Article

Mosaic Deficiency in Mitochondrial OxidativeMetabolismPromotesCardiacArrhythmia duringAging

Graphical Abstract

Highlightsd Cardiomyocytes steadily accumulate mitochondrial DNA

deletions with aging

d This leads to the development of a tissue mosaic of

mitochondrial deficiency

d Impaired mitochondrial function in few cells promotes

cardiac arrhythmia

Authors

Olivier R. Baris, Stefan Ederer, ...,

Jan W. Schrickel, Rudolf J. Wiesner

Correspondencerudolf.wiesner@uni-koeln.de

In BriefBaris et al. test the idea of mosaic

respiratory chain deficiency contributing

to aging-related human cardiac disease.

Using a genetic mouse model of

accelerated accumulation of mtDNA

deletions in the heart, they show that a

few cardiomyocytes with mitochondrial

DNA deletions progressively accumulate

during aging, leading to cardiac

arrhythmias.

Baris et al., 2015, Cell Metabolism 21, 667–677May 5, 2015 ª2015 Elsevier Inc.http://dx.doi.org/10.1016/j.cmet.2015.04.005

Cell Metabolism

Article

Mosaic Deficiency in Mitochondrial OxidativeMetabolismPromotesCardiacArrhythmiaduringAgingOlivier R. Baris,1 Stefan Ederer,1 Johannes F.G. Neuhaus,1 Jurgen-Christoph von Kleist-Retzow,2

ClaudiaM.Wunderlich,3,4,5 Martin Pal,3,4,5 F. ThomasWunderlich,3,4,5 Viktoriya Peeva,6 Gabor Zsurka,6WolframS. Kunz,6

Tilman Hickethier,7 Alexander C. Bunck,7 Florian Stockigt,8 Jan W. Schrickel,8 and Rudolf J. Wiesner1,4,5,*1Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, University of Koln, Koln 50931, Germany2Department of Pediatrics, University Hospital of Koln, Koln 50924, Germany3Max Planck Institute for Metabolism Research, Koln 50931, Germany4Center for Molecular Medicine Cologne (CMMC), University of Koln, Koln 50931, Germany5Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Koln 50674, Germany6Department of Epileptology and Life and Brain Center, University of Bonn, Bonn 53105, Germany7Department of Radiology, University Hospital of Koln, Koln 50931, Germany8University Hospital of Bonn, Department of Medicine II–Cardiology, Bonn 53105, Germany*Correspondence: rudolf.wiesner@uni-koeln.dehttp://dx.doi.org/10.1016/j.cmet.2015.04.005

SUMMARY

Aging is a progressive decline of body function, dur-ing which many tissues accumulate few cells withhigh levels of deleted mitochondrial DNA (mtDNA),leading to a defect of mitochondrial functions.Whether this mosaic mitochondrial deficiency con-tributes to organ dysfunction is unknown. Toinvestigate this, we generated mice with an acceler-ated accumulation of mtDNA deletions in themyocardium, by expressing a dominant-negativemutant mitochondrial helicase. These animals accu-mulated few randomly distributed cardiomyocyteswith compromised mitochondrial function, whichled to spontaneous ventricular premature contrac-tions and AV blocks at 18 months. These symptomswere not caused by a general mitochondrialdysfunction in the entire myocardium, and werenot observed in mice at 12 months with significantlylower numbers of dysfunctional cells. Therefore, ourresults suggest that the disposition to arrhythmiatypically found in the aged human heart mightbe due to the random accumulation of mtDNA dele-tions and the subsequent mosaic respiratory chaindeficiency.

INTRODUCTION

It is well-established that during aging, tissues of mammalsbecome mosaics of many normal and few cells with severemitochondrial dysfunction (summarized in Larsson, 2010). Thiswas first demonstrated in hearts from aged human individuals,in which cardiomyocytes lacking cytochrome c oxidase activity(COXneg cells) are randomly distributed (Muller-Hocker, 1989).The reason for this defect is the accumulation of mitochondrialDNA (mtDNA) mutations, as single cardiomyocytes isolatedfrom hearts of old humans contain a high burden of large mtDNA

deletions (Khrapko et al., 1999). The mitochondrial genome,which is present with hundreds to thousands of copies inmost cells, codes for 13 crucial subunits of the enzyme com-plexes of the mitochondrial electron transport chain (ETC),including cytochrome c oxidase, and contains the genes for22 tRNAs and 2 ribosomal RNAs. Thus, large deletions ofmtDNA, even in the presence of wild-type (WT) molecules(called a heteroplasmic state), will consequently reduce theamount of newly synthesized subunits due to the decreasedabundance of all three types of RNAs. Also, truncated or fusedsubunits may be generated, further impairing ETC function(Hornig-Do et al., 2012) and, as a consequence, affecting gener-ation of ATP or other essential mitochondrial metabolic func-tions such as Ca2+ handling (von Kleist-Retzow et al., 2007). Inpatients who carry a heavy burden of such mtDNA deletions,either generated by mutation events in their mother’s germlineor due to Mendelian inheritance of mutations in nuclear-encoded proteins essential for replication and maintenance ofmtDNA, a threshold of deleted molecules has to be surpassedbefore an ETC defect occurs in a cell (reviewed in Rossignolet al., 2003). Accumulation of few such cells in a tissue leadsto the typical mosaic pattern, but their potential involvement inaging-associated organ dysfunction has not yet been deter-mined experimentally.Heart appears as an obvious choice to investigate this ques-

