Mitochondrial Cardiomyopathy Caused by Elevated Reactive Oxygen Species and Impaired Cardiomyocyte Proliferation

Abstract

Rationale

Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood.

Objective

To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (Transcription Factor A, Mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication.

Methods and Results

Tfam inactivation by Nkx2.5^Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation.

Conclusions

Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial dysfunction.

INTRODUCTION

Mitochondria are a central hub of cellular metabolism and energy production. Mutations in genes encoded in both the nuclear and mitochondrial genomes can disrupt mitochondrial function, resulting in diseases with myriad manifestations, including cardiac hypertrophy, non-compaction, and failure¹. While the need for mitochondria to produce an adequate supply of energy is the most obvious link between mitochondrial diseases and heart failure, other metabolic derangements may be equally if not more important for muscle cell dysfunction². Moreover, the mechanisms by which mitochondrial abnormalities cause aberrant cardiac morphogenesis, such as left ventricular non-compaction, are largely unknown. Greater understanding of the role of mitochondria in cardiac morphogenesis and maturation may provide insights into the pathogenesis of mitochondrial cardiomyopathies and identify productive therapeutic avenues to ameliorate the consequences of mitochondrial disease.

Few studies have examined the role of mitochondria in governing cardiac development and maturation. Porter et al. implicated mitochondria, and specifically mitochondrial reactive oxygen species (ROS) signaling, in promoting fetal cardiomyocyte (CM) maturation³. Cardiac ablation of both Ppargc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and Ppargc1b (Peroxisome proliferator-activated receptor gamma coactivator 1-beta), nuclear transcriptional co-activators required for mitochondrial biogenesis and expression of nuclearly encoded mitochondrial genes, caused late gestational defects in cardiac function and CM maturation⁴, providing further evidence that mitochondria are required for normal CM maturation. On the other hand, cardiac-specific inactivation of genes encoding core components of the mitochondrial electron transport chain (e.g., Ndufs4⁵ or Ndufs6⁶) caused lethal postnatal cardiomyopathy but ostensibly did not impact embryonic survival or cardiac development.

Mitochondrial transcription factor A (TFAM) is a nucleus-encoded protein that is required for mitochondrial DNA (mtDNA) transcription⁷. Ablation of Tfam prevents expression of the 13 polypeptides encoded in the mitochondrial genome, all components of enzyme complexes required for oxidative phosphorylation. TFAM is also required to maintain mtDNA stability, both by stimulating its replication and by binding it to form nucleoids⁷. Although Tfam inactivation reduced mitochondrial number and mass, in some contexts it elevated ROS production, likely as a result of electron transport chain impairment⁸. Conditional Tfam inactivation in CMs by Myh6-Cre⁹ or MCK-Cre¹⁰ was previously shown to cause lethal dilated cardiomyopathy. Survival to birth was reportedly normal, and most Tfam^fl/fl; Myh6-Cre neonates died in the first week. However, the effect on cardiac development or CM maturation was not investigated.

Here, we inactivated Tfam in CMs to disrupt mitochondrial function and evaluate the consequences on cardiac development, CM maturation, and cardiac function. We found that Tfam inactivation in fetal CMs severely impaired CM proliferation, associated with severe myocardial hypoplasia and fetal demise. Impaired CM proliferation was linked to ROS-mediated activation of the DNA damage response pathway. Postnatally, Tfam inactivation impaired neonatal CM proliferation but morphologically did not alter CM maturation. Cardiomyopathy caused by neonatal Tfam inactivation was ameliorated by inhibition of ROS or the DNA damage response pathway, suggesting a potential therapeutic strategy for mitochondrial cardiomyopathies.

METHODS

See Online Supplement for detailed methods.

Animal

All animal procedures were performed following protocols approved by the Institutional Animal Care and Use Committee of Boston Children’s Hospital. Tfam^fl/fl, Nkx2-5^IRES-CRE/+, ROSA26^CreERT2, and Rosa26^tdTomato mice and AAV9-TnT-Cre were described previously^9,11–14. AAV was injected subcutaneously at P1 or P8 at 4.0×10¹⁰ vg/g (high dose) or 8.0×10⁹ vg/g (low dose).

Fetal CM culture

Embryonic day 15.5 (E15.5) CMs were isolated using the Neomyt Kit (Cellutron, NC-6031).

Adenovirus expressing Cre (Ad:Cre) or LacZ (Ad:LacZ) was added on day 1.

Mitochondrial function was measured using a Seahorses Biosciences XF96e analyzer.

Histology

Tissues were fixed in 4% paraformaldehye and 8 µm cryosections were immunostained using antibodies listed in the detailed methods (Online Supplement). Apoptosis was measured using the TUNEL Assay Kit (Abcam, ab66108).

Adult CM characterization

Adult CMs were isolated by Langendorff perfusion as described previously¹⁵. In situ T-tubule imaging was performed as described¹⁵. T-tubule organization was quantified using AutoTT^15,16. Contraction of isolated ventricular CMs was measured using an IonOptix system. Intracellular Ca²⁺ recordings were obtained by line scan imaging of CMs loaded with Fluo-4 AM (Life Technologies).

RNA-seq and gene expression

Polyadenylated RNA was isolated and converted into stranded RNA-seq libraries using Script-Seq v2 (Illumina). Real-time PCR was performed using primer sequences are listed in Online Table I.

Statistics

Unless otherwise specified, bar graphs present data as mean ± SD. Significance of intergroup differences was tested using Student’s t-test or the Mann-Whitney test as indicated (SPSS 20.0).

RESULTS

Cardiac-specific deletion of Tfam causes embryonic lethality at E15.5

To disrupt mitochondrial function in fetal CMs early in cardiac development, we used Nkx2.5^IRES-Cre to inactivate a conditional Tfam allele⁹ (Tfam^fl/fl; Nkx2.5^IRES-Cre/+, abbreviated Tfam^NK). Tfam^fl/+; Nkx2-5^IRES-Cre/+ littermates were used as controls. We analyzed embryos from E13.5 to birth, and found that Tfam^NK mutants were present at a normal Mendelian ratio at E13.5 and E15.5, but no viable mutants were recovered at E16.5 or birth (Fig. 1A). At E15.5, Tfam^NK mutants had strikingly thin myocardial walls (Fig. 1B). E13.5 hearts likewise showed thinner myocardial walls (Online Fig. I, A). Quantitative RT-PCR demonstrated reduction of Tfam mRNA in Tfam^NK mutants (Fig. 1C), and TFAM immunostaining confirmed protein depletion (Fig. 1D). Immunoblotting also demonstrated the reduction of TFAM and ATP5B, a nuclear encoded mitochondrial protein, in Tfam^NK mutants (Online Fig. I, B). Furthermore, mtDNA copy number relative to nuclear DNA was reduced by 9.2-fold compared to control (Online Fig. I, C).

