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Tafazzin knockdown causes hypertrophy of neonatal ventricular myocytes

Quan He

Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 29 January 2010; accepted in final form 23 March 2010

He Q. Tafazzin knockdown causes hypertrophy of neonatal ventricularmyocytes. Am J Physiol Heart Circ Physiol 299: H210H216, 2010. Firstpublished March 26, 2010; doi:10.1152/ajpheart.00098.2010.Mutation ofthe mitochondrial protein tafazzin causes dilated cardiomyopathy inBarth syndrome. We employed an adenovirus as a vector to transfertafazzin small hairpin RNA (shRNA) into neonatal ventricular myo-cytes (NVMs) to investigate the effects of tafazzin knockdown. Thetafazzin shRNA adenovirus consistently knocked down tafazzinmRNA and lowered cardiolipin while significantly decreasing theproduction of ATP by the mitochondria. The phosphorylation ofAMP-activated protein kinase and mitochondrial density were bothincreased in tafazzin knockdown NVMs compared with scrambledshRNA controls. When we tested whether tafazzin knockdown causeshypertrophy in vitro, we found that the surface area of NVMs infectedwith tafazzin shRNA adenovirus was significantly increased, as werethe protein synthesis and expression of the hypertrophic marker gene,brain natriuretic peptide. Taken together, our data support the conceptthat a decreased tafazzin expression causes cardiomyocyte hypertro-phy in vitro.

cardiolipin; adenosine 5=-triphosphate; dilated cardiomyopathy

TAFAZZIN IS A MITOCHONDRIAL protein encoded by the G4.5 genein humans (7). It is a phospholipid transacylase (54), and itsmutation results in decreased cardiolipin (18, 4749, 53), aphospholipid predominantly present in the inner membrane ofthe mitochondria. Cardiolipin is initially synthesized as apremature form, which is deacylated by a phospholipase togenerate monolysocardiolipin, and this lipid is finally reacy-lated by tafazzin to form the mature fully functional cardiolipin(17, 54). Cardiolipin is required for 1) optimal activity ofrespiratory chain complexes and ADP-ATP translocase (31),which are involved in ATP and reactive oxygen species pro-duction; 2) cytochrome c attachment within the mitochondrialintermembrane space (39), which is associated with mitochon-dria-initiated cell death; 3) maintaining inner membrane fluid-ity and osmotic stability (11), which are associated with theopening of mitochondrial permeability transition pores andmitochondria-gated cell death; and 4) mitochondrial proteinimport (30), which is associated with mitochondrial biogenesis.

Tafazzin mutation causes Barth syndrome, a rare X-linkedgenetic disorder characterized by dilated cardiomyopathy

(DCM) with or without hypertrophy, skeletal myopathy, neu-tropenia, growth retardation, and 3-methylglutaconic aciduria(4, 5, 45). End-stage heart failure resulting from DCM is amajor cause of death among these patients during infancy andearly childhood.

DCM, the most common type of cardiomyopathy, is char-acterized by ventricular chamber enlargement and systolicdysfunction, commonly resulting in congestive heart failure(35). An eccentric cardiomyocyte hypertrophy (increased

length-to-width ratio) and an energy depletion are characteris-tic of DCM (15, 20, 21, 36).

A deletion of tafazzin in yeast causes mitochondrial dys-function, including changes in energy transformation and os-motic properties of the mitochondria (18, 34). A mutation oftafazzin in Drosophila causes a pathological dysfunction ofmitochondria and motor weakness (53). Tafazzin knockdownin zebrafish led to bradycardia and retarded cardiac develop-ment (32). The decreased expression of tafazzin has beenshown in spontaneously hypertensive failing hearts (42). How-ever, we do not know how tafazzin knockdown affects mam-malian cardiomyocytes. We here present evidence that tafazzinknockdown results in ATP deficiency and hypertrophy in

cultured neonatal cardiomyocytes.METHODS

All animal experiments were approved by the Henry Ford HealthSystem Institutional Animal Care and Use Committee.

