1
Online Data Supplements
Detailed Methods
Neonatal rat cardiac myocyte culture
Neonatal Wistar rats (1 to 2 days old) were anesthetized with ethyl ether, and
primary neonatal rat cardiac ventricular myocyte cultures were prepared as previously
described1 with minor modifications. Using this method, we routinely obtained cultures
containing more than 95% cardiac myocytes, as determined by immunohistochemical
staining with a monoclonal antibody against sarcomeric -actin (Sigma-Aldrich, Inc., St.
Louis, MO).
Subcellular fractionation and western blot analysis for TFAM and TFB2M
Mitochondria-rich subcellular fraction was isolated from neonatal rat cardiac
myocytes by the mitochondria isolation kit (MITOISO2, Sigma-Aldrich, Inc.). Briefly,
cultured cardiac myocytes were trypsinized and collected from culture dishes. The cell
pellet was lysed and serially centrifuged to isolate mitochondrial proteins. Nuclear
proteins were also fractionated by the Nuclei EZ Prep Nuclei Isolation kit
(Sigma-Aldrich, Inc.). Purity of each fraction was determined by western blotting using
antibodies for each marker protein, namely, COX IV for mitochondria and Histone-H3
for nuclei. For whole-cell lysate, cells were lysed with ice-cold
radioimmunoprecipitation (RIPA) buffer. Western blot analysis for TFAM and TFB2M
2
was performed using rabbit polyclonal TFAM or TFB2M antibody. Western blot
analysis for SERCA2a and calsequestrin, a loading control of sarcoplasmic reticulum
protein, was performed using rabbit polyclonal SERCA2a antibody (Badrilla, Leeds,
UK) and rabbit polyclonal calsequestrin antibody (Affinity Bioreagents, Inc., Golden,
CO). Mitochondrial and nuclear protein fractions were electrophoresed on 12.5% SDS
polyacrylamide gels, and transferred to 0.20-mm nitrocellulose membranes. Specific
binding of antibodies was detected by enhanced chemiluminescence (GE Healthcare,
Tokyo, Japan).
TFAM and TFB2M overexpression and plasmids
Rat Tfam and Tfb2m expression vectors produced by RT-PCR using pcDNA 3.1
His vector. Each expression vector (1 g) was introduced into neonatal rat cardiac
myocytes using FuGENE 6 transfection reagent. In the experiments shown as Figures
4A and 4B, sequences of our TFAM- and TFB2M-overexpressing plasmids cover the
entire protein coding region, which preserves mitochondria-targeted signal sequences in
the N-terminal end of TFAM and TFB2M, as well as amino acids for the nuclear
localization of TFAM2. Therefore, even for episomal expression, protein transport to the
nucleus and mitochondria should not be significantly different from that of native
TFAM and TFB2M proteins. Cells were incubated for 24 h and then total RNA was
extracted using the Total RNA Extraction Kit (Promega Corp., Madison, WI) for
Northern blot or the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) for
quantitative RT-PCR.
RNA interference (RNAi) assay
3
Pre-designed siRNA duplexes composed of 21 nucleotides for rat Tfam and
Tfb2m were obtained from Qiagen; sequences are shown in Supplemental Table 1. A
scramble oligo-ribonucleotide duplex that is not homologous to any mammalian genes
was utilized as the control. Transfection of siRNA was accomplished with the HVJ
envelope vector, an inactivated viral genome-free HVJ particle (Ishihara Sangyo Kaisha,
Inc., Osaka, Japan) according to the manufacturer’s protocol. Preliminary experiments
using FITC-labeled siRNA revealed that approximately 95% of cells were transfected
by the HVJ envelope vector.
Northern blot analysis
Total RNA was extracted from rat heart and cultured rat cardiac myocytes.
For Northern blot analysis, random-primed labeled cDNAs for rat genes, Serca2a, Tfam,
Tfb2m, cytochrome b, mitochondrial ATPase 6/8, and Bnp, were used as probes for
hybridization.
Fluorescence correlation spectroscopy
For the determination of the TFAM and TFB2M binding sites in the
5’-regulatory region of the Serca2 gene, fluorescence correlation spectroscopy was
performed using the MF20 molecular interaction analytical system (Olympus, Tokyo,
Japan)3. Recombinant rat TFAM and TFB2M proteins were produced using the
pGEX-6P-1 vector (GE Healthcare, Buckinghamshire, England) or pCold TF DNA
vector (Takara Bio, Inc., Shiga, Japan) in E. coli and purified using Glutathione
Sepharose 4B beads (GE Healthcare) or Ni-NTA His-Bind resin (Merck KGaA,
Darmstadt, Germany), respectively, according to manufacturers’ protocols. For the rat
4
Serca2 5’-regulatory region, 30-bp double-stranded probes were produced as follows.
