardiac involvement, termed the mitochondrial cardiomyopathies, and myosin heavy chain-promoter driven Cre recombinase (Myh6-Cre) mice
can be devastating, increasing the mortality rates of children suering (Jackson Labs, Bar Harbor, ME, strain #011038) have been backcrossed
from these disorders three-fold [5]. For our studies, such decient into a C57BL/6J background [6,23]. All procedures were approved by
OXPHOS provides a unique opportunity to examine how the heart the Institutional Animal Care and Use Committee in accordance with
modulates mitochondrial Ca2 + uxes when energy production is institutional guidelines. Animals used were 1015 days old except
failing. One such mouse model was created by a cardiac-specic dele- where noted.
tion of the main transcription factor for mtDNA (transcription factor A,
mitochondrial, Tfam) [6], leading to depletion of electron transport 2.2. Mitochondrial isolation
chain (ETC) subunits encoded by mtDNA. In humans, two infants with
TFAM mutations died within months of liver failure, had mitochondrial For all assays, knockout and littermate controls were processed in
abnormalities on muscle biopsies, and in one case developed clinical parallel. Mice were euthanized with CO2 and hearts rapidly removed. A
heart failure [7]. mitochondrial fraction was subsequently isolated by dierential cen-
In mice, cardiac Tfam deletion recapitulates the central problem trifugation [24].
caused by the heterogeneous group of mutations causing mitochondrial
cardiomyopathies: mtDNA depletion and subsequent OXPHOS dys- 2.3. Mitochondrial enzyme assays
function. Multiple studies have revealed that these mice develop car-
diac dysfunction with many of the same clinical, biochemical, and ul- We processed 20 g of mitochondrial fractions using the Citrate
trastructural features found in human mitochondrial cardiomyopathies, Synthase Activity Kit (Sigma, St. Louis, MO). For subsequent assays of
such as an early decline in energetic function and abnormal cytoplasmic complex IIV activity, we determined the citrate synthase activity in a
Ca2 + handling [810]. Although these mice have been otherwise sample of Tfam heterozygous mitochondria and adjusted amounts of
characterized, regulation of their cardiac mitochondrial Ca2 + remains wild-type and knockout littermate mitochondria to match that level.
unexplored. Complex IIV were analyzed as detailed previously [25], using assay
Studying mitochondrial Ca2 + regulation in this model of mi- rates in the presence or absence of an inhibitor to determine the com-
tochondrial dysfunction may also provide broader clinical insight, as plex-specic activity.
mitochondrial dysfunction is widespread in heart failure. OXPHOS
dysfunction and mtDNA damage are prevalent in dilated cardiomyo- 2.4. Inductively coupled plasma-mass spectrometry (ICP-MS)
pathies, and indicate worse prognosis [1114]. In animal models of
ischemic [15], cardiotoxic [16], diabetic [17], tachycardia-induced Mitochondria isolated by dierential centrifugation were incubated
[18], and pressure-overload [19] cardiomyopathies, reductions in in 40 M digitonin to permeabilize compartments other than the mi-
TFAM, mtDNA, and OXPHOS are common. In a proteomic analysis, tochondrial matrix, prior to nal pelleting for analysis. Mitochondria
TFAM was the second-most downregulated protein in failing hearts were processed by the ICP-MS metals lab at the University of Utah.
[20]. Conversely, overexpressing TFAM improves function after cardiac
injury [21], and is the subject of therapies targeted towards cardio- 2.5. Fluorescent imaging of mitochondrial inner membrane voltage gradient
protection [22]. However, the late occurrence of clinically evident () and Ca2 + transport
OXPHOS deciency has made dening regulatory mechanisms dicult
in common forms of heart failure, whereas it drives heart failure in We performed protocols as previously described using 50 g of the
mitochondrial cardiomyopathies. mitochondrial fractions, except for Ca2 +-induced tetra-
Here, we study mitochondrial Ca2 + transport in the second post- methylrhodamine methyl ester (TMRM) depolarization, where we used
natal week in mice with cardiac-specic Tfam deletion. As OXPHOS 20 g [26].
declines, these animals develop a dilated cardiomyopathy. In these
failing hearts, we nd that mitochondria become far more Ca2 + avid, 2.6. Whole-mitoplast electrophysiology
taking up Ca2 + at twice the rate as controls. This occurs via increased
activity of the mitochondrial Ca2 + uniporter, the main channel trans- We prepared mitoplasts from mitochondrial fractions and subse-
porting Ca2 + from cytoplasm to mitochondrial matrix. In addition, quently performed voltage-clamp analysis using the Kirichok protocol
mitochondria from these mice release matrix Ca2 + at half the rate as [24].
controls, indicating reduced activity of the sodium-calcium (Na+-
Ca2 +) exchanger. The increase in Ca2 + inux and reduction in Ca2 + 2.7. Oxygen consumption analysis
eux reect corresponding changes in protein levels for the mi-
tochondrial Ca2 + uniporter and Na+-Ca2 + exchanger. Though the Oxygen consumption was determined with a Clark electrode
decrease in Na+-Ca2 + exchanger levels occurs at the transcript level, (Hansatech Instruments, Norfolk, UK), or with the uorescent oxygen
the mechanism for increased uniporter function appears to be post- probe MitoXpress Xtra (Luxcel Biosciences, Cork, Ireland). For
transcriptional, as mRNA levels are reduced despite higher protein MitoXpress Xtra, we used uorescence lifetime imaging of mineral oil-
amounts. Finally, we nd substantial inhibition of mitochondrial re- sealed wells in a 96-well microplate [27].
spiration in these mice after Ca2 + depletion, whereas Ca2 + incubation
boosts respiration to levels comparable to controls. 2.8. Statistical analysis

Microsoft Excel and R were used for data analysis. We rejected the
null hypothesis for P values < 0.05. For multiple comparison tests on