Supplies and chemicals. Phosphatase and proteinase inhibitor cock-tail tablets (PhosSTOP and Complete Mini) were obtained fromRoche Applied Science (Indianapolis, IN). A pSilencer adeno 1.0-CMV system was purchased from Ambion (Austin, TX). Primaryantibodies against phospho-AMP-activated protein kinase (phospho-AMPK) (Thr172) and AMPK and an horseradish peroxidase-conju-gated secondary antibody against rabbit IgG were obtained from CellSignaling Technology (Boston, MA). -Actin antibody and FITC-conjugated anti-goat IgG were purchased from Santa Cruz (SantaCruz, CA). Coomassie protein assay and SuperSignal West Pico

chemiluminescent substrate kits and Restore Plus Western blot strip-ping buffer were purchased from Thermo Scientific (Rockford, IL).MitoTracker Red, Taq DNA polymerase kits, DMEM medium andcell culture supplements, precast Tris-glycine polyacrylamide gels,and polyvinylidene fluoride membranes were obtained from Invitro-gen (San Diego, CA). Laminin-coated coverslips were purchasedfrom BD Sciences (San Jose, CA). ATP bioluminescent assay kits and4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (St.Louis, MO). [32P]phosphate and [3H]leucine were purchased fromPerkinElmer (Waltham, MA); Whatman TLC plates (LK5D silica gel,150 ) were from VWR (Batavia, IL); RNeasy fibrous tissue minikits, QIAamp DNA Micro Kit, random primers, and Omniscriptreverse transcriptase were from Qiagen (Valencia, CA); and SYBRgreen dye was from SA Biosciences (Frederick, MD). Custom primerswere designed by TIB MolBiol (Adelphia, NJ). The HEK293 cell line

was obtained from ATCC (Manassas, VA), luciferase assay reagentswere from Promega (Madison, WI), and other routine supplies andchemicals were from Fisher and Sigma.

Construction of tafazzin small hairpin RNA adenovirus. The smallhairpin sequences recognize tafazzin coding regions 5=-CAGCT-GTGGAGATGCGGAA-3= Taz1 and 5=-AGAGGAATTCCAGCG-GCT-3= Taz2 with restriction endonuclease XhoI and SpeI sites added ateach end to facilitate subcloning. The small hairpin RNA (shRNA)oligonucleotides and scrambled sequence 5=-CAGTCCAGCTAGCTC-TACT-3= were synthesized by TIB MolBiol. Equal amounts of the upperand lower oligonucleotide were annealed by boiling and cooled to makethe double-stranded shRNA. This was subcloned into a shuttle vector(pSilencer adeno 1.0-CMV), andthe insertion was confirmed by sequenc-ing. The virus DNA containing the insertion was packaged inside

Address for reprint requests and other correspondence: Q. He, Hypertensionand Vascular Research Div., Henry Ford Hospital, 2799 W. Grand Blvd.,Detroit, MI 48202-2689 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 299: H210H216, 2010.First published March 26, 2010; doi:10.1152/ajpheart.00098.2010.

0363-6135/10 Copyright 2010 the American Physiological Society http://www.ajpheart.orgH210


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HEK293 cells. The adenovirus was expanded and the virus concentration(expressed as viral particles per milliliter) was estimated by spectropho-tometry following Ambions protocol.

Neonatal cardiac myocyte culture. Neonatal ventricular myocytes(NVMs) were isolated from 1- to 2-day-old Sprague-Dawley rats fromCharles River as we described previously (24). They were placed on6-well (1 million per well) or 12-well plates (0.5 million per well) inDMEM containing 10% FBS and incubated for 40 h. The medium waschanged to serum-, glucose-, and pvruvate-free medium supplementedwith insulin, transferrin, and selenium; glutamine (2 mM); penicillin-streptomycin; and bromodeoxyuridine (0.1 mM). The adenovirus wasadded, and samples were incubated for 48 h. When the cell size wasmeasured, they were plated at a lower density (6-well plates, 2.5 105 cells per well) to improve the visualization of each individual cell.