Figure 1. Abnormal development of Tfam^NK heart.

A. Distribution of genotypes at indicated gestational ages. NB, newborn. B. Morphology of Tfam^NK and control heart at E15.5, shown in H&E stained transverse sections. Boxed areas are enlarged in the insets. Bar, 200 µm. C. Relative Tfam expression in control and Tfam^NK embryos by qPCR. n=6. D. Representative E15.5 heart sections stained for TFAM, demonstrating reduced immunoreactivity in Tfam^NK heart. Bar, 50 µm. E. Representative E15.5 heart sections stained for CM marker sarcomeric alpha actin (SAA), M-phase marker phosphohistone H3 (pH3), and apoptosis marker Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Bar, 100 µm. Filled yellow arrowheads, pH3⁺ CM. Open white arrowhead, TUNEL⁺ CM. F. Quantification of the frequency of pH3⁺ CMs. n=6. t-test: **, P<0.01.

Tfam is essential for mitochondrial morphogensis and function

To investigate the effect of Tfam inactivation on mitochondria, we analyzed mitochondrial morphology in E15.5 hearts by electron microscopy. Tfam^NK mitochondria had abnormal morphology and organization (Online Fig. II, A). Cristae within mitochondria also appeared more sparse and irregular in Tfam^NK CMs. Quantitative analysis showed that average mitochondrial size was lower in the Tfam^NK CMs (Online Fig. II, B). The mitochondrial area fraction was also reduced in Tfam^NK CMs (Online Fig. II, C). These data indicate that Tfam inactivation disrupts mitochondrial biogenesis and morphology.

We next assessed the effect of Tfam ablation on mitochondrial function. We cultured CMs dissociated from E15.5 Tfam^fl/fl; Rosa26^tdTomato hearts. To deplete Tfam and activate the Cre-dependent tdTomato reporter, we transduced the cultured CMs with adenovirus expressing Cre (Ad:Cre), resulting in activation of tdTomato in >90% of CMs. Ad:LacZ transduced CMs served as controls. Immunostaining 48 hours after virus treatment showed markedly reduced TFAM immunoreactivity in tdTomato⁺ Cre-recombined cells, consistent with effective gene inactivation (Online Fig. III). Furthermore, staining for mitochondrial marker ATP5B demonstrated decreased mitochondrial abundance (Online Fig. III) as well as perinuclear predominance, in contrast to control CMs in which mitochondria were distributed throughout the cytoplasm (Online Fig. III).

Having established an efficient system for in vitro Tfam ablation in fetal CMs, we next assessed the effect on mitochondrial function using a microfluidic extracellular flux analyzer (Online Fig. IV, A–C). Tfam inactivation increased basal respiration, which was attributable to greater F1F0-ATPase-linked respiration and to a lesser extent on increased proton leak (Online Fig. IV, A–C). Moreover, maximal respiratory rate was significantly lower in Tfam-deleted CMs, indicative of reduced maximal electron transport chain activity (Online Fig. IV, A–C). While Tfam deficient CMs exhibited impaired respiration, their extracellular acidification rate, a measure of glycolytic activity, was elevated (Online Fig. IV, D–E).

Functional mitochondria have hyperpolarized inner mitochondrial membranes. We assessed mitochondrial membrane potential with JC-1, which stains normal, hyperpolarized mitochondria red, and impaired, depolarized mitochondria green. Confocal imaging demonstrated increased green and decreased red staining of mitochondria in Tfam^fl/fl CMs treated with Ad:Cre, indicative of mitochondrial depolarization (Online. Fig. V, A). Quantitative data obtained by flow cytometry confirmed decreased red/green fluorescence ratio in Tfam-depleted CMs (Online Fig. V, B). Indeed, the red/green ratio in Tfam-deficient CMs approached the level observed when CMs were treated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (also known as FCCP), which abrogates mitochondrial membrane potential (Online Fig. V, B). These data demonstrate that Tfam inactivation depolarizes mitochondria.

To assess the effect of Tfam knockout on cellular energy reserves, we measured the ADP/ATP ratio in both CMs freshly dissociated from fetal hearts and cultured CMs. In E15.5 CMs and cultured CMs 3 and 7 days after Ad:Cre treatment, ADP/ATP ratio was lower in Tfam-deficient samples (Online. Fig. VI). This suggests that Tfam-deficient CMs surprisingly did not have depletion of energy stores, potentially due to increased glycolytic activity.

Tfam is required cell autonomously for fetal CM proliferation

A major effect of Tfam inactivation on fetal heart development was myocardial hypoplasia (Fig. 1B). To assess the underlying cellular mechanism, we analyzed CM proliferation and apoptosis at E13.5 and E15.5. At both stages, the fraction of CMs expressing the M phase marker phosphohistone H3 (pH3) was markedly and significantly decreased in Tfam^NK, indicative of depressed CM proliferation (Fig. 1E, F). Measurement of Ki67 further confirmed reduced CM proliferation at E15.5 (Online Fig. VII, A–B). TUNEL staining showed that TUNEL⁺ CMs were significantly more frequent in Tfam^NK hearts at E13.5 but not E15.5 (Fig. 1E and Online Fig. VII, C). However, in both groups TUNEL⁺ CMs were infrequent. Together these data suggest that myocardial hypoplasia was due to both reduced CM proliferation and increased CM apoptosis, with the change in proliferation likely making a relatively larger contribution.