23
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

samples < 15, we established an overall P-value using a Kruskal-Wallis these were reduced in Tfam KO hearts (Fig. S1DF, Fig. 5). We then
test, and for P < 0.05 proceeded to a Dunn's multiple comparison test. measured ATP production using a luciferin-luciferase assay (Fig.
For multiple comparisons on samples > 15, we established an overall S1GeI), integrating OXPHOS and ATP transport. Using this measure,
P-value using a one-way ANOVA, and for P < 0.05 proceeded to two- we found that, as with respiration, Tfam KO mice had a lower rate of
tailed, unequal variance, Student's t-tests. For multiple comparison ATP production (0.73 0.07). Subsequent to blocking the F1-FO-ATP
tests, nal P-values between groups were Bonferroni-corrected. synthase with oligomycin, we found no dierence between genotypes.
Such deciencies in electron transport chain activity and ATP synthesis
3. Results are notable in the development of heart failure, including in the Tfam
KO animals [6,9,30].
3.1. The dilated cardiomyopathy produced by cardiac-specic deletion of Further analyses conrmed that the cardiomyopathy in P10-P15
Tfam is evident in the second postnatal week animals had not produced end-stage tissue remodeling. A prior study
demonstrated enhanced oxidative stress and apoptotic cell death, but
For our studies, we crossed the previously-developed Tfam loxP not extensive brosis, in 23 week old Tfam KO animals [8]. For oxi-
mice with mice bearing a Cre recombinase gene under the control of the dative stress, we measured H2O2 production using an Amplex Red assay
-myosin heavy chain promoter (Myh6-Cre), which becomes active (Fig. S2AeC). These showed a slight increase in ADP-stimulated H2O2
during embryogenesis in a cardiomyocyte-specic manner [28]. For our production in Tfam KO hearts (1.23 0.03), whereas maximal anti-
studies, we compared knockout animals (TfamloxP/loxP; +/Myh6-Cre, mycin A-induced H2O2 production was reduced (0.32 0.07), con-
[Tfam KO]) to littermate Tfam heterozygotes (TfamloxP/+; +/Myh6-Cre, sistent with diminished complex III activity. For cell death (Fig. S2D,E),
[Tfam HET]) and wild-type animals (TfamloxP/loxP; +/+ or TfamloxP/+; we saw an increase in terminal deoxynucleotidyl transferase dUTP nick
+/+ [Tfam WT]). end labeled (TUNEL) nuclei in Tfam KO hearts. Similarly, we found that
We conducted our experiments on P10-P15 animals, an age range in in 3 of 4 Tfam KO, but no control animals, we could detect cardio-
which most Tfam KO mice were alive, yet hearts were large enough to myocytes staining for activated caspase 3 (Fig. S2F). Despite these in-
yield sucient mitochondria for functional characterization of in- dicators of cell death, Masson's trichrome staining did not reveal any
dividual animals. By this age, Tfam deletion was essentially complete, evidence of increased brosis in this age range (Fig. S2G,H). These
reected in the near absence of mRNA and protein in knockout hearts results are congruent with the prior report [8].
(Fig. 1AC; numbers are average standard error fold changes com- As dilated cardiomyopathies progress, cardiac mitochondria ssion
pared to controls here and throughout: 0.12 0.02 mRNA, and become more abundant, with increased mitochondrial mass seen in
0.05 0.02 protein). The Tfam KO mice experienced early mortality, both human disease and animal models [6,9,11]. We thus examined
with animals dying mostly from postnatal week 2 onwards (Fig. 1D). mitochondrial abundance in P10-15 Tfam KO mice using several in-
This is consistent with prior reports of cardiac Tfam deletion [6]. In one dependent methods. First, complex II activity, which does not depend
study using TfamloxP/loxP; +/Myh6-Cre animals, > 75% of knockout on TFAM, was preserved. Second, Tfam KO mitochondrial ultra-
animals died in the rst postnatal week [29]. However, those animals structure under electron microscopy remained comparable to that of
were in a mixed background, and the Myh6-Cre transgene construct controls, as were mitochondrial size distributions and volume density
diered from the one used here. (Fig. 3AC). Similarly, the mitochondrial yield from each heart was not
All Tfam KO animals assayed had a dilated cardiomyopathy within dierent between Tfam KO and controls (Fig. 3D). Finally, we found no
this period (Fig. 1E,F). Cardiac function on echocardiography was im- dierence in citrate synthase activity, a marker of mitochondrial mass
paired, with severely reduced fractional shortening and dilated left (Fig. 3E).
ventricles (Fig. 1G,J). Tfam HET animals, despite having an inter- We then examined , the voltage gradient driving ATP synthesis.
mediate reduction in TFAM, had normal cardiac function at this age. We were unable to enzymatically dissociate high-quality cardiomyo-
cytes for single-cell imaging from animals < P17. In P17 Tfam KO an-
3.2. Tfam KO cardiac mitochondria have impaired OXPHOS activity but imals, single cardiomyocytes loaded with the voltage-sensitive dye
preserved numbers and in the second postnatal week TMRM (20 nM) uoresced less intensely than controls (0.74 0.02),
suggesting an abnormal, depolarized (Fig. S3A,B). However, since
Next, we examined mitochondrial function in Tfam KO animals in the rest of our analyses were performed on mice aged P10P15, we
this timespan. First, we studied the TFAM-mediated transcription of sought alternative means to test at younger ages. After mechani-
mtDNA. Quantitative reverse transcription polymerase chain reaction cally disrupting cells, mitochondrial fractions were enriched by dier-
(qPCR) analysis of a subset of mtDNA genes revealed minimal mRNA ential centrifugation and incubated in TMRM at high concentration
levels in Tfam KO compared to controls (Fig. 2A). Such disruption was (20 M), which quenched uorescence. Adding 5 M carbonyl cyanide
replicated in enzymatic assays of ETC function. We found marked in- 4-(triuoromethoxy)phenylhydrazone (FCCP) to this suspension un-
hibition of complex I, III, and IV but not complex II activity (Fig. 2BE; couples mitochondria, and the total TMRM uorescence increase as-
complex I: 0.23 0.06, complex II: 1.18 0.12, complex III: sociated with dequenching is a very sensitive measure of [31].
0.72 0.07, complex IV: 0.64 0.07). This pattern reects the en- Using this technique, Tfam KO mitochondria had indistinguishable
coding of ETC subunits in nuclear versus mtDNA, as, unlike the other from controls at younger ages (Fig. 3F). Together, these data reveal that
complexes, complex II subunits are wholly nuclear-encoded. Ad- the P10P15 period features disease caused by energetic failure, but has
ditionally, the absence of heart failure in Tfam HET animals at this age, not progressed to a late phase characterized by extensive tissue re-
despite intermediate reductions in ETC activity, is consistent with the modeling, impaired mitochondrial ultrastructure, and abnormal enzy-
signicant reserve capacity of cardiac mitochondria. Next, we mea- matic function.
sured respiration on complex I (glutamate/malate) or complex II sub-
strates (rotenone/succinate), using a Clark electrode (Fig. S1A). Con- 3.3. Tfam KO cardiac mitochondria feature higher baseline Ca2 +,
sistent with the qPCR and isolated complex measurements, complex I increased Ca2 + inux, and diminished Ca2 + eux
respiration was reduced in the Tfam KO (Fig. S1B; 0.6 0.1), whereas
respiration on complex II was unchanged compared to controls (Fig. We next turned to an analysis of mitochondrial Ca2 +. First, total
S1C; 1.3 0.2). Finally, we assayed ATP production. For the F1-FO mitochondrial Ca2 + content was measured by applying ICP-MS to mi-
ATP synthase, beyond the mtDNA-encoded mt-Atp6 and mt-Atp8 genes tochondrial pellets [32]. Tfam KO hearts had a 2.2 0.5-fold increase
(Fig. 2A), we quantied the expression of the nuclear-encoded ATP5A1, over controls in mitochondrial Ca2 + content (Fig. 4A). This elevation
ATP5F1, ATP5G1/2/3, and ATP5O subunits, and found that several of was specic to Ca2 +, as there was no dierence in mitochondrial

24
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Fig. 1. Myh6-Cre deletes Tfam in cardiac tissue and leads to a dilated cardiomyopathy in 1015 day old animals. A. qPCR analysis of Tfam expression, displayed as fold change relative to
wild-type animals. Myh6-Cre produces mice that are wild-type (WT), heterozygous (HET), or knockout (KO) for cardiomyocyte Tfam. Here and throughout grey points represent samples
from individual mice, summary bars show mean standard error, and numbers listed are animals used per genotype, unless otherwise noted (n = 910). B. Exemplar Western blot with
each lane showing cardiac TFAM expression (top) and -actin (loading control, bottom) from a mouse with genotype listed above. C. Summary data showing ratio of TFAM to -actin
chemiluminescence on Western, normalized to WT (n = 12). D. Mouse survival data (n = 2671). E. Left, photograph of mice on postnatal day 10. Right, hearts extracted from the
corresponding animal. Scale, 1 cm. F. Ratio of heart to body weight for individuals of listed genotype (n = 4586). G. Exemplar transthoracic echocardiograph M-mode recordings in left
ventricular short axis of mouse hearts. Calculated fractional shortening (H), left ventricular dimension in diastole (LVID, I), and calculated left ventricular (LV) mass (J) on transthoracic
echocardiography (n = 56). P < 0.05 (*), < 0.01 (**), < 0.001 (***), > 0.05 (NS) here and throughout.

potassium content (Fig. S4A). Next, we measured electrophoretic Ca2 + so this faster rate was due to increased uptake by Tfam KO mitochon-
uptake in mitochondrial fractions isolated by dierential centrifuga- dria. Next, we examined the predominant eux pathway for Ca2 +, the
tion. Mitochondrial suspensions were incubated in a Ca2 +-sensitive dye mitochondrial Na+-Ca2 + exchanger [34]. Following a pulse to load
restricted to the media [33]. A Ca2 + pulse produces a large uores- mitochondria with Ca2 + in Na+-free media, an injection of Ru360 and
cence increase that declines as mitochondria take up the Ca2 + bolus Na+ respectively blocks further transport through the uniporter and
(Fig. 4B). Notably, with this assay Tfam KO cardiac mitochondria se- activates Ca2 + eux. A nal injection of the Na+-Ca2 + exchange
questered Ca2 + at twice the rate as littermate controls (Fig. 4C, blocker CGP-37157 largely abolishes further eux, and allows mea-
2.0 0.1), suggesting enhanced activity of the uniporter. Importantly, surement of the Na+-Ca2 + exchanger-specic rate (Fig. 4D). We found
the total Ca2 + pulse amplitude was comparable across trials (Fig. S4B), that eux rates in Tfam KO mitochondria were reduced to 0.3 0.1