tion, given its strong reliance on mitochondrial metabolism, re-flected by a high content in mitochondria, and its well-describedaging-related loss of function. Indeed, the incidence of arrhyth-mias, namely supraventricular and ventricular premature beats,conduction disturbances including AV blocks, and atrial fibrilla-tion increases drastically with age (Lok and Lau, 1996; Treschand Thakur, 1998) and importantly contributes to morbidityand mortality in the elderly population, but the mechanisms arenot clear. Cellular hyperplasia compensating an ongoing lossof cardiomyocytes, fibrosis, and disruption of intracellular Ca2+

regulation, respectively, are discussed as possible reasons (An-versa et al., 1990; Anyukhovsky et al., 2005, 2002; Chow et al.,2012), as reviewed recently (de Jong et al., 2011; Hatch et al.,2011). However, as (1) accumulation of mtDNA deletions in theheart, (2) the development of mosaic respiratory deficiency,

Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc. 667

Figure 1. Generation of K320E-TwinkleMyo Mice(A) Targeting strategy was as follows: homologous recombination (HR) between the R26-K320E-TwinkleloxP targeting vector and the ROSA26 wild-type (WT)

allele was identified in ESCs by Southern blot analysis of EcoRI-digested clonal DNA using the ROSA26 probe (16 kb, WT allele; 6 kb, targeted allele). Gray

triangles, loxP sequence; black ovals, Flp-FRT sites.

(B) Polymerase chain reaction (PCR) genotyping and verification of cre-recombination using DNA extracted from different organs. Animals with the genotype

R26-K320E-TwinkleloxP/+ +/Ckm-cre show cre-recombination only in heart and skeletal muscle.

(legend continued on next page)

668 Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc.

and (3) an increase in the incidence of cardiac arrhythmias areconcomitant phenomena during aging, it is tempting to specu-late that they are causally linked. Several observations supportthis hypothesis. First, cardiac arrhythmias have been reportedin patients suffering from diseases caused by the massive accu-mulation of mtDNA deletions, such as Kearns-Sayre syndrome(KSS) or progressive external ophthalmoplegia (PEO) (Bateset al., 2012; Fratter et al., 2010; Hubner et al., 1986; Suomalainenet al., 1992). Second, a mosaic pattern of respiratory deficiencyhas been observed in the heart of a 26-year-old KSS patient withdilated cardiomyopathy (Muller-Hocker et al., 1992). Third, asignificant part of patients with postoperative atrial fibrillationwere found to suffer from mitochondrial defects in the myocar-dium (Montaigne et al., 2013). Last, mice lacking mitochondrialtranscription factor A (TFAM) specifically in heart and skeletalmuscle, displaying almost completemtDNA loss and a large pro-portion of COXneg cardiomyocytes, die from atrioventricularblocks (AV blocks), thus emphasizing the importance of mito-chondrial function for proper action potential propagation inthe heart (Wang et al., 1999). However, the latter model is notappropriate to determine whether the presence of only few car-diomyocytes with mitochondrial dysfunction, as observed innormal human aging, is sufficient to cause aging-related cardiacdisorders.To answer this question, we have generated a mouse model

that allows accelerated accumulation of mtDNA deletions in aspecific cell type, thus recapitulating on the short timescale ofa mouse life a situation that is the result of decades-longprocesses during human aging. These mice carry a targetedinsertion of a dominant-negative mutant of the mitochondrialreplicative helicase Twinkle (K320E-Twinkle), whose expressionis prevented by an upstream loxP-flanked STOP cassette. Bycrossing these mice with the CKM-cre line (Bruning et al.,1998), we have generated animals expressing the mutant heli-case in myocardium (K320E-TwinkleMyo).

RESULTS

K320E-TwinkleMyo Mice Are Viable and Show NoObvious PhenotypeMice expressing the K320E-Twinkle transgene (Figure 1A) wereborn in normal litter sizes according to Mendelian distribution.WeconfirmedbyPCR that cre-mediated recombination occurredin targeted tissues, heart and skeletal muscle, but not in other or-gans (Figure 1B).QuantitativePCRshowed thatTwinkle transcriptlevels, derived from both WT and R26-K320E-Twinkle alleles,were about 3-fold higher in K320E-TwinkleMyo mice (p < 0.05,Figure 1C). Immunohistochemistry further showed that GFP, asurrogate marker encoded downstream of the K320E-Twinklesequence, was expressed in the myocardium of K320E-TwinkleMyo mice (Figure 1D). Animals never showed any obviousbehavior phenotype throughout their lifetime, and no prematuredeath was observed, as all mutants survived until the latest time

point set for sacrifice (24 months). Thus, since the expression ofthe mutant helicase in the heart as well as in skeletal muscle isobviously well-tolerated, this model is suitable for studying late-onset, truly aging-related symptoms.