We used two independent approaches to assess whether decreased proliferation was a direct, cell-autonomous effect of Tfam inactivation, as opposed to a secondary or indirect consequence of impaired embryo health or myocardial dysfunction. First, we used a tamoxifen-inducible Cre allele (Rosa26^CreERT2) and a low dose of tamoxifen to inactivate Tfam in a minor fraction of cells (Online Fig. VIII, A). In pilot experiments, we titrated the amount of tamoxifen administered at E8.5 so that CreERT2 activated the Cre-dependent Rosa26^tdTomato reporter in ~30% of CMs by E15.5 (Online Fig. VIII, B–C). When this tamoxifen dose was administered to Tfam^fl/fl; Rosa26^{CreERT2/tdTomato} embryos, by immunostaining 80% of tdTomato⁺ CMs lacked detectable TFAM immunoreactivity (Online. Fig. VIII, D–E), indicating that the large majority of tdTomato⁺ CMs lack TFAM. We then analyzed tdTomato⁺ and tdTomato⁻ cells from tamoxifen-treated Tfam^fl/fl; Rosa26^{CreERT2/tdTomato} or Tfam^fl/+; Rosa26^{CreERT2/tdTomato} for cell cycle activity, as measured by pH3 staining (Fig. 2A). As expected, in tdTomato⁻ cells, which were not recombined by CreERT2, there was no significant difference in the frequency of pH3⁺ CMs (Fig. 2B). In contrast, in tdTomato⁺ cells, which were recombined by CreERT2, pH3⁺ CMs from Tfam^fl/fl embryos were significantly less frequent than from Tfam^fl/+ littermates (Fig. 2B). This genetic mosaic analysis demonstrated that Tfam is required cell autonomously for normal CM proliferation. This result makes potential confounding effects from poor cardiac function or low embryo viability less likely, because the embryos were phenotypically normal as a result of the low fraction of cells that were mutated.

Figure 2. Tfam inactivation cell autonomously reduced CM proliferation.

A. Representative images of E15.5 heart sections stained for CM marker (cardiac Troponin T, cTNT), Cre marker (Tomato), and M phase marker (pH3). Insets show magnification of boxed regions with cTNT staining indicated in white. Filled yellow arrowheads, pH3⁺, tdTomato⁺ CMs. Open white arrowheads, pH3⁺, tdTomato⁻ cells. Bar, 100 µm. Boxed regions are magnified in inset with cTNT staining shown in white. B. Quantification of the percentage of pH3⁺ CMs from staining described in (A). 3 sections were stained and imaged per heart and 5 independent hearts were studied. C–F. CMs cultured as in C and exposed to EdU for 24 hours were stained for pH3 or EdU. Representative confocal images are shown in C and E, and boxed regions are magnified in insets on the right. Bar, 100 µm. Results are quantified in D and F. n=6. *, P<0.05. **, P<0.01.

Second, we studied the effect of Tfam inactivation on CM proliferation in cultured fetal CMs. We measured CM proliferation using both uptake of the nucleotide analog EdU (passage through S phase) and immunostaining for pH3 (M phase). Both measures of cell cycle activity were reduced in Tfam-depleted CMs (Fig. 2C–F). Because the intrauterine environment has lower oxygen tension, we repeated these experiments under hypoxic conditions (7% Oxygen). Cell cycle activity of both control and Tfam-depleted groups was higher in hypoxia compared to normoxia, consistent with recent studies¹⁷, but the proliferation rate of hypoxic Tfam-depleted CMs remained lower than hypoxic controls (Online Fig IX).

Collectively, these data demonstrate that Tfam inactivation cell autonomously reduces fetal CM proliferation.

Tfam ablation elevates ROS levels and activates DNA damage response pathways

To further assess the mechanisms by which Tfam ablation inhibits cell cycle activity, we performed RNA-seq on Tfam^NK and control hearts (n=3 biological replicates per group, 5 hearts per replicate). Hearts were collected at E13.5, prior to the onset of severe morphological abnormalities. There were 346 upregulated and 449 downregulated genes (adjusted P-value < 0.05; Fig. 3A). Gene set enrichment analysis¹⁸ of the RNA-seq data (Fig. 3B) showed that the mutant is enriched for terms related to glycolysis and metabolism, consistent with the central role of mitochondria in cellular metabolism. Muscle contraction gene sets were downregulated in mutants, suggesting a link between mitochondrial function and expression of contractile genes. Interestingly, DNA damage response terms, most notably the p53 DNA damage response pathway, was enriched in Tfam^NK. Key upregulated genes in this pathway included Cdkn1a (encoding the cell cycle inhibitor p21), Gadd45a, a gene induced by the DNA damage response pathway, and Ddit3, a gene that mediates cell cycle arrest and that is induced by DNA damage (Fig. 3C).

Figure 3. RNA-seq analysis of Tfam inactivation in fetal heart.

RNA-seq was performed on Tfam^NK and control hearts at E13.5 (n=3 per group). A. Scatterplot of gene expression in Tfam^NK and control. Selected differentially expressed genes are labeled. B. Summary of Gene Set Enrichment Analysis (GSEA) for RNA-seq data. Selected pathways significantly enriched in Tfam^NK or control are listed. Leading edge analysis of the p53 pathway is shown. C. Relative expression of selected genes differentially expressed in p53 pathway based on the RNA-seq results. **, P<0.01.

At birth, exposure to increased oxygen tension results in greater mitochondrial ROS production in normal CMs, and this has been linked to cell cycle exit through activation of DNA damage response pathways¹⁷. Elevated ROS and activation of the p53 pathway has also been implicated in damage to telomeres, and the gene set “Packaging of telomere ends” was down-regulated in Tfam^NK. Therefore we hypothesized that elevated ROS production by Tfam-depleted cells¹⁹ activates DNA damage response pathways, resulting in cell cycle exit.

