Transcriptional coactivators PGC-1?
and PGC-l? control overlapping
programs required for perinatal
maturation of the heart
Ling Lai,1,2,7Teresa C. Leone,1,2,7Christoph Zechner,1,2Paul J. Schaeffer,1,2,3Sean M. Kelly,1,2
Daniel P. Flanagan,1,2Denis M. Medeiros,4Attila Kovacs,1,2and Daniel P. Kelly1,2,5,6,8
1Center for Cardiovascular Research, Washington University School of Medicine, St Louis, Missouri 63110, USA;
2Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA;3Department
of Zoology, Miami University, Oxford, Ohio 45056, USA;4Kansas State University, Manhattan, Kansas 66506, USA;
5Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, Missouri 63110,
USA;6Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri 63110, USA
Oxidative tissues such as heart undergo a dramatic perinatal mitochondrial biogenesis to meet the high-energy
demands after birth. PPAR? coactivator-1 (PGC-1) ? and ? have been implicated in the transcriptional control
of cellular energy metabolism. Mice with combined deficiency of PGC-1? and PGC-1? (PGC-1??−/−mice)
were generated to investigate the convergence of their functions in vivo. The phenotype of PGC-1?−/−mice
was minimal under nonstressed conditions, including normal heart function, similar to that of PGC-1?−/−
mice generated previously. In striking contrast to the singly deficient PGC-1 lines, PGC-1??−/−mice died
shortly after birth with small hearts, bradycardia, intermittent heart block, and a markedly reduced cardiac
output. Cardiac-specific ablation of the PGC-1? gene on a PGC-1?-deficient background phenocopied the
generalized PGC-1??−/−mice. The hearts of the PGC-1??−/−mice exhibited signatures of a maturational
defect including reduced growth, a late fetal arrest in mitochondrial biogenesis, and persistence of a fetal
pattern of gene expression. Brown adipose tissue (BAT) of PGC-1??−/−mice also exhibited a severe
abnormality in function and mitochondrial density. We conclude that PGC-1? and PGC-1? share roles that
collectively are necessary for the postnatal metabolic and functional maturation of heart and BAT.
[Keywords: Transcriptional regulation; heart development; mitochondria; energy metabolism]
Supplemental material is available at http://www.genesdev.org.
Received February 11, 2008; revised version accepted May 16, 2008.
The transcriptional coactivator peroxisome proliferator-
activated receptor ? (PPAR?) coactivator-1? (Ppargc1a, or
commonly called PGC-1?) was discovered based on its
functional interaction with the nuclear receptor PPAR?
in brown adipocytes (Puigserver et al. 1998). There-
after, two related transcriptional coactivators, PGC-1?
(Ppargc1b) and PGC-1-related coactivator (Pprc1, or com-
monly called PRC), were identified (Andersson and
Scarpulla 2001; Kressler et al. 2002; Lin et al. 2002a).
PGC-1? and PGC-1? exhibit the greatest degree of ho-
mology among the PGC-1 family members and are pref-
erentially expressed in tissues with high-capacity mito-
chondrial function such as heart, slow-twitch skeletal
muscle, and brown adipose tissue (BAT). PGC-1 coacti-
vators dock to specific target transcription factors, pro-
viding a platform for the recruitment of protein com-
plexes that exert powerful effects on target gene tran-
scription by remodeling chromatin and enabling access
by the RNA polymerase II machinery (Puigserver et al.
1999; Wallberg et al. 2003; Sano et al. 2007). Studies fo-
cused largely on PGC-1? have shown that it exerts its
biologic actions by coactivating a variety of nuclear re-
ceptor (e.g., PPAR?, PPAR?, estrogen-related receptor
[ERR] ?), and non-nuclear receptor (e.g., nuclear respira-
tory factors [NRFs], FOXO1) transcription-factor targets
(Wu et al. 1999; Vega et al. 2000; Huss et al. 2002; Puig-
server et al. 2003; Schreiber et al. 2003).
As opposed to the majority of known transcriptional
coactivators, the expression of PGC-1? and, to a lesser
extent PGC-1?, is highly inducible in response to devel-
opmental stage-specific and physiological cues (Puig-
server et al. 1998; Wu et al. 1999; Lehman et al. 2000;
7These authors contributed equally to this work.
E-MAIL firstname.lastname@example.org; FAX (407) 745-2001.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1661708.
1948 GENES & DEVELOPMENT 22:1948–1961 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org
Finck and Kelly 2006; Handschin and Spiegelman 2006).
PGC-1? expression is induced by cold exposure, exer-
cise, and fasting in tissue-specific patterns. Studies of
murine heart have shown that PGC-1? is induced at
birth and during postnatal stages coincident with a dra-
matic energy metabolic maturation that involves a ro-
bust mitochondrial biogenic response and switch to re-
liance on fatty acids as the chief fuel (Lehman et al. 2000;
Lehman and Kelly 2002; Taha and Lopaschuk 2007; Bu-
roker et al. 2008). Overexpression studies in mammalian
cells in culture or in transgenic mice have shown that
both PGC-1? and PGC-1? are capable of activating the
expression of a cascade of genes involved in mitochon-
drial biogenesis and respiratory function in adipocytes,
cardiac myocytes, and myogenic cells (Wu et al. 1999;
Lehman et al. 2000; Lin et al. 2002a; St-Pierre et al.
2003). In addition, gain-of-function studies have shown
that PGC-1? activates gene regulatory programs in-
volved in hepatic gluconeogenesis, (Herzig et al. 2001;
Yoon et al. 2001; Puigserver and Spiegelman 2003; Koo et
al. 2004), muscle glucose uptake (Michael et al. 2001;
Wende et al. 2007), and slow-twitch muscle fiber type
determination (Lin et al. 2002b).
More recently, loss-of-function studies have been con-
ducted in mice in an attempt to define specific biological
roles for the PGC-1 coactivators and to determine the
necessity of these molecules in the regulation of biologic
processes in vivo. Two independent mouse lines with
generalized inactivation of the PGC-1? gene have been
generated (Lin et al. 2004; Leone et al. 2005). PGC-1?−/−
mice exhibit a surprisingly minimal phenotype under
basal physiologic conditions, indicating that it is dis-
pensable for the fundamental process of mitochondrial
biogenesis or fetal development. However, physiological
conditions that impose increased energy demands such
as cold exposure, fasting, or exercise, precipitate pheno-
types in the PGC-1?-deficient mice (Lin et al. 2004; Le-
one et al. 2005). The baseline cardiac phenotype of PGC-
1?-deficient mice is remarkably minimal given the im-
portance of a high-capacity mitochondrial system for
this organ. However, PGC-1?-deficient mice develop
ventricular dysfunction after prolonged pressure over-
load (Arany et al. 2006).
Recently, three independent lines of generalized PGC-
1?-deficient mice were generated. PGC-1?−/−mice ex-
hibit stress-induced phenotypes that are generally milder
but very similar to PGC-1?−/−mice (Lelliott et al. 2006;
Vianna et al. 2006; Sonoda et al. 2007). These results
suggest the possibility that PGC-1? and PGC-1? control
a subset of overlapping targets and are, therefore, capable
of compensating for the loss of the other factor in the
PGC-1 loss-of-function mice. To address this question
and to learn more about the role of PGC-1 coactivators in
vivo, we generated mice that are doubly deficient in
PGC-1? and PGC-1? (PGC-1??−/−) by targeting all four
alleles. As described by others, we found that our inde-
pendent line of PGC-1?-deficient mice exhibits a mild
basal phenotype similar to that of PGC-1?-deficient
mice. In striking contrast, PGC-1??−/−mice die shortly
after birth as a result of heart failure related to a perinatal
developmental arrest in cardiac maturation including a
block in mitochondrial biogenesis.