K320E-TwinkleMyo Mice Accumulate mtDNA Deletionsin the Heart with AgingLong-range PCR amplification of mtDNA revealed many bandsindicating the presence of multiple mtDNA deletions in heart ho-mogenates from 12-month-old K320E-TwinkleMyomice, but veryfew such PCR products were seen in controls (Figure 2A). Wethen followed the accumulation over time of three definedmtDNA deletions using quantitative PCR, as recently described(Neuhaus et al., 2014). This revealed that the proportion of thesethree deletions is approximately 60 times higher in mutantanimals compared to age-matched controls (e.g., 18 months,0.23% ± 0.03% versus 0.003% ± 0.003%; p < 0.001, Figure 2B).Based on these results, we estimated that the deletion load in-creases 6-fold between 12 and 18 months in K320E-TwinkleMyo

mice. Using single-molecule PCR to precisely determine the pro-portion of all deleted mtDNA species in heart homogenate sam-ples at 12 and 18 months, we could confirm this estimation, aswe found a 5-fold increase in global deletion load between thetwo time points (proportion of deleted species at 18 months,10.92% ± 9.5%; at 12 months, 2.18% ± 1.3%). Last, we de-tected mtDNA sublimons (Kajander et al., 2000), i.e., low-abun-dance rearranged mtDNA molecules (see Figure S1 availableonline) in the mutant hearts at 18 months, as observed in the de-letor Twinkle mouse expressing a different, more benign Twinkletransgene in the whole body (Tyynismaa et al., 2005). Overall,these results show that K320E-TwinkleMyo mice display anaccelerated accumulation of mtDNA deletions in the heart duringaging.

K320E-TwinkleMyo Mice Accumulate COX-NegativeCardiomyocytes with AgingTo investigate whether these mtDNA deletions lead to randomaccumulation of cells with mitochondrial dysfunction, we stainedfor activities of cytochrome c oxidase (COX, complex IV; 3mtDNA-encoded subunits) and succinate dehydrogenase(SDH, complex II; no mtDNA-encoded subunit). No COXneg cellscould ever be observed in control animals, even at 18 months,implying that the lifespan of a mouse is too short to accumulatesuch cells in the heart. COXneg cells were also not observed at6 months in the K320E-TwinkleMyo mice, which, together withthe absence of detectable mtDNA deletions, prompted us toexclude this time point from subsequent analysis. However, in12-month-oldmutants, 0.17%± 0.12%of the cells were COXneg,and this value increased three times in 18-month-old animals to0.56% ± 0.34%, i.e., 1 in about 200 cells (Figure 3A). We nextinvestigated whether this aging-associated increase in the pro-portion of COXneg cells was caused by the accumulation ofmtDNA deletions. Single COXneg and COX-positive (COXpos)

(C) Quantification of Twinkle mRNA levels in heart samples from 18-month-old mice. Mean values ± SD from 5 mice per group (*p < 0.05).

(D) Immunofluorescence showing expression of eGFP in the heart of K320E-TwinkleMyo animals. Scale bar, 100 mm. CAG, CAG promoter; NeoR, neomycin

resistance gene;WSS,Westphal stop sequence; IRES, internal ribosomal entry site; EGFP, enhanced green fluorescent protein; HR, homologous recombination;

CR, cre-mediated recombination.

Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc. 669

cardiomyocytes from 18-month-old hearts were harvested bylaser-capture microdissection, collected in pools of about 30cells, and analyzed by long-range PCR, single-molecule PCR,and quantitative PCR. Indeed, we observed multiple PCR prod-ucts derived from mtDNA deletions in COXneg cells (Figure 3B),while such bands were found rarely in COXpos cells from controlanimals. In addition, we regularly observed few such PCR prod-ucts also in COXpos cells from K320E-TwinkleMyo mice, suggest-ing the presence of cells in the pools which had accumulatedsignificant amounts of deleted species, but not enough yet tocause mitochondrial dysfunction. Interestingly, as previouslyobserved in COXneg segments of skeletal muscle (Herbst et al.,2007), mtDNA copy number was higher in mutant COXneg cells(Figure 3C) than in control COXpos cells. This shows that mito-chondrial dysfunction observed in these cardiomyocytes is notcaused by depletion of mtDNA but rather leads to a compensa-tory increase in copy number. No statistical difference in mtDNAcopy number could be observed between COXpos cells frommutant and control animals at both checked time points. Pointmutation rates remain as yet unknown in the context of our study,but previous Twinkle mouse models showed no increased inci-dence of point mutations in heart, muscle, and neural progenitorcells (Ahlqvist et al., 2012; Tyynismaa et al., 2005). Moreover,quantification of the total deletion loads in three differentpools from COXpos and COXneg cells of 18-month-old K320E-TwinkleMyo mice by single-molecule PCR revealed percentagesof 85%± 18%of mutatedmtDNA in the COXneg cells and 2.3%±0.6% in the COXpos cells (p < 0.05). Therefore, since hetero-plasmy levels above 60% are sufficient to explain substantialfunctional impairment of oxidative phosphorylation (Kraytsberget al., 2006; Rossignol et al., 2003), these quantitative datashow that the mitochondrial dysfunction observed in COXneg

cardiomyocytes of K320E-TwinkleMyo mice is caused by accu-mulation of mtDNA deletions.

Overall, this shows that the K320E-TwinkleMyo mouse modelmimics the development of an aging-associated mosaicof normal and COXneg cells in the heart due to the accumula-tion of mtDNA deletions, recapitulating what has been reportedfor aging human heart (Khrapko et al., 1999; Muller-Hocker,1989).

