Journal of Molecular and Cellular Cardiology 113 (2017) 2232

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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:
Mitochondrial calcium uniporter
Mitochondrial sodium-calcium exchanger
Whole-mitoplast electrophysiology
Cardiac metabolism
Respiratory-chain decient cardiomyopathy
OXPHOS decient cardiomyopathy
Calcium (Ca2 +) inux into the mitochondrial matrix stimulates ATP synthesis. Here, we investigate whether
mitochondrial Ca2 + transport pathways are altered in the setting of decient mitochondrial energy synthesis, as
increased matrix Ca2 + may provide a stimulatory boost. We focused on mitochondrial cardiomyopathies, which
feature such dysfunction of oxidative phosphorylation. We study a mouse model where the main transcription
factor for mitochondrial DNA (transcription factor A, mitochondrial, Tfam) has been disrupted selectively in
cardiomyocytes. By the second postnatal week (1015 day old mice), these mice have developed a dilated
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
Heart failure leads to a profound reprogramming of energetic pro-
duction in cardiomyocytes [1]. As heart failure progresses, energetic
supply fails to meet demand, and individuals with more severe supply-
demand mismatch have reduced survival [2]. Amongst the pathways
altered in heart failure, our interest focuses on calcium (Ca2 +), which is
central for excitation-contraction coupling, and is increasingly re-
cognized as both an enhancer and inhibitor of mitochondrial function.
Upon entering the mitochondrial matrix, Ca2 + can stimulate ATP
production [3], but excess Ca2 + entry disrupts mitochondrial function.
Because Ca2 + has bimodal eects, deciphering its contribution to en-
ergetic homeostasis during heart failure has remained dicult. Perhaps
such diculties arise from the relatively late occurrence of clinically-
evident energetic depletion during heart failure [2], with mechanisms
regulating mitochondrial Ca2 + becoming obscured by other homeo-
static pathways triggered during heart failure progression. To identify
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.
disease model that features energetic failure as an early phenotype. We
expected that early onset of energetic dysfunction during heart failure
would allow us to examine mitochondrial Ca2 + regulation in a rela-
tively isolated manner.
A spectrum of diseases featuring energetic imbalance arise from a
heterogeneous group of mutations in proteins encoded in either the
nuclear or mitochondrial genome (mtDNA) that are involved in oxi-
dative phosphorylation (OXPHOS) [4]. These disorders often feature
cardiac involvement, termed the mitochondrial cardiomyopathies, and
can be devastating, increasing the mortality rates of children suering
from these disorders three-fold [5]. For our studies, such decient
OXPHOS provides a unique opportunity to examine how the heart
modulates mitochondrial Ca2 + uxes when energy production is
failing. One such mouse model was created by a cardiac-specic dele-
tion of the main transcription factor for mtDNA (transcription factor A,
mitochondrial, Tfam) [6], leading to depletion of electron transport
chain (ETC) subunits encoded by mtDNA. In humans, two infants with
TFAM mutations died within months of liver failure, had mitochondrial
abnormalities on muscle biopsies, and in one case developed clinical
heart failure [7].
In mice, cardiac Tfam deletion recapitulates the central problem
caused by the heterogeneous group of mutations causing mitochondrial
cardiomyopathies: mtDNA depletion and subsequent OXPHOS dys-
function. Multiple studies have revealed that these mice develop car-
diac dysfunction with many of the same clinical, biochemical, and ul-
trastructural features found in human mitochondrial cardiomyopathies,
such as an early decline in energetic function and abnormal cytoplasmic
Ca2 + handling [810]. Although these mice have been otherwise
characterized, regulation of their cardiac mitochondrial Ca2 + remains
unexplored.