Generation and general characterization
of PGC-1?-null mice
Previously, three research groups have reported and char-
acterized PGC-1?-deficient mice (Lelliott et al. 2006; Vi-
anna et al. 2006; Sonoda et al. 2007). We generated an
independent line of PGC-1?-deficient mice. Briefly, a
cre-lox strategy was used to delete exons 4–6 of the mu-
rine PGC-1? gene (Supplemental Fig. 1A). The targeted
deletion introduced a predicted amino acid frameshift,
resulting in a premature stop codon in exon 7. The effi-
cacy of the gene targeting event and generation of the
mutant transcript was confirmed by PCR, Southern blot-
ting, RNA blotting, and immunoblotting studies (Sup-
plemental Fig. 1B–D).
Heterozygous mice (PGC-1?+/−) were bred to generate
PGC-1?−/−offspring. As described previously (Sonoda et
al. 2007), survival rates were modestly, but significantly
reduced (17% observed, 25% expected; Supplemental
Table 1). The surviving mice appeared normal. Given
that PGC-1? is necessary for adaptive physiological re-
sponses to stressors that demand increased mitochon-
drial oxidative capacity (Lin et al. 2004; Leone et al.
2005), the PGC-1?−/−mice were subjected to short-term
cold exposure and exercise. Six-week-old PGC-1?−/−
mice were subjected for 4 h to a cold environment (4°C)
without food. The PGC-1?−/−mice were unable to main-
tain core body temperature to the same degree as sex-
and weight-matched wild-type controls (Supplemental
Electron microscopic analysis of the BAT of PGC-
1?−/−mice did not reveal any significant abnormalities
in mitochondrial volume density or ultrastructure (data
not shown). Exercise performance was assessed using a
low-intensity, run-to-exhaustion exercise protocol on a
motorized treadmill. The mean running duration for the
PGC-1?−/−mice was less than that of PGC-1?+/+mice
(164 ± 14 vs. 202 ± 10 min, P < 0.05) (Supplemental Fig.
2B). Histologic analyses and electron microscopic stud-
ies of soleus muscles did not reveal any overt cellular or
ultrastructural abnormalities in the PGC-1?−/−mice
(data not shown). However, mean state 3 respiration
rates of mitochondria isolated from hindlimb muscle
were modestly but significantly decreased in PGC-1?−/−
mice compared with PGC-1?+/+controls (73.93 ± 6.12
vs. 102.58 ± 3.22 nmol O2per minute per milligram of
protein, P < 0.05) (Supplemental Fig. 2C). This finding of
reduced muscle state 3 respiratory rates in the PGC-
1?−/−mice is consistent with the results of recent studies
demonstrating that the expression of genes involved in
mitochondrial oxidative phosphorylation (OXPHOS) are
down-regulated in skeletal muscle (Lelliott et al. 2006;
Vianna et al. 2006; Sonoda et al. 2007) and BAT (Lelliott
et al. 2006; Sonoda et al. 2007) in PGC-1?-deficient
mouse lines. Taken together, these results demonstrate
PGC-1?/? deficiency causes cardiac death
GENES & DEVELOPMENT1949
that the muscle and BAT phenotypes of the PGC-1?−/−
mice are similar to that of PGC-1?-deficient mice (Lin et
al. 2004; Leone et al. 2005) indicating that both PGC-1?
and PGC-1? are necessary for adaptive thermogenesis to
cold and exertional exercise.
To determine the necessity of PGC-1? for cardiac
function, echocardiographic studies were performed on
age-matched PGC-1?−/−and PGC-1?+/+control mice at 2
mo of age. Mean left ventricular (LV) mass (64.87 ± 3.52
vs. 73.33 ± 4.13 mg) and cardiac systolic function (% LV
fractional shortening, 64.96 ± 3.53 vs. 58.32 ± 0.99) were
not different between the groups. To assess the cardiac
response to stress, exercise echocardiography was per-
formed. The post-exercise heart rate response and LV
fractional shortening was similar between the groups
(data not shown). Tissue histologic studies of the PGC-
1?−/−ventricle did not reveal any significant fibrosis or
cellular abnormalities (data not shown). Electron micro-
scopic analyses of PGC-1?−/−papillary muscle revealed
normal sarcomeric architecture, mitochondrial mor-
phology, and mitochondrial volume density (Supplemen-
tal Fig. 3). Collectively, these results indicate that, as we
reported previously for PGC-1? (Leone et al. 2005), PGC-
1? is dispensable for normal cardiac development, struc-
ture, and function.
PGC-1? compensates for the loss of PGC-1?
The lack of a cardiac phenotype in PGC-1?−/−mice un-
der nonstressed conditions strongly suggested that com-
pensatory mechanisms are activated, possibly through
the actions of the related coactivator, PGC-1?. As an
initial step to explore this possibility, levels of PGC-1?
gene expression were determined in several relevant
PGC-1?−/−tissues. Under basal conditions, PGC-1?
mRNA levels in PGC-1?−/−heart were not significantly
different from that of littermate PGC-1?+/+controls (Fig.
1A). However, after short-term starvation, a condition
that increases demands on mitochondrial oxidation of
fatty acids and ketones in heart, PGC-1? mRNA levels
were induced to higher levels in PGC-1?−/−hearts com-
pared with controls (Fig. 1A). In addition, mean levels of
BAT PGC-1? protein were higher in PGC-1?−/−mice
compared with PGC-1?+/+mice under basal conditions
and following exposure to cold (Fig. 1B). Thus, PGC-1?
gene expression is induced in the heart and BAT of PGC-
1?−/−mice, particularly in the context of physiological
stressors that increase requirements for mitochondrial
We next sought to determine whether the PGC-1 co-
activators share gene targets in cardiac myocytes given
the minimal cardiac phenotype of the PGC-1?−/−and
PGC-1?−/−mice. To this end, gene expression profiling
was conducted using RNA isolated from neonatal rat
cardiac myocytes (NRCM) infected with adenovirus
overexpressing PGC-1?, PGC-1?, or GFP alone (adeno-
viral backbone control). NRCM were used because, in
culture conditions, the myocytes assume a late fetal
metabolic phenotype including minimal expression of
PGC-1 coactivators and targets (Lehman et al. 2000).
Pathway analysis revealed that 38 pathways were up-
regulated by PGC-1?, 26 of which were also regulated by
PGC-1? (Fig. 1C). As expected, mitochondrial-associated
pathways were predominantly targeted by both coacti-
vators (Supplemental Table 2). Mitochondrial targets
shared by the coactivators include genes involved in
fatty acid oxidation (FAO), the TCA cycle, OXPHOS,
and glucose oxidation (Supplemental Table 3). Notably,
subset of overlapping gene regulatory programs. (A,
left) Representative autoradiograph of a Northern
blot using RNA isolated from heart of PGC-1?+/+
and PGC-1?−/−mice on standard chow (Fed) or post
36 h fast (Fast) is shown using a full-length PGC-1?
cDNA as a probe. PGC-1? transcripts are denoted by
the arrows. Ethidium bromide staining of 28s ribo-
somal RNA is shown at the bottom as a loading
control. (Right) Quantitative RT–PCR (TaqMan) of
total RNA from heart was used to characterize the
level of PGC-1? gene expression in fed (gray bar) and
fasted (black bars) PGC-1?+/+and PGC-1?−/−hearts.