K320E-TwinkleMyoMiceDevelop Ventricular Arrhythmiawith AgingIn order to answer the question of whether these fewCOXneg car-diomyocytes could cause aging-associated arrhythmias, long-

term-ECG telemetric devices were implanted in 18-month-oldmice, which displayed the highest proportion of COXneg cells,and ECGs were recorded over a period of 24 hr. A strong ten-dency toward an elevated rate of spontaneous ventricular pre-mature contractions was found at rest in K320E-TwinkleMyo

mice (6.3 ± 9.73 per hour versus controls, 0.73 ± 1.49 perhour; p = 0.122; Figures 4A and 4B), which was dramaticallyincreased while performing a swimming stress test (K320E-TwinkleMyo, 94.0 ± 108.3 per hour versus controls, 14.6 ± 15.4per hour; p < 0.0001). In addition, spontaneous AV blocksoccurred more frequently (events/hr, 33.0 ± 19.6 versus 12.9 ±12.7; p < 0.0132; Figures 4B and 4C). Intracardiac ECG record-ings revealed equal supra- and infra-Hissian conduction timesin K320E-TwinkleMyo and control mice, indicating no generalAV nodal conduction defect (Table 1). In addition, we checkedwhether there was a preferential accumulation of COXneg cellsin regions of the conduction system, which would be a plausibleexplanation for the observed arrhythmic events, by staining foracetylcholinesterase activity, an enzyme highly expressed insuch cells, and COX/SDH, on consecutive sections (Figure S2).We could identify cells from the AV node and the bundle ofHis, which showed a weaker COX staining, in accordance withtheir lower mitochondrial content (Christoffels et al., 2010). How-ever, no preferential accumulation of COXneg cells could beobserved in these regions (0.64% ± 0.52% versus 0.56% ±0.32% in ventricular wall at 18 months, nonsignificant), showingthat our model truly mimics random accumulation of COXneg

cardiomyocytes.Furthermore, functional testing did not show differences in si-

nus- or AV-nodal function, or in atrial or ventricular refractory pe-riods. No differences in heart rate, PQ, QRS, or QTc intervalswere observed (Figure S3A), further showing that the few inter-spersed COXneg cells did not lead to alterations in basic electro-physiological conduction parameters.In order to show that the cardiac symptoms are indeed caused

by the accumulation of a significant number of COXneg cardio-myocytes, animals were also investigated at 12 months, wherethe proportion of such cells is three times lower. No prematureventricular contractions were ever observed under resting condi-tion (Figures S3B and S3C). Under stress conditions, we didobserve such events and also spontaneous AV blocks but atsimilar levels in both control and K320E-TwinkleMyo hearts (Fig-ures S3C and S3D). This shows that below a certain threshold,the presence of COXneg cells does not disturb normal actionpropagation in the myocardium.

Figure 2. K320E-TwinkleMyo Mice Accumu-late Mitochondrial DNA Deletions in theHeart during Aging(A) Long-range PCR gel showing multiple prod-

ucts derived from mtDNA deletions in heart sam-

ples of 12-month-old K320E-TwinkleMyo mice.

(B) Levels of three selected mtDNA deletions in

heart samples of 12- and 18-month-old K320E-

TwinkleMyo and control mice. Values obtained for

the three analyzed deletions were added to give a

total value. Mean values ± SD from 4 mice per

group (*p < 0.05; **p < 0.01).

670 Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc.

Cardiac Arrhythmia in K320E-TwinkleMyo Mice Is NotCaused by a General Mitochondrial DefectIn order to show that the cardiac symptoms we observed in18-month-old K320E-TwinkleMyo mice are not caused by a gen-eral mitochondrial dysfunction, since the K320E mutant helicaseand GFP are expressed in all cardiomyocytes, we analyzedmtDNA copy number, as well as abundance and functionalityof the respiratory chain complexes in heart homogenates. Thecopy number of mtDNA was unchanged (Figure 5A). Also, theabundance of index subunits of the five complexes of the oxida-tive phosphorylation system, which likely represent the fullyassembled complexes (Figure 5B), as well as their enzymatic ac-tivity (Figure 5C), was similar in all hearts. These results were alsotrue for 12-month-old hearts (Figures S4A–S4C). Thus, express-ing K320E-TWINKLE in the whole myocardium did not lead to ageneral mitochondrial dysfunction. Additionally, the heart weightto body weight ratios (12 months, Ctrl 7.93 ± 1.38 mg/g versusK320E-TwinkleMyo 8.07 ± 1.33 mg/g; 18 months, Ctrl 6.83 ±1.51 mg/g versus K320E-TwinkleMyo 6.45 ± 1.28 mg/g) as wellas the average cardiomyocyte size (Figures S4D and S4E)were similar to controls, demonstrating that no cardiac hypertro-phy occurred, which is a reliable sign for general myocardialdysfunction (Figure 5D). In another effort to check whether ourintervention caused general heart failure, we measured cardiacfunction in vivo usingmagnetic resonance imaging, but we couldnot detect any dysfunction of mutant hearts compared to con-trols (Figures 5E–5G). Moreover, no enhanced cardiac fibrosiswas detected, while we could observe the previously describedaging-related increase of fibrotic areas (Stein et al., 2008) (Fig-ure S4F). Finally, TUNEL staining revealed that apoptosis is anextremely rare event, as only one to two positive cells per sectionwere observed at 18 months in both groups (data not shown),implying that our intervention did not lead to increased apoptosisof cardiomyocytes in K320E-TwinkleMyo hearts. Overall, theseresults confirm that the observed arrhythmias are not due to ageneral mitochondrial defect in all cardiomyocytes, but ratherto the few defective cells.

DISCUSSION

Tissues of aged mammals display respiratory mosaicism, i.e.,few cells with severe mitochondrial dysfunction embedded intonormal tissue. This was shown for heart, skeletal muscle of thelimbs and extraocular muscle, substantia nigra, and liver (re-viewed in Larsson, 2010). However, it was unclear whether thismosaic phenotype is responsible for causing any of the typicalaging-related symptoms of organ dysfunction.