Studying mitochondrial Ca2 + regulation in this model of mi-
tochondrial dysfunction may also provide broader clinical insight, as
mitochondrial dysfunction is widespread in heart failure. OXPHOS
dysfunction and mtDNA damage are prevalent in dilated cardiomyo-
pathies, and indicate worse prognosis [1114]. In animal models of
ischemic [15], cardiotoxic [16], diabetic [17], tachycardia-induced
[18], and pressure-overload [19] cardiomyopathies, reductions in
TFAM, mtDNA, and OXPHOS are common. In a proteomic analysis,
TFAM was the second-most downregulated protein in failing hearts
[20]. Conversely, overexpressing TFAM improves function after cardiac
injury [21], and is the subject of therapies targeted towards cardio-
protection [22]. However, the late occurrence of clinically evident
OXPHOS deciency has made dening regulatory mechanisms dicult
in common forms of heart failure, whereas it drives heart failure in
mitochondrial cardiomyopathies.
Here, we study mitochondrial Ca2 + transport in the second post-
natal week in mice with cardiac-specic Tfam deletion. As OXPHOS
declines, these animals develop a dilated cardiomyopathy. In these
failing hearts, we nd that mitochondria become far more Ca2 + avid,
taking up Ca2 + at twice the rate as controls. This occurs via increased
activity of the mitochondrial Ca2 + uniporter, the main channel trans-
porting Ca2 + from cytoplasm to mitochondrial matrix. In addition,
mitochondria from these mice release matrix Ca2 + at half the rate as
controls, indicating reduced activity of the sodium-calcium (Na+-
Ca2 +) exchanger. The increase in Ca2 + inux and reduction in Ca2 +
eux reect corresponding changes in protein levels for the mi-
tochondrial Ca2 + uniporter and Na+-Ca2 + exchanger. Though the
decrease in Na+-Ca2 + exchanger levels occurs at the transcript level,
the mechanism for increased uniporter function appears to be post-
transcriptional, as mRNA levels are reduced despite higher protein
amounts. Finally, we nd substantial inhibition of mitochondrial re-
spiration in these mice after Ca2 + depletion, whereas Ca2 + incubation
boosts respiration to levels comparable to controls.
23
Journal of Molecular and Cellular Cardiology 113 (2017) 2232
2. Methods
Full description of methods is available in the Supplementary ma-
terials.
2.1. Animal use
The oxed Tfam (a kind gift of Dr. Nils-Gran Larsson) and -
myosin heavy chain-promoter driven Cre recombinase (Myh6-Cre) mice
(Jackson Labs, Bar Harbor, ME, strain #011038) have been backcrossed
into a C57BL/6J background [6,23]. All procedures were approved by
the Institutional Animal Care and Use Committee in accordance with
institutional guidelines. Animals used were 1015 days old except
where noted.
2.2. Mitochondrial isolation
For all assays, knockout and littermate controls were processed in
parallel. Mice were euthanized with CO2 and hearts rapidly removed. A
mitochondrial fraction was subsequently isolated by dierential cen-
trifugation [24].
2.3. Mitochondrial enzyme assays
We processed 20 g of mitochondrial fractions using the Citrate
Synthase Activity Kit (Sigma, St. Louis, MO). For subsequent assays of
complex IIV activity, we determined the citrate synthase activity in a
sample of Tfam heterozygous mitochondria and adjusted amounts of
wild-type and knockout littermate mitochondria to match that level.
Complex IIV were analyzed as detailed previously [25], using assay
rates in the presence or absence of an inhibitor to determine the com-
plex-specic activity.
2.4. Inductively coupled plasma-mass spectrometry (ICP-MS)
Mitochondria isolated by dierential centrifugation were incubated
in 40 M digitonin to permeabilize compartments other than the mi-
tochondrial matrix, prior to nal pelleting for analysis. Mitochondria
were processed by the ICP-MS metals lab at the University of Utah.
2.5. Fluorescent imaging of mitochondrial inner membrane voltage gradient
() and Ca2 + transport
We performed protocols as previously described using 50 g of the
mitochondrial fractions, except for Ca2 +-induced tetra-
methylrhodamine methyl ester (TMRM) depolarization, where we used
20 g [26].
2.6. Whole-mitoplast electrophysiology
We prepared mitoplasts from mitochondrial fractions and subse-
quently performed voltage-clamp analysis using the Kirichok protocol
[24].