The mRNA levels were normalized to 36?4 mRNA
content, and are shown relative to the PGC-1?+/+fed
values (=1.0). (*) P < 0.05 compared with the fed
group of the same genotype; (†) P < 0.05 compared
with the fasted group of the PGC-1?+/+. (B) Western
blot analysis of whole-cellprotein extracts preparedfromBAT of PGC-1?+/+and PGC-1?−/−mice at room temperature or at 4°C. The top
arrow designates the full-length PGC-1? protein. A nonspecific band (NS) is shown as a loading control. (C–D) NRCM in culture were
infected with Ad-GFP, Ad-PGC-1?, or Ad-PGC-1?, and gene expression array analysis was performed using the Affymetrix Rat
Expression Set 230 chip. (C) Venn diagram showing the Gene Ontology pathways that were up-regulated by either PGC-1? or PGC-1?
compared with Ad-GFP-infected cells. The left circle represents the pathways up-regulated by PGC-1?, and right circle represents
those up-regulated by PGC-1? with overlapping portion (black) representing pathways up-regulated by both. (D) The pie chart represents
genes in the Gene Ontology category “mitochondrion” that were up-regulated at least 1.5-fold by PGC-1? compared with Ad-GFP-infected
cells (P < 0.05). The white portion represents those that were also up-regulated by PGC-1? by at least 1.5-fold (P < 0.05).
PGC-1? and PGC-1? drive a significant
Lai et al.
1950GENES & DEVELOPMENT
70.5% of genes involved in mitochondrial metabolism
up-regulated by PGC-1? were also induced by PGC-1?
(Fig. 1D). These results suggest that there is significant
overlap in the cardiac metabolic gene targets regulated
by PGC-1? and PGC-1?.
We next sought to evaluate the functional redundancy
of the two coactivators by generating mice with targeted
deactivation of all four PGC-1? and PGC-1? alleles
(PGC-1??−/−mice). For these studies, PGC-1?+/−mice on
a PGC-1?-null background (PGC-1?−/−?+/−) were gener-
ated and intercrossed to generate double-deficient off-
spring (PGC-1??−/−). Of 109 pups generated from the
PGC-1?−/−?+/−× PGC-1?−/−?+/−crosses, no double ho-
mozygous null mice (PGC-1??−/−) were found at wean-
ing. Furthermore, the PGC-1?−/−?+/−mice that survived
until weaning were not found in the expected 2:1 ratio
with PGC-1?−/−mice (Table 1, top), indicating that the
PGC-1??−/−genotype is lethal. To determine the age of
death in the PGC-1??−/−group, timed breedings were
performed. Inspection of birth sacs during the late fetal
period did not reveal any evidence of embryonic lethal-
ity. At birth, all of the PGC-1??−/−mice were viable and
genotyping revealed the expected Mendelian ratio (Table
1, bottom), although their birth weights were less than
the other genotypes (Supplemental Fig. 4). However, the
majority (∼70%) of PGC-1??−/−pups died within 24 h of
birth and all died within 14 d (Fig. 2). These results dem-
onstrate the importance of having at least one PGC-1? or
PGC-1? allele for survival following birth.
The PGC-1??−/−pups exhibited a labored breathing
pattern during the first hours after birth, suggestive of a
metabolic or cardiopulmonary crisis. Blood glucose val-
ues were not significantly different among the four geno-
types with the exception that glucose levels in PGC-
1??−/−mice were modestly but significantly lower than
PGC-1??+/+controls, yet similar to that of PGC-1?−/−
and PGC-1?−/−mice (Supplemental Fig. 5A). In addition,
blood lactate levels were not different across all four
genotypes following a 4-h fast (Supplemental Fig. 5B).
Postmortem histologic analyses of the PGC-1??−/−lungs
revealed evidence of alveolar collapse, whereas sections
taken before death were normal, suggesting that the ab-
normalities were secondary, possibly to congestive heart
failure (Supplemental Fig. 6A). In addition, postmortem
gross and histologic analyses of the PGC-1??−/−mice did
not reveal overt abnormalities in brain, liver, and kidney
(Supplemental Fig. 6B,C; data not shown). The PGC-
1??−/−hearts were significantly smaller than the hearts
from the other genotypes, but did not exhibit any gross
morphologic abnormalities; all four chambers were
present, the great vessels were intact and in the proper
orientation, and the ductus arteriosus was appropriately
closed post-birth (data not shown).
PGC-1 loss-of-function studies conducted in isolated
adipocytes have strongly suggested that the presence of
either PGC-1? or PGC-1? is necessary for the differen-
tiation and mitochondrial maturation of brown adipo-
cytes in vitro (Uldry et al. 2006). Accordingly, we char-
acterized BAT structure and function in the doubly mu-
tant mice. Lipid droplet size and density appeared greater
in brown adipocytes of PGC-1?−/−, PGC-1?−/−, and PGC-
1??−/−mice compared with the wild-type control, with
the PGC-1??−/−group being slightly more abnormal
(Supplemental Fig. 7A). Triglyceride content in BAT
from all four genotypes paralleled the histologic results
(Supplemental Fig. 7B). Electron microscopy coupled
with quantitative morphometry demonstrated that
whereas BAT mitochondrial volume density was not dif-
ferent in PGC-1?−/−and PGC-1?−/−BAT compared with
wild-type control, it was significantly lower in PGC-
1??−/−mice (Fig. 3A,B). The mitochondria of PGC-1??−/−
BAT also exhibited reduced cristae density (Fig. 3A).
Consistent with the ultrastructural findings, BAT ex-
pression of key PGC-1 mitochondrial gene targets (cyto-
chrome c, somatic, Cycs; cytochrome oxidase 4, Cox4;
and ATP synthase, H+ transporting, mitochondrial F1
complex, ? polypeptide, Atp5b) was significantly re-
duced in PGC-1??−/−mice compared with the other
genotypes (Fig. 3C). To assess functional correlates of the
BAT histologic and gene expression results, cold expo-
sure studies were conducted with 5- to 6-wk-old PGC-
1?−/−?+/−mice. Triple allele mutants were used because
the PGC-1??−/−mice did not survive. After 2 h, the core
temperature of PGC-1?−/−?+/−mice dropped signifi-
cantly lower than age- and sex-matched wild-type, PGC-
1?−/−, or PGC-1?−/−mice (Fig. 3D). Taken together, these
results suggest that all four alleles are necessary for a
normal adaptive thermogenic response, but that some
Survival rates of the PGC-1??−/−mice
? −/−, ?+/−, ?+/−
P < 0.01
?−/−, ?+/−, ?+/−
Results are expressed as the total number of animals found with
either genotype and the percentage of the total. Percentage
(rounded) is given in parentheses.
Mortality curve depicting the percent survival of male and fe-
male PGC-1?−/−(diamonds, n = 55) and PGC-1??-deficient
(PGC-1??−/−, squares, n = 31) pups 28 d after birth.
Deficiency of both PGC-1? and PGC-1? is lethal.
PGC-1?/? deficiency causes cardiac death
GENES & DEVELOPMENT1951
functional overlap exists between the two PGC-1 coac-
tivator proteins for mitochondrial biogenesis.
PGC-1??−/−mice die of heart failure
To assess cardiac structure and function in the PGC-
1??−/−mice, echocardiographic and Doppler studies
were performed at ∼12 h after birth. Both absolute LV
mass (LVM) and LVM corrected for body weight (LVMI)
were significantly reduced in PGC-1??−/−mice com-
pared with wild-type, PGC-1?−/−, and PGC-1?−/−groups
consistent with a growth defect (Table 2). PGC-1??−/−
hearts also exhibited a significant reduction in mean LV
internal diameter (LVID) during diastole consistent with
reduced heart size (Table 2). Mean heart rate was signifi-
cantly lower in the PGC-1??−/−mice compared with the
other genotypes (Table 2). Careful inspection of the heart
rate and Doppler velocity waveforms representing the
passive (E wave) and atrial contraction (A wave) compo-
nents of diastolic LV filling revealed that they were not
always coupled to a systolic LV outflow jet, consistent
with intermittent second-degree heart block, which oc-
curred in a variety of A:V conduction patterns including
2:1, 3:1, 4:1, 6:1, and 8:1 (Supplemental Fig. 8).