25
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Fig. 2. Disruption of Tfam inhibits ETC activity in KO hearts. A. qPCR analysis of mtDNA gene expression (n = 5). B. Spectrophotometric assay of complex I activity measured via decline
in absorbance at 340 nm (Abs340/min) produced by NADH oxidation (n = 5). C. Complex II activity measured via decline in absorbance at 600 nm (Abs600/min) produced by 2,6-
dichlorophenolindophenol reduction (n = 5). D. Complex III activity measured via increase in absorbance at 550 nm (Abs550/min) produced by cytochrome c reduction (n = 5). E.
Complex IV activity measured via decrease in absorbance at 550 nm (Abs550/min) produced by cytochrome c oxidation (n = 5).

of control rates (Fig. 4E). mitochondrial currents were minuscule (Fig. 4F) [36]. To isolate the
Ca2 + inux depends not only on the activity of the uniporter but uniporter-mediated current, the total Ca2 + current was subtracted from
other factors such as the amount of intramitochondrial buering and the fraction left after uniporter inhibition with ruthenium red (RuR,
the magnitude of driving uptake. It is unlikely that a change in Fig. 4G). In fact, the uniporter-mediated current was 5.0 1.0-fold
caused the enhanced uptake, as this would require a larger than larger in mitochondria from Tfam KO mice compared to controls
controls. Yet, we observed no dierence at this age (Fig. 3F) and a (Fig. 4H). The slight disparity in magnitude of Ca2 + enhancement be-
decline in with disease progression (Fig. S3). Thus, we hypothesized tween this measure and other assays may be due to a few outlier values
that the increase in uptake was due to the uniporter. To conrm this, we present in the voltage clamp data. Without these, our results remained
turned to whole-mitoplast electrophysiology, the most accurate method signicant but the fold change was 3.7 0.5. Thus, direct measure-
to directly measure uniporter current [24,35]. ment of uniporter currents conrms that the enhanced mitochondrial
To measure uniporter current, we isolated cardiac mitoplasts, which Ca2 + uptake seen in these animals is mediated by increased transport
are mitochondria with outer membranes stripped away. After accessing through the uniporter.
the matrix, was clamped across the entire inner membrane (whole- Next, we examined several parameters to establish whether the
mitoplast mode). As in prior reports, we found that mouse cardiac change in Ca2 + uptake was specic or might reect generalized

Fig. 3. Mitochondrial ultrastructure and are preserved. A. Exemplar transmission electron photomicrographs showing cardiac mitochondrial ultrastructure. Scale, 2 m. B.
Distribution of mitochondrial cross-sectional area in electron microscopy images across genotypes (means: WT, 0.34 0.01 m2; HET, 0.35 0.01 m2; KO, 0.31 0.02 m2, for
n > 220 mitochondria per condition, with no dierence between genotypes). C. Mitochondrial volume density in electron microscopy images (n = 1324 images per condition). D.
Mitochondrial yield measured as ratio of total protein in mitochondrial fraction (g) to heart weight (mg, n = 1921). E. Citrate synthase activity measured as increase in absorbance at
412 nm (Abs412/min) produced by formation of 5,5-dithiobis-(2-nitrobenzoic acid) (n = 67). F. assessment via FCCP-induced depolarization, measured as the total change in
uorescence in isolated cardiac mitochondria incubated in 20 M TMRM (TMRM, arbitrary units, A.U., n = 810).

26
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Fig. 4. Mitochondrial Ca2 + is increased in Tfam KO hearts. A. ICP-MS analysis of mitochondrial Ca2 + content (n = 45). B. Exemplar traces display Ca2 + uptake of cardiac mitochondria
following a 50 M pulse (arrow). C. Summary of Ca2 + uptake measured as linear slope t to 1575 s after Ca2 + pulse (n = 510) D. Exemplar traces display protocol for measuring Ca2 +
eux rates. Cardiac mitochondria are pulsed with 15 M Ca2 + in Na+-free media, followed by addition of 5 mM Na+ and 1 M uniporter blocker (Ru360), followed by addition of a
blocker of Na+-Ca2 + exchange (CGP-37157, 5 M) as indicated. Inset shows Ca2 + eux traces shifted vertically to start from the same point. E. Summary of Ca2 + eux measured as
linear slope t following Na+ addition minus residual slope after CGP-37157 addition (n = 9). F. Exemplar whole-mitoplast current density traces in 100 mM Ca2 +, with or without
ruthenium red (RuR, 0.1 M). The inwards mitochondrial Ca2 + current is indicated (IMiCa). Voltage ramp protocol is shown above the traces. G. Ca2 + current density at 160 mV with or
without RuR. H. Summary data shows uniporter-specic Ca2 + current density at 160 mV, calculated by subtracting the + RuR from Ca2 + values (G, H: n = 1217 mitoplasts per
condition).

mitochondrial dysfunction not captured in our prior assays (Fig. 3). in our preparations compared to controls. Though an increased con-
First, we measured another transport pathway present in cardiac mi- tribution of broblast mitochondria may partly explain dierences in
tochondria, chloride (Cl) ux through the inner membrane anion Ca2 + uptake, they are unlikely to be the main reason. Fibroblast
channel (IMAC) [37]. This redox-sensitive channel becomes active numbers are lowest in young hearts, and the mitochondrial mass in
during periods of stress, depolarizing cardiac mitochondria [38]. Under cardiomyocytes far exceeds that of broblasts, demonstrated by the
our recording conditions, IMAC current becomes maximal at depolar- trivial amount of TFAM left in the KO animals (Fig. 1AC). Never-
ized potentials, and its magnitude was unchanged between Tfam KO theless, to examine this in more detail, we quantied the levels of a
and control mitoplasts (Fig. S5A,B). In addition, mitoplast sizes, mea- broblast marker, vimentin, in Tfam KO versus control hearts, and
sured indirectly via capacitance values during electrophysiology, were found no dierence (Fig. S5D). Finally, we examined whether the
comparable between Tfam KO and controls (Fig. S5C). Next, we ex- change in Ca2 + transport altered sensitivity to permeability transition,
amined if Tfam KO animals had higher levels of broblast mitochondria measured by pulsing mitochondria repeatedly with 10 M Ca2 +

27
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Fig. 5. Changes in Ca2 + transporter protein expression underlie altered mitochondrial Ca2 + ux. A. qPCR analysis of expression of mitochondrial Ca2 + uniporter subunits, other Ca2 +
handling proteins, including the Na+-Ca2 + exchanger (Nclx) and Ca2 +-H+ exchanger (Letm1), and other mitochondrial proteins, relative to GAPDH mRNA (n = 5). B. Summary of
Western blot quantitation expressed as normalized ratio of protein to GAPDH loading control (n = 8). C. Representative samples from Western blot data. D. For the subset of animals that
contributed both protein and mRNA expression data, the graph plots the protein:GAPDH ratio on Western against the relative expression level on qPCR for each animal. Grey circles, data
from non-uniporter genes across genotypes, and uniporter genes for WT and HET animals. Black squares, data from core uniporter genes for Tfam KO animals.