2.7. Oxygen consumption analysis
Oxygen consumption was determined with a Clark electrode
(Hansatech Instruments, Norfolk, UK), or with the uorescent oxygen
probe MitoXpress Xtra (Luxcel Biosciences, Cork, Ireland). For
MitoXpress Xtra, we used uorescence lifetime imaging of mineral oil-
sealed wells in a 96-well microplate [27].
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
S. Sommakia et al.
samples < 15, we established an overall P-value using a Kruskal-Wallis
test, and for P < 0.05 proceeded to a Dunn's multiple comparison test.
For multiple comparisons on samples > 15, we established an overall
P-value using a one-way ANOVA, and for P < 0.05 proceeded to two-
tailed, unequal variance, Student's t-tests. For multiple comparison
tests, nal P-values between groups were Bonferroni-corrected.
3. Results
3.1. The dilated cardiomyopathy produced by cardiac-specic deletion of
Tfam is evident in the second postnatal week
For our studies, we crossed the previously-developed Tfam loxP
mice with mice bearing a Cre recombinase gene under the control of the
-myosin heavy chain promoter (Myh6-Cre), which becomes active
during embryogenesis in a cardiomyocyte-specic manner [28]. For our
studies, we compared knockout animals (TfamloxP/loxP; +/Myh6-Cre,
[Tfam KO]) to littermate Tfam heterozygotes (TfamloxP/+; +/Myh6-Cre,
[Tfam HET]) and wild-type animals (TfamloxP/loxP; +/+ or TfamloxP/+;
+/+ [Tfam WT]).
We conducted our experiments on P10-P15 animals, an age range in
which most Tfam KO mice were alive, yet hearts were large enough to
yield sucient mitochondria for functional characterization of in-
dividual animals. By this age, Tfam deletion was essentially complete,
reected in the near absence of mRNA and protein in knockout hearts
(Fig. 1AC; numbers are average standard error fold changes com-
pared to controls here and throughout: 0.12 0.02 mRNA,
0.05 0.02 protein). The Tfam KO mice experienced early mortality,
with animals dying mostly from postnatal week 2 onwards (Fig. 1D).
This is consistent with prior reports of cardiac Tfam deletion [6]. In one
study using TfamloxP/loxP; +/Myh6-Cre animals, > 75% of knockout
animals died in the rst postnatal week [29]. However, those animals
were in a mixed background, and the Myh6-Cre transgene construct
diered from the one used here.
All Tfam KO animals assayed had a dilated cardiomyopathy within
this period (Fig. 1E,F). Cardiac function on echocardiography was im-
paired, with severely reduced fractional shortening and dilated left
ventricles (Fig. 1G,J). Tfam HET animals, despite having an inter-
mediate reduction in TFAM, had normal cardiac function at this age.
3.2. Tfam KO cardiac mitochondria have impaired OXPHOS activity but
preserved numbers and in the second postnatal week
Next, we examined mitochondrial function in Tfam KO animals in
this timespan. First, we studied the TFAM-mediated transcription of
mtDNA. Quantitative reverse transcription polymerase chain reaction
(qPCR) analysis of a subset of mtDNA genes revealed minimal mRNA
levels in Tfam KO compared to controls (Fig. 2A). Such disruption was
replicated in enzymatic assays of ETC function. We found marked in-
hibition of complex I, III, and IV but not complex II activity (Fig. 2BE;
complex I: 0.23 0.06, complex II: 1.18 0.12, complex III:
0.72 0.07, complex IV: 0.64 0.07). This pattern reects the en-
coding of ETC subunits in nuclear versus mtDNA, as, unlike the other
complexes, complex II subunits are wholly nuclear-encoded. Ad-
ditionally, the absence of heart failure in Tfam HET animals at this age,
despite intermediate reductions in ETC activity, is consistent with the
signicant reserve capacity of cardiac mitochondria. Next, we mea-
sured respiration on complex I (glutamate/malate) or complex II sub-
strates (rotenone/succinate), using a Clark electrode (Fig. S1A). Con-
sistent with the qPCR and isolated complex measurements, complex I
respiration was reduced in the Tfam KO (Fig. S1B; 0.6 0.1), whereas
respiration on complex II was unchanged compared to controls (Fig.