Cardiac output measurements were made in the neo-
natal mice by measuring aortic diameter and blood flow
velocity via Doppler in the proximal portion of the de-
scending aorta. Cardiac output was markedly reduced in
the PGC-1??−/−mice compared with the other geno-
types (Fig. 4A,B), despite preservation of LV fractional
shortening (Table 2). To further analyze cardiac perfor-
mance, the Tei index, a noninvasively derived parameter
of combined systolic and diastolic function, based on the
timing of events during the cardiac cycle, was deter-
mined using the pulse wave Doppler spectra of the trans-
mitral and LV outflow tract velocities (Tei et al. 1995).
To account for the potential confounding effect of heart
rate differences among the groups, the heart rate of wild-
ficient mice. (A) Representative electron
micrographs of BAT at PD0.5 from wild-
type (??+/+), PGC-1?−/−(?−/−), PGC-1?−/−
(?−/−), and PGC-1??−/−(??−/−) mice at two
different magnifications. Each genotype la-
bel denotes the vertical column below it.
Bars: top row, 2 µm; bottom row, 500 nm.
(B) Quantitative morphometric measure-
ments of the cellular volume density for the
mitochondrial fraction based on analysis
of electron micrographs. Bars represent
mean ± SEM. (*) P < 0.05 compared with
??+/+. (C) Quantitative real-time RT–PCR
analysis of RNA extracted from hearts
of PD0.5 wild-type, PGC-1?−/−, PGC-1?−/−,
and PGC-1??−/−mice for the following:
oxidative phosphorylation-cytochrome c,
(Cox4), ATP synthase, H+ transporting, mi-
tochondrial F1 complex, ? polypeptide
(Atp5b). The mRNA levels were normal-
ized to 18s rRNA content, and expressed
relative to PGC-1??+/+values. Bars repre-
sent mean ± SEM. (*) P < 0.05 compared
with ??+/+; (†) P < 0.05 compared with ?−/−;
(#) P < 0.05 compared with ?−/−. (D) Thirty-
six-day-old to 42-d-old PGC-1??+/+(open
squares, n = 11), PGC-1?−/−(open triangle,
n = 7), PGC-1?−/−(open circle, n = 13), and
PGC-1?−/−?+/−mice (black squares, n = 13)
were subjected to cold (4°C). The change in
core temperature ± SEM is shown in the
graph as a function of time. (*) P < 0.05,
compared with ??+/+; (†) P < 0.05 compared
with ?−/−; (#) P < 0.05 compared with ?−/−.
BAT phenotype in PGC-1??-de-
Lai et al.
1952GENES & DEVELOPMENT
type mice was lowered to that measured in the PGC-
1??−/−mice by the sinus node inhibitor, Zatebradine.
The mean Tei index was abnormal in the PGC-1??−/−
mice compared with the other genotypes (Fig. 4A), re-
flecting a significant abnormality in each of the three
components of the index (increased isovolumic contrac-
tion time and isovolumic relaxation time [IVRT], and
decreased ejection time, depicted in Fig. 4B). Interest-
ingly, the PGC-1?-deficient heart exhibited a significant
decrease in performance compared with the PGC-1??+/+
heart, albeit not as severe as that of the PGC-1??−/−
mice. Lastly, Doppler-derived parameters of diastolic
filling showed decreased E/A ratio and prolonged IVRT
in PGC-1??−/−mice, consistent with impaired ventricu-
lar diastolic relaxation (Fig. 4A). Taken together, the car-
diac function results indicate that the PGC-1??−/−mice
have markedly reduced postnatal cardiac output, likely
due to an inappropriately low heart rate combined with
reduced contractile and diastolic function.
Despite our findings of a significant cardiac phenotype
in the PGC-1??−/−mice, it was possible that extracardiac
effects including, but not limited to, abnormal thermo-
genesis contributed to the early postnatal lethality. To
address this, mice were generated in which the PGC-1?
gene was deleted specifically in heart on a generalized
PGC-1?-deficient background (PGC-1?−/−?f/f/MHC-Cre)
via Cre-recombinase-mediated excision of exons 4–6 us-
ing the same parent targeting vector used to generate the
Supplemental Fig. 9A). Cardiac specificity of the PGC-1?
gene deletion was achieved through the use of ?MHC-
Cre mice (Agah et al. 1997), which expresses Cre recom-
binase specifically in cardiac myocytes driven by the car-
diac ? myosin heavy chain promoter (Supplemental Fig.
9B; data not shown). Combined PGC-1?−/−and cardiac-
specific PGC-1?-deficient mice (PGC-1?−/−?f/f/MHC-Cre)
were generated by breeding. Among the four geno-
types expected in the offspring, three were viable and
produced with the expected 1:1:1 ratio (Supplemental
Table 4). Similar to the generalized PGC-1??−/−mice,
PGC-1?−/−?f/f/MHC-Cremice were born alive, 67% of the
pups died within 24 h of birth, and all were dead within
7 d (Supplemental Fig. 10). The mean echocardiographic-
derived cardiac output measurements with the neonatal
PGC-1?−/−?f/f/MHC-Cremice (controls, 920 ± 40 vs. PGC-
1?−/−?f/f/MHC-Cre, 479 ± 28 µL per minute) were strik-
ingly similar to that of the PGC-1??−/−mice (??+/+,
852 ± 52 vs. ??−/−, 342 ± 47 µL per minute). These results
mice(strategy shown in
Echocardiographic parameters of the PGC-1??−/−mice at birth
429 ± 14
0.28 ± 0.005
0.189 ± 0.008
1.59 ± 0.04
0.49 ± 0.02
1.04 ± 0.06
6.19 ± 0.29
4.16 ± 0.21
0.35 ± 0.01
34.7 ± 2.6
413 ± 27
0.27 ± 0.008
0.185 ± 0.006
1.52 ± 0.06
0.46 ± 0.2
1.01 ± 0.03
5.59 ± 0.52
3.79 ± 0.31
0.36 ± 0.01
33.5 ± 1.4
411 ± 20
0.27 ± 0.006
0.196 ± 0.009
1.59 ± 0.04
0.53 ± 0.02†
0.90 ± 0.03*†
5.94 ± 0.28
4.26 ± 0.25
0.35 ± 0.02
43.4 ± 1.8*†
316 ± 13*†#
0.26 ± 0.007
0.189 ± 0.006
1.27 ± 0.03*†#
0.44 ± 0.01#
0.82 ± 0.02*†
3.86 ± 0.25*†#
2.80 ± 0.11*†#
0.41 ± 0.07*†#
35.8 ± 1.5#
(*) P < 0.05 compared with PGC-1??+/+mice; (†) P < 0.05 compared with PGC-1?−/−mice; (#) P < 0.05 compared with PGC-1?−/−mice.
(HR) Heart rate; (LVPW) LV posterior wall (diastole or systole); (LVPWdi) LV posterior wall diastole index; (LVID) LV internal diameter
(diastole or systole); (LVM) LV mass; (LVMI) LV mass index; (RWT) relative wall thickness = (LVPWd + IVSd)/LVIDd; (FS) fractional
Figure 4. Evidence for cardiac failure in PGC-1??-de-
ficient mice. To evaluate cardiac function noninva-
sively in all four genotypes, high-resolution echocar-
diography was performed within a few hours after
birth. (A) Bar graphs show representative indices of
systolic (cardiac output), diastolic (E/A ratio, IVRT),
and combined (Tei index) left ventricular perfor-
mance. (B) Representative images of the trans-mitral/
left ventricular outflow tract (LVOT) Doppler spectra
from wild-type (??+/+) and PGC-1??−/−(??−/−) mice
demonstrate markedly altered cardiac time intervals
and reduced LVOT velocities in the double null mice.