Figure 3. K320E-TwinkleMyo Mice Accumulate Cardiomyocytes withMitochondrial Dysfunction during Aging(A) Double staining for cytochrome c oxidase (COX) and succinate dehydro-

genase (SDH) enzyme activity revealing a mosaic pattern in hearts of 12- and

18-month-old K320E-TwinkleMyo mice. Cardiomyocytes deficient for COX

activity (COXneg) appear as blue, while those showing activity for bothCOX and

SDH (COXpos) appear as brown. Scale bar, 200 mm.

(B) Long-range PCR gel showing multiple products derived from mtDNA

deletions in pools of microdissected COXneg cardiomyocytes from K320E-

TwinkleMyo mice. Note the presence of PCR products from single mtDNA

deletions also in pools of COXpos cells from mutant hearts, but not in controls

(n = 3 pools of cells per condition).

(C) Relative mtDNA copy number in laser-microdissected COXpos and COXneg

cells of mutant and control mice. The mtDNA region for ND1 was used to

determine the copy number in a given dissected area. To allow interage

comparison, the control COXpos value at 12 months was set to 1. Mean

values ± SD from 6–8 mice per group (*p < 0.05; **p < 0.01).

Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc. 671

Here we tested the hypothesis that few cardiomyocytes withseverely impaired ETC function may interfere with the regularpropagation of action potentials in the myocardium and maycause arrhythmia. To accelerate aging-related accumulationof mtDNA deletions and, consequently, the development ofsuch a mosaicism in mice, we have generated a strain allowingfor the conditional expression of a dominant-negative mu-tant form of the mitochondrial helicase TWINKLE (K320E-TWINKLE). These mice were crossed to CKM-Cre mice andthus express the K320E-TWINKLE construct in the myocardiumand skeletal muscle. Very few COXneg skeletal muscle fiberswere found in muscle of 18-month-old mice (data not shown),which may lead to a mild myopathy, as reported previously inthe transgenic deletor Twinkle mice (Tyynismaa et al., 2005).This point mutation (K319E in humans) was shown to causemassive accumulation of mtDNA deletions in muscle andCOXneg skeletal muscle fiber segments, and a severe neurolog-ical syndrome in a heterozygous germline mosaic patient (sen-sory ataxic neuropathy, dysarthria, and ophthalmoparesis,SANDO; Hudson et al., 2005). Cardiac problems have not

been reported; however, the patient died already at the ageof 41 years.Indeed, we detected an elevated rate of spontaneous ventric-

ular premature contractions and AV blocks under stress condi-tions in K320E-TwinkleMyo mice, in which hearts contained only0.6% of COXneg cardiomyocytes. Nevertheless, one may arguethat these events are not caused by the presence of these fewCOXneg cells, but may be due to a general cellular defect inducedby overexpression of the K320E-TWINKLE helicase or the long-term expression of GFP. However, we believe that we havestrong evidence that this is not the case, since (1) no enhancedincidence of arrhythmia was observed in 12-month-old K320E-TwinkleMyo mice displaying only 0.2% of COXneg cells, indicatingthat transgene expression is well tolerated; (2) no hypertrophy, asensitive marker for cardiomyocyte dysfunction, as well as noimpairment of pump function, was observed; and (3) mtDNAcopy number, abundance, and activity of respiratory chain com-plexes were similar to control hearts at 12 and 18 months, con-trasting with the compensatory increase of these parameterscommonly observed in hearts with mitochondrial dysfunction

Figure 4. K320E-TwinkleMyo Mice Develop Ventricular Arrhythmia with Aging(A) Long-term ECG recordings in 18-month-old mice did not show spontaneous arrhythmias in the control group during rest or during the stress test. However,

K320E-TwinkleMyo exhibited premature ventricular contractions (VPCs), ventricular couplets, nonsustained ventricular tachycardias, and AV blocks. Note that

during the stress test the isoelectric line appears unsteady due to the excessive body movement of the animals.

(B) Spontaneous VPCs were slightly elevated in mutant mice at rest, though statistically not significant. However, a marked increase in VPCs could be registered

during the stress test in K320E-TwinkleMyo.

(C) Analogously, after initiation of physical stress we detected a significant higher number of spontaneous AV blocks in mutant mice compared to controls. Mean

values ± SD from 6–8 mice per group.

672 Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc.

(Hansson et al., 2004; Heddi et al., 1999; Sebastiani et al., 2007).We have also excluded increased fibrosis as the reason forenhanced arrhythmias in K320E-TwinkleMyo mice, which isthought to be an important arrhythmogenic substrate in theaged myocardium (de Jong et al., 2011; Stein et al., 2008), andwhich may indeed be responsible for the increased incidenceof such events in the hearts of our old control mice. We alsocould exclude an increased rate of cardiomyocyte apoptosis,as well as a preferential accumulation of COXneg cells in regionsof the conduction system. Therefore, our data suggest that few,randomly distributed COXneg cardiomyocytes are sufficient topromote spontaneous as well as stress-induced arrhythmias.What could be the molecular mechanism causing few cardio-

myocytes to initiate (1) premature ventricular contractions and (2)AV blocks? Ventricular arrhythmias are the most common causefor sudden cardiac death and cause around 20% of total deathsper year in the United States (Lopshire and Zipes, 2006). A majorsuspect for premature ventricular contractions during aging isthe disruption of intracellular Ca2+ regulation (summarized inHatch et al., 2011), driven by kinetic changes of L-type Ca2+

