Transcriptional Coactivators PGC-1 and PGC-L

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Transcriptional coactivators PGC-1␣ and PGC-l␤ control overlapping programs required for perinatal maturation of the heart

Ling Lai,1,2,7 Teresa C. Leone,1,2,7 Christoph Zechner,1,2 Paul J. Schaeffer,1,2,3 Sean M. Kelly,1,2 Daniel P. Flanagan,1,2 Denis M. Medeiros,4 Attila Kovacs,1,2 and 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- vators dock to specific target transcription factors, pro- activated receptor ␥ (PPAR␥) coactivator-1␣ (Ppargc1a, or viding a platform for the recruitment of protein com- commonly called PGC-1␣) was discovered based on its plexes that exert powerful effects on target gene tran- functional interaction with the nuclear receptor PPAR␥ scription by remodeling chromatin and enabling access in brown adipocytes (Puigserver et al. 1998). There- by the RNA polymerase II machinery (Puigserver et al. after, two related transcriptional coactivators, PGC-1␤ 1999; Wallberg et al. 2003; Sano et al. 2007). Studies fo- (Ppargc1b) and PGC-1-related coactivator (Pprc1, or com- cused largely on PGC-1␣ have shown that it exerts its monly called PRC), were identified (Andersson and biologic actions by coactivating a variety of nuclear re- Scarpulla 2001; Kressler et al. 2002; Lin et al. 2002a). ceptor (e.g., PPAR␥, PPAR␣, estrogen-related receptor PGC-1␣ and PGC-1␤ exhibit the greatest degree of ho- [ERR] ␣), and non-nuclear receptor (e.g., nuclear respira- mology among the PGC-1 family members and are pref- tory factors [NRFs], FOXO1) transcription-factor targets erentially expressed in tissues with high-capacity mito- (Wu et al. 1999; Vega et al. 2000; Huss et al. 2002; Puig- chondrial function such as heart, slow-twitch skeletal server et al. 2003; Schreiber et al. 2003). muscle, and brown adipose tissue (BAT). PGC-1 coacti- As opposed to the majority of known transcriptional coactivators, the expression of PGC-1␣ and, to a lesser 7These authors contributed equally to this work. extent PGC-1␤, is highly inducible in response to devel- 8Corresponding author. E-MAIL [email protected]; FAX (407) 745-2001. opmental stage-specific and physiological cues (Puig- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1661708. server et al. 1998; Wu et al. 1999; Lehman et al. 2000;

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PGC-1␣/␤ deficiency causes cardiac death

Finck and Kelly 2006; Handschin and Spiegelman 2006). developmental arrest in cardiac maturation including a PGC-1␣ expression is induced by cold exposure, exer- block in mitochondrial biogenesis. 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- Results matic energy metabolic maturation that involves a ro- Generation and general characterization bust mitochondrial biogenic response and switch to re- of PGC-1␤-null mice liance on fatty acids as the chief fuel (Lehman et al. 2000; Lehman and Kelly 2002; Taha and Lopaschuk 2007; Bu- Previously, three research groups have reported and char- roker et al. 2008). Overexpression studies in mammalian acterized PGC-1␤-deficient mice (Lelliott et al. 2006; Vi- cells in culture or in transgenic mice have shown that anna et al. 2006; Sonoda et al. 2007). We generated an both PGC-1␣ and PGC-1␤ are capable of activating the independent line of PGC-1␤-deficient mice. Briefly, a expression of a cascade of genes involved in mitochon- cre-lox strategy was used to delete exons 4–6 of the mu- drial biogenesis and respiratory function in adipocytes, rine PGC-1␤ gene (Supplemental Fig. 1A). The targeted cardiac myocytes, and myogenic cells (Wu et al. 1999; deletion introduced a predicted amino acid frameshift, Lehman et al. 2000; Lin et al. 2002a; St-Pierre et al. resulting in a premature stop codon in exon 7. The effi- 2003). In addition, gain-of-function studies have shown cacy of the gene targeting event and generation of the that PGC-1␣ activates gene regulatory programs in- mutant transcript was confirmed by PCR, Southern blot- volved in hepatic gluconeogenesis, (Herzig et al. 2001; ting, RNA blotting, and immunoblotting studies (Sup- Yoon et al. 2001; Puigserver and Spiegelman 2003; Koo et plemental Fig. 1B–D). al. 2004), muscle glucose uptake (Michael et al. 2001; Heterozygous mice (PGC-1␤+/−) were bred to generate Wende et al. 2007), and slow-twitch muscle fiber type PGC-1␤−/− offspring. As described previously (Sonoda et determination (Lin et al. 2002b). al. 2007), survival rates were modestly, but significantly More recently, loss-of-function studies have been con- reduced (17% observed, 25% expected; Supplemental ducted in mice in an attempt to define specific biological Table 1). The surviving mice appeared normal. Given roles for the PGC-1 coactivators and to determine the that PGC-1␣ is necessary for adaptive physiological re- necessity of these molecules in the regulation of biologic sponses to stressors that demand increased mitochon- processes in vivo. Two independent mouse lines with drial oxidative capacity (Lin et al. 2004; Leone et al. generalized inactivation of the PGC-1␣ gene have been 2005), the PGC-1␤−/− mice were subjected to short-term generated (Lin et al. 2004; Leone et al. 2005). PGC-1␣−/− cold exposure and exercise. Six-week-old PGC-1␤−/− mice exhibit a surprisingly minimal phenotype under mice were subjected for4htoacold environment (4°C) basal physiologic conditions, indicating that it is dis- without food. The PGC-1␤−/− mice were unable to main- pensable for the fundamental process of mitochondrial tain core body temperature to the same degree as sex- biogenesis or fetal development. However, physiological and weight-matched wild-type controls (Supplemental conditions that impose increased energy demands such Fig. 2A). as cold exposure, fasting, or exercise, precipitate pheno- Electron microscopic analysis of the BAT of PGC- types in the PGC-1␣-deficient mice (Lin et al. 2004; Le- 1␤−/− mice did not reveal any significant abnormalities one et al. 2005). The baseline cardiac phenotype of PGC- in mitochondrial volume density or ultrastructure (data 1␣-deficient mice is remarkably minimal given the im- not shown). Exercise performance was assessed using a portance of a high-capacity mitochondrial system for low-intensity, run-to-exhaustion exercise protocol on a this organ. However, PGC-1␣-deficient mice develop motorized treadmill. The mean running duration for the ventricular dysfunction after prolonged pressure over- PGC-1␤−/− mice was less than that of PGC-1␤+/+ mice load (Arany et al. 2006). (164 ± 14 vs. 202 ± 