(IVRT) Isovolumic relaxation time; (IVCT) isovolumic
contraction time; (ET) ejection time.
PGC-1?/? deficiency causes cardiac death
GENES & DEVELOPMENT1953
indicate that the PGC-1?−/−?f/f/MHC-Cremice phenocopy
the generalized PGC-1??-deficient mice, providing fur-
ther support for the conclusion that the PGC-1??−/−
mice die of heart failure.
Evidence for a mitochondrial maturational arrest
in the hearts of PGC-1??−/−mice
We next sought to investigate the basis for the cardiac
failure in the PGC-1??−/−mice. Evidence for apoptosis or
fibrotic changes was absent based on TUNEL, caspase,
and trichrome staining (data not shown). However, elec-
tron microscopic studies demonstrated dramatic mito-
chondrial abnormalities in the hearts of PGC-1??−/−
mice, most prominent of which was a significant dimi-
nution in mitochondrial number and size, consistent
with a defect in mitochondrial biogenesis (Fig. 5A). Car-
diac mitochondria of the PGC-1??−/−mice also exhib-
ited a variety of ultrastructural abnormalities including
vacuoles and reduced cristae density, suggesting a defect
in biogenesis or swelling (Fig. 5A). Quantitative mor-
phometry confirmed a significant reduction in mean cel-
lular mitochondrial volume density despite normal myo-
fibrillar volume density in the cardiac myocytes of the
PGC-1??−/−mice (Fig. 5B). Importantly, the myocyte mi-
tochondrial volume density of PGC-1?−/−and PGC-1?−/−
hearts was not reduced. This conclusion was also sup-
ported by the results of mitochondrial DNA quantifica-
tion (Supplemental Fig. 11).
A variety of cellular ultrastructural abnormalities
were also noted in the cardiac ventricles of PGC-1??−/−
mice. A subset of myocytes that were largely or partially
devoid of mitochondria, sarcomeres, and other cellular
organelles was noted (Supplemental Fig. 12A,B). Simi-
lar cellular and mitochondrial derangements, includ-
ing a marked reduction in mitochondrial number and
size, were also seen in the postnatal hearts of the
PGC-1?−/−?f/f/MHC-Cremice (data not shown).
The mitochondrial abnormalities in hearts of PGC-
1??−/−mice suggested a mitochondrial maturation ar-
rest. A major surge in cardiac mitochondrial biogenesis
occurs in heart during the late fetal period and continues
through the early postnatal stages (Hallman 1971; Smol-
ich et al. 1989; Marin-Garcia et al. 2000). To determine
whether this perinatal mitochondrial biogenic response
was defective in the PGC-1??−/−mice, electron micros-
copy studies were conducted on heart samples from mice
at embryonic days 16.5 and 17.5 (E16.5 and E17.5) com-
pared with that of postnatal day 0.5 (PD0.5) across all
four genotypes. At E16.5, mitochondria were small and
sity and structure in hearts of PGC-1??−/−
mice. (A) Representative electron micro-
graphs of cardiac muscle (LV free wall) at
PD0.5 from wild-type (??+/+), PGC-1?−/−
(?−/−), PGC-1?−/−(?−/−), and PGC-1??−/−
(??−/−) mice at three different magnifica-
tions. Each genotype label denotes the ver-
tical column below it. Bars: top row, 2 µm;
middle row, 500 nm; bottom row, 100 nm.
Arrows indicate vacuolar abnormalities
within mitochondria of the PGC-1??−/−
mice. (B) Quantitative morphometric mea-
surements of the cellular volume density
for the mitochondrial (left) and myofibrilar
(right) fractions based on analysis of elec-
tron micrographs. Bars represent mean ± SEM.
(*) P < 0.05 compared with ??+/+.
Abnormal mitochondrial den-
Lai et al.
1954 GENES & DEVELOPMENT
sparse compared with postnatal ventricular sections in
all four genotypes (Fig. 6A). In striking contrast, at PD0.5
a marked cardiac biogenic response occurred in PGC-
1??+/+, PGC-1?−/−, and PGC-1?−/−mice, but not the
PGC-1??−/−group (Fig. 6A). At E17.5, a modest increase
in mitochondrial density occurs in all genotypes except
the PGC-1??−/−group, indicative of a block in this late
prenatal surge of mitochondrial biogenesis in the doubly
mutant mice. Mitochondrial DNA measurements also
revealed the same pattern (Supplemental Fig. 13). Expres-
sion of the PGC-1? and PGC-1? genes is coordinately
induced in wild-type murine heart from E15.5 to PD0.5,
in parallel with the observed mitochondrial biogenic re-
sponse (Fig. 6B) providing additional evidence supporting
a role for these coactivators in the perinatal mitochon-
drial biogenic surge.
The small heart size and mitochondrial biogenic arrest
noted in PGC-1??−/−mice strongly suggested a general-
ized defect in cardiac maturation. To further explore this
possibility, gene regulatory signatures of postnatal car-
diac myocyte maturation were assessed in the PGC-
1??−/−mice and compared with the other genotypes.
Known metabolic markers of terminal maturation in-
clude PGC-1 target genes involved in mitochondrial oxi-
dative pathways such as FAO and OXPHOS. Quantita-
tive RT–PCR analyses revealed that the expression of
genes involved in FAO (acetyl-Coenzyme A dehydroge-
nase, medium chain [Acadm], acyl-Coenzyme A dehy-
drogenase, very long chain [Acadvl], carnitine palmitoyl-
transferase 1b [Cpt1b], carnitine palmitoyltransferase 2
[Cpt2]), and OXPHOS (Cycs, Cox4, and Atp5b) were sig-
nificantly reduced in the PGC-1??−/−hearts compared
with wild-type controls (Fig. 7). Glucose metabolic
markers were also analyzed. Hexokinase 2 (Hk2) was in-
creased and pyruvate dehydrogenase kinase 4 (Pdk4) was
significantly decreased in the PGC-1??−/−hearts, sug-
gesting that the programs directing the switch from re-
liance on glucose during the fetal period to fatty acids as
the preferred fuel after birth was blocked. In addition,
expression of several fetal cardiac gene markers not di-
rectly involved in cellular energy metabolism, including
atrial natriuretic factor (Nppa) and brain natriuretic pep-
tide (Nppb), remained elevated in the PGC-1??−/−car-
diac ventricles (Fig. 7), whereas expression of ? myosin
heavy chain (Mhy6), an adult sarcomeric isoform, was
reduced in PGC-1??−/−as well as PGC-1?−/−hearts.
These gene marker results are consistent with a general
arrest in cardiac maturation and suggest a regulatory link
between metabolic pathways and the broad program of
The mammalian heart functions as a constant pump
throughout the life of the organism. Following birth, the
myocardium burns tremendous amounts of ATP daily to
meet the energy demands of postnatal life. During the
fetal period, the heart uses mainly glucose and lactate to
muscle sections (LV free wall) at E16.5 (top panels), E17.5 (middle panels), and PD0.5 (bottom panels) from wild-type (??+/+), PGC-1?−/−
(?−/−), PGC-1?−/−(?−/−), and PGC-1??−/−(??−/−) mice. Bar, 2 µm. (B) Quantitative real-time RT–PCR analysis of RNA extracted from
hearts of E15.5, E17.5, E18.5, and PD0.5 C57BL6/J mice for the expression of PGC-1? (white bars) and PGC-1? (black bars) genes. The
mRNA levels were normalized to 36B4 mRNA levels, and expressed relative to E15.5 values (=1.0). Quantitative PCR of total DNA
from heart was performed to quantify mitochondrial DNA (gray bars) using primers for NADH dehydrogenase (ND1) and genomic
DNA using primers for lipoprotein lipase (LPL). The ND1 levels were normalized to LPL DNA content, and expressed relative to E15.5
values (=1.0). Bars represent mean ± SEM. (*) P < 0.05 compared with E15.5.