S1C; 1.3 0.2). Finally, we assayed ATP production. For the F1-FO
ATP synthase, beyond the mtDNA-encoded mt-Atp6 and mt-Atp8 genes
(Fig. 2A), we quantied the expression of the nuclear-encoded ATP5A1,
ATP5F1, ATP5G1/2/3, and ATP5O subunits, and found that several of
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Journal of Molecular and Cellular Cardiology 113 (2017) 2232
these were reduced in Tfam KO hearts (Fig. S1DF, Fig. 5). We then
measured ATP production using a luciferin-luciferase assay (Fig.
S1GeI), integrating OXPHOS and ATP transport. Using this measure,
we found that, as with respiration, Tfam KO mice had a lower rate of
ATP production (0.73 0.07). Subsequent to blocking the F1-FO-ATP
synthase with oligomycin, we found no dierence between genotypes.
Such deciencies in electron transport chain activity and ATP synthesis
are notable in the development of heart failure, including in the Tfam
KO animals [6,9,30].
Further analyses conrmed that the cardiomyopathy in P10-P15
animals had not produced end-stage tissue remodeling. A prior study
demonstrated enhanced oxidative stress and apoptotic cell death, but
not extensive brosis, in 23 week old Tfam KO animals [8]. For oxi-
dative stress, we measured H2O2 production using an Amplex Red assay
(Fig. S2AeC). These showed a slight increase in ADP-stimulated H2O2
production in Tfam KO hearts (1.23 0.03), whereas maximal anti-
mycin A-induced H2O2 production was reduced (0.32 0.07), con-
sistent with diminished complex III activity. For cell death (Fig. S2D,E),
we saw an increase in terminal deoxynucleotidyl transferase dUTP nick
end labeled (TUNEL) nuclei in Tfam KO hearts. Similarly, we found that
in 3 of 4 Tfam KO, but no control animals, we could detect cardio-
myocytes staining for activated caspase 3 (Fig. S2F). Despite these in-
dicators of cell death, Masson's trichrome staining did not reveal any
evidence of increased brosis in this age range (Fig. S2G,H). These
results are congruent with the prior report [8].
As dilated cardiomyopathies progress, cardiac mitochondria ssion
and become more abundant, with increased mitochondrial mass seen in
both human disease and animal models [6,9,11]. We thus examined
mitochondrial abundance in P10-15 Tfam KO mice using several in-
dependent methods. First, complex II activity, which does not depend
on TFAM, was preserved. Second, Tfam KO mitochondrial ultra-
structure under electron microscopy remained comparable to that of
controls, as were mitochondrial size distributions and volume density
(Fig. 3AC). Similarly, the mitochondrial yield from each heart was not
dierent between Tfam KO and controls (Fig. 3D). Finally, we found no
dierence in citrate synthase activity, a marker of mitochondrial mass
(Fig. 3E).
We then examined , the voltage gradient driving ATP synthesis.
We were unable to enzymatically dissociate high-quality cardiomyo-
cytes for single-cell imaging from animals < P17. In P17 Tfam KO an-
imals, single cardiomyocytes loaded with the voltage-sensitive dye
TMRM (20 nM) uoresced less intensely than controls (0.74 0.02),
suggesting an abnormal, depolarized (Fig. S3A,B). However, since
the rest of our analyses were performed on mice aged P10P15, we
sought alternative means to test at younger ages. After mechani-
cally disrupting cells, mitochondrial fractions were enriched by dier-
ential centrifugation and incubated in TMRM at high concentration
(20 M), which quenched uorescence. Adding 5 M carbonyl cyanide
4-(triuoromethoxy)phenylhydrazone (FCCP) to this suspension un-
couples mitochondria, and the total TMRM uorescence increase as-
sociated with dequenching is a very sensitive measure of [31].
Using this technique, Tfam KO mitochondria had indistinguishable
from controls at younger ages (Fig. 3F). Together, these data reveal that
the P10P15 period features disease caused by energetic failure, but has
not progressed to a late phase characterized by extensive tissue re-
modeling, impaired mitochondrial ultrastructure, and abnormal enzy-
matic function.