Semiquantitative and real-time RT-PCR. Total RNA isolation fromNVMs was performed as described previously (25). RNA (2 g) wasreverse-transcribed into cDNA in 20 l reaction buffer containing 1unit reverse transcriptase, 2 g random primer, 0.5 mM dNTP, and 10units RNasin at 37C for 1 h. The reverse transcription mixture wasdenatured at 95C for 10 min. For semiquantitative PCR, tafazzin wasamplified with 0.5 M primers (upper, 5=-AGCACGGTGATTTTCT-GTCC-3=; and lower, 5=-GGCAAATGTGTGCCTGTATG-3=) in 20l reaction volume containing 0.25 mM dNTP and 1 unit Taq DNApolymerase for 22 cycles. The PCR products were run on 2% agarosegel, and DNA bands were visualized by ethidium bromide staining.The gels were imaged by transillumination and DNA bands quantifiedwith Density software corrected by GAPDH (upper, 5=-ATTCAACG-GCACAGTCAAGG-3=; and lower: 5=-TGGATGCAGGGATGAT-GTTC-3=), which was amplified for 18 cycles. Tafazzin mRNA levelswere expressed as a percentage of control. For real-time PCR, 2 l ofthe products of the reverse transcription reaction were amplified usingSYBR dye (SA Biosciences) along with the same primers (includingtafazzin and GAPDH as normalizer) in a Roche LightCycler (v. 2) aswe described previously (20). Tafazzin mRNA levels were deter-mined using the Ct method as described by Winer et al. (51) andexpressed as a percentage of control.

Phospholipid metabolic labeling. NVMs were placed on six-wellplates (1 million cells per well). [32P]phosphate (1 Ci/well) was

added 24 h after the adenovirus, and the samples were incubated for24 h. The cells were harvested for phospholipids and analyzed byone-dimensional TLC as we described previously (22). After exposureto X-ray film, the cardiolipin bands were scanned, quantified, andexpressed as a percentage of control (NVMs infected with a scram-bled shRNA adenovirus).

ATP assay. Cells were lysed in 0.2% SDS, and ATP was measuredwith a kit from Sigma. ATP content was corrected by total protein,which was determined using a kit from Thermo Fisher with BSA as astandard. ATP content was expressed as relative light units permicrogram protein.

Western blot analysis. Western blot analysis was performed as wedescribed previously (23) using 50 g total cell extract protein.Protein concentration was determined with the Coomassie proteinassay kit using BSA as the standard.

Mitochondrial staining. At the end of the experiment, the cellswere incubated with 200 nM MitoTracker red in the dark for 20 min.They were then washed with PBS and fixed in 37% formaldehyde for30 min at room temperature in the dark. The cells were washed withPBS and counterstained with DAPI for 3 min and then washed andmounted. Five images were obtained from each field using fluores-cence microscopy with red and blue filters and merged with SPOTsoftware. The red fluorescence intensity representing mitochondrialdensity was quantified using MicroSuite software and expressed asfluorescent intensity per cell.

Mitochondrial DNA assay. Total DNA including mitochondrialDNA was isolated from NVMs using QIAamp DNA Micro Kit fromQiagen. Mitochondrial NADH dehydrogenase 1 (ND1) gene contentwas determined by real-time PCR, as described in Semiquantitative

and real-time RT-PCR, with10 ng total DNA as the template usingprimers upper 5=-ATGGCCTTCCTCACCCTAGT-3= and lower 5=-AGAGGGCGTATGGGTTCTTT-3 =. ND1 was normalized withgenomic gene GAPDH using the same set of primers described inSemiquantitative and real-time RT-PCR.

Cardiac myocyte surface area. NVMs were plated onto laminin-coated coverslips in six-well plates at a density of 0.25 million cellsper well. After we treated them with the adenovirus, they wereimmunostained with a -actin antibody and visualized with an FITC-conjugated secondary antibody. The nuclei were counterstained byDAPI. Five images of each field were captured with fluorescencemicroscopy using DAPI and FITC filters and merged with SPOTsoftware. The surface area (expressed as m2) was measured withMicroSuite software.

[3H]leucine incorporation assay. The rate of protein synthesis byNVMs was estimated by [3H]leucine incorporation as described byHarding et al. (21). NVMs were placed on six-well plates at a densityof one million cells per well. After a 40-h incubation in DMEMcontaining 10% FBS, the medium was changed to serum-, glucose-,and pvruvate-free DMEM containing 1 Ci [3H]leucine, and theadenovirus was added. After 48 h, the cells were harvested fortrichloroacetic acid precipitates, which were counted for 3H activity(counts/min of [3H]leucine incorporation) in a scintillation counter.3H activity of NVMs infected with a tafazzin shRNA adenovirusrepresents protein synthesis, expressed as a percentage of control(NVMs infected with a scrambled adenovirus).