Perinatal mitochondrial biogenesis is blocked in PGC-1??−/−hearts. (A) Representative electron micrographs of cardiac
PGC-1?/? deficiency causes cardiac death
GENES & DEVELOPMENT1955
generate ATP (Fisher et al. 1980, 1981; Girard et al.
1992). The enormous energy demands of the adult heart
are met, in large part, by the oxidation of fatty acids in
mitochondria (Bing 1955; Itoi and Lopaschuk 1993; Taha
and Lopaschuk 2007). Accordingly, to meet the rigors of
postnatal life, the heart undergoes a perinatal metabolic
maturation that involves a fuel “switch” concordant
with a dramatic increase in mitochondrial functional ca-
pacity (Marin-Garcia et al. 2000; Taha and Lopaschuk
2007). Herein, we show that the collective actions of
PGC-1? and PGC-1? comprise a critical component of
the molecular circuitry that drives the perinatal mito-
chondrial biogenesis necessary for metabolic and func-
tional maturation of the murine heart and BAT.
Our results indicate that key late fetal and perinatal
cardiac developmental events are not activated in the
PGC-1??−/−mice. The heart chambers and great vessels
of the PGC-1??−/−mice were overtly normal, suggesting
that major early fetal developmental events were unim-
paired. However, the hearts of the double mutant ani-
mals are small and the dramatic mitochondrial biogenic
response known to occur at the time of birth was found
to be completely absent in the PGC-1??−/−mice. The
doubly deficient animals also exhibited evidence of an
immature conduction system, including bradycardia and
heart block. Consistent with this conclusion, mice defi-
cient for ERR?, a known target of PGC-1-mediated co-
activation, were recently shown to exhibit gene regula-
tory signatures consistent with a block in the cardiac
perinatal switch from relying on glucose to oxidative
metabolism (Alaynick et al. 2007). Taken together, these
results indicate that whereas PGC-1? and PGC-1? are
not required for early formation of mitochondria, they
are necessary for programs directing late fetal and post-
natal cardiac maturation. It is possible that the related
factor PRC is compensating for early fetal development
processes. In addition, given that our original PGC-1?
gene ablation strategy did not exclude the possible pro-
duction of a smaller mutant PGC-1? protein (Leone et al.
2005), it is theoretically possible that a small amount of
residual PGC-1? activity compensates during early de-
velopmental stages in the PGC-1??−/−mice.
Our results strongly suggest that PGC-1? and PGC-1?
share a subset of key gene targets and functions, at least
in heart and BAT. This conclusion is supported by the
following lines of evidence: (1) PGC-1? gene expression
is induced in BAT and heart of PGC-1?-deficient mice,
suggesting a compensatory response in this context; (2)
the severity of the cold intolerance phenotype and BAT
mitochondrial derangements increases in parallel with
the number of deleted PGC-1? and PGC-1? alleles, re-
sults that are consistent with the findings of a previous
sistent with a block in fetal–adult transition. Quan-
titative real-time RT–PCR analysis of RNA extracted
from hearts of PD0.5 wild-type (??+/+), PGC-1?−/−
(?−/−), PGC-1?−/−(?−/−), and PGC-1??−/−(??−/−) mice
for the following: oxidative phosphorylation-cyto-
chrome c, somatic (Cycs), cytochrome oxidase 4
(Cox4), ATP synthase, H+ transporting, mitochon-
drial F1 complex, ? polypeptide (Atp5b); fatty acid
oxidation-acetyl-Coenzyme A dehydrogenase, me-
dium chain (Acadm), acyl-Coenzyme A dehydroge-
nase, very long chain (Acadvl), carnitine palmitoyl-
transferase 1b (Cpt1b), carnitine palmitoyltransferase
2 (Cpt2); Glycolysis/Glucose oxidation-hexokinase 2
(Hk2), phosphofructokinase (Pfk), pyruvate dehydrog-
enase kinase 4 (Pdk4); General adult cardiac gene
markers-atrial natriuretic factor (ANF), brain natri-
uretic peptide (BNP), ATPase, Ca2+transporting, car-
diac muscle, slow twitch 2 (Serca2a), and ?-myosin
heavy chain (Myh6). The mRNA levels were normal-
ized to ?-actin mRNA content, and expressed rela-
tive to PGC-1??+/+values. Bars represent mean ± SEM.
(*) P < 0.05 compared with ??+/+; (†) P < 0.05 com-
pared with ?−/−; (#) P < 0.05 compared with ?−/−.
Cardiac gene expression markers are con-
Lai et al.
1956GENES & DEVELOPMENT
study conducted in isolated brown adipocytes (Uldry et
al. 2006); (3) gene expression profiling in cardiac myo-
cytes demonstrated a high degree of overlap in PGC-1?
and PGC-1? target pathways, especially those involved
in mitochondrial metabolism; and (4) whereas the PGC-
1?-deficient mice and PGC-1?-deficient mice survive
with a minimal cardiac phenotype, combined deficiency
results in 100% postnatal lethality due to heart failure.
Further analysis of our gene expression array results also
provided some idea about which of the downstream tran-
scription factors may be involved in the shared PGC-1
functions. A significant subset of genes activated by both
coactivators (Supplemental Tables 2, 3) are also direct
targets for ERR? and ERR?, known PGC-1 coactivating
targets in cardiac myocytes based on gene expression
profiling and ChIP-chip promoter occupation assays (Du-
four et al. 2007). This comparative analysis reveals that a
significant number of ERR target genes involved in mi-
tochondrial FAO, respiration, and ATP synthesis were
also shown to be activated by PGC-1? and PGC-1? in
this study. These results suggest that the profound car-
diac mitochondrial phenotype that occurs in the PGC-
1??−/−mice is related to deactivation of the ERR gene
Although these results indicate significant gene target
and functional redundancy, evidence for complementary
PGC-1 coactivator-specific roles also exist. For example,
the stress-induced phenotypes of the single PGC-1-null
mice indicate that both coactivators are necessary to
meet the full range of physiological demands imposed on
postnatal life. Cold exposure, treadmill exercise, or fast-
ing precipitate phenotypes in the single PGC-1 gene de-
letion mice (Lin et al. 2004; Leone et al. 2005; Lelliott et
al. 2006; Vianna et al. 2006; Sonoda et al. 2007). In addi-
tion, the results of studies focused on noncardiac tissues
have suggested that PGC-1? drives a subset of programs
distinct from that of PGC-1?, including hepatic lipogen-
esis and cholesterol metabolism (Lin et al. 2005) and
skeletal muscle IIx fiber type determination (Mortensen
et al. 2006; Arany et al. 2007). Single and combined PGC-
1? and PGC-1? loss-of-function studies conducted in
brown adipocytes in culture have also revealed comple-
mentary effects on mitochondrial function (Uldry et al.
2006). It is likely that the two coactivators are regulated
by distinct upstream circuits (Lin et al. 2002a) providing
for the regulation of common and distinct gene targets in
coactivator-specific patterns based on the specific physi-
ological stimulus among tissues or cell types.
