Tafazzin knockdown causes hypertrophy of neonatal ventricular myocytes

Quan He
Am J Physiol Heart Circ Physiol 299:H210-H216, 2010. doi:10.1152/ajpheart.00098.2010 You might find this additional info useful... Supplemental material for this article can be found at: http://ajpheart.physiology.org/content/suppl/2010/05/12/ajpheart.00098.2010.DC1.html This article cites 56 articles, 33 of which can be accessed free at: http://ajpheart.physiology.org/content/299/1/H210.full.html#ref-list-1 Updated information and services including high resolution figures, can be found at: http://ajpheart.physiology.org/content/299/1/H210.full.html Additional material and information about AJP - Heart and Circulatory Physiology can be found at: http://www.the-aps.org/publications/ajpheart
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Am J Physiol Heart Circ Physiol 299: H210H216, 2010. First published March 26, 2010; doi:10.1152/ajpheart.00098.2010.

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 nal form 23 March 2010

He Q. Tafazzin knockdown causes hypertrophy of neonatal ventricular myocytes. Am J Physiol Heart Circ Physiol 299: H210H216, 2010. First published March 26, 2010; doi:10.1152/ajpheart.00098.2010.Mutation of the mitochondrial protein tafazzin causes dilated cardiomyopathy in Barth syndrome. We employed an adenovirus as a vector to transfer tafazzin small hairpin RNA (shRNA) into neonatal ventricular myocytes (NVMs) to investigate the effects of tafazzin knockdown. The tafazzin shRNA adenovirus consistently knocked down tafazzin mRNA and lowered cardiolipin while signicantly decreasing the production of ATP by the mitochondria. The phosphorylation of AMP-activated protein kinase and mitochondrial density were both increased in tafazzin knockdown NVMs compared with scrambled shRNA controls. When we tested whether tafazzin knockdown causes hypertrophy in vitro, we found that the surface area of NVMs infected with tafazzin shRNA adenovirus was signicantly increased, as were the protein synthesis and expression of the hypertrophic marker gene, brain natriuretic peptide. Taken together, our data support the concept that a decreased tafazzin expression causes cardiomyocyte hypertrophy in vitro. cardiolipin; adenosine 5=-triphosphate; dilated cardiomyopathy
TAFAZZIN IS A MITOCHONDRIAL

length-to-width ratio) and an energy depletion are characteristic of DCM (15, 20, 21, 36). A deletion of tafazzin in yeast causes mitochondrial dysfunction, including changes in energy transformation and osmotic properties of the mitochondria (18, 34). A mutation of tafazzin in Drosophila causes a pathological dysfunction of mitochondria and motor weakness (53). Tafazzin knockdown in zebrash led to bradycardia and retarded cardiac development (32). The decreased expression of tafazzin has been shown in spontaneously hypertensive failing hearts (42). However, we do not know how tafazzin knockdown affects mammalian cardiomyocytes. We here present evidence that tafazzin knockdown results in ATP deciency and hypertrophy in cultured neonatal cardiomyocytes.
METHODS

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protein encoded by the G4.5 gene in humans (7). It is a phospholipid transacylase (54), and its mutation results in decreased cardiolipin (18, 47 49, 53), a phospholipid predominantly present in the inner membrane of the mitochondria. Cardiolipin is initially synthesized as a premature form, which is deacylated by a phospholipase to generate monolysocardiolipin, and this lipid is nally reacylated by tafazzin to form the mature fully functional cardiolipin (17, 54). Cardiolipin is required for 1) optimal activity of respiratory chain complexes and ADP-ATP translocase (31), which are involved in ATP and reactive oxygen species production; 2) cytochrome c attachment within the mitochondrial intermembrane space (39), which is associated with mitochondria-initiated cell death; 3) maintaining inner membrane uidity and osmotic stability (11), which are associated with the opening of mitochondrial permeability transition pores and mitochondria-gated cell death; and 4) mitochondrial protein import (30), which is associated with mitochondrial biogenesis. Tafazzin mutation causes Barth syndrome, a rare X-linked genetic disorder characterized by dilated cardiomyopathy (DCM) with or without hypertrophy, skeletal myopathy, neutropenia, growth retardation, and 3-methylglutaconic aciduria (4, 5, 45). End-stage heart failure resulting from DCM is a major cause of death among these patients during infancy and early childhood. DCM, the most common type of cardiomyopathy, is characterized by ventricular chamber enlargement and systolic dysfunction, commonly resulting in congestive heart failure (35). An eccentric cardiomyocyte hypertrophy (increased