3.3. Tfam KO cardiac mitochondria feature higher baseline Ca2 +,
increased Ca2 + inux, and diminished Ca2 + eux
We next turned to an analysis of mitochondrial Ca2 +. First, total
mitochondrial Ca2 + content was measured by applying ICP-MS to mi-
tochondrial pellets [32]. Tfam KO hearts had a 2.2 0.5-fold increase
over controls in mitochondrial Ca2 + content (Fig. 4A). This elevation
was specic to Ca2 +, as there was no dierence in mitochondrial
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 +
uptake in mitochondrial fractions isolated by dierential centrifuga-
tion. Mitochondrial suspensions were incubated in a Ca2 +-sensitive dye
restricted to the media [33]. A Ca2 + pulse produces a large uores-
cence increase that declines as mitochondria take up the Ca2 + bolus
(Fig. 4B). Notably, with this assay Tfam KO cardiac mitochondria se-
questered Ca2 + at twice the rate as littermate controls (Fig. 4C,
2.0 0.1), suggesting enhanced activity of the uniporter. Importantly,
the total Ca2 + pulse amplitude was comparable across trials (Fig. S4B),
so this faster rate was due to increased uptake by Tfam KO mitochon-
dria. Next, we examined the predominant eux pathway for Ca2 +, the
mitochondrial Na+-Ca2 + exchanger [34]. Following a pulse to load
mitochondria with Ca2 + in Na+-free media, an injection of Ru360 and
Na+ respectively blocks further transport through the uniporter and
activates Ca2 + eux. A nal injection of the Na+-Ca2 + exchange
blocker CGP-37157 largely abolishes further eux, and allows mea-
surement of the Na+-Ca2 + exchanger-specic rate (Fig. 4D). We found
that eux rates in Tfam KO mitochondria were reduced to 0.3 0.1

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

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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).
First, we measured another transport pathway present in cardiac mi-
tochondria, chloride (Cl) ux through the inner membrane anion
channel (IMAC) [37]. This redox-sensitive channel becomes active
during periods of stress, depolarizing cardiac mitochondria [38]. Under
our recording conditions, IMAC current becomes maximal at depolar-
ized potentials, and its magnitude was unchanged between Tfam KO
and control mitoplasts (Fig. S5A,B). In addition, mitoplast sizes, mea-
sured indirectly via capacitance values during electrophysiology, were
comparable between Tfam KO and controls (Fig. S5C). Next, we ex-
amined if Tfam KO animals had higher levels of broblast mitochondria
in our preparations compared to controls. Though an increased con-
tribution of broblast mitochondria may partly explain dierences in
Ca2 + uptake, they are unlikely to be the main reason. Fibroblast
numbers are lowest in young hearts, and the mitochondrial mass in
cardiomyocytes far exceeds that of broblasts, demonstrated by the
trivial amount of TFAM left in the KO animals (Fig. 1AC). Never-
theless, to examine this in more detail, we quantied the levels of a
broblast marker, vimentin, in Tfam KO versus control hearts, and
found no dierence (Fig. S5D). Finally, we examined whether the
change in Ca2 + transport altered sensitivity to permeability transition,
measured by pulsing mitochondria repeatedly with 10 M Ca2 +

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

28
S. Sommakia et al.
our qPCR analysis. Consistent with the reduced Nclx transcripts, protein
levels for the Na+-Ca2 + exchanger were also reduced in Tfam KO mi-
tochondria (0.65 0.05). For core uniporter subunits, (MCU, MICU1,
EMRE, and MCUB), a 1.51.9 fold increase in protein levels was de-
tected (range: 1.51.9 0.10.2). The only uniporter-associated pro-
tein showing a decrease, MCUR1, appears to be a regulator rather than
a core channel component [26,4648]. Taken together, our data sug-
gest that the enhanced Ca2 + uptake reects a post-transcriptional
mechanism increasing or stabilizing the amount of uniporter present in
mitochondria. This eect can be visualized in Fig. 5D, where we map
cardiac protein and qPCR levels for all the genes tested across controls
and Tfam KO animals. For all genes except for the uniporter subunits
from Tfam KO animals, transcript versus protein levels are directly
correlated (grey circles, Fig. 5D). The Tfam KO uniporter subunits
(black squares, Fig. 5D) clearly form an outlier group with excess
protein expression compared to their transcript levels. Thus, the en-
hancement in mitochondrial Ca2 + appears to result from coincident
changes in protein levels of the mitochondrial Ca2 + uniporter and Na+-
Ca2 + exchanger.