Extensive phenotyping revealed that the early postna-
tal death of the PGC-1??−/−mice is due to severe cardiac
dysfunction. The PGC-1??−/−hearts exhibited intermit-
tent second-degree AV block and generated a very low
cardiac output. The basis for the low cardiac output
likely relates to the relatively small size of the heart,
bradycardia, and reduced contractile function. The con-
tractile dysfunction is likely due, at least in part, to re-
duced capacity for mitochondrial ATP production re-
lated to immature mitochondria and a block in the post-
natal induction of genes involved in FAO, the chief
source of energy in the postnatal period. In support of
this latter conclusion, inborn errors in mitochondrial
FAO enzymes are an important cause of inherited car-
diomyopathy in children (Kelly and Strauss 1994). In ad-
dition, mice with targeted ablation of the gene encoding
ERR?, a known target of PGC-1? and ? (Huss et al. 2002;
Kamei et al. 2003; Schreiber et al. 2003), die early after
birth with a small heart and reduced expression of
genes involved in myocardial FAO (Alaynick et al. 2007).
Lastly, mice with cardiac-specific deficiency of PGC-1?
in a generalized PGC-1?-deficient background (PGC-
1?−/−?f/f/MHC-Cremice) phenocopy the generalized PGC-
1??−/−mice, providing additional support for the conclu-
sion that the lethal phenotype is caused by cardiac de-
rangements. However, given the severe abnormalities
found in the BAT of the PGC-1??−/−mice, it is possible
that this phenotype is also incompatible with postnatal
Materials and methods
Generation of generalized PGC-1?-null and cardiac-specific
Sv129 genomic DNA was used as a template to create three
amplicons using PCR, which were subsequently inserted into
pGKNeo-p1339 (GenBank Accession #AF335420). The 3? am-
plicon also contained an engineered LoxP site that was used to
excise exons 4, 5, and 6 via Cre recombinase. The construct was
linearized with XhoI and electroporated into SCC10 ES cells
(derived from RW4) using G418 selection. The clones were
screened by Southern blotting and PCR. One clone out of 216
screened was positive for the recombination event. This clone
was injected into a C57BL6/J blastocyst. Germ-line transmis-
sion was confirmed by coat color as well as PCR analysis of tail
DNA. The female mice containing the targeted allele were bred
with the male EIIa-Cre mice to generate both complete and
conditional knockout mice. The offspring were screened by
Southern blotting and PCR. Mice that contained the recombi-
nation allele with the exon 4–6 cassette as well as the neomycin
cassette removed were chosen for generation of the generalized
“knockout” (PGC-1?−/−). Mice that had only the neomycin cas-
sette removed were chosen to create the conditional knockout.
Mice were maintained in the hybrid background, C57BL6/
J × sv129. Littermates were used whenever possible (as indi-
cated in the text) to control for strain effects.
Generation of PGC-1a? double-deficient mice
Mice with a single gene deletion of either PGC-1? (Leone et al.
2005) or PGC-1? were bred to generate compound heterozygous
(PGC-1?+/−?+/−) mice, which in turn were crossed to generate
mice deficient for PGC-1? and heterozygous for PGC-1? (PGC-
1?−/−?+/−). These mice were used as breeders to generate the
offspring used in this study. Mice were maintained in a hybrid
background (C57BL6/J × sv129) and littermates for PGC-1?−/−
and PGC-1??−/−were used for comparative analysis to control
for genetic background effects. PGC-1?−/−and PGC-1??+/+mice
were generated with separate breeding pairs.
Animal phenotyping studies
All animal experiments and euthanasia protocols were con-
ducted in strict accordance with the National Institutes of
PGC-1?/? deficiency causes cardiac death
GENES & DEVELOPMENT1957
Health guidelines for humane treatment of animals and were
reviewed and approved by the Institutional Animal Care and
Use Committee of Washington University School of Medicine.
Animals were weighed at different time points between 2 and
8 wk of age and compared directly to their sex-matched litter-
mates. For cold exposure experiments, male and female PGC-
1?+/+and PGC-1?−/−mice were singly housed and placed for 3–4
h at 4°C without food. Core body temperatures were monitored
by rectal probe at baseline and every hour thereafter. Mice were
monitored at least every 30 min to check for lethargy. At the
end of 4 h, mice were sacrificed and tissues harvested for RNA
and protein extraction. For fasting studies, animals were singly
housed and given water ad libitum. Food was removed from
cages in the morning and tissues harvested at 36 h for RNA and
histology. For prenatal and mortality curve studies, timed
breedings were performed, and pregnancy was determined by
detection of a vaginal plug (E0.5). The time of birth was closely
monitored, and newborns were counted and genotyped within
12 h after birth for ?2analysis. Postnatal survival was followed
daily for 28 d. Blood glucose in newborns was measured with a
One-Touch Ultra glucometer (LifeScan, Inc.). Blood lactate in
newborns was measured with a Lactate Pro blood lactate test
meter (ARKRAY, Inc.).
For the low-intensity exercise studies, 9-wk-old female PGC-
1?+/+(n = 6) and PGC-1?−/−(n = 6) mice were run to exhaustion
using a motorized, speed-controlled modular treadmill system
(Columbus Instruments). The treadmill was equipped with an
electric shock stimulus and an adjustable inclination angle.
Running velocity was set at 10 m/min for an hour, and in-
creased by 2 m/min increments every 15 min until exhaustion
For echocardiographic studies performed on adult mice,
2-mo-old female PGC-1?+/+(n = 5) and PGC-1?−/−(n = 5) mice
were lightly anesthetized with an intraperitoneal injection of
3% avertin (tribromoethanol, 0.01 mL/g). Cardiac ultrasound
studies were performed as described previously (Rogers et al.
1999). Exercise echocardiography was performed on a motorized
treadmill as described previously at a duration tolerated by the
PGC-1?−/−mice (1–1.5 min) (Leone et al. 2005).
For neonatal echocardiography, male and female pups were
imaged within 12 h after birth using a Vevo 770 ultrasound
system (Visual Sonics, Inc.). Unanesthetized mice were placed
on an imaging table under a heating lamp, and were lightly
restrained in a left lateral decubitus position. Parasternal long-
and short-axis images of the heart were obtained from standard
echocardiographic views. Semi-apical long-axis views of the LV
were used to interrogate the combined trans-mitral and LV out-
flow tract blood flow velocities. Basal short-axis views were
used to image the pulmonary artery and the proximal portion of
the descending thoracic aorta. Pulse wave Doppler sample vol-
ume was placed parallel with the direction of blood flow, and
aortic diameter was measured at the same level. Cardiac output
was measured as the product of aortic area and velocity time
integral of the Doppler tracing. The Tei index was calculated as
(IVCT + IVRT)/LVET, where IVCT is isovolumic contraction
time (period between mitral valve closure and aortic valve open-
ing), IVRT is isovolumic relaxation time (period between aortic
valve closure and mitral valve opening), and LVET is the LV
ejection period (time between aortic valve opening and closure).
RNA, DNA, protein, and tissue triglyceride analyses
Total RNA was isolated from various mouse tissues using the
RNAzol method (Tel-Test). Northern blotting (Kelly et al. 1989)
and quantitative real-time RT–PCR were performed as de-
scribed (Huss et al. 2004). In brief, total RNA was isolated and
reverse transcribed with Taqman reverse transcription reagents
(Applied Biosystems). PCR reactions were performed in tripli-
cate in a 96-well format using a Prism 7500 Sequence Detector
(Applied Biosystems). The mouse-specific primer-probe sets
used to detect specific gene expression can be found in Supple-
mental Table 5. Either ?-actin (Applied Biosystems), 36B4, or
18s primer-probe sets (Supplemental Table 5) was included in a
separate well (in triplicate) and used to normalize the gene ex-
pression data as noted in the figure legends.