The effect of Tfam ablation on ROS levels is cell context dependent: Tfam inactivation elevated ROS in adipocytes⁸, but reduced ROS in keratinocytes¹⁹. Therefore as a first step towards testing this hypothesis, we evaluated the effect of Tfam ablation on fetal CM ROS levels. Tfam^NK heart sections had elevated levels of 4-hydroxynonenal (4HNE), a product of lipid peroxidation by ROS, at E13.5 and E15.5 (Fig. 4A, B). In cultured E15.5 Tfam^fl/fl; Rosa26^tdTomato CMs, we measured cellular ROS levels using CellRox, a fluorogenic live cell ROS probe. Positive control cells treated with 50 µM H₂O₂ exhibited high CellRox fluorescence, as expected (Fig. 4C, left panel). In Ad:LacZ treated CMs, most cells had low CellRox signal, consistent with low ROS levels (Fig. 4C, middle panel). In contrast, tdTomato⁺, Ad:Cre-transduced Tfam^fl/fl; Rosa26^tdTomato CMs exhibited markedly increased CellRox fluorescence (Fig. 4C, right panel, upper right quadrant), whereas there the frequency of CellRox positive, tdTomato⁻ cells was not substantially affected (Fig. 4C, right panel, lower right quadrant). Quantitative analysis across replicates confirmed that Tfam ablation markedly increased CM ROS production (Fig. 4D). To determine if the excess ROS originated from mitochondria, we used the mitochondrially targeted ROS fluorescent probe Mitosox. Tfam-depleted CMs exhibited greater Mitosox fluorescent intensity than control CMs (Online Fig. X), supporting elevated mitochondrial ROS production in these cells.

Figure 4. Tfam inactivation elevates CM ROS levels.

A. Representative control and Tfam^NK tissue sections stained for lipid peroxidation product 4HNE, CM marker (cTNT), and DAPI on tissue sections at E13.5 and E15.5. B. Quantitative comparison of 4HNE staining intensity between control and Tfam^NK heart sections. 3 sections were stained and imaged per heart and 4 independent hearts were studied. C. Cultured Tfam^fl/fl; Rosa26^tdTomato E15.5 CMs were treated with H₂O₂ (positive control), Ad:LacZ, and Ad:Cre. The fraction of CellRox⁺ positive cells was measured by FACS. D. Quantification of CellRox⁺ cells between Ad:LacZ and Ad:Cre treated groups. Since LacZ control cells did not activate the Tomato reporter, we compared total CellRox⁺ cells between groups. *, P<0.05. **, P<0.01.

Together these data indicate that mitochondria in Tfam-depleted CMs produce excess ROS by impairing electron transport chain function²⁰. Elevation of ROS and the transcriptomic signature of activated DNA damage response in Tfam-deficient CMs suggested the hypothesis that ROS activation of the DNA damage response reduces their cell cycle activity.

Rescue of cell cycle activity of Tfam-deficient CMs by ROS or WEE1 kinase inhibition

DNA oxidation by ROS triggers phosphorylation of histone H2A.X (γH2A.X), which accumulates at sites of damage and serves as a platform that activates the DNA damage checkpoint to progression from G2 to M phase of the cell cycle²¹. In cultured fetal CMs, Tfam ablation induced increased γH2A.X (Online Fig. XI, A,B), consistent with ROS-mediated DNA damage and activation of DNA damage response pathways. In hypoxic culture conditions (7% O2), overall frequency of γH2A.X CMs was lower, but Tfam-depleted CMs continued to have elevated γH2A.X compared to control (Online Fig. XI, C,D).

To test the hypothesis that elevated ROS production from Tfam-deficient CMs contributed to reduced CM proliferation, we treated cultured CMs with the mitochondrially targeted ROS scavenger mitoTEMPO (MT). MT normalized γH2A.X levels in Tfam-depleted cultured fetal CMs (Fig. 5A, B). This effect of MT was accompanied by increased cell cycle activity, as measured by Ki67 staining (Fig. 5C, D). These data suggest that elevated ROS in Tfam-depleted CMs activates the DNA damage response to reduce CM cell cycle activity.

Figure 5. Rescue of cell cycle activity of Tfam-deficient CMs by ROS or WEE1 kinase inhibition.

Cultured Tfam^fl/fl; Rosa26^tdTomato E15.5 CMs were treated with Ad:Cre on the 1st day after seeding. Then MitoTEMPO (MT) or MK-1775 (MK) were added, and CMs were fixed and stained on the 5th day. A. Representative images of CMs treated with Cre, Cre+MT, Cre+MK, or LacZ (control, Con) and stained for γH2A.X and a CM marker (cTNT, cardiac troponin T). Bar, 100 µm. B. Quantification of the percentage of γH2AX⁺ CMs in (A). C. Representative images of CMs treated with Cre, Cre+MT, Cre+MK, or LacZ (Con) and stained for Ki67 and a CM marker (SAA). Bar, 100 µm. D. Quantification of the percentage of Ki67⁺ positive CMs in (B). t-test: *, P<0.05. **, P<0.01.

Cell cycle progression through the G2/M checkpoint depends upon CDC25-mediated dephosphorylation of CDK1 at tyrosine-15²². WEE1 kinase counterbalances CDC25 by phosphorylating this residue. Activation of DNA damage response pathways inhibit CDC25 activity, and thereby prevent progression through the G2/M checkpoint. We reasoned that DNA-damage mediated cell cycle inhibition could be ameliorated by inhibiting WEE1 kinase¹⁷. Treatment of Tfam-deficient, cultured fetal CMs with MK-1755 (MK), a selective WEE1 kinase inhibitor, increased cell cycle activity, as measured by Ki67 staining (Fig. 5C,D). Remarkably, MK-1755-treated, Tfam-deficient CMs had cell cycle activity that was not significantly different from control CMs. MK-1755 did not reduce γH2A.X levels in Tfam-deficient CMs (Fig. 5A,B), consistent with WEE1 kinase inhibition acting distal to this DNA damage signal.

Collectively, these data indicate that Tfam deficiency causes ROS-mediated DNA damage, which inhibits CM cell cycle activity through activation of the G2/M checkpoint. ROS scavenging or WEE1 kinase inhibition rescue CM proliferation by reducing ROS or by relieving G2/M checkpoint cell cycle inhibition.