Address for reprint requests and other correspondence: Q. He, Hypertension and Vascular Research Div., Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202-2689 (e-mail: qhe1@hfhs.org). H210

All animal experiments were approved by the Henry Ford Health System Institutional Animal Care and Use Committee. Supplies and chemicals. Phosphatase and proteinase inhibitor cocktail tablets (PhosSTOP and Complete Mini) were obtained from Roche Applied Science (Indianapolis, IN). A pSilencer adeno 1.0CMV system was purchased from Ambion (Austin, TX). Primary antibodies against phospho-AMP-activated protein kinase (phosphoAMPK) (Thr172) and AMPK and an horseradish peroxidase-conjugated secondary antibody against rabbit IgG were obtained from Cell Signaling Technology (Boston, MA). -Actin antibody and FITCconjugated anti-goat IgG were purchased from Santa Cruz (Santa Cruz, CA). Coomassie protein assay and SuperSignal West Pico chemiluminescent substrate kits and Restore Plus Western blot stripping buffer were purchased from Thermo Scientic (Rockford, IL). MitoTracker Red, Taq DNA polymerase kits, DMEM medium and cell culture supplements, precast Tris-glycine polyacrylamide gels, and polyvinylidene uoride membranes were obtained from Invitrogen (San Diego, CA). Laminin-coated coverslips were purchased from BD Sciences (San Jose, CA). ATP bioluminescent assay kits and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (St. Louis, MO). [32P]phosphate and [3H]leucine were purchased from PerkinElmer (Waltham, MA); Whatman TLC plates (LK5D silica gel, 150 ) were from VWR (Batavia, IL); RNeasy brous tissue mini kits, QIAamp DNA Micro Kit, random primers, and Omniscript reverse transcriptase were from Qiagen (Valencia, CA); and SYBR green dye was from SA Biosciences (Frederick, MD). Custom primers were designed by TIB MolBiol (Adelphia, NJ). The HEK293 cell line was obtained from ATCC (Manassas, VA), luciferase assay reagents were from Promega (Madison, WI), and other routine supplies and chemicals were from Fisher and Sigma. Construction of tafazzin small hairpin RNA adenovirus. The small hairpin sequences recognize tafazzin coding regions 5=-CAGCTGTGGAGATGCGGAA-3= Taz1 and 5=-AGAGGAATTCCAGCGGCT-3= Taz2 with restriction endonuclease XhoI and SpeI sites added at each end to facilitate subcloning. The small hairpin RNA (shRNA) oligonucleotides and scrambled sequence 5=-CAGTCCAGCTAGCTCTACT-3= were synthesized by TIB MolBiol. Equal amounts of the upper and lower oligonucleotide were annealed by boiling and cooled to make the double-stranded shRNA. This was subcloned into a shuttle vector (pSilencer adeno 1.0-CMV), and the insertion was conrmed by sequencing. The virus DNA containing the insertion was packaged inside
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HEK293 cells. The adenovirus was expanded and the virus concentration (expressed as viral particles per milliliter) was estimated by spectrophotometry following Ambions protocol. Neonatal cardiac myocyte culture. Neonatal ventricular myocytes (NVMs) were isolated from 1- to 2-day-old Sprague-Dawley rats from Charles River as we described previously (24). They were placed on 6-well (1 million per well) or 12-well plates (0.5 million per well) in DMEM containing 10% FBS and incubated for 40 h. The medium was changed to serum-, glucose-, and pvruvate-free medium supplemented with insulin, transferrin, and selenium; glutamine (2 mM); penicillinstreptomycin; and bromodeoxyuridine (0.1 mM). The adenovirus was added, and samples were incubated for 48 h. When the cell size was measured, 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 from NVMs was performed as described previously (25). RNA (2 g) was reverse-transcribed into cDNA in 20 l reaction buffer containing 1 unit reverse transcriptase, 2 g random primer, 0.5 mM dNTP, and 10 units RNasin at 37C for 1 h. The reverse transcription mixture was denatured at 95C for 10 min. For semiquantitative PCR, tafazzin was amplied with 0.5 M primers (upper, 5=-AGCACGGTGATTTTCTGTCC-3=; and lower, 5=-GGCAAATGTGTGCCTGTATG-3=) in 20 l reaction volume containing 0.25 mM dNTP and 1 unit Taq DNA polymerase for 22 cycles. The PCR products were run on 2% agarose gel, and DNA bands were visualized by ethidium bromide staining. The gels were imaged by transillumination and DNA bands quantied with Density software corrected by GAPDH (upper, 5=-ATTCAACGGCACAGTCAAGG-3=; and lower: 5=-TGGATGCAGGGATGATGTTC-3=), which was amplied for 18 cycles. Tafazzin mRNA levels were expressed as a percentage of control. For real-time PCR, 2 l of the products of the reverse transcription reaction were amplied using SYBR dye (SA Biosciences) along