5’-Terminal carboxytetramethyl rhodamine (TAMRA)-labeled sense strand and
unlabeled antisense strand oligonucleotides were synthesized by Greiner Bio-One
(Frickenhausen, Germany). Double-stranded oligonucleotides were prepared by
annealing each oligonucleotide. Resting single-stranded oligonucleotides were
hydrolyzed by exonuclease I (New England Biolabs, Inc., Ipswich, MA), and
hydrolyzed nucleotides, free dyes and salts were removed using the Nucleotide
Removal Kit (Qiagen).
Fluorescence correlation spectroscopy was performed using the MF20
molecular interaction analytical system (Olympus, Tokyo, Japan). Recombinant TFAM
or TFB2M protein was incubated with TAMRA-labeled double-stranded DNA for 10
min at 25°C in 20 l binding buffer (10 mmol/L Tris-HCl [pH 7.5], 1.09 mol/L [10%]
glycerol, 50 mmol/L NaCl, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, and 0.5 mmol/L
dithiothreitol). Measurements of translational diffusion time were repeated 5 times per
sample, and all experiments were performed under identical conditions with a data
acquisition time of 10 seconds per measurement. Elongation of translational diffusion
time indicates the direct binding of TFAM or TFB2M protein with the Serca2 gene
probe. For the determination of the TFAM and TFB2M binding site in the 5’-regulatory
region of the Serca2 gene, mutations were introduced into the TAMRA-labeled probe
that encodes from -140 to -111 nt in this region (Serca2 -140 wild) as shown in Figure
4A. Because this region is guanine and cytosine (GC)-rich, every 3 nucleotides of GC
residues were replaced by 3 alanine residues.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication No. 85-23, revised
5
1996). All experimental protocols were approved by the Animal Care Committee of the
Gunma University Graduate School of Medicine.
Measurement of intracellular ATP concentration
ATP concentration in the adult rat heart muscle and neonatal rat cardiac
myocytes were measured by ATP bioluminescence assay kit HS II (Roche Applied
Science, Manheim, Germany). Briefly, adult rat heart muscle tissue was homogenized
by glass homogenizer in the 25 mM Tricine buffer (pH 7.75) and then lysed by
detergent. Neonatal rat cardiac myocytes were directly lysed by detergent. The ATP
dependency of the light emitting reaction of D-luciferin was used to measure the ATP
content.
Rat myocardial infarction model
Experiments were carried out in adult male Wister rats (0.25 kg) obtained from
Charles River Laboratories Japan, Inc. (Yokohama, Japan). Ligation of the left anterior
descending coronary artery was performed to 21 rats. Eight sham-operated rats were
prepared in the same manner but did not undergo coronary artery ligation. The hearts
were excised 5 months after induction of myocardial infarction and sham operation, and
total RNA was isolated using the Total RNA Extraction Kit (Promega Corp.).
Five months after coronary occlusion, hemodynamic parameters were
measured using a pressure-volume (PV) catheter. Rats were anesthetized with 108
mmol/L air (2% in air) isoflurane, and a microtip PV catheter (SPR-838, Millar
Instruments, Houston, TX) was introduced into the left ventricle through the right
carotid artery. Absolute volume was calibrated, and PV data were measured at a steady
6
state and during transient reduction of venous return as described previously4.
Hemodynamic parameters were calculated by the PVAN ver 3.1 algorism (Millar
Instruments). The measurement of Emax is performed by altering preload through
inferior vena caval occlusion. The slope of line which connects end-systolic
pressure-volume points under different preload was defined as Emax. Tau was
calculated from the decline in the left ventricular pressure trace according to the
equation Pt=P0e(-t/) where Pt is left ventricular pressure at any time (t), and P0 is left
ventricular pressure at time 0.
Luciferase assay
Serial deletion constructs of the 5’ upstream region of the rat Serca2 gene
(nucleotides -1829, -1351, -850, -479, -327, -171 and -88 to +123 bp relative to the
transcription initiation site) were used to determine the responsive site of TFAM and
TFB2M. Each construct (0.5 g each) was transfected with Tfam and/or Tfb2m
expression vector (1 g each) into neonatal rat myocytes using FuGENE 6 reagent.
Cells were incubated for 24 h and then harvested using the Luciferase Assay System
(Promega Corp.).