boluses. In this assay, permeability transition becomes visible as a rise analyzed mRNA expression of genes encoding the uniporter, mi-
in the extra-mitochondrial Ca2 + uorescence, which is blocked by in- tochondrial Na+-Ca2 + exchanger, and other mitochondrial genes
cubating mitochondria with cyclosporin A (Fig. S6A). Tfam KO mi- (Fig. 5A). The exchanger is encoded by the Nclx gene [39]. The mi-
tochondria required a similar total Ca2 + load to trigger permeability tochondrial Ca2 + uniporter is a multi-subunit channel composed of a
transition compared to controls (Fig. S6B). Thus, the enhanced Ca2 + main pore-forming subunit (Mcu) [24,33,40,41], core accessory sub-
transport seen in Tfam KO hearts appears to be a specic regulatory units that target the channel and confer Ca2 + sensitivity and gating
mechanism active in cardiomyocytes during energetic failure. (Micu1-3, Emre, Mcub) [4245], and other associated proteins that alter
mitochondrial Ca2 + handling (Mcur1) [26,46,47].
Nclx mRNA expression was 0.4 0.1 of control levels (Fig. 5A).
3.4. Altered Ca2 + transport in Tfam KO cardiac mitochondria is caused by Surprisingly, and contrary to the functional data showing enhanced
changes in protein expression Ca2 + uptake, transcripts of subunits forming the mitochondrial Ca2 +
uniporter were also reduced (range: 0.490.65 0.040.07). To ex-
Having established that Tfam KO cardiac mitochondria take up amine if the qPCR data reected protein levels, we measured band in-
Ca2 + more avidly and release it more slowly than controls, we sought tensity after Western blotting, a semi-quantitative means to gauge
potential mechanisms for this eect. The most straightforward ex- candidate protein expression (Fig. 5B,C). We used the same loading
planation for altered Ca2 + transport rates would be correlated changes control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as in
in the expression of the uniporter and exchanger. To test this, we

28
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

our qPCR analysis. Consistent with the reduced Nclx transcripts, protein uorophore (MitoXpress Xtra dye), which allowed assays on smaller
levels for the Na+-Ca2 + exchanger were also reduced in Tfam KO mi- mitochondrial quantities (20 g) for prolonged periods. Oxygen
tochondria (0.65 0.05). For core uniporter subunits, (MCU, MICU1, quenching of the dye shortens its uorescence lifetime (FL), allowing
EMRE, and MCUB), a 1.51.9 fold increase in protein levels was de- measurement of mitochondrial oxygen consumption in the sealed
tected (range: 1.51.9 0.10.2). The only uniporter-associated pro- chamber as an increase in FL. Before studying the eects of Ca2 +, we
tein showing a decrease, MCUR1, appears to be a regulator rather than performed several quality control assays. Maximal oxygen consump-
a core channel component [26,4648]. Taken together, our data sug- tion, induced by uncoupling mitochondria in 0.5 M FCCP, was indis-
gest that the enhanced Ca2 + uptake reects a post-transcriptional tinguishable between Tfam KO and controls (Fig. 6D,E). Thus, despite
mechanism increasing or stabilizing the amount of uniporter present in compromised ETC activity, cardiac mitochondria from KO animals have
mitochondria. This eect can be visualized in Fig. 5D, where we map substantial reserve capacity. Furthermore, oxygen consumption stimu-
cardiac protein and qPCR levels for all the genes tested across controls lated by 0.5 mM ADP was mitochondrial, as it ceases with inhibition of
and Tfam KO animals. For all genes except for the uniporter subunits the ATP synthase in 1 M oligomycin (Fig. 6F,G).
from Tfam KO animals, transcript versus protein levels are directly Next, we measured ADP-stimulated respiration in mitochondrial
correlated (grey circles, Fig. 5D). The Tfam KO uniporter subunits suspensions containing either the Ca2 + chelator EGTA (1 mM, Ca2 +
(black squares, Fig. 5D) clearly form an outlier group with excess depleted) or 10 M EGTA +12 M Ca2 +. As expected, for WT or HET
protein expression compared to their transcript levels. Thus, the en- cardiac mitochondria, Ca2 + enhanced respiration, visible as a faster
hancement in mitochondrial Ca2 + appears to result from coincident rise in FL (Fig. 6H). For these controls, Ca2 + increased oxygen con-
changes in protein levels of the mitochondrial Ca2 + uniporter and Na+- sumption 1.7 0.1 fold (Fig. 6I,J), consistent with prior reports [49].
Ca2 + exchanger. Tfam KO mitochondria had a markedly dierent response. After de-
pletion of mitochondrial Ca2 +, Tfam KO mitochondria respired at half
3.5. Ca2 + stimulates respiration in Tfam KO mitochondria more than the control rates (Figs. 6H,I, S5A; 0.45 0.12), and in some instances,
control was almost absent (Fig. 6I, far right-most exemplar). When incubated
with Ca2 +, however, mitochondrial respiration increased 2.5 0.2
Next, we examined the eects of mitochondrial Ca2 + on OXPHOS. fold, approaching control rates (Fig. 6I,J). Thus, the increased Ca2 + in
Physiological Ca2 + concentrations appear not to aect ETC complex Tfam KO mitochondria produces a robust activation of respiration.
IIV activity directly but can alter OXPHOS in two important ways [3].
Ca2 + entry mildly uncouples mitochondria, increasing the stimulus for 4. Discussion
electron ow through complex IIV. Ca2 + also stimulates pyruvate
dehydrogenase phosphatase, several dehydrogenases of the citric acid 4.1. Conclusion
cycle, and the F1-FO-ATP synthase, all ultimately enhancing NADH
production and ATP synthesis [3,49]. We show here that a mouse model of mitochondrial cardiomyo-
We tested these two eects separately. First, we subjected mi- pathy features increased mitochondrial Ca2 + levels, using multiple
tochondria incubated in 20 M TMRM (dequenching mode) to a Ca2 + independent techniques: analysis of mitochondrial Ca2 + content, ima-
pulse to measure changes in . This pulse should depolarize mi- ging of Ca2 + transport, direct electrophysiological measurement of
tochondria as Ca2 + enters the matrix, visible as a rise in TMRM uniporter currents, and assays of Ca2 + transporter protein expression.
uorescence with dequenching (Fig. 6A). Subsequently, enhanced ETC Such increases can improve decits in OXPHOS in in vitro assays.
function should repolarize , visible as a reduction in uorescence. Whether such increased matrix Ca2 + compensates for decient energy
We measured these parameters as the maximal deection in TMRM synthesis or ultimately leads to mitochondrial dysfunction in vivo re-
uorescence (Fig. 6B), an index for Ca2 +-induced uncoupling, and the mains to be established, and will require further studies where the
slope of TMRM uorescence recovery (Fig. 6C), an index for the balance genes responsible for mitochondrial Ca2 + transport, such as Mcu, or
between ETC activity and depolarization associated with continued overload can be selectively deleted in Tfam KO animals.
Ca2 + entry. In this test, Ca2 + pulses produced greater depolarization in
Tfam KO mitochondria (Fig. 6B), reecting enhanced Ca2 + uptake in 4.2. Regulation of mitochondrial Ca2 + transport in cardiac or skeletal
these samples. Subsequently, Tfam KO mitochondria also repolarized myopathies
faster than their control counterparts (Fig. 6C). Thus, the direct un-
coupling eects of Ca2 +, though larger in the Tfam KO mice, were Besides describing the Ca2 + phenotype present in these animals, we
largely complete within a few minutes, consistent with prior reports show that the cardiomyopathy features an increase in the mitochon-
[49]. drial Ca2 + uniporter. Although several prior studies have shown in-
Next, we studied the eect of Ca2 + on ATP synthesis. Directly creased mitochondrial Ca2 + content in the setting of disease, typically
comparing synthesis rates in the presence or absence of Ca2 + is dicult in muscle or nervous system, such increases have not implicated en-
[49], as divalent binding alters the kinetics of the luciferase reaction hanced uniporter expression. Much of this prior evidence comes from
[50]. Therefore, as with our analysis in the absence of Ca2 +, we nor- skeletal muscle. The prevalence of mitochondrial Ca2 + overload in
malized ATP synthesis rates to the WT level for each set of samples run skeletal muscle disorders may reect the larger magnitude of uniporter-
in parallel. Remarkably, we found that the deciency in Tfam KO ATP mediated transport. Uniporter current density in skeletal muscle is 30
synthesis noted in Fig. S1G,H was rescued by incubating mitochondria times larger than in heart [36], and becomes active at lower cyto-
in Ca2 + (Fig. S7AC). ATP synthesis rates in Tfam KO cardiac mi- plasmic Ca2 + levels [51]. Thus, given the already-large ux, skeletal
tochondria were comparable to WT, despite the larger transient un- muscle mitochondria may be far more prone to pathological Ca2 +
coupling noted previously. overload, and not achieve any benet by enhancing the ux further.
To more robustly quantify the stimulation exerted by matrix Ca2 + Indeed, in the analysis of skeletal muscle myopathy caused by muscle-
in Tfam KO hearts, we analyzed mitochondrial respiration. To avoid the specic Tfam deletion [52], mitochondrial Ca2 + overload during re-
eects of Ca2 + uncoupling, respiration was monitored over a longer petitive stimulation was attributed to the permeability transition pore.
period, as largely repolarized within 5 min after a single Ca2 + pulse Similarly, the tendency to deleterious mitochondrial Ca2 + overload in
(Fig. 6A) [49]. This assay minimizes the direct eects associated with skeletal muscle is seen in malignant hypothermia [32], and myopathies
increased Ca2 + accumulation in Tfam KO mitochondria, allowing us to caused by MICU1 mutations [53].
focus on the steady-state eects of such uptake. To measure respiration, Mitochondrial Ca2 + overload also appears to cause cardiac injury in
we incubated mitochondria in media containing an oxygen-sensitive heart failure [54,55]. However, there has been a lack of evidence for