Transfection and luciferase assay. NVMs were transfected byelectroporation using a cuvette with a 2-mm gap (BTX, San Diego,CA) in 0.4 ml PBS buffer containing 0.1% glucose and 1 g1818hBNPluc plasmid DNA per million cells. The cells were placedon 12-well plates and maintained in DMEM containing 10% FBS for40 h. The medium was then changed to serum-, glucose-, andpvruvate-free DMEM; the shRNA adenovirus (100 vp/cell) wasadded; and the samples were maintained for 48 h. Finally, the cellswere harvested for luciferase activity (23), which represents humanbrain natriuretic peptide (hBNP) promoter activity and is expressed asfold increase vs. control (cells infected with a scrambled shRNAvirus). The 1818hBNPluciferase construct (33) was graciously pro-

vided by Dr. M. C. LaPointe (Henry Ford Hospital, Detroit, MI).Statistical analysis. Data are expressed as means SE. Differences

in mean values were analyzed by two-tailed t-test or one-wayANOVA using the Student-Newman-Keuls method for pairwise mul-tiple comparisons. A value of P 0.05 was considered significant.

RESULTS

Construction of tafazzin shRNA adenovirus. To test theefficiency of our tafazzin shRNA adenovirus in knocking downtafazzin mRNA, we used doses ranging from 0.1 to 100 viralparticles per myocyte (vp/cell) of two different constructs(Taz1 and Taz2). We found that 100 vp/cell gave the bestresults without affecting cell growth (Fig. 1A). Some NVMs

were killed and the others displayed cell abnormal morphologywhen treated with 800 vp/cell shRNA adenovirus (data notshown). Whereas both Taz1 and Taz2 significantly knockeddown tafazzin mRNA, Taz1 reduced the expression by 70%compared with only 28% for Taz2 (Fig. 1B). The internalcontrol gene GAPDH was not affected by the shRNA adeno-virus. Based on our findings, we used a dose of 100 vp/cellTaz1 shRNA adenovirus for all experiments.

Tafazzin knockdown decreases cardiolipin. Tafazzin muta-tion decreases cardiolipin content (18, 43, 48). We then testedwhether tafazzin knockdown in NVMs affects cardiolipin con-tent and found that tafazzin knockdown significantly decreasedcardiolipin in NVMs (Fig. 2) compared with scrambled con-

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trol, as determined by metabolic labeling and thin-layer chro-matography analysis. In contrast, other phospholipids were notaffected by tafazzin knockdown. Thus tafazzin knockdown inNVMs mimics cardiolipin deficiency in Barth syndrome.

Tafazzin knockdown decreases intracellular ATP. The pri-mary function of the mitochondria is the production of ATP,which is critically important for maintaining the pumpingfunction of the heart. ATP depletion has been associated withcardiomyopathy and heart failure (2, 3, 8, 36). Barth syndrome,which is characterized by DCM, causes metabolic decompen-

sation and energy deficiency (12, 34, 56). We infected NVMswith the tafazzin shRNA adenovirus for 48 h in a mediumcontaining glutamine as the sole energy molecule and foundthat intracellular ATP was significantly reduced compared withcells infected with a scrambled shRNA adenovirus (Fig. 3).This ATP must have come from the mitochondria, becauseglutamine must be metabolized by oxidative phosphorylationwithin the mitochondria to generate ATP (38); thus tafazzinknockdown decreases mitochondrial ATP production.

Tafazzin knockdown activates AMPK and increases mito-chondrial density. AMPK has been postulated as the fuelgauge of the cell and is regulated by intracellular ATP (19). Itis activated by a decrease in the ATP-to-AMP ratio. One of the

long-term responses to AMPK activation is an enhanced mi-tochondrial biogenesis (29). We investigated the effects oftafazzin on AMPK activation and mitochondrial biogenesisand found that tafazzin knockdown dramatically increasedAMPK phosphorylation (Fig. 4A) compared with a scrambledvirus control. As visualized by MitoTracker red (Fig. 4, B andC), the mitochondrial density (red stains) around the nucleus,which was stained in blue by DAPI, was significantly in-

Fig. 1. Tafazzin mRNA is knocked down by the tafazzin small hairpin(shRNA) adenovirus. A: representative images obtained by semiquantitativeRT-PCR. Tafazzin mRNA was knocked down by the tafazzin shRNA adeno-virus in a dose-dependent manner. TAZ, amplified tafazzin gene (278 bp);GAPDH, 302 bp. Lanes 1, 4, 7, and 10 represent neonatal ventricular myocytes(NMVs) treated with the scrambled virus; lanes 2, 5, 8, and 11 representNVMs treated with the Taz1 shRNA adenovirus; and lanes 3, 6, 9, and 12represent NVMs treated with the Taz2 shRNA adenovirus. MM, molecularmarker; vp, viral particles. B: quantification of A. Tafazzin mRNA wasexpressed as a percentage of cells infected with a scrambled shRNA adeno-virus (SCR). Data represent means SE from 14 independent experiments.#P 0.01 vs. SCR; *P 0.05 vs. SCR.