Inhibition of ROS-activated DNA damage response mitochondrial rescues cardiomyopathy in vivo

To determine if ROS suppression or WEE1 kinase inhibition protect CM proliferation from Tfam depletion in vivo, we developed and studied a model of neonatal Tfam deficiency, since neonates are more accessible to interventions than embryos. This model is also more relevant to clinical scenarios of mitochondrial abnormalities, which typically present postnatally. Tfam^fl/fl; Rosa26^tdTomato/+ mice were treated with AAV9-cTNT-Cre (abbreviated AAV9-Cre), in which cardiotropic adeno-associated virus 9 expresses Cre from the cardiac troponin T promoter¹⁴. At postnatal day 0 (P0), we delivered two different doses of AAV9-cTNT-Cre (AAV9-Cre; 4 × 10¹⁰ vg or 8 × 10⁹ vg), referred to as High and Low groups, respectively) to Tfam^fl/fl; Rosa26^tdTomato mice (Fig. 6A). Control mice with the genotype Tfam^fl/+; Rosa26^tdTomato were treated with AAV9-Cre at the high dose. Three days after AAV treatment, ~55% and 30% of CMs expressed the tdTomato Cre-dependent reporter (Fig. 6B and Online Fig. XII, A). To confirm that AAV9-Cre effectively inactivates Tfam in Tfam^fl/fl; Rosa26^tdTomato mice, we performed immunostaining on dissociated CMs. We observed reduced TFAM immunoreactivity in tdTomato⁺ CMs (Fig. 6C and Online Fig. XII, B). We confirmed elevated levels of 8-oxo-guanine, DNA damage induced by ROS, as well as increased 4HNE, P21 cell cycle inhibitor, WEE1 kinase, and γH2A.X in Tfam-deficient CMs (Online Fig. XII, C–D).

Figure 6. Inhibition of ROS-activated DNA damage response ameliorates cardiomyopathy in vivo.

A. Experimental set-up and timeline. AAV9-Cre was injected at P0. Experiment A examined the effect of AAV dose. Experiment B examined the effect of treatment with MT or MK during either the first or second post-natal week (groups 1 and 2, respectively). Echocardiography and tissue analyses were performed at the indicated times. B. The percentage of tdTomato⁺ CMs observed after AAV9-Cre injected. Tfam^fl/fl; Rosa26^tdTomato mice were treated with high or low dose AAV9-Cre, while Tfam^fl/+; Rosa26^tdTomato mice were treated with high dose (CON). C. Representative images of P28 CMs isolated from Tfam^fl/+; Rosa26^tdTomato or Tfam^fl/fl; Rosa26^tdTomato mice and control mice treated with AAV9-Cre at P0. CMs are stained for TFAM and CAV3, a T-tubule marker. D–E. Echocardiographic parameters from CON, low dose, and high dose mice. FS, fraction shortening, LVID;d, left ventricular internal diameter at end diastole. Shaded regions indicate SEM at each time point. F–G. Effect of MT or MK treatment on heart function when given in postnatal week 1 (group 1) or 2 (group 2). H–I. Representative heart tissue sections (H) stained for Ki67 at P5 after treatment with Cre+Vehicle, Cre+MT or Cre+MK. Quantification of the percentage of Ki67⁺ CMs is shown in I. n=4. Bar, 50 µm. J–K. Representative heart tissue sections (J) from Cre+Vehicle, Cre+MT or Cre+MK treated mice. Sections were stained for WGA and DAPI and imaged for endogenous tdTomato fluorescence at 8 weeks. Bar, 200 µm. Quantification of the percentage of tdTomato+ CMs is shown in K. n=4. t-test: *, P<0.05. **, P<0.01.

Mice were followed by serial echocardiography until 8 weeks of age. Consistent with prior cardiac knockout of Tfam using Myh6-Cre⁹, high dose mice with a greater fraction of CM transduction developed progressive ventricular dysfunction and dilatation, as measured by echocardiography (Fig. 6D–E). In contrast, the low dose group had heart function that was not distinguishable from control (Fig. 6D–E). These data show that Tfam inactivation in a large fraction of CMs causes progressive cardiomyopathy, while its inactivation in a small fraction of CMs is well tolerated and does not cause organ-level evidence of dysfunction.

Murine CMs continue to proliferate until about postnatal day 7 (P7), by which time they largely exit the cell cycle^23,24. We hypothesized that Tfam ablation during this time period contributed to heart dysfunction by impairing cell cycle activity, and that this effect of Tfam ablation could be ameliorated by either ROS or WEE1 kinase inhibition. To test this hypothesis, mice were treated with AAV9-Cre at the high dose and also with MT (ROS scavenger), MK (WEE1 inhibitor), or vehicle for the first postnatal week (Fig. 6F). Remarkably, treatment with either MT or MK during the first postnatal week, when CMs retain proliferative competence, ameliorated ventricular dysfunction at 8 weeks (Fig. 6F).

We further tested this hypothesis using two independent approaches. In one approach, we treated a second cohort of mice with MT or MK in the second postnatal week (Fig. 6G). Delaying therapy to this period outside of the period of CM proliferative competence resulted in it being ineffective. In a second approach, we asked if inactivation of Tfam after CMs lose proliferative competence has a less deleterious effect than when it is inactivated during active CM proliferation. Tfam^fl/fl; Rosa26^tdTomato and Tfam^fl/+; Rosa26^tdTomato mice at P8, and Tfam^fl/fl; Rosa26^tdTomato mice at P1, were injected with the same weight-adjusted dose of AAV9-Cre. Serial echocardiography confirmed progressive, severe systolic dysfunction caused by AAV9-Cre delivery at P1 (Online Fig. XIII, A). In comparison, AAV9-Cre delivery at P8 caused relatively less severe systolic dysfunction (Online Fig. XIII, A). Together, these data suggest that impaired CM proliferation contributes to the progressive cardiomyopathy caused by neonatal Tfam knockout. Furthermore, the data point to a limited therapeutic window that corresponds to the time when CMs are actively cycling.