29
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

Fig. 6. Increasing Ca2 + boosts mitochondrial respiration in Tfam KO hearts more than controls. A. Exemplar trace of response to a 15 M Ca2 + bolus, measured via TMRM
dequenching as in Fig. 3F. B. Maximal change in TMRM signal following Ca2 + bolus (n = 814). C. Kinetics of measured as linear slope t to 30150 s after Ca2 + pulse (n = 814).
D. Exemplar raw traces of mitochondrial O2 consumption stimulated by 0.5 M FCCP, visible as an increase in FL of an O2-sensing uorophore. After maximal consumption of O2 in the
sealed chamber, the FL values plateau. E. Peak relative oxygen consumption rates (OCR) stimulated by incubation with 0.5 M FCCP, calculated from the rising portion of FL curves as in
(D) (n = 913). F. Exemplar raw traces showing inhibition of O2 consumption, stimulated by 0.5 mM ADP, in mitochondria incubated with 1 M oligomycin. G. Oligomycin summary
data (n = 56). H. Exemplar raw traces of ADP-stimulated mitochondrial O2 consumption in the presence or absence of Ca2 +. I. Summary of Ca2 + eects on ADP-stimulated OCR
(n = 913). J. Fold change in OCR stimulated by Ca2 + (n = 913).

regulation of the uniporter, and investigators have yet to establish if be obscured, especially if such feedback regulation ultimately leads to
mitochondrial Ca2 + transport is regulated in other forms of cardiac mitochondrial Ca2 + overload and selective injury to those very cardi-
energetic failure. In another model of cardiac mitochondrial disease, omyocytes that took up the most Ca2 +. Indeed, the eect of deleting
mice with complex I deciency have normal lifespan and cardiac Mcu variably aects cardiac injury depending on the exact mouse and
function, but develop accelerated heart failure with stress [56]. Mi- injury model [5759]. Moreover, in end-stage failing human hearts,
tochondria from these animals were more sensitive to Ca2 + overload, mitochondrial Ca2 + uptake appears to be blunted [60]. Given this
but the investigators did not assess whether Ca2 + uptake rates changed complexity in mitochondrial Ca2 + transport in cardiac injury, mi-
during heart failure progression. Here, we found no sensitization to tochondrial cardiomyopathies present a unique opportunity to study
Ca2 +-induced permeability transition in the Tfam KO mice. Never- regulation of this pathway and these diseases may best respond to
theless, Tfam KO may still be more likely to undergo permeability therapies targeting mitochondrial Ca2 + transport.
transition, as enhanced baseline Ca2 + and Ca2 + inux, and diminished
Ca2 + eux all may be driving mitochondrial Ca2 + closer to activating 4.3. Mechanism regulating mitochondrial Ca2 + transport in Tfam KO
the permeability transition. hearts
By studying Ca2 + transport via whole-mitoplast voltage clamping
relatively early in the cardiomyopathy, we bypassed several con- The regulation of mitochondrial Ca2 + described here is unlikely to
founding factors associated with analyses of end-stage failure, such as be due to direct transcriptional control by TFAM, as it is a transcription
an increased mass of ssioned mitochondria and reduced . In the factor for mtDNA and not nuclear-encoded genes, such as the uniporter
late stages of heart failure, enhanced mitochondrial Ca2 + uptake may or exchanger subunits. In fact, recent genome-wide studies show that