channel currents, transient outward K+ channel current (Lakattaand Sollott, 2002), and reduced activity of the SERCA Ca2+

pump (Lompre et al., 1991). In addition, a compensatoryincreased expression of the Na+/Ca2+ exchanger was shown(Dai et al., 2009), a combination of mechanisms which is finallyhighly proarrhythmogenic (Morgan et al., 1990; Fares and Howl-ett, 2010). Proper mitochondrial function is essential for myocar-dial Ca2+ handling, since mitochondria provide ATP and alsosequester Ca2+ in close vicinity to the ER (Dorn and Maack,2013). Therefore, we postulate that the combination of these ag-ing-induced alterations in Ca2+ handling together with severemitochondrial dysfunction in a few cells is probably the explana-tion for the increased incidence of arrhythmias. Moreover, recentexperimental data suggest that regional mitochondrial dysfunc-tion activates ATP-sensitive potassium channels, consequentlyleading to local shortening of the action potential whichalso increases the probability for re-entry circuits (Brown andO’Rourke, 2010; Zhou et al., 2014).AV blocks could result from defects in the conduction system,

and indeed one study reports a preferential accumulation of de-

letions in the cardiac conduction system in patients with KSSwith large-scale, germline-acquired mtDNA deletions (Muller-Hocker et al., 1998). In contrast, in our mice, no preferential con-centration of defective cells was found in the AV node and bundleof His. Since this is a very small compartment in mouse heart,the few randomly accumulated cells may still contribute to theincreased incidence of AV blocks, which is also observed inother mitochondrial diseases (Bates et al., 2012; Limongelliet al., 2012). However, given the fact that conducting fibers arethree-dimensional bundles of Purkinje cells which are electricallyconnected not only vertically but also horizontally, it is unlikelythat they would be solely responsible for the arrhythmic eventsobserved in our mice.In summary, the disposition to arrhythmia typically found in the

aging human heart might indeed be caused by the accumulationof mtDNA deletions in single cells and the subsequent mosaicrespiratory chain deficiency, thus aggravating the proarrhythmo-genic state due to increased Ca2+ concentrations and fibrosis.

EXPERIMENTAL PROCEDURES

Mouse Tissue PreparationAll animal studies were approved by local government authorities (Landesamt

fur Naturschutz, LANUV, Recklinghausen). Mice of both genders were used in

the study. Mouse hearts were removed directly after sacrifice, weighted, and

either snap-frozen in liquid nitrogen for subsequent DNA and protein isolation

or frozen in cooled isopentane and embedded in O.C.T. compound for

cryosections (Tissue Tek, Vogel). Other removed organs were immediately

snap-frozen in liquid nitrogen.

Generation of R26-K320E-TwinkleloxP/+ MiceThe pCR-Blunt II Topo vector (Life Technologies) including the sequence

encoding the mouse K320E mutant of TWINKLE (equivalent to the human

K319E mutation described in Hudson et al. (2005) was a kind gift from S. Gof-

fart and J.N. Spelbrink. By PCR, we produced a K320E-Twinkle insert with

added AscI restriction sites at both 50 and 30 ends and a Kozak sequence at

the 50 end. This PCR fragment was then subcloned into a pJET1.2 vector

(Thermo Scientific) for sequencing, and subsequently inserted into the AscI

site of the STOP-EGFP-ROSA-CAG targeting vector (Belgardt et al., 2008).

Bruce4 embryonic stem cells (ESCs) (Kontgen et al., 1993) of C57BL/6J origin

were transfected with the linearized targeting vector, and positive clones were

identified by Southern blot after EcoRI digestion of their DNA, using a probe for

the ROSA26 locus (see Figure 1A). Positive ESC clones were used to generate

the R26-K320E-TwinkleloxP/+ mouse strain. Mice were genotyped by PCR

using genomic DNA isolated from tail biopsies. Primer sequences and PCR

conditions are available upon request.

Generation of K320E-TwinkleMyo MiceK320E-TwinkleMyo mice were generated by crossing CKM-cre mice (Bruning

et al., 1998) with R26-K320E-TwinkleloxP/+ mice. The specificity of cre-recom-

bination was checked by PCR using total tissue DNA extracted with the

DNeasy Blood & Tissue Kit (QIAGEN). Littermate mice with the genotype

R26-K320E-TwinkleloxP/+ were used as controls. Expression levels of Twinkle

mRNA were determined by RT-qPCR using the Superscript VILO kit (Life

Technologies) and the QuantiTect SYBR Green kit (QIAGEN). Values were

normalized to the expression levels of the housekeeping gene Rps9. Immuno-

fluorescence for GFP was performed using an anti-GFP Rabbit polyclonal

antibody (A-6455, Molecular Probes). Primer sequences and PCR conditions

are available upon request.