We investigated the mechanisms underlying the response to MT and MK during the first postnatal week. Western blotting for TFAM or ATP5B indicated that MT and MK did not reduce the extent of TFAM or mitochondrial depletion (Online Fig. XIV, A). MT and MK likewise did not rescue expression of mt-Atp6, mt-Nd1, or mt-Co1, genes encoded by the mitochondrial genome that require TFAM for transcription (Online Fig. XIV, B). We assessed neonatal CM proliferation at P5 by immunostaining tissue sections for Ki67 (Fig. 6H–I) or pH3 (Online Fig. XIV, C–D). Consistent with the results from cultured fetal CMs, Tfam ablation strongly reduced neonatal CM proliferation (Fig. 6H–I, Cre+Veh group). Treatment with MT increased cell cycle activity of Cre-recombined CMs (Fig. 6H–I, Cre+MT vs Cre+Veh in tdTomato⁺ CMs; Online Fig. XIV, C–D). Interestingly, MT also increased cell cycle activity of non-recombined (tdTomato⁻) CMs (Fig. 6H–I, Cre+MT vs Cr+Veh in tdTomato⁻ CMs), in keeping with reports that physiological increases in ROS during the neonatal period promote cell cycle exit¹⁷. WEE1 inhibition with MK did not enhance cell cycle activity of tdTomato⁻ CMs, but it strongly increased cell cycle activity of tdTomato⁺ CMs, restoring the Ki67⁺ CMs to levels comparable to tdTomato⁻ controls (Fig. 6H). These data indicate that MT and MK treatment inhibit ROS and DNA damage to rescue proliferation without ameliorating the effect of Tfam ablation on mitochondrial abundance or gene expression.

This result was corroborated by an independent approach. The dose of AAV9-Cre that we administered created a genetic mosaic in which tdTomato⁺ CMs lack Tfam and tdTomato⁻ CMs are replete in Tfam. Therefore the fraction of tdTomato⁺ CMs is an independent measure of the relative proliferation of these populations. In heart sections from mice treated with AAV9-Cre at P0 and analyzed at 8 weeks of age, the tdTomato⁺ CM fraction was lowest in the Cre group and significantly higher in both the Cre+MT and Cre+MK groups (Fig. 6J–K), consistent with the measurements of cell cycle activity. In contrast, in heart sections from mice treated with AAV9-Cre at P8 and analyzed at 8 weeks of age, the proportion of tdTomato⁺ CMs was similar between control and Tfam mutant hearts (Online Fig. XIII, B), consistent with Tfam inactivation at this stage having little effect on CM cell cycle activity.

These data collectively support a model in which mitochondrial dysfunction triggered by Tfam inactivation decreases neonatal CM proliferation through ROS-mediated DNA damage and activation of the G2/M cell cycle checkpoint. This impaired neonatal CM cell cycle activity contributes to adult ventricular dysfunction. Alleviating the inhibition of CM cell cycle activity, either by ROS scavenging or WEE1 kinase inhibition, improves CM cell cycle activity and thereby partially rescues heart function.

Mitochondria are dispensible for postnatal CM maturation

During the neonatal period, CMs undergo a dramatic phenotypic switch that includes tremendous physiological CM hypertrophy, increased sarcomere organization, and formation of T-tubules^25,26. Concurrent with these structural changes is a switch from glycolytic to oxidative metabolism. In CMs derived from stem cells, metabolic maturation influences CM differentiation²⁷ and a similar role in neonatal CM maturation has been hypothesized^28,29. Since Tfam is essential for normal CM metabolic maturation, we tested this hypothesis by assessing the postnatal maturation of Tfam-deficient CMs. Previously, we showed that inactivation of genes in a fraction of CMs is a powerful approach to study cell autonomous function without impairing viability or eliciting non-specific effects caused by cardiac dysfunction^14,15. Low dose AAV9-Cre ablated Tfam in transduced CMs marked by tdTomato expression (Fig. 6C and Online Fig. XII, B) without measurably impairing cardiac function (Fig. 6D–E). Therefore we took advantage of this model to interrogate the effect of Tfam depletion on CM maturation.

Tfam^fl/+; Rosa26^tdTomato and Tfam^fl/fl; Rosa26^tdTomato mice were treated with low dose AAV9-Cre at P0. We characterized the kinetics of mitochondrial dysfunction in these mice by measuring the expression of mt-Atp6, mt-Nd1 and mt-Co1, genes encoded by the mitochondrial genome that require TFAM for transcription. After heart dissociation, tdTomato⁺ CMs were isolated by flow cytometry, and gene expression was measured by qRTPCR. By P14, all three transcripts were reduced by over 50%, and by P28 their expression was reduced by 85%, 80%, and 80%, respectively, compared to littermate Tfam^fl/+ controls (Online Fig. XV, A). These data suggest that mitochondrial dysfunction occurs rapidly in this model, within a window that would be anticipated to impact perinatal CM maturation.

We studied the cell autonomous effect of mosaic Tfam ablation on CM maturation at 4 weeks of age. We used in situ confocal imaging, in which intact hearts are optically sectioned by confocal imaging, to examine T-tubule structure. Mitochondria and T-tubules were imaged by perfusion with mitoTracker and the voltage-sensitive membrane dye FM4-64. These data showed that tdTomato⁺ cells from Tfam^fl/fl hearts had disrupted mitoTracker staining, consistent with effective Tfam depletion and mitochondrial disruption, yet had preserved T-tubule morphology (Fig. 7A). We quantitatively analyzed T-tubule morphology of tdTomato⁺ cells from low dose AAV9-Cre-treated Tfam^fl/fl and Tfam^fl/+ hearts using AutoTT software¹⁶ (Fig. 7B, D–G). There was no significant difference between genotypes. These data show that development of mature T-tubules, often considered a hallmark of CM maturation, does not require normal mitochondrial function.

Figure 7. Mitochondria are dispensable for morphological postnatal CM maturation.

A. In situ confocal live imaging of mitochondria and T-tubules. Control and mosaic TFAM-depleted P28 hearts were perfused with Mitotracker and FM4-64 and optically sectioned. B. In situ confocal live imaging of T-tubules in FM4-64 loaded P28 hearts after control or mosaic TFAM depletion. The T-Tubule patterns in white boxes were enlarged and skeletonized using AutoTT. C. Imaging of sarcomeres in P28 CMs dissociated from hearts after control or mosaic TFAM depletion. Sarcomeres were visualized by sarcomeric α-actinin (SAA) staining. The boxed areas were enlarged (3rd row) and skeletonzied (4th row) using AutoTT. D–G. AutoTT quantification of T-Tubule patterns visualized by in situ imaging. H–I. AutoTT quantification of sacomere organization in isolated CMs. J–K. Quantification of CM dimensions. Length and width of SAA-stained dissociated CMs was determined using ImageJ. Violin plots show the distribution of values as well as the median (circle), 25th and 75th percentiles (thick line), and 1.5 times the interquartile range (thin lines). Sample sizes are indicated by numbers above the abscissa. P-values of intergroup comparisons using the Mann-Whitney U-test are shown.