30
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

while TFAM coats mitochondrial DNA, it fails to bind nuclear DNA, and [4] A.W. El-Hattab, F. Scaglia, Mitochondrial Cardiomyopathies, Front. Cardiovasc.
Med. 3 (2016) 25.
is essentially absent in the nucleus [61]. In addition, Tfam HET animals [5] D. Holmgren, H. Wahlander, B.O. Eriksson, A. Oldfors, E. Holme, M. Tulinius,
at this age had no observable change in mitochondrial Ca2 + transport Cardiomyopathy in children with mitochondrial disease; clinical course and car-
despite reduced transcription of mtDNA-encoded ETC subunits, eects diological ndings, Eur. Heart J. 24 (3) (2003) 280288.
[6] J. Wang, H. Wilhelmsson, C. Gra, H. Li, A. Oldfors, P. Rustin, J.C. Bruning,
mediated directly by TFAM. Thus, increased mitochondrial Ca2 + in C.R. Kahn, D.A. Clayton, G.S. Barsh, P. Thoren, N.G. Larsson, Dilated cardiomyo-
Tfam KO animals is not due to a direct alteration in uniporter or ex- pathy and atrioventricular conduction blocks induced by heart-specic inactivation
changer transcription caused by TFAM, but rather to the heart failure of mitochondrial DNA gene expression, Nat. Genet. 21 (1) (1999) 133137.
[7] A.R. Stiles, M.T. Simon, A. Stover, S. Eftekharian, N. Khanlou, H.L. Wang,
that develops in its absence. S. Magaki, H. Lee, K. Partynski, N. Dorrani, R. Chang, J.A. Martinez-Agosto,
The mechanism for enhanced mitochondrial Ca2 + in Tfam KO J.E. Abdenur, Mutations in TFAM, encoding mitochondrial transcription factor A,
hearts appears to vary based on the transport pathway. The reduction in cause neonatal liver failure associated with mtDNA depletion, Mol. Genet. Metab.
119 (12) (2016) 9199.
Ca2 + eux appears mediated by a reduction in Nclx transcription,
[8] J. Wang, J.P. Silva, C.M. Gustafsson, P. Rustin, N.G. Larsson, Increased in vivo
which leads to consequent reduction in Na+-Ca2 + exchanger protein apoptosis in cells lacking mitochondrial DNA gene expression, Proc. Natl. Acad. Sci.
levels. Such a reduction may ultimately predispose to sudden death via U. S. A. 98 (7) (2001) 40384043.
mitochondrial Ca2 + overload, as seen in other forms of heart failure [9] A. Hansson, N. Hance, E. Dufour, A. Rantanen, K. Hultenby, D.A. Clayton,
R. Wibom, N.G. Larsson, A switch in metabolism precedes increased mitochondrial
[62]. The situation for the mitochondrial Ca2 + uniporter is more biogenesis in respiratory chain-decient mouse hearts, Proc. Natl. Acad. Sci. U. S. A.
complicated, since we nd decreased transcripts but increased protein 101 (9) (2004) 31363141.
expression of channel subunits. Such discrepancy between transcript [10] P. Tavi, A. Hansson, S.J. Zhang, N.G. Larsson, H. Westerblad, Abnormal Ca(2 +)
release and catecholamine-induced arrhythmias in mitochondrial cardiomyopathy,
and protein levels can occur for up to 20% of genes assayed in heart Hum. Mol. Genet. 14 (8) (2005) 10691076.
failure versus control animals [20]. However, the changes we see here [11] P. Ahuja, J. Wanagat, Z. Wang, Y. Wang, D.A. Liem, P. Ping, I.A. Antoshechkin,
likely reect a regulatory pathway rather than heart failure epipheno- K.B. Margulies, W.R. Maclellan, Divergent mitochondrial biogenesis responses in
human cardiomyopathy, Circulation 127 (19) (2013) 19571967.
mena, as we found similar magnitudes and concordant changes across [12] E. Arbustini, M. Diegoli, R. Fasani, M. Grasso, P. Morbini, N. Banchieri, O. Bellini,
all the core uniporter subunits assayed. Potential mechanisms for reg- B. Dal Bello, A. Pilotto, G. Magrini, C. Campana, P. Fortina, A. Gavazzi, J. Narula,
ulating the uniporter include altering transcript expression [63], M. Vigano, Mitochondrial DNA mutations and mitochondrial abnormalities in di-
lated cardiomyopathy, Am. J. Pathol. 153 (5) (1998) 15011510.
changing channel conductance [64,65], changing channel oligomer-
[13] N.A. Diakos, S. Navankasattusas, E.D. Abel, J. Rutter, L. McCreath, P. Ferrin,
ization state [6668], or changing channel sensitivity to cytoplasmic S.H. McKellar, D.V. Miller, S.Y. Park, R.S. Richardson, R. Deberardinis, J.E. Cox,
Ca2 + [51]. Future studies will need to establish the regulatory me- A.G. Kfoury, C.H. Selzman, J. Stehlik, J.C. Fang, D.Y. Li, S.G. Drakos, Evidence of
glycolysis up-regulation and pyruvate mitochondrial oxidation mismatch during
chanism active in Tfam KO hearts and whether it stabilizes the channel
mechanical unloading of the failing human heart: implications for cardiac reloading
or modies its underlying behavior. and conditioning, JACC 1 (6) (2016) 432444.
[14] S. Neubauer, M. Horn, M. Cramer, K. Harre, J.B. Newell, W. Peters, T. Pabst, G. Ertl,
Disclosures D. Hahn, J.S. Ingwall, K. Kochsiek, Myocardial phosphocreatine-to-ATP ratio is a
predictor of mortality in patients with dilated cardiomyopathy, Circulation 96 (7)
(1997) 21902196.
None. [15] R. Yue, X. Xia, J. Jiang, D. Yang, Y. Han, X. Chen, Y. Cai, L. Li, W.E. Wang, C. Zeng,
Mitochondrial DNA oxidative damage contributes to cardiomyocyte ischemia/re-
perfusion-injury in rats: cardioprotective role of lycopene, J. Cell. Physiol. 230 (9)
Sources of funding (2015) 21282141.
[16] D. Lebrecht, B. Setzer, U.P. Ketelsen, J. Haberstroh, U.A. Walker, Time-dependent
This work was supported by the Heart, Lung, and Blood Institute and tissue-specic accumulation of mtDNA and respiratory chain defects in chronic
doxorubicin cardiomyopathy, Circulation 108 (19) (2003) 24232429.
and Institute of Neurological Disorders and Stroke of the NIH under [17] A. Kanazawa, Y. Nishio, A. Kashiwagi, H. Inagaki, R. Kikkawa, K. Horiike, Reduced
awards R00HL124070 (D.C.), T32HL007572 (P.R.H.), and activity of mtTFA decreases the transcription in mitochondria isolated from diabetic
T32NS007473 (P.R.H.), American Heart Association under award rat heart, Am. J. Physiol. Endocrinol. Metab. 282 (4) (2002) E77885.
[18] Z. Gao, A.S. Barth, D. DiSilvestre, F.G. Akar, Y. Tian, A. Tanskanen, D.A. Kass,
13FTF16890003 (D.C.), and the Nora Eccles Treadwell Foundation R.L. Winslow, G.F. Tomaselli, Key pathways associated with heart failure devel-
(D.C.). The content is solely the responsibility of the authors and does opment revealed by gene networks correlated with cardiac remodeling, Physiol.
not necessarily represent the ocial views of the funding agencies. Genomics 35 (3) (2008) 222230.
[19] A. Garnier, D. Fortin, C. Delomenie, I. Momken, V. Veksler, R. Ventura-Clapier,
Depressed mitochondrial transcription factors and oxidative capacity in rat failing
Acknowledgements cardiac and skeletal muscles, J. Physiol. 551 (Pt 2) (2003) 491501.
[20] D.B. Foster, T. Liu, K. Kammers, R. O'Meally, N. Yang, K.N. Papanicolaou, C.C.
We thank Nils-Gran Larsson and C. Ronald Kahn for providing Talbot, Jr., R.N. Cole, B. O'Rourke, Integrated omic analysis of a guinea pig model
of heart failure and sudden cardiac death, J. Proteome Res. 15(9) (2016) 300928.
Tfam/ mice, Kenneth Spitzer for assistance with cardiomyocyte ima- [21] M. Ikeuchi, H. Matsusaka, D. Kang, S. Matsushima, T. Ide, T. Kubota, T. Fujiwara,
ging, Diego Fernandez and Christopher Anderson for ICP-MS core ser- N. Hamasaki, A. Takeshita, K. Sunagawa, H. Tsutsui, Overexpression of mi-
vices (Geology and Geophysics, University of Utah), Maria Ericsson for tochondrial transcription factor a ameliorates mitochondrial deciencies and car-
diac failure after myocardial infarction, Circulation 112 (5) (2005) 683690.
electron microscopy core services (Harvard Medical School), and sta [22] G.H. Kunkel, P. Chaturvedi, S.C. Tyagi, Mitochondrial pathways to cardiac re-
at the Research Histology core (Huntsman Cancer Institute, University covery: TFAM, Heart Fail. Rev. 21 (5) (2016) 499517.
of Utah and ARUP Research institute) for tissue processing. [23] R. Agah, P.A. Frenkel, B.A. French, L.H. Michael, P.A. Overbeek, M.D. Schneider,
Gene recombination in postmitotic cells. Targeted expression of Cre recombinase
provokes cardiac-restricted, site-specic rearrangement in adult ventricular muscle
Appendix A. Supplementary data in vivo, J. Clin. Invest. 100 (1) (1997) 169179.
[24] D. Chaudhuri, Y. Sancak, V.K. Mootha, D.E. Clapham, MCU encodes the pore
conducting mitochondrial calcium currents, elife 2 (2013) e00704.
Supplementary data to this article can be found online at https:// [25] M. Spinazzi, A. Casarin, V. Pertegato, L. Salviati, C. Angelini, Assessment of mi-
doi.org/10.1016/j.yjmcc.2017.09.009. tochondrial respiratory chain enzymatic activities on tissues and cultured cells, Nat.
Protoc. 7 (6) (2012) 12351246.
[26] D. Chaudhuri, D.J. Artiga, S.A. Abiria, D.E. Clapham, Mitochondrial calcium uni-
References
porter regulator 1 (MCUR1) regulates the calcium threshold for the mitochondrial
permeability transition, Proc. Natl. Acad. Sci. U. S. A. 113 (13) (2016)
[1] T. Doenst, T.D. Nguyen, E.D. Abel, Cardiac metabolism in heart failure: implications E1872E1880, http://dx.doi.org/10.1073/pnas.1602264113.
beyond ATP production, Circ. Res. 113 (6) (2013) 709724. [27] Y. Will, J. Hynes, V.I. Ogurtsov, D.B. Papkovsky, Analysis of mitochondrial function
[2] J.S. Ingwall, R.G. Weiss, Is the failing heart energy starved? On using chemical using phosphorescent oxygen-sensitive probes, Nat. Protoc. 1 (6) (2006)
energy to support cardiac function, Circ. Res. 95 (2) (2004) 135145. 25632572.
[3] R.S. Balaban, The role of Ca(2+) signaling in the coordination of mitochondrial [28] V. Gaussin, T. Van de Putte, Y. Mishina, M.C. Hanks, A. Zwijsen, D. Huylebroeck,
ATP production with cardiac work, Biochim. Biophys. Acta 1787 (11) (2009) R.R. Behringer, M.D. Schneider, Endocardial cushion and myocardial defects after
13341341. cardiac myocyte-specic conditional deletion of the bone morphogenetic protein