3.5. Ca2 + stimulates respiration in Tfam KO mitochondria more than
control
Next, we examined the eects of mitochondrial Ca2 + on OXPHOS.
Physiological Ca2 + concentrations appear not to aect ETC complex
IIV activity directly but can alter OXPHOS in two important ways [3].
Ca2 + entry mildly uncouples mitochondria, increasing the stimulus for
electron ow through complex IIV. Ca2 + also stimulates pyruvate
dehydrogenase phosphatase, several dehydrogenases of the citric acid
cycle, and the F1-FO-ATP synthase, all ultimately enhancing NADH
production and ATP synthesis [3,49].
We tested these two eects separately. First, we subjected mi-
tochondria incubated in 20 M TMRM (dequenching mode) to a Ca2 +
pulse to measure changes in . This pulse should depolarize mi-
tochondria as Ca2 + enters the matrix, visible as a rise in TMRM
uorescence with dequenching (Fig. 6A). Subsequently, enhanced ETC
function should repolarize , visible as a reduction in uorescence.
We measured these parameters as the maximal deection in TMRM
uorescence (Fig. 6B), an index for Ca2 +-induced uncoupling, and the
slope of TMRM uorescence recovery (Fig. 6C), an index for the balance
between ETC activity and depolarization associated with continued
Ca2 + entry. In this test, Ca2 + pulses produced greater depolarization in
Tfam KO mitochondria (Fig. 6B), reecting enhanced Ca2 + uptake in
these samples. Subsequently, Tfam KO mitochondria also repolarized
faster than their control counterparts (Fig. 6C). Thus, the direct un-
coupling eects of Ca2 +, though larger in the Tfam KO mice, were
largely complete within a few minutes, consistent with prior reports
[49].
Next, we studied the eect of Ca2 + on ATP synthesis. Directly
comparing synthesis rates in the presence or absence of Ca2 + is dicult
[49], as divalent binding alters the kinetics of the luciferase reaction
[50]. Therefore, as with our analysis in the absence of Ca2 +, we nor-
malized ATP synthesis rates to the WT level for each set of samples run
in parallel. Remarkably, we found that the deciency in Tfam KO ATP
synthesis noted in Fig. S1G,H was rescued by incubating mitochondria
in Ca2 + (Fig. S7AC). ATP synthesis rates in Tfam KO cardiac mi-
tochondria were comparable to WT, despite the larger transient un-
coupling noted previously.
To more robustly quantify the stimulation exerted by matrix Ca2 +
in Tfam KO hearts, we analyzed mitochondrial respiration. To avoid the
eects of Ca2 + uncoupling, respiration was monitored over a longer
period, as largely repolarized within 5 min after a single Ca2 + pulse
(Fig. 6A) [49]. This assay minimizes the direct eects associated with
increased Ca2 + accumulation in Tfam KO mitochondria, allowing us to
focus on the steady-state eects of such uptake. To measure respiration,
we incubated mitochondria in media containing an oxygen-sensitive
29
Journal of Molecular and Cellular Cardiology 113 (2017) 2232
uorophore (MitoXpress Xtra dye), which allowed assays on smaller
mitochondrial quantities (20 g) for prolonged periods. Oxygen
quenching of the dye shortens its uorescence lifetime (FL), allowing
measurement of mitochondrial oxygen consumption in the sealed
chamber as an increase in FL. Before studying the eects of Ca2 +, we
performed several quality control assays. Maximal oxygen consump-
tion, induced by uncoupling mitochondria in 0.5 M FCCP, was indis-
tinguishable between Tfam KO and controls (Fig. 6D,E). Thus, despite
compromised ETC activity, cardiac mitochondria from KO animals have
substantial reserve capacity. Furthermore, oxygen consumption stimu-
lated by 0.5 mM ADP was mitochondrial, as it ceases with inhibition of
the ATP synthase in 1 M oligomycin (Fig. 6F,G).