COX/SDH Staining and COX-Negative Cell CountingSlices of frozen hearts (10mm)were stained for COX (complex IV) and succinate

dehydrogenase (SDH, complex II) activity as previously described (Sciacco

and Bonilla, 1996). Blue cardiomyocytes with low COX activity (COXneg cells)

were quantitated (ImageJ software, v1.47) on five nonconsecutive sections

Table 1. In Vivo Transvenous Electrophysiological Investigation

WT (n = 4) K320E-TwinkleMyo (n = 5) p

CL (ms) 133.8 ± 10.7 134.8 ± 9.4 0.8800

AV (ms) 50.8 ± 11.8 51.8 ± 8.8 0.8828

AH (ms) 41.0 ± 11.9 38.8 ± 8.1 0.7657

HV (ms) 10.8 ± 1.5 10.5 ± 1.3 0.8090

WBP (ms) 91.8 ± 2.9 96.0 ± 6.5 0.3283

2:1-block (ms) 55.0 ± 0.0 56.0 ± 4.1 0.7024

SNRT (ms) 197.0 ± 43.9 210.2 ± 81.7 0.8084

AVNRP (ms) 43.3 ± 5.7 45.0 ± 0.0 0.5191

ARP (ms) 23.3 ± 5.7 26.3 ± 7.5 0.6018

VRP (ms) 38.8 ± 4.8 40.0 ± 4.1 0.7049

CL, cycle length; AH, time from first atrial to His signal; HV, time from His

to first ventricular signal; WBP, Wenckebach time; SNRT, sinus node re-

covery time; AVNRP, AV-nodal refractory period; ARP, atrial refractory

period; VRP, ventricular refractory period.

Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc. 673

from different regions of the heart (n = 7 animals per group). To determine the

proportion of COXneg cells, we first estimated for each heart the average num-

ber of cells permm2using hematoxylin/eosin staining and counting the nuclei in

length-defined visual fields. We then determined the average section area (in

mm2) to obtain the average total number of cells per section and the number

was divided by two to take into account noncardiomyocytic cells (Banerjee

et al., 2007). Last, the average number of COXneg cells per section was deter-

mined to obtain their average percentage.

Abundance and Activity of ETC ComplexesAbundance of ETC complexes was evaluated by western blot analysis of five

index subunits using a primary antibody cocktail raised in mice against sub-

units NDUFB8, SDHB, UQCR2, MTCO1, and ATP5A of the ETC (Mitoscien-

ces), and subsequently with a primary rabbit antiserum against cardiac

Troponin I as a loading control (TNNI3) (Pineda Antikorper-Service). Signals

were detected using the enhanced chemiluminescence western blotting

detection reagent ECL (Perkin Elmer Inc.) and quantitated (ImageJ software,

v1.47).

Figure 5. K320E-TwinkleMyo Hearts Do NotShow a General Mitochondrial Defect(A) Quantification of mtDNA copy number in

heart homogenates of 18-month-old mice. Mean

values ± SD from 3–4 mice per group.

(B) Immunoblot showing protein levels of five

subunits of the mitochondrial respiratory chain in

heart homogenates.

(C) Spectrophotometric measurement of enzyme

activities of succinate-quinone-dichlorophenol

indophenol-reductase (complex II, C II), succinate-

cytochrome c-reductase (C II+III), decylubiquinol-

cytochrome c-reductase (C III), COX (C IV), and

citrate synthase in heart homogenates. Mean

values ± SD from 5 mice per group.

(D) Heart weight/body weight ratios of K320E-

TwinkleMyo hearts and age-matched controls.

Mean values ± SD from 5–8 mice per group.

(E) Magnetic resonance imaging of K320E-

TwinkleMyo hearts and age-matched controls

(24 months, n = 5 control mice, 4 mutant mice).

Scale bar, 5 mm.

(F and G) Heart pumping function parameters

show no statistical difference between control and

mutant mice at 24 months. EDV, end-diastolic

volume; ESV, end-systolic volume; SV, stroke

volume; EF, ejection fraction. Mean values ± SD

(n = 5 control mice, 4 mutant mice).

Spectrophotometric measurements to deter-

mine enzyme activities of succinate-quinone-di-

chlorophenol indophenol-reductase (complex II,

C II), succinate-cytochrome c-reductase (C II+III),

decylubiquinol-cytochrome c-reductase (C III),

COX (C IV), and citrate synthase were carried

out using a Varian Cary 50 Scan spectrophoto-

meter (Varian Inc. Spectroscopy Instruments).

All measurements were performed as previously

described (Rustin et al., 1994).

Detection of mtDNA Deletions by Long-Range PCRLong-range PCR was performed to detect large-

scale mtDNA deletions using the LA Taq Hot Start

DNA polymerase (Takara Bio Inc.) under the

following conditions: 95!C for 2.5 min, 10 cycles

of 92!C for 20 s and 55!C for 14 min, 20 cycles

of 92!C for 25 s and 55!C for 16 min, and 72!C for 10 min. Primer sequences

are available upon request.

Quantification of mtDNA Deletions and mtDNA Copy Number inHeart HomogenatesTotalDNA frommyocardial tissuewaspreparedwith theDNeasyBlood&Tissue

Kit (QIAGEN). Deletions were quantified using a qPCR method as described

elsewhere (Neuhaus et al., 2014). In short, three different mtDNA deletions re-

ported to accumulate in agedmice (Tanhauser andLaipis, 1995)were quantified

and compared to a PCR fragment from the nondeleted D loop region. mtDNA

copy numbers were measured comparing a part of the ND1 region (Taqman-

probe, Life Technologies) to a single copy nuclear gene (Tert; DNAcopy number

assay, Life Technologies). Primer sequences are available upon request.