31
S. Sommakia et al. Journal of Molecular and Cellular Cardiology 113 (2017) 2232

receptor ALK3, Proc. Natl. Acad. Sci. U. S. A. 99 (5) (2002) 28782883. [51] D. Vecellio Reane, F. Vallese, V. Checchetto, L. Acquasaliente, G. Butera, V. De
[29] H. Li, J. Wang, H. Wilhelmsson, A. Hansson, P. Thoren, J. Duy, P. Rustin, Filippis, I. Szabo, G. Zanotti, R. Rizzuto, A. Raaello, A MICU1 splice variant
N.G. Larsson, Genetic modication of survival in tissue-specic knockout mice with confers high sensitivity to the mitochondrial Ca2 + uptake machinery of skeletal
mitochondrial cardiomyopathy, Proc. Natl. Acad. Sci. U. S. A. 97 (7) (2000) muscle, Mol. Cell 64 (4) (2016) 760773.
34673472. [52] J. Aydin, D.C. Andersson, S.L. Hanninen, A. Wredenberg, P. Tavi, C.B. Park,
[30] Q. Long, K. Yang, Q. Yang, Regulation of mitochondrial ATP synthase in cardiac N.G. Larsson, J.D. Bruton, H. Westerblad, Increased mitochondrial Ca2 + and de-
pathophysiology, Am. J. Cardiovasc. Dis. 5 (1) (2015) 1932. creased sarcoplasmic reticulum Ca2 + in mitochondrial myopathy, Hum. Mol.
[31] S.G. Voronina, S.L. Barrow, O.V. Gerasimenko, O.H. Petersen, A.V. Tepikin, Eects Genet. 18 (2) (2009) 278288.
of secretagogues and bile acids on mitochondrial membrane potential of pancreatic [53] C.V. Logan, G. Szabadkai, J.A. Sharpe, D.A. Parry, S. Torelli, A.M. Childs, M. Kriek,
acinar cells: comparison of dierent modes of evaluating DeltaPsim, J. Biol. Chem. R. Phadke, C.A. Johnson, N.Y. Roberts, D.T. Bonthron, K.A. Pysden, T. Whyte,
279 (26) (2004) 2732727338. I. Munteanu, A.R. Foley, G. Wheway, K. Szymanska, S. Natarajan, Z.A. Abdelhamed,
[32] C. Giulivi, C. Ross-Inta, A. Omanska-Klusek, E. Napoli, D. Sakaguchi, G. Barrientos, J.E. Morgan, H. Roper, G.W. Santen, E.H. Niks, W.L. van der Pol, D. Lindhout,
P.D. Allen, I.N. Pessah, Basal bioenergetic abnormalities in skeletal muscle from A. Raaello, D. De Stefani, J.T. den Dunnen, Y. Sun, I. Ginjaar, C.A. Sewry,
ryanodine receptor malignant hyperthermia-susceptible R163C knock-in mice, J. M. Hurles, R. Rizzuto, U.K. Consortium, M.R. Duchen, F. Muntoni, E. Sheridan,
Biol. Chem. 286 (1) (2011) 99113. Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to
[33] J.M. Baughman, F. Perocchi, H.S. Girgis, M. Plovanich, C.A. Belcher-Timme, primary alterations in mitochondrial calcium signaling, Nat. Genet. 46 (2) (2014)
Y. Sancak, X.R. Bao, L. Strittmatter, O. Goldberger, R.L. Bogorad, V. Koteliansky, 188193.
V.K. Mootha, Integrative genomics identies MCU as an essential component of the [54] H. Nakayama, X. Chen, C.P. Baines, R. Klevitsky, X. Zhang, H. Zhang, N. Jaleel,
mitochondrial calcium uniporter, Nature 476 (7360) (2011) 341345. B.H. Chua, T.E. Hewett, J. Robbins, S.R. Houser, J.D. Molkentin, Ca2 +- and mi-
[34] P.J. Doonan, H.C. Chandramoorthy, N.E. Homan, X. Zhang, C. Cardenas, tochondrial-dependent cardiomyocyte necrosis as a primary mediator of heart
S. Shanmughapriya, S. Rajan, S. Vallem, X. Chen, J.K. Foskett, J.Y. Cheung, failure, J. Clin. Invest. 117 (9) (2007) 24312444.
S.R. Houser, M. Madesh, LETM1-dependent mitochondrial Ca2 + ux modulates [55] G. Santulli, W. Xie, S.R. Reiken, A.R. Marks, Mitochondrial calcium overload is a
cellular bioenergetics and proliferation, FASEB J. 28 (11) (2014) 49364949. key determinant in heart failure, Proc. Natl. Acad. Sci. U. S. A. 112 (36) (2015)
[35] Y. Kirichok, G. Krapivinsky, D.E. Clapham, The mitochondrial calcium uniporter is 1138911394.
a highly selective ion channel, Nature 427 (6972) (2004) 360364. [56] G. Karamanlidis, C.F. Lee, L. Garcia-Menendez, S.C. Kolwicz Jr., W. Suthammarak,
[36] F. Fieni, S. Bae Lee, Y.N. Jan, Y. Kirichok, Activity of the mitochondrial calcium G. Gong, M.M. Sedensky, P.G. Morgan, W. Wang, R. Tian, Mitochondrial complex I
uniporter varies greatly between tissues, Nat. Commun. 3 (2012) 1317. deciency increases protein acetylation and accelerates heart failure, Cell Metab.
[37] J. Borecky, P. Jezek, D. Siemen, 108-pS channel in brown fat mitochondria might 18 (2) (2013) 239250.
Be identical to the inner membrane anion channel, J. Biol. Chem. 272 (31) (1997) [57] J.Q. Kwong, X. Lu, R.N. Correll, J.A. Schwanekamp, R.J. Vagnozzi, M.A. Sargent,
1928219289. A.J. York, J. Zhang, D.M. Bers, J.D. Molkentin, The mitochondrial calcium uni-
[38] M.A. Aon, S. Cortassa, C. Maack, B. O'Rourke, Sequential opening of mitochondrial porter selectively matches metabolic output to acute contractile stress in the heart,
ion channels as a function of glutathione redox thiol status, J. Biol. Chem. 282 (30) Cell Rep. 12 (1) (2015) 1522.
(2007) 2188921900. [58] T.S. Luongo, J.P. Lambert, A. Yuan, X. Zhang, P. Gross, J. Song, S. Shanmughapriya,
[39] R. Palty, W.F. Silverman, M. Hershnkel, T. Caporale, S.L. Sensi, J. Parnis, C. Nolte, E. Gao, M. Jain, S.R. Houser, W.J. Koch, J.Y. Cheung, M. Madesh, J.W. Elrod, The
D. Fishman, V. Shoshan-Barmatz, S. Herrmann, D. Khananshvili, I. Sekler, NCLX is mitochondrial calcium uniporter matches energetic supply with cardiac workload
an essential component of mitochondrial Na+/Ca2 + exchange, Proc. Natl. Acad. during stress and modulates permeability transition, Cell Rep. 12 (1) (2015) 2334.
Sci. U. S. A. 107 (1) (2010) 436441. [59] X. Pan, J. Liu, T. Nguyen, C. Liu, J. Sun, Y. Teng, M.M. Fergusson, I.I. Rovira,
[40] D. De Stefani, A. Raaello, E. Teardo, I. Szabo, R. Rizzuto, A forty-kilodalton pro- M. Allen, D.A. Springer, A.M. Aponte, M. Gucek, R.S. Balaban, E. Murphy, T. Finkel,
tein of the inner membrane is the mitochondrial calcium uniporter, Nature 476 The physiological role of mitochondrial calcium revealed by mice lacking the mi-