Next, we measured ADP-stimulated respiration in mitochondrial
suspensions containing either the Ca2 + chelator EGTA (1 mM, Ca2 +
depleted) or 10 M EGTA +12 M Ca2 +. As expected, for WT or HET
cardiac mitochondria, Ca2 + enhanced respiration, visible as a faster
rise in FL (Fig. 6H). For these controls, Ca2 + increased oxygen con-
sumption 1.7 0.1 fold (Fig. 6I,J), consistent with prior reports [49].
Tfam KO mitochondria had a markedly dierent response. After de-
pletion of mitochondrial Ca2 +, Tfam KO mitochondria respired at half
the control rates (Figs. 6H,I, S5A; 0.45 0.12), and in some instances,
was almost absent (Fig. 6I, far right-most exemplar). When incubated
with Ca2 +, however, mitochondrial respiration increased 2.5 0.2
fold, approaching control rates (Fig. 6I,J). Thus, the increased Ca2 + in
Tfam KO mitochondria produces a robust activation of respiration.
4. Discussion
4.1. Conclusion
We show here that a mouse model of mitochondrial cardiomyo-
pathy features increased mitochondrial Ca2 + levels, using multiple
independent techniques: analysis of mitochondrial Ca2 + content, ima-
ging of Ca2 + transport, direct electrophysiological measurement of
uniporter currents, and assays of Ca2 + transporter protein expression.
Such increases can improve decits in OXPHOS in in vitro assays.
Whether such increased matrix Ca2 + compensates for decient energy
synthesis or ultimately leads to mitochondrial dysfunction in vivo re-
mains to be established, and will require further studies where the
genes responsible for mitochondrial Ca2 + transport, such as Mcu, or
overload can be selectively deleted in Tfam KO animals.
4.2. Regulation of mitochondrial Ca2 + transport in cardiac or skeletal
myopathies
Besides describing the Ca2 + phenotype present in these animals, we
show that the cardiomyopathy features an increase in the mitochon-
drial Ca2 + uniporter. Although several prior studies have shown in-
creased mitochondrial Ca2 + content in the setting of disease, typically
in muscle or nervous system, such increases have not implicated en-
hanced uniporter expression. Much of this prior evidence comes from
skeletal muscle. The prevalence of mitochondrial Ca2 + overload in
skeletal muscle disorders may reect the larger magnitude of uniporter-
mediated transport. Uniporter current density in skeletal muscle is 30
times larger than in heart [36], and becomes active at lower cyto-
plasmic Ca2 + levels [51]. Thus, given the already-large ux, skeletal
muscle mitochondria may be far more prone to pathological Ca2 +
overload, and not achieve any benet by enhancing the ux further.
Indeed, in the analysis of skeletal muscle myopathy caused by muscle-
specic Tfam deletion [52], mitochondrial Ca2 + overload during re-
petitive stimulation was attributed to the permeability transition pore.
Similarly, the tendency to deleterious mitochondrial Ca2 + overload in
skeletal muscle is seen in malignant hypothermia [32], and myopathies
caused by MICU1 mutations [53].
Mitochondrial Ca2 + overload also appears to cause cardiac injury in
heart failure [54,55]. However, there has been a lack of evidence for
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
mitochondrial Ca2 + transport is regulated in other forms of cardiac
energetic failure. In another model of cardiac mitochondrial disease,
mice with complex I deciency have normal lifespan and cardiac
function, but develop accelerated heart failure with stress [56]. Mi-
tochondria from these animals were more sensitive to Ca2 + overload,
but the investigators did not assess whether Ca2 + uptake rates changed
during heart failure progression. Here, we found no sensitization to
Ca2 +-induced permeability transition in the Tfam KO mice. Never-
theless, Tfam KO may still be more likely to undergo permeability
transition, as enhanced baseline Ca2 + and Ca2 + inux, and diminished
Ca2 + eux all may be driving mitochondrial Ca2 + closer to activating
the permeability transition.