For RNAi experiments, 21-nt siRNA duplexes against Tfam and Tfb2m gene
(200 pmol each) were simultaneously incorporated with Serca2 promoter-luciferase
vector (0.5 g) into cardiac myocytes using Lipofectamine 2000 reagent (Invitrogen
Co.).
Mutations in the -171 to +123 nt construct of the rat Serca2 5’-regulatory
region were introduced using the QuickChange Site-Directed Mutagenesis Kit (Agilent
Technologies, Santa Clara, CA). The fidelity of introduced mutations was confirmed by
7
nucleotide sequencing.
mRNA expression microarray
The mRNA expression profiles in rat cardiac myocytes after Tfam and Tfb2m
gene expression at 24 h were compared with those after empty vector expression, using
the Affymetrix GeneChip expression analysis system (Rat genome 230 2.0 array;
Affymetrix, Santa Clara, CA).
Chromatin immunoprecipitation (ChIP) assay
Binding of TFAM and TFB2M to the 5’-regulatory region of the Serca2 gene
and the D-loop region of mitochondrial gene was determined by chromatin
immunoprecipitation (ChIP) assay. ChIP assay was performed using rabbit polyclonal
anti-TFAM, goat polyclonal anti-TFB2M, anti-acetylated histone H3 in the 9th and 14th
lysine residues (Millipore, Billerica, MA), anti-methylated histone H3 in the 4th lysine
residue (Abcam Plc.) and anti-histone H3 (Abcam Plc.) antibodies according to the
Chromatin Immunoprecipitation Assay Kit (Millipore) protocol. Briefly, myocytes were
fixed with 330 mmol/L (1%) formaldehyde for 10 min at 25°C. Cells were washed with
ice cold PBS, and cell pellets were resuspended with SDS lysis buffer (34.6 mmol/L
[1%] SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl [pH 8.1]). Soluble chromatin was
prepared by sonication, and incubated with antibodies and protein G agarose with
rotation at 4°C. Protein/DNA complexes were eluted, and crosslinks of the complexes
were reversed to free DNA. Precipitated DNA was purified using a spin column for
PCR. PCR primers were designed for the -479 to -1 region of the rat Serca2
5’-regulatory region. Information regarding forward and reverse primer sequences is
8
denoted in Supplemental Table 1.
9
Legend for Supplemental Figures
Supplemental Figure 1 Western blot analysis showing changes in the amount of
TFAM and TFB2M in the mitochondria and nuclei under oxidative stress and after
carvedilol treatment.
TFAM and TFB2M expression was quantified in the nuclear (A), mitochondrial (B) and
whole-cell (C) fractions of neonatal rat cardiac myocytes exposed to 40 mmol/L
hydrogen peroxide, 5 mmol/L carvedilol or both reagents for 24 h. Histone-H3 and
COX-IV are specific markers for nuclear and mitochondrial proteins. The amount of
TFAM, TFB2M and SERCA2a in the vehicle group in each fraction was designated as 1
to calculate the relative amount of these proteins in each experimental group.
Calsequestrin (CSQ) was used as a loading control for sarcoplasmic reticulum proteins.
Values in the bar graphs represent the means  SD of five independent measurements.
TFAM and TFB2M do not preferentially accumulate in either nuclei or mitochondria
under any experimental condition.
Supplemental Figure 2 Effect of overexpression and RNAi of Tfam and Tfb2m on
their mRNA expression levels.
A. Expression level of Tfam and Tfb2m mRNA after 24-h transfection of 1 g empty,
and Tfam and Tfb2m expression vectors in neonatal rat cardiac myocytes. Tfam and
Tfb2m mRNA levels in the empty vector group were designated as 1. Tfam and Tfb2m
mRNA levels were quantified by quantitative PCR only in their respective
overexpression group. Values represent the means  SD of five independent
measurements.
10
B. Northern blot showing the effect of two different constructs of Tfam and Tfb2m
siRNA. Neonatal rat cardiac myocytes were transfected for 24 h with 100 nmol/L
siRNAs designated in Supplemental Table 1.
C. Densitometric measurements of the Tfam and Tfb2m mRNA expression levels.
Expression levels are shown relative to Tfam and Tfb2m mRNA levels in the scramble
siRNA-transfected group, which were designated as 1. Values represent the means  SD
of five independent measurements.
Supplemental Figure 3 TFAM and TFB2M binding sites in the -1,829 to –1,350
region of the Serca2 gene determined by fluorescence correlation spectroscopy.
Changes in the translational diffusion time were measured after binding 5 mmol/L
recombinant TFAM (A) or TFB2M (B) protein to the TAMRA-labeled 30-bp
double-stranded oligonucleotide of the 5’-regulatory region of the Serca2 gene.