with the same primers (including tafazzin and GAPDH as normalizer) in a Roche LightCycler (v. 2) as we described previously (20). Tafazzin mRNA levels were determined using the Ct method as described by Winer et al. (51) and expressed as a percentage of control. Phospholipid metabolic labeling. NVMs were placed on six-well plates (1 million cells per well). [32P]phosphate (1 Ci/well) was added 24 h after the adenovirus, and the samples were incubated for 24 h. The cells were harvested for phospholipids and analyzed by one-dimensional TLC as we described previously (22). After exposure to X-ray lm, the cardiolipin bands were scanned, quantied, and expressed as a percentage of control (NVMs infected with a scrambled shRNA adenovirus). ATP assay. Cells were lysed in 0.2% SDS, and ATP was measured with a kit from Sigma. ATP content was corrected by total protein, which was determined using a kit from Thermo Fisher with BSA as a standard. ATP content was expressed as relative light units per microgram protein. Western blot analysis. Western blot analysis was performed as we described previously (23) using 50 g total cell extract protein. Protein concentration was determined with the Coomassie protein assay kit using BSA as the standard. Mitochondrial staining. At the end of the experiment, the cells were incubated with 200 nM MitoTracker red in the dark for 20 min. They were then washed with PBS and xed in 37% formaldehyde for 30 min at room temperature in the dark. The cells were washed with PBS and counterstained with DAPI for 3 min and then washed and mounted. Five images were obtained from each eld using uorescence microscopy with red and blue lters and merged with SPOT software. The red uorescence intensity representing mitochondrial density was quantied using MicroSuite software and expressed as uorescent intensity per cell. Mitochondrial DNA assay. Total DNA including mitochondrial DNA was isolated from NVMs using QIAamp DNA Micro Kit from Qiagen. Mitochondrial NADH dehydrogenase 1 (ND1) gene content was determined by real-time PCR, as described in Semiquantitative
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and real-time RT-PCR, with10 ng total DNA as the template using primers upper 5=-ATGGCCTTCCTCACCCTAGT-3= and lower 5=AGAGGGCGTATGGGTTCTTT-3=. ND1 was normalized with genomic gene GAPDH using the same set of primers described in Semiquantitative and real-time RT-PCR. Cardiac myocyte surface area. NVMs were plated onto laminincoated coverslips in six-well plates at a density of 0.25 million cells per well. After we treated them with the adenovirus, they were immunostained with a -actin antibody and visualized with an FITCconjugated secondary antibody. The nuclei were counterstained by DAPI. Five images of each eld were captured with uorescence microscopy using DAPI and FITC lters and merged with SPOT software. The surface area (expressed as m2) was measured with MicroSuite software. [3H]leucine incorporation assay. The rate of protein synthesis by NVMs was estimated by [3H]leucine incorporation as described by Harding et al. (21). NVMs were placed on six-well plates at a density of one million cells per well. After a 40-h incubation in DMEM containing 10% FBS, the medium was changed to serum-, glucose-, and pvruvate-free DMEM containing 1 Ci [3H]leucine, and the adenovirus was added. After 48 h, the cells were harvested for trichloroacetic acid precipitates, which were counted for 3H activity (counts/min of [3H]leucine incorporation) in a scintillation counter. 3 H activity of NVMs infected with a tafazzin shRNA adenovirus represents protein synthesis, expressed as a percentage of control (NVMs infected with a scrambled adenovirus). Transfection and luciferase assay. NVMs were transfected by electroporation using a cuvette with a 2-mm gap (BTX, San Diego, CA) in 0.4 ml PBS buffer containing 0.1% glucose and 1 g 1818hBNPluc plasmid DNA per million cells. The cells were placed on 12-well plates and maintained in DMEM containing 10% FBS for 40 h. The medium was then changed to serum-, glucose-, and pvruvate-free DMEM; the shRNA adenovirus (100 vp/cell) was added; and the samples were maintained for 48 h. Finally, the cells were harvested for luciferase activity (23), which represents human brain natriuretic peptide (hBNP) promoter activity and is expressed as fold increase vs. control (cells infected with a scrambled shRNA virus). The 1818hBNPluciferase construct (33) was graciously provided 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-way ANOVA using the Student-Newman-Keuls method for pairwise multiple comparisons. A value of P 0.05 was considered signicant.
RESULTS