(7360) (2011) 336340. tochondrial calcium uniporter, Nat. Cell Biol. 15 (12) (2013) 14641472.
[41] K.J. Kamer, V.K. Mootha, The molecular era of the mitochondrial calcium uni- [60] G. Michels, I.F. Khan, J. Endres-Becker, D. Rottlaender, S. Herzig, A. Ruhparwar,
porter, Nat. Rev. Mol. Cell Biol. 16 (9) (2015) 545553. T. Wahlers, U.C. Hoppe, Regulation of the human cardiac mitochondrial Ca2 +
[42] F. Perocchi, V.M. Gohil, H.S. Girgis, X.R. Bao, J.E. McCombs, A.E. Palmer, uptake by 2 dierent voltage-gated Ca2 + channels, Circulation 119 (18) (2009)
V.K. Mootha, MICU1 encodes a mitochondrial EF hand protein required for Ca(2 +) 24352443.
uptake, Nature 467 (7313) (2010) 291296. [61] Y.E. Wang, G.K. Marinov, B.J. Wold, D.C. Chan, Genome-wide analysis reveals
[43] M. Plovanich, R.L. Bogorad, Y. Sancak, K.J. Kamer, L. Strittmatter, A.A. Li, coating of the mitochondrial genome by TFAM, PLoS One 8 (8) (2013) e74513.
H.S. Girgis, S. Kuchimanchi, J. De Groot, L. Speciner, N. Taneja, J. Oshea, [62] T.S. Luongo, J.P. Lambert, P. Gross, M. Nwokedi, A.A. Lombardi,
V. Koteliansky, V.K. Mootha, MICU2, a paralog of MICU1, resides within the mi- S. Shanmughapriya, A.C. Carpenter, D. Kolmetzky, E. Gao, J.H. van Berlo, E.J. Tsai,
tochondrial uniporter complex to regulate calcium handling, PLoS One 8 (2) (2013) J.D. Molkentin, X. Chen, M. Madesh, S.R. Houser, J.W. Elrod, The mitochondrial
e55785. Na+/Ca2 + exchanger is essential for Ca2 + homeostasis and viability, Nature 545
[44] Y. Sancak, A.L. Markhard, T. Kitami, E. Kovacs-Bogdan, K.J. Kamer, N.D. Udeshi, (7652) (2017) 9397.
S.A. Carr, D. Chaudhuri, D.E. Clapham, A.A. Li, S.E. Calvo, O. Goldberger, [63] L. Pan, B.J. Huang, X.E. Ma, S.Y. Wang, J. Feng, F. Lv, Y. Liu, Y. Liu, C.M. Li,
V.K. Mootha, EMRE is an essential component of the mitochondrial calcium uni- D.D. Liang, J. Li, L. Xu, Y.H. Chen, MiR-25 protects cardiomyocytes against oxi-
porter complex, Science 342 (6164) (2013) 13791382. dative damage by targeting the mitochondrial calcium uniporter, Int. J. Mol. Sci. 16
[45] A. Raaello, D. De Stefani, D. Sabbadin, E. Teardo, G. Merli, A. Picard, (3) (2015) 54205433.
V. Checchetto, S. Moro, I. Szabo, R. Rizzuto, The mitochondrial calcium uniporter is [64] M.L. Joiner, O.M. Koval, J. Li, B.J. He, C. Allamargot, Z. Gao, E.D. Luczak,
a multimer that can include a dominant-negative pore-forming subunit, EMBO J. 32 D.D. Hall, B.D. Fink, B. Chen, J. Yang, S.A. Moore, T.D. Scholz, S. Strack,
(17) (2013) 23622376. P.J. Mohler, W.I. Sivitz, L.S. Song, M.E. Anderson, CaMKII determines mitochon-
[46] K. Mallilankaraman, C. Cardenas, P.J. Doonan, H.C. Chandramoorthy, K.M. Irrinki, drial stress responses in heart, Nature 491 (7423) (2012) 269273.
T. Golenar, G. Csordas, P. Madireddi, J. Yang, M. Muller, R. Miller, J.E. Kolesar, [65] F. Fieni, D.E. Johnson, A. Hudmon, Y. Kirichok, Mitochondrial Ca2 + uniporter and
J. Molgo, B. Kaufman, G. Hajnoczky, J.K. Foskett, M. Madesh, MCUR1 is an es- CaMKII in heart, Nature 513 (7519) (2014) E12.
sential component of mitochondrial Ca(2 +) uptake that regulates cellular meta- [66] J. O-Uchi, B.S. Jhun, S. Xu, S. Hurst, A. Raaello, X. Liu, B. Yi, H. Zhang, P. Gross,
bolism, Nat. Cell Biol. 14 (12) (2012) 13361343. J. Mishra, A. Ainbinder, S. Kettlewell, G.L. Smith, R.T. Dirksen, W. Wang,
[47] D. Tomar, Z. Dong, S. Shanmughapriya, D.A. Koch, T. Thomas, N.E. Homan, R. Rizzuto, S.S. Sheu, Adrenergic signaling regulates mitochondrial Ca2 + uptake
S.A. Timbalia, S.J. Goldman, S.L. Breves, D.P. Corbally, N. Nemani, through Pyk2-dependent tyrosine phosphorylation of the mitochondrial Ca2 +
J.P. Fairweather, A.R. Cutri, X. Zhang, J. Song, F. Jana, J. Huang, C. Barrero, uniporter, Antioxid. Redox Signal. 21 (6) (2014) 863879.
J.E. Rabinowitz, T.S. Luongo, S.M. Schumacher, M.E. Rockman, A. Dietrich, [67] Z. Dong, S. Shanmughapriya, D. Tomar, N. Siddiqui, S. Lynch, N. Nemani,
S. Merali, J. Caplan, P. Stathopulos, R.S. Ahima, J.Y. Cheung, S.R. Houser, S.L. Breves, X. Zhang, A. Tripathi, P. Palaniappan, M.F. Riitano, A.M. Worth,
W.J. Koch, V. Patel, V.M. Gohil, J.W. Elrod, S. Rajan, M. Madesh, MCUR1 is a A. Seelam, E. Carvalho, R. Subbiah, F. Jana, J. Sobolo, Y. Peng, J.Y. Cheung,
scaold factor for the MCU complex function and promotes mitochondrial bioe- S.K. Joseph, J. Caplan, S. Rajan, P.B. Stathopulos, M. Madesh, Mitochondrial Ca2 +
nergetics, Cell Rep. 15 (8) (2016) 16731685. uniporter is a mitochondrial luminal redox sensor that augments MCU channel
[48] V. Paupe, J. Prudent, E.P. Dassa, O.Z. Rendon, E.A. Shoubridge, CCDC90A (MCUR1) activity, Mol. Cell 65 (6) (2017) 10141028, http://dx.doi.org/10.1016/j.molcel.
is a cytochrome c oxidase assembly factor and not a regulator of the mitochondrial 2017.01.032.
calcium uniporter, Cell Metab. 21 (1) (2015) 109116. [68] T. Konig, S.E. Troder, K. Bakka, A. Korwitz, R. Richter-Dennerlein, P.A. Lampe,
[49] P.R. Territo, V.K. Mootha, S.A. French, R.S. Balaban, Ca(2 +) activation of heart M. Patron, M. Muhlmeister, S. Guerrero-Castillo, U. Brandt, T. Decker, I. Lauria,
mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase, Am. J. A. Paggio, R. Rizzuto, E.I. Rugarli, D. De Stefani, T. Langer, The m-AAA protease
Physiol. Cell Physiol. 278 (2) (2000) C42335. associated with neurodegeneration limits MCU activity in mitochondria, Mol. Cell
[50] R.T. Lee, J.L. Denburg, W.D. McElroy, Substrate-binding properties of rey luci- 64 (1) (2016) 148162.
ferase. II. ATP-binding site, Arch. Biochem. Biophys. 141 (1) (1970) 3852.

32