By studying Ca2 + transport via whole-mitoplast voltage clamping
relatively early in the cardiomyopathy, we bypassed several con-
founding factors associated with analyses of end-stage failure, such as
an increased mass of ssioned mitochondria and reduced . In the
late stages of heart failure, enhanced mitochondrial Ca2 + uptake may
be obscured, especially if such feedback regulation ultimately leads to
mitochondrial Ca2 + overload and selective injury to those very cardi-
omyocytes that took up the most Ca2 +. Indeed, the eect of deleting
Mcu variably aects cardiac injury depending on the exact mouse and
injury model [5759]. Moreover, in end-stage failing human hearts,
mitochondrial Ca2 + uptake appears to be blunted [60]. Given this
complexity in mitochondrial Ca2 + transport in cardiac injury, mi-
tochondrial cardiomyopathies present a unique opportunity to study
regulation of this pathway and these diseases may best respond to
therapies targeting mitochondrial Ca2 + transport.
4.3. Mechanism regulating mitochondrial Ca2 + transport in Tfam KO
hearts
The regulation of mitochondrial Ca2 + described here is unlikely to
be due to direct transcriptional control by TFAM, as it is a transcription
factor for mtDNA and not nuclear-encoded genes, such as the uniporter
or exchanger subunits. In fact, recent genome-wide studies show that

30
S. Sommakia et al.
while TFAM coats mitochondrial DNA, it fails to bind nuclear DNA, and
is essentially absent in the nucleus [61]. In addition, Tfam HET animals
at this age had no observable change in mitochondrial Ca2 + transport
despite reduced transcription of mtDNA-encoded ETC subunits, eects
mediated directly by TFAM. Thus, increased mitochondrial Ca2 + in
Tfam KO animals is not due to a direct alteration in uniporter or ex-
changer transcription caused by TFAM, but rather to the heart failure
that develops in its absence.
The mechanism for enhanced mitochondrial Ca2 + in Tfam KO
hearts appears to vary based on the transport pathway. The reduction in
Ca2 + eux appears mediated by a reduction in Nclx transcription,
which leads to consequent reduction in Na+-Ca2 + exchanger protein
levels. Such a reduction may ultimately predispose to sudden death via
mitochondrial Ca2 + overload, as seen in other forms of heart failure
[62]. The situation for the mitochondrial Ca2 + uniporter is more
complicated, since we nd decreased transcripts but increased protein
expression of channel subunits. Such discrepancy between transcript
and protein levels can occur for up to 20% of genes assayed in heart
failure versus control animals [20]. However, the changes we see here
likely reect a regulatory pathway rather than heart failure epipheno-
mena, as we found similar magnitudes and concordant changes across
all the core uniporter subunits assayed. Potential mechanisms for reg-
ulating the uniporter include altering transcript expression [63],
changing channel conductance [64,65], changing channel oligomer-
ization state [6668], or changing channel sensitivity to cytoplasmic
Ca2 + [51]. Future studies will need to establish the regulatory me-
chanism active in Tfam KO hearts and whether it stabilizes the channel
or modies its underlying behavior.
Disclosures
None.
Sources of funding
This work was supported by the Heart, Lung, and Blood Institute
and Institute of Neurological Disorders and Stroke of the NIH under
awards R00HL124070 (D.C.), T32HL007572 (P.R.H.), and
T32NS007473 (P.R.H.), American Heart Association under award
13FTF16890003 (D.C.), and the Nora Eccles Treadwell Foundation
(D.C.). The content is solely the responsibility of the authors and does
not necessarily represent the ocial views of the funding agencies.
Acknowledgements
We thank Nils-Gran Larsson and C. Ronald Kahn for providing
Tfam/ mice, Kenneth Spitzer for assistance with cardiomyocyte ima-
ging, Diego Fernandez and Christopher Anderson for ICP-MS core ser-
vices (Geology and Geophysics, University of Utah), Maria Ericsson for
electron microscopy core services (Harvard Medical School), and sta
at the Research Histology core (Huntsman Cancer Institute, University
of Utah and ARUP Research institute) for tissue processing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.yjmcc.2017.09.009.
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