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Construction of tafazzin shRNA adenovirus. To test the efciency of our tafazzin shRNA adenovirus in knocking down tafazzin mRNA, we used doses ranging from 0.1 to 100 viral particles per myocyte (vp/cell) of two different constructs (Taz1 and Taz2). We found that 100 vp/cell gave the best results without affecting cell growth (Fig. 1A). Some NVMs were killed and the others displayed cell abnormal morphology when treated with 800 vp/cell shRNA adenovirus (data not shown). Whereas both Taz1 and Taz2 signicantly knocked down tafazzin mRNA, Taz1 reduced the expression by 70% compared with only 28% for Taz2 (Fig. 1B). The internal control gene GAPDH was not affected by the shRNA adenovirus. Based on our ndings, we used a dose of 100 vp/cell Taz1 shRNA adenovirus for all experiments. Tafazzin knockdown decreases cardiolipin. Tafazzin mutation decreases cardiolipin content (18, 43, 48). We then tested whether tafazzin knockdown in NVMs affects cardiolipin content and found that tafazzin knockdown signicantly decreased cardiolipin in NVMs (Fig. 2) compared with scrambled con299 JULY 2010

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Fig. 1. Tafazzin mRNA is knocked down by the tafazzin small hairpin (shRNA) adenovirus. A: representative images obtained by semiquantitative RT-PCR. Tafazzin mRNA was knocked down by the tafazzin shRNA adenovirus in a dose-dependent manner. TAZ, amplied 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 represent NVMs treated with the Taz1 shRNA adenovirus; and lanes 3, 6, 9, and 12 represent NVMs treated with the Taz2 shRNA adenovirus. MM, molecular marker; vp, viral particles. B: quantication of A. Tafazzin mRNA was expressed as a percentage of cells infected with a scrambled shRNA adenovirus (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: TLC image representing 3 separate experiments. NVMs were infected with the shRNA adenovirus and metabolically labeled with [32P]phosphate. Phospholipids were isolated and analyzed on TLC plates as described in METHODS. Taz, tafazzin shRNA; CL, cardiolipin; PE, phosphatidylelamine; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine. B: quantication of cardiolipin from A. *P 0.05 vs. SCR; n 3 separate experiments.