To interrogate sarcomere structure, we dissociated CMs by collagenase perfusion and immunostained them for sarcomeric α-actinin (SAA), which labels Z-lines (Fig. 7C). Sarcomeric organization was also quantified using AutoTT. We found no significant difference in sarcomere regularity or spacing between AAV9-Cre treated Tfam^fl/fl and Tfam^fl/+ tdTomato⁺ CMs (Fig. 7H–I). These results indicate that normal mitochondrial function is not required for attaining mature sarcomere organization.

Physiological hypertrophy of CMs is another hallmark of CM maturation. We measured the length, length/width ratio, and area of dissociated, tdTomato⁺ CMs from Tfam^fl/fl or Tfam^fl/+ hearts (Fig. 7J–L). As with T-tubule and sarcomere organization, these parameters of CM hypertrophy were no different between genotypes, indicating that normal mitochondrial function is not required for physiological CM growth.

Switching of sarcomere isoforms is another hallmark of postnatal CM maturation²⁶. We assessed the exchange of Myh7 (immature) to Myh6 (mature), and the switch of Tnni1 (immature) to Tnni3 (mature). This latter isoform switch is specific to maturation and insensitive to CM stress³⁰. We measured Tnni3, Tnni1, Myh6, and Myh7 transcript levels by qRTPCR (Online Fig. XV, B). Expression of these genes, as well as the Tnni3/Tnni1 and Myh6/Myh7 ratio, were not significantly changed by Tfam inactivation, indicating that these gene expression markers of CM maturation were not perturbed by Tfam ablation.

While Tfam inactivation had minimal effect on structural aspects of CM maturation, it did significantly impact two aspects of CM function: intracellular Ca²⁺ handling and contraction. To evaluate the effect of Tfam inactivation on intracellular Ca²⁺ handling, we treated Tfam^fl/fl; Rosa26^tdTomato and Tfam^fl/+; Rosa26^tdTomato mice with low dose AAV9-Cre at P1, dissociated CMs at P28, and loaded them with the Ca²⁺ sensitive dye Fluo-4. Ca²⁺ transients were measured by confocal line scan during 1 Hz pacing (Fig. 8A). Tfam-deficient CMs exhibited significantly lower Ca²⁺ transient amplitude (F/F₀; Fig. 8A, B). On the other hand, time-to-peak was not significantly different in Tfam-deficient CMs (Fig. 8A, C), consistent with the preserved T-tubule structure that we observed in these cells. Tfam inactivation also did not significantly affect time-to-50%-decay (Fig. 8A, D), a measure of the kinetics of cytosolic Ca²⁺ return to diastolic levels.

Figure 8. Cell autonomous effect of Tfam inactivation on CM Ca²⁺ handling and contraction.

A–D. Analysis of Ca²⁺ handling. P28 dissociated CMs were loaded with the Ca²⁺sensitive dye Fluo-4 AM and imaged by confocal line scan during 1 Hz pacing. A, Representative Ca²⁺ transient profiles are shown. Ca²⁺ transient magnitude, time-to-peak, and time-to-50% recovery were quantitative analyzed (B–D). E–I. Analysis of contraction. Bright field images of dissociated P28 CMs contracting during 1 Hz pacing were recorded. Image analysis yielded sarcomere length of live cells (E) and length-time relationship during contraction (F). Analysis of the length-time relationship yielded the fractional CM shortening (G), departure velocity (H), and return velocity (I). Violin plots show the distribution of values as well as the median (circle), 25th and 75th percentiles (thick line), and 1.5 times the interquartile range (thin lines). Numbers within graph legend indicate the number of CMs analyzed. These CMs were isolated from 4 hearts per group. Groups were compared by Mann-Whitney U-test. *P<0.05. **P<0.01. ***P<0.001.

To assess the effect of Tfam inactivation on CM contraction, we prepared dissociated Tfam^fl/fl; Rosa26^tdTomato and Tfam^fl/+; Rosa26^tdTomato CMs as described for the Ca²⁺ handling experiments. Bright-field images of the CMs were obtained during 1 Hz pacing. Consistent with immunostaining of fixed cells, sarcomere length from these live cells was not different between Tfam-deficient and control CMs (Fig. 8E). However, Tfam-deleted CMs had significantly reduced shortening and departure velocity compared to the other groups (Fig. 8F–H), consistent with reduced contractility and lower Ca²⁺ transient amplitude. Tfam-deficient CMs did not have significantly altered return velocity, suggesting that CM relaxation was not significantly affected (Fig. 8I). These data indicate that Tfam-deficient CMs have reduced contractility compared to control CMs.

Collectively, these data support the surprising conclusion that morphological CM maturation is not dependent upon their metabolic maturation. However, Tfam is required for normal CM Ca²⁺ handling and contraction.

DISCUSSION

Although cardiomyopathy and abnormal myocardial morphologies such as left ventricular non-compaction and hypertrophic cardiomyopathy are associated with mitochondrial disease, the mechanisms linking mitochondria to these morphological findings have been understudied. While energy depletion likely contributes to these phenotypes, our study highlights the effect that mitochondrial dysfunction has on another basic process of cardiac development, namely CM proliferation. We observed that mitochondrial dysfunction triggered by Tfam ablation induces cell cycle arrest that contributes to the resulting mitochondrial cardiomyopathy. Protecting CMs from cell cycle arrest induced by mitochondrial dysfunction improved cardiac function for weeks beyond the treatment period, suggesting a potential therapeutic strategy for mitochondrial cardiomyopathies.

While Tfam inactivation reduced mitochondrial mass, we observed that it increased mitochondrial ROS in CMs. The effect of Tfam inactivation on mitochondrial ROS appears to be cell type dependent, since it was previously observed to elevate ROS in adipocytes⁸ and reduce ROS in keratinocytes¹⁹. The cell types in which it elevated ROS (CMs and adipocytes) are normally rich in mitochondria, suggesting a potential factor responsible for the difference between cell types. A potential mechanism that accounts for elevated ROS in Tfam-deficient CMs is the markedly reduced expression of components of the electron transport chain encoded by the mitochondrial genome⁸. The defective electron transport chain of Tfam-deficient CMs may be predisposed to produce ROS. In normal mitochondria, membrane depolarization accelerates electron transit through the electron transport chain and thereby reduces ROS production³¹; however, increased activity of the defective electron transport chain of Tfam-deficient CMs may elevate ROS production.