Fig. 2. Tafazzin shRNA adenovirus decreases cardiolipin in NVMs. A: TLCimage representing 3 separate experiments. NVMs were infected with theshRNA adenovirus and metabolically labeled with [32P]phosphate. Phospho-lipids were isolated and analyzed on TLC plates as described in METHODS. Taz,tafazzin shRNA; CL, cardiolipin; PE, phosphatidylelamine; PS, phosphatidyl-serine; PI, phosphatidylinositol; PC, phosphatidylcholine. B: quantification ofcardiolipin from A. *P 0.05 vs. SCR; n 3 separate experiments.

Fig. 3. Tafazzin knockdown decreases intracellular ATP. NVMs were treated,harvested, and assayed for ATP as described in METHODS. ATP content wasexpressed as relative light units per microgram protein. Data represent means SE from 4 separate experiments. *P 0.05 vs. SCR.

H212 TAFAZZIN AND CARDIOMYOCYTE HYPERTROPHY



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creased. The concentration of mitochondrial DNA is propor-tional to the number of mitochondria per cell (37). An en-

hanced mitochondrial biogenesis was further confirmed by theincreased ND1 gene content as assayed by real-time PCR (Fig.4D). These data suggest that decreased mitochondrial ATPproduction activates AMPK, which in turn enhances mitochon-drial biogenesis.

Tafazzin knockdown causes cardiac myocyte hypertrophy.We next studied the effect of tafazzin knockdown on NVMhypertrophy. We found that tafazzin knockdown significantlyincreased NVM size compared with the SCR control (Fig. 5, Aand B). Protein synthesis as represented by [3H]leucine incor-poration was also significantly increased (Fig. 5C).

We also investigated whether tafazzin knockdown inducesthe hypertrophic marker gene, BNP expression. As shown in

Fig. 6, A and B, tafazzin knockdown significantly increased theexpression of BNP as determined by semiquantitative RT-PCR. The activation of BNP gene transcription was assayed bymeasuring a hBNP promoter coupled to a luciferase reportergene. Tafazzin knockdown significantly increased BNP pro-moter activity compared with the scrambled virus control (Fig.6C). Thus tafazzin knockdown induced hypertrophy in NVMs.

DISCUSSION

Ablation or decreased expression of tafazzin has been shownto reduce cardiolipin in several types of cells and modelsystems (18, 43, 44, 47, 48, 53). Our current finding thattafazzin knockdown decreases cardiolipin in NVMs is consis-

tent with previous cell studies. Cardiolipin deficiency due totafazzin knockdown resulted in an impaired mitochondrial

function (seen as reduced ATP production) as observed inyeast (34), as well as the energy depletion seen in patients withBarth syndrome.

The depletion of ATP in cardiac myocytes has severaldetrimental effects, including contractile dysfunction, hyper-trophy, and cell death. Acutely, ATP depletion switches on theATP-producing catabolic pathways such as fatty acid oxidationand glycolysis and switches off the ATP-consuming anabolicpathways such as lipogenesis and glyconeogenesis (41). Onelong-term response to ATP depletion is enhanced mitochon-drial biogenesis, which is mediated by the activation of thefuel gauge AMPK (19). Jager et al. (29) reported that AMPKdirectly phosphorylates and activates PGC-1, the key regula-

tor of mitochondrial biogenesis. During strength and endurancetraining, more ATP is consumed, and this coincides withincreased mitochondrial volume in the muscle (40). Our datashowing that tafazzin knockdown increased mitochondrial den-sity in NVMs support the concept that ATP depletion enhancesmitochondrial biogenesis. This is also consistent with theincreased number of mitochondria found in the lymphocytes ofpatients with Barth syndrome (55).

Our data show that tafazzin knockdown causes NVM hy-pertrophy as indicated by increased cell surface area, proteinsynthesis, and expression of the hypertrophic marker geneBNP. Several factors may be involved in the cardiomyocytehypertrophy induced by tafazzin knockdown. First, increased

Fig. 4. Tafazzin knockdown activates AMP-activated protein kinase (AMPK) and increasesmitochondrial density in NVMs. A: NVMs weretreated and assayed for phospho-AMPK (p-AMPK) as described in METHODS. p-AMPK wascorrected for total AMPK and expressed as foldincrease vs. SCR control (arbitrarily set at 1),which was NVMs treated with the scrambledvirus. #P 0.01 vs. SCR; n 4 separate exper-iments. B: images taken from 3 separate experi-ments show that tafazzin knockdown increasedmitochondrial density in NVMs. C: quantifica-tion of red fluorescence from B, expressed asintensity per cell. #P 0.01 vs. SCR; n 3separate experiments. D: mitochondrial DNAcontent. NADH dehydrogenase 1 (ND1) gene

content was determined by real-time PCR asdescribed in METHODS. #P 0.01 vs. SCR; n5 separate experiments.




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mitochondrial density contributes to increased NVM volumebecause cardiac myocytes have a very high mitochondrialdensity (16), occupying about 40% of the cytoplasmic volumein mice (13). Previous studies also showed that mitochondrial

volume and density increase in proportion to increased cellvolume (26, 28, 46). Second, the depletion of ATP maydirectly cause cardiomyocyte hypertrophy, since ATP defi-ciency reduces contractile efficiency and this leads to compen-

satory hypertrophy. Finally, as we observed in yeast (9),impaired mitochondrial function due to a deletion of tafazzincauses increased oxidative stress that contributes to cardio-myocyte hypertrophy (1, 6, 9, 10, 27, 52). Without conducting

pressure loading, we could not differentiate between eccentric(increased length-to-width ratio) and concentric hypertrophy(increased cross-sectional area); however, eccentric cardio-myocyte hypertrophy is characteristic of DCM based on pre-

Fig. 5. Tafazzin knockdown causes hypertro-phy of NVMs. A: images of tafazzin knock-down NVMs. NVMs were cultured on cov-erslips, transduced with the shRNA adenovi-rus, and treated with an immunofluorescentstain as described in METHODS. Cell surfacearea is shown in B. #P 0.01 vs. SCR; n 3 separate experiments. C: tafazzin knock-down increases protein synthesis by NVMs.NVMs were transduced with the shRNA ad-enovirus and metabolically labeled with[3H]leucine as described in METHODS. Proteinsynthesis is represented by counts per minute(CPM) of [3H]leucine incorporation. Datarepresent means SE from 4 separate ex-periments. *P 0.05 vs. SCR.

Fig. 6. Tafazzin knockdown increases expression of the hypertrophic marker brain natriuretic peptide (BNP). A: representative images obtained bysemiquantitative RT-PCR. Lane 1, molecular marker (lane reordered); lane 2, scrambled shRNA virus; lane 3, tafazzin shRNA virus. The bands from A werescanned, quantified, and summarized in B. Data represent means SE from 3 separate experiments. *P 0.05 vs. SCR. C: tafazzin knockdown activates theBNP promoter. NVMs were transfected with a luciferase reporter gene controlled by a human BNP promoter and infected with shRNA adenovirus; BNP promoteractivity is represented by luciferase activity, expressed as fold increase vs. SCR control. Data represent means SE from 4 separate experiments. *P 0.05vs. SCR.




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vious studies (14, 15, 20). The fact that tafazzin knockdowninduces hypertrophy of NVMs is relevant in vivo becausecardiac myocytes occupy 75% of the myocardial structuralspace even though they constitute only one third of the cellpopulation (50). Undoubtedly, cardiomyocyte hypertrophy is amajor factor in cardiomyopathy.

In summary, our data indicate that tafazzin knockdown

causes hypertrophy in NVMs. Decreased tafazzin expressionlowers cardiolipin in NVMs, and this in turn blunts ATPproduction by the mitochondria. The decrease in ATP activatesAMPK and enhances mitochondrial biogenesis, which contrib-utes to the increased volume of NVMs. We believe our find-ings establish a link between tafazzin mutation and cardio-myocyte hypertrophy.

ACKNOWLEDGMENTS

I thank Drs. Pamela Harding, Margot LaPointe, Xiao-Ping Yang, andMiriam Greenberg for providing reagents and helpful discussions and ZizhengHou for excellent technical assistance.

GRANTS

This work was supported by a grant from the Barth Syndrome Foundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author.

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