Analysis and Quantification of mtDNA Deletions and mtDNA CopyNumber in Laser-Microdissected CardiomyocytesSingle COXneg or COXpos cardiomyocytes were laser-microdissected and

collected in pools of approximately 30 cells. Total DNA was then extracted

674 Cell Metabolism 21, 667–677, May 5, 2015 ª2015 Elsevier Inc.

using the QIAamp DNA Micro Kit (QIAGEN). mtDNA deletions were detected

using the long-range and quantitative PCR protocols described above,

respectively. The relative proportion of deleted mtDNA molecules was deter-

mined by single-molecule PCR as described previously (Kornblum et al.,

2013). Briefly, template DNA was diluted so that only a subset of identical mul-

tiple reactions resulted in amplification products (ideally less than 50%). Under

these conditions, each positive reaction originates with high probability from a

single mtDNAmolecule. Single mtDNA deletions and total mtDNA copies were

amplified using different primer pairs. Amplification conditions were the same

as for long-range PCR, but with 42 cycles. Deletion ratios were calculated

by comparing the dilution numbers that resulted in single-molecule amplifica-

tion for deletions versus total mtDNA. Primer sequences are available upon

request.

To determine mtDNA copy number in COXneg and COXpos cells, 10 mm sec-

tions were stained for COX/SDH, cells of both categories were laser-micro-

dissected (collected area, 20,000–40,000 mm2), and their DNA was extracted

as described above. The mtDNA region for ND1 was amplified as an indicator

for mtDNA copy number, using a Taqman probe (Life Technologies). A stan-

dard of known ND1 target concentrations was amplified as well in every run,

to confirm exponential amplification of each sample. The determined ND1

values were then related to the collected area, as it matches closely the

actual mitochondria content in a cell, to define the mtDNA copy number

per mm2.

Long-Term Telemetric Electrocardiographic RecordingsLong-term ECG analysis was performed using telemetry devices (Model EA-

F20; DataSciences International) that were implanted subcutaneously as

described before (Stockigt et al., 2014). The analogous ECG signals were re-

corded with a telemetry receiver (PhysioTel Receiver RPC-1, DataSciences

International) and digitized using a data acquisition system (AD Instruments,

Powerlab 8/30) with 12-bit precision at a sampling rate of 1 kHz. ECG param-

eters were measured using standard criteria (Mitchell et al., 1998). A physical

stress test (10 min swimming) was performed at the end of the baseline

recording under continuation of ECG recording as described before (Knoll-

mann et al., 2003).

Magnetic Resonance Imaging of Mouse HeartsCardiac MRIs were performed on anesthetized mice as previously described

(Bunck et al., 2009). After positioning of the mouse in the isocenter of the mag-

net gradient echo, scout images were acquired in a horizontal orientation for

localization of the heart. For cine imaging we used a retrospectively ECG-trig-

gered, segmented gradient echo sequence. Slice thickness was 1mm, and in-

plane resolution was set to 0.33 0.3 mm. The reconstructed spatial resolution

was 0.153 0.153 1 mm. The effective temporal resolution was 100 frames/s,

i.e., one image every 10 ms. Standard imaging planes for LV assessment were

obtained according to the recommendations by Schneider et al. (Schneider

et al., 2006). For functional analysis a complete set of contiguous short-axis sli-

ces covering the entire left ventricle (9–10 slices) was acquired. With scanning

being performed at free breathing, respiratory motion was compensated for by

averaging of three signals (number of signal averages, NSA = 3).

StatisticsNo statistical method was used to estimate sample size. No randomization

or blinding was used in this study. Due to the size of some groups of samples

(n = 3–5), normality of the distribution could not always be assessed. There-

fore, statistical analysis was performed with the assumption that all data

were normally distributed. All data are expressed as mean ± 1 SD. ECG

data were analyzed by one-way ANOVA; Bonferroni’s multiple comparison

tests were used to assess significant differences between the groups. Discrete

variables were analyzed by two-sided Chi-square test. All other comparisons

were performed using unpaired Student’s t test with unequal variance. p < 0.05

was regarded as statistically significant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes four figures and Supplemental Experi-

mental Procedures and can be found with this article at http://dx.doi.org/10.

1016/j.cmet.2015.04.005.

AUTHOR CONTRIBUTIONS

The authors contributed to this work in the following ways: O.R.B. performed

conception and design, experiments, data analysis and interpretation, and

manuscript writing; S.E., J.F.G.N., J.-C.v.K.-R., C.M.W., M.P., V.P., T.H.,

and F.S. performed experiments, data analysis, and interpretation; F.T.W.,

G.Z., and W.S.K. performed conception and design, financial support, data

analysis, and interpretation; A.C.B. and J.W.S. performed conception and

design, data analysis, and interpretation; and R.J.W. performed conception

and design, financial support, data analysis and interpretation, manuscript

writing, and final approval of manuscript.

ACKNOWLEDGMENTS

We acknowledge the gift of the pCR-Blunt II Topo vector containing the

K320E-Twinkle sequence by Steffi Goffart and Hans Spelbrink. We thank

Daniel Bestgen, Maria Bust, Florian Kupper, Katrin Lanz, Alexander Muller,

Petra Kirschner, and Henning Panatzek for excellent technical assistance.

This work was funded by grants from the Deutsche Forschungsgemeinschaft

(DFGWi 889/6-2, Cluster of Excellence CECAD and SFB 728/C2 to R.J.W.; KU

911/21-1 to W.S.K.; and ZS 99/3-1 to G.Z.) and the Center for Molecular Med-

icine Cologne (CMMC to R.J.W. and F.T.W.).

Received: September 27, 2014

Revised: February 4, 2015

Accepted: March 31, 2015

Published: May 5, 2015

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