Genomic/mitochondrial DNA was isolated using RNAzol,
followed by back extraction with 4 M guanidine thiocyanate, 50
mM sodium citrate, and 1 M tris, and an alcohol precipitation.
Mitochondrial DNA content was determined by SYBR green
analysis (Applied Biosystems). To this end, the levels of NADH
dehydrogenase subunit 1 (mitochondrial DNA) were normal-
ized to the levels of lipoprotein lipase (genomic DNA). The
primer sequences are noted in Supplemental Table 5.
For Southern blot analysis, 5 µg of genomic DNA was di-
gested with SpeI, electrophoresed on a 0.8% TAE gel, and trans-
ferred to Gene Screen (Perkin Elmer) membrane for hybridiza-
tion. Western blotting was performed as described (Cresci et al.
1996) using Super Signal West Dura Extended Duration Sub-
strate (Pierce) for detection. The polyclonal PGC-1? antibody
was a generous gift provided by Dr Anastasia Kralli. The PGC-
1? antibody has been previously described (Lehman et al. 2000).
Total tissue triglyceride analysis was performed by the Ani-
mal Model Research Core at Washington University School of
Medicine (supported by the CNRU) from frozen tissue using a
modified Bligh and Dyer technique as described previously
(Bligh and Dyer 1959).
Mitochondrial respiration studies
Mitochondrial respiration was assessed in isolated mitochon-
dria from the hindlimb muscle with pyruvate as substrate as
described previously (Bhattacharya et al. 1991). In brief, 3-mo-
old male mice were euthanized by CO2inhalation. The entire
hindlimb was dissected from the bone and minced well, fol-
lowed by a 5-min incubation in Ionic Medium (IM) plus Nagarse
(100 mM sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46 mM Kcl
at pH 7.4, 10 mg nagarse). Samples were homogenized using an
Eberbach homogenizer, centrifuged at 500g for 10 min at 4°C,
and supernatant transferred to a clean tube. The supernatant
was centrifuged at 12,000g and the pellet was resuspended in
IM + 0.5% BSA. The sample was spun again and the pellet re-
suspended in Suspension Buffer (230 mM mannitol, 70 mM su-
crose, 0.02 mM EDTA, 20 mM Tris-HCl, 5 mM K2HPO4at pH
7.4). Total protein was quantified by a BCA assay (Pierce) and
respiration was performed at 25°C using an optical probe (Oxy-
gen FOXY Probe, Ocean Optics). Following measurement of
basal respiration, maximal (ADP-stimulated) respiration was
determined by exposing the mitochondria to 1 mM ADP. Un-
coupled respiration was evaluated following addition of oligo-
mycin (1 µg/mL). The solubility of oxygen in the respiration
buffer at 25°C was taken as 246.87 nmol O2per milliliter. Res-
piration rates were expressed as “nmol O2? min−1? mg pro-
Histology and electron microscopy
Adult mice were anesthetized and perfused with Karnovsky’s
fixative (2% glutaraldehyde, 1% paraformaldehyde, and 0.08%
sodium cacodylate) to avoid artifact. Neonates were euthanized
and hearts were fixed in Karnovsky’s fixative. Cardiac papillary
muscle (adult), LV free wall (neonates), BAT, soleus, and EDL
muscle were dissected and postfixed in 1% osmium tetroxide,
Lai et al.
1958GENES & DEVELOPMENT
dehydrated in graded ethanol, embedded in Poly Bed plastic
resin, and sectioned for electron microscopy. Mitochondrial and
myofibrillar volume densities were determined from electron
micrographs as described previously (Russell et al. 2004). For
each animal, three different fields at the magnification of 7500×
were quantified in blinded fashion. Data were expressed as
mean volume density of mitochondria or myofibrils in each
H&E staining was performed by the Morphology Core at
Washington University School of Medicine (DDRCC). The tis-
sues were fixed with 10% buffered formalin overnight, dehy-
drated in graded concentrations of alcohol, and embedded in
paraffin from which 5-µm sections were prepared.
Gene expression profiling
Total RNA from cultured NRCM post adenoviral infection with
either Ad-GFP, Ad-PGC-1?, or Ad-PGC-1? was reverse tran-
scribed using Superscript II (Invitrogen Corp.) and primed with
T7 promoter-polyA primer (T7T24), followed by second-strand
synthesis according to the manufacturer’s protocol. Biotin-la-
beled cRNA was synthesized using T7-coupled ENZO BioArray
High Yield RNA Transcript Labeling Kit (ENZO Diagnostics,
Inc.). The Alvin J. Siteman Cancer Center’s Bioinformatics Core
at Washington University School of Medicine performed hy-
bridization to Affymetrix Rat Expression Set 230 chip. Af-
fymetrix MAS 5.0 software was used for initial analysis and
background normalization, and Z score calculation and subse-
quent data analysis were performed using Spotfire DecisionSite
for Functional Genomics 9.0. Probe sets called “absent” by
MAS 5.0 in all Ad-GFP control, Ad-PGC-1?, and Ad-PGC-1?
were excluded. Two independent trials were performed. Stu-
dent’s t-test was performed and a P-value <0.05 was used to
determine genes significantly up-regulated. Signal intensity ra-
tios were averaged from both trials and were calculated as ei-
ther Ad-PGC-1?/Ad-GFP or Ad-PGC-1?/Ad-GFP to determine
changes due to exogenous expression of either PGC-1? or PGC-
1?. A gene with a calculated fold change ?1.5 was considered an
up-regulated gene target in cultured NRCM. For pathway analy-
sis, the filtered data sets were uploaded into GenMAPP software
to review the biopathways using the Gene Ontology database.
GenMAPP produced a ranked list of Gene Ontology biological
categories based on the following criteria: (1) at least five regu-
lated genes in selected GO terms; (2) >50% of genes regulated in
selected pathways (>50 “percent changed”). The “percent
changed” was calculated by “the genes meeting the criteria/
genes measured * 100.” The Z score was calculated by subtract-
ing the expected number of genes meeting the criteria from the
observed number, and then dividing by the standard deviation of
the observed number of genes.
Data were analyzed using t-tests or ANOVA where appropriate.
The level of significance was set at P < 0.05 in all cases. Data are
reported as mean values ± the standard error of the mean, unless
We thank Dr. Anastasia Kralli (Scripps) for providing the PGC-
1? antibody; Juliet Fong, Michelle Trusgnich, and Alicia Wallis
for help with mouse husbandry; William Kraft for expert tech-
nical assistance with electron microscopy; Mike Courtois for
assistance with echocardiography; Dr. Suellen Greco, Dr. Erica
Crouch, Dr. Feng Chen, and Dr. Robert Schmidt for careful
analysis of the histopathology; Dr. Jeffrey Saffitz (Beth Israel
Deaconess Medical Center) for consultative advice on electron
microscopy; Dr. Patrick Jay for help with assessing cardiac mor-
phology; and Mary Wingate for assistance with manuscript
preparation. L.L. is supported by the AHA Fellowship award
(0525743Z). C.Z. is a recipient of the Deutsche Forschungsge-
meinschaft research Fellowship ZE 796/2-1. This work was sup-
ported by NIH grants RO1 DK045416, RO1 HL058427, Diges-
tive Diseases Research Core Center (P30 DK052574), Diabetes
Research and Training Center (P60 DK020579), Alvin J. Siteman
Cancer Center Bioinformatics Core and Embryonic Stem Cell
Core (P30 CA91842), and Clinical Nutrition Research Unit
Core Center (P30 DK56341).
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PGC-1?/? deficiency causes cardiac death
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