Nucleotide numbers in the figure indicate the boundary of 30-bp oligonucleotides.
Values represent the means  S.D. of five independent measurements. Statistical
significance was determined by comparing the TFAM(+) group with the TFAM(-) group
in each sequence.
Supplemental Figure 4 Effect of Tfam and Tfb2m overexpression on the ATP
content in rat neonatal cardiac myocytes.
The amount of ATP produced by cardiac myocytes after transfection of 1 g Tfam and
Tfb2m expression vectors (A) or 100 nmol/L Tfam siRNA and Tfb2m siRNA (B) was
determined. Values in the bar graphs represent the means  SD of five independent
measurements.
11
Supplemental Figure 5 Correlation of Tfam, Tfb2m, cytochrome b and
mitochondrial ATPase 6/8 mRNA levels in the failing rat heart at 5 months after
myocardial infarction.
Myocardial infarction and sham operation was introduced into 21 and 8 rats,
respectively.
A, B. Correlation between Tfam mRNA levels and cytochrome b (A) and mitochondrial
ATPase 6/8 (B) mRNA levels, determined by linear regression analysis.
C, D. Correlation between Tfb2m mRNA levels and cytochrome b (C) and
mitochondrial ATPase 6/8 (D) mRNA levels, determined by linear regression analysis.
Supplemental Figure 6 Relationship of SP1 transcription factor, TFAM and
TFB2M on Serca2 gene transcription.
A. Fluorescence correlation spectroscopy showing the binding site of SP1 transcription
factor. Changes in the translation diffusion time were determined after binding 200
nmol/L recombinant SP1 protein (Promega Corp.) to the TAMRA-labeled 30-bp
double-stranded oligonucleotide corresponding to the designated sequence of the
5’-regulatory region of the Serca2 gene. Values represent the means  S.D. of five
independent measurements. Statistical significance was determined by comparing the
SP1(+) group with the SP1(-) group in each sequence.
B. Effect of overexpression of TFAM, TFB2M and SP1 on Serca2 gene transcription
using the wild-type Serca2 gene reporter construct (Serca2 Luc wild) and constructs
with 3-nt mutations in the TFAM/TFB2M binding site (-122 to -114 nt region) of the
5’-regulatory region of the Serca2 gene (Serca2 Luc mutation -122, -119 and -116).
12
Details of the mutations in each construct are depicted in Figure 3D.
C. Effect of overexpression of TFAM and SP1 on Serca2 gene transcription. Empty,
TFAM-expressing, and SP1-expressing vectors were co-transfected with a Serca2 gene
reporter vector. *P < 0.05 vs. the empty vector group. Values represent the means  S.D.
of five independent experiments.
D. Fluorescence correlation spectroscopy on direct binding of TFAM and SP1 proteins.
TAMRA dye was introduced to TFAM protein using the Protein Labeling Kit (Olympus,
Tokyo, Japan). Recombinant SP1 protein did not increase in translation diffusion time,
suggesting that SP1 does not bind to TFAM. Values represent the means  S.D. of five
independent experiments.
Supplemental Figure 7 Northern blot showing the effect of enodothelin-1 (ET-1)
and norepinephrine (NE) on mRNA levels of Tfam, Tfb2m, Serca2a and Bnp.
Neonatal rat cardiac myocytes were treated with designated concentrations of ET-1 and
NE for 24 h.
Supplemental Figure 8 Scheme showing novel function of TFAM and TFB2M.
TFAM and TFB2M regulate the Serca2 gene as well as mtDNA.
13
References for supplemental data
1.
Yokoyama T, Sekiguchi K, Tanaka T, Tomaru K, Arai M, Suzuki T et al.
Angiotensin II and mechanical stretch induce production of tumor necrosis
factor in cardiac fibroblasts. Am J Physiol 1999;276:H1968-1976.
2.
Pastukh V, Shokolenko I, Wang B, Wilson G, Alexeyev M. Human
mitochondrial transcription factor A possesses multiple subcellular targeting
signals. FEBS J 2007;274:6488-6499.
3.
Kobayashi T, Okamoto N, Sawasaki T, Endo Y. Detection of protein-DNA
interactions in crude cellular extracts by fluorescence correlation spectroscopy.
Anal Biochem 2004;332:58-66.
4.
Pacher P, Mabley JG, Liaudet L, Evgenov OV, Marton A, Hasko G et al. Left
ventricular pressure-volume relationship in a rat model of advanced
aging-associated heart failure. Am J Physiol Heart Circ Physiol
2004;287:H2132-2137.