trol, as determined by metabolic labeling and thin-layer chromatography analysis. In contrast, other phospholipids were not affected by tafazzin knockdown. Thus tafazzin knockdown in NVMs mimics cardiolipin deciency in Barth syndrome. Tafazzin knockdown decreases intracellular ATP. The primary function of the mitochondria is the production of ATP, which is critically important for maintaining the pumping function of the heart. ATP depletion has been associated with cardiomyopathy and heart failure (2, 3, 8, 36). Barth syndrome, which is characterized by DCM, causes metabolic decompensation and energy deciency (12, 34, 56). We infected NVMs with the tafazzin shRNA adenovirus for 48 h in a medium containing glutamine as the sole energy molecule and found that intracellular ATP was signicantly reduced compared with cells infected with a scrambled shRNA adenovirus (Fig. 3). This ATP must have come from the mitochondria, because glutamine must be metabolized by oxidative phosphorylation within the mitochondria to generate ATP (38); thus tafazzin knockdown decreases mitochondrial ATP production. Tafazzin knockdown activates AMPK and increases mitochondrial density. AMPK has been postulated as the fuel gauge of the cell and is regulated by intracellular ATP (19). It is activated by a decrease in the ATP-to-AMP ratio. One of the
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long-term responses to AMPK activation is an enhanced mitochondrial biogenesis (29). We investigated the effects of tafazzin on AMPK activation and mitochondrial biogenesis and found that tafazzin knockdown dramatically increased AMPK phosphorylation (Fig. 4A) compared with a scrambled virus control. As visualized by MitoTracker red (Fig. 4, B and C), the mitochondrial density (red stains) around the nucleus, which was stained in blue by DAPI, was signicantly in-

Fig. 3. Tafazzin knockdown decreases intracellular ATP. NVMs were treated, harvested, and assayed for ATP as described in METHODS. ATP content was expressed as relative light units per microgram protein. Data represent means SE from 4 separate experiments. *P 0.05 vs. SCR.
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Fig. 4. Tafazzin knockdown activates AMPactivated protein kinase (AMPK) and increases mitochondrial density in NVMs. A: NVMs were treated and assayed for phospho-AMPK (pAMPK) as described in METHODS. p-AMPK was corrected for total AMPK and expressed as fold increase vs. SCR control (arbitrarily set at 1), which was NVMs treated with the scrambled virus. #P 0.01 vs. SCR; n 4 separate experiments. B: images taken from 3 separate experiments show that tafazzin knockdown increased mitochondrial density in NVMs. C: quantication of red uorescence from B, expressed as intensity per cell. #P 0.01 vs. SCR; n 3 separate experiments. D: mitochondrial DNA content. NADH dehydrogenase 1 (ND1) gene content was determined by real-time PCR as described in METHODS. #P 0.01 vs. SCR; n 5 separate experiments.

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creased. The concentration of mitochondrial DNA is proportional to the number of mitochondria per cell (37). An enhanced mitochondrial biogenesis was further conrmed by the increased ND1 gene content as assayed by real-time PCR (Fig. 4D). These data suggest that decreased mitochondrial ATP production activates AMPK, which in turn enhances mitochondrial biogenesis. Tafazzin knockdown causes cardiac myocyte hypertrophy. We next studied the effect of tafazzin knockdown on NVM hypertrophy. We found that tafazzin knockdown signicantly increased NVM size compared with the SCR control (Fig. 5, A and B). Protein synthesis as represented by [3H]leucine incorporation was also signicantly increased (Fig. 5C). We also investigated whether tafazzin knockdown induces the hypertrophic marker gene, BNP expression. As shown in Fig. 6, A and B, tafazzin knockdown signicantly increased the expression of BNP as determined by semiquantitative RTPCR. The activation of BNP gene transcription was assayed by measuring a hBNP promoter coupled to a luciferase reporter gene. Tafazzin knockdown signicantly increased BNP promoter 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 shown to reduce cardiolipin in several types of cells and model systems (18, 43, 44, 47, 48, 53). Our current nding that tafazzin knockdown decreases cardiolipin in NVMs is consisAJP-Heart Circ Physiol VOL