Elevated ROS in Tfam-depleted CMs induced the DNA damage response, including deposition of γH2A.X. As a result, inhibitory cell cycle checkpoints were activated, resulting in reduced CM cell cycle activity. Elevated CM ROS triggered by increased mitochondrial oxidative phosphorylation in the oxygen-rich post-natal environment has been proposed to trigger physiological cell cycle arrest of neonatal CMs through a similar mechanism¹⁷. That WEE1 kinase inhibition alleviated cell cycle exit induced by Tfam inactivation suggests that the main checkpoint occurred at the G2/M transition, where cell cycle progression requires CDC25 dephosphorylation of CKD1 to overcome its phosphorylation by WEE1 kinase²².

Our data suggest that inhibition of CM cell cycle activity may be an important contributor to some forms of mitochondrial cardiomyopathy. In our model, mitochondrial dysfunction was triggered in neonatal mice by delivery of AAV9-Cre at P0 or P1. Remarkably, transient treatment of neonatal mice during the first postnatal week with either ROS scavenger or WEE1 inhibitor ameliorated heart dysfunction as much as seven weeks later. That this same treatment did not have benefit when given in the second postnatal week suggests that these treatments primarily work by maintaining CM cell cycle activity, since the therapeutic window coincides with the period during which neonatal CMs normally retain cell cycle activity. A corollary to this interpretation is that increased abundance of Tfam-deficient CMs supports increased cardiac contraction. Indeed, we observed that Tfam-deficient CMs do contract, albeit more weakly than littermate control CMs. The important contribution of reduced CM proliferation to the Tfam mutant cardiomyopathic phenotype was further corroborated by the observation that Tfam inactivation at P8 (after CM cell cycle exit) was less deleterious to heart function that its inactivation at P1 (during CM proliferation). In future work it will be important to determine the durability of the improvement in heart function induced by MT or MK treatment. It is interesting to consider that some human cardiomyopathies, such as those caused by a subset of mitochondrial diseases, might exhibit a similar window during which myocardial outcome could be improved by neonatal therapy. Further research will be needed to evaluate the relevance of our observations in the Tfam-knockout model to human mitochondrial cardiomyopathy.

Neonatal CMs undergo many phenotypic changes that convert them from proliferative fetal cells with relatively low pumping capability to mature, terminally differentiated, adult CMs with much higher pumping capacity^25,26. Accompanying these changes is a metabolic switch from glycolysis to oxidative phosphorylation, and this metabolic switch has been hypothesized to help to drive the other phenotypic changes^27–29. Using postnatal mosaic Tfam inactivation, we critically tested this hypothesis. Surprisingly, we found that CM growth, sarcomeric organization, and T-tubule formation were unaffected by neonatal Tfam inactivation. These data suggest that mitochondrial expansion and robust function are not prerequisites for morphological CM maturation in vivo, although they are required for normal Ca²⁺ handling and contraction. However, this result does not exclude the possibility that enhancing metabolic maturation of iPSC-CMs will expedite their morphological and functional maturation.

NOVELTY AND SIGNIFICANCE.

What Is Known?

• Mutations in genes essential for mitochondrial function often affect cardiac morphogenesis and postnatal cardiac function.

• Postnatal cardiomyocyte maturation involves striking changes in both morphology and metabolism, leading to the hypothesis at metabolic maturation could enhance morphological maturation.

• The nuclear encoded mitochondrial protein TFAM is required for mitochondrial gene expression and DNA replication.

What New Information Does This Article Contribute?

• Early stage inactivation of a conditional Tfam allele in cardiomyocytes caused lethal defects in cardiac development associated with ROS-induced cardiomyocyte cell cycle activity.

• Postnatal mitochondrial dysfunction induced by neonatal cardiomyocyte-selective Tfam inactivation likewise reduced neonatal cardiomyocyte proliferation and caused progressive heart failure.

• In this neonatal Tfam cardiomyocyte inactivation model, suppressing ROS or kinases responsible for the G2/M cell cycle checkpoint in the neonatal period delayed the progression of heart failure.

• Mosaic Tfam inactivation in neonatal cardiomyocytes did not have a cell autonomous effect on their morphological maturation.

Gene mutations that impair mitochondrial function often disrupt cardiac morphogenesis and function, but the mechanistic links between mitochondria, cardiac morphogenesis, and heart failure are incompletely understood. Here we study both fetal and neonatal inactivation of Tfam, which is required for mitochondrial gene expression and DNA replication. Tfam inactivation at either stage elevated reactive oxygen species (ROS) and activated the DNA damage response, resulting in reduced cardiomyocyte cell cycle activity. Suppressing ROS or blocking WEE kinase, required for the G2/M cell cycle checkpoint, restored cardiomyocyte cell cycle activity. In the neonatal Tfam inactivation model, ROS suppression or WEE kinase inhibition delayed the progression of heart failure. Using a mosaic Tfam inactivation approach, we further demonstrate that mitochondrial dysfunction caused by Tfam depletion did not affect postnatal cardiomyocyte morphological maturation. Together our data demonstrate that reduced cardiomyocyte cell cycle activity is one mechanism that links mitochondrial dysfunction to abnormal cardiac morphogenesis and function. In addition, our study points out that the neonatal period may offer a therapeutic window during which intervention can ameliorate later cardiac dysfunction.

Nonstandard Abbreviations and Acronyms

4HNE

4-hydroxynonenal

CM

cardiomyocyte

EdU

5-ethynyl-2´-deoxyuridine

mtDNA

mitochondrial DNA

MK

MK-1755

MT

mitoTEMPO

PH3

phosphohistone H3

ROS

reactive oxygen species

SAA

sarcomeric alpha actinin

TFAM

mitochondrial transcription factor A

Tfam^NK

Tfam^fl/fl inactivated by Nkx2-5^Cre

OCR

oxygen consumption rate

ECAR

extracellular acidification rate

AAV

adeno-associated virus

T-tubule

transverse tubule