tent with previous cell studies. Cardiolipin deciency due to tafazzin knockdown resulted in an impaired mitochondrial function (seen as reduced ATP production) as observed in yeast (34), as well as the energy depletion seen in patients with Barth syndrome. The depletion of ATP in cardiac myocytes has several detrimental effects, including contractile dysfunction, hypertrophy, and cell death. Acutely, ATP depletion switches on the ATP-producing catabolic pathways such as fatty acid oxidation and glycolysis and switches off the ATP-consuming anabolic pathways such as lipogenesis and glyconeogenesis (41). One long-term response to ATP depletion is enhanced mitochondrial biogenesis, which is mediated by the activation of the fuel gauge AMPK (19). Jager et al. (29) reported that AMPK directly phosphorylates and activates PGC-1 , the key regulator of mitochondrial biogenesis. During strength and endurance training, more ATP is consumed, and this coincides with increased mitochondrial volume in the muscle (40). Our data showing that tafazzin knockdown increased mitochondrial density in NVMs support the concept that ATP depletion enhances mitochondrial biogenesis. This is also consistent with the increased number of mitochondria found in the lymphocytes of patients with Barth syndrome (55). Our data show that tafazzin knockdown causes NVM hypertrophy as indicated by increased cell surface area, protein synthesis, and expression of the hypertrophic marker gene BNP. Several factors may be involved in the cardiomyocyte hypertrophy induced by tafazzin knockdown. First, increased
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Fig. 5. Tafazzin knockdown causes hypertrophy of NVMs. A: images of tafazzin knockdown NVMs. NVMs were cultured on coverslips, transduced with the shRNA adenovirus, and treated with an immunouorescent stain as described in METHODS. Cell surface area is shown in B. #P 0.01 vs. SCR; n 3 separate experiments. C: tafazzin knockdown increases protein synthesis by NVMs. NVMs were transduced with the shRNA adenovirus and metabolically labeled with [3H]leucine as described in METHODS. Protein synthesis is represented by counts per minute (CPM) of [3H]leucine incorporation. Data represent means SE from 4 separate experiments. *P 0.05 vs. SCR.

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mitochondrial density contributes to increased NVM volume because cardiac myocytes have a very high mitochondrial density (16), occupying about 40% of the cytoplasmic volume in mice (13). Previous studies also showed that mitochondrial volume and density increase in proportion to increased cell volume (26, 28, 46). Second, the depletion of ATP may directly cause cardiomyocyte hypertrophy, since ATP deciency reduces contractile efciency and this leads to compen-

satory hypertrophy. Finally, as we observed in yeast (9), impaired mitochondrial function due to a deletion of tafazzin causes increased oxidative stress that contributes to cardiomyocyte 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 cardiomyocyte hypertrophy is characteristic of DCM based on pre-

Fig. 6. Tafazzin knockdown increases expression of the hypertrophic marker brain natriuretic peptide (BNP). A: representative images obtained by semiquantitative RT-PCR. Lane 1, molecular marker (lane reordered); lane 2, scrambled shRNA virus; lane 3, tafazzin shRNA virus. The bands from A were scanned, quantied, and summarized in B. Data represent means SE from 3 separate experiments. *P 0.05 vs. SCR. C: tafazzin knockdown activates the BNP promoter. NVMs were transfected with a luciferase reporter gene controlled by a human BNP promoter and infected with shRNA adenovirus; BNP promoter activity is represented by luciferase activity, expressed as fold increase vs. SCR control. Data represent means SE from 4 separate experiments. *P 0.05 vs. SCR. AJP-Heart Circ Physiol VOL
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vious studies (14, 15, 20). The fact that tafazzin knockdown induces hypertrophy of NVMs is relevant in vivo because cardiac myocytes occupy 75% of the myocardial structural space even though they constitute only one third of the cell population (50). Undoubtedly, cardiomyocyte hypertrophy is a major factor in cardiomyopathy. In summary, our data indicate that tafazzin knockdown causes hypertrophy in NVMs. Decreased tafazzin expression lowers cardiolipin in NVMs, and this in turn blunts ATP production by the mitochondria. The decrease in ATP activates AMPK and enhances mitochondrial biogenesis, which contributes to the increased volume of NVMs. We believe our ndings establish a link between tafazzin mutation and cardiomyocyte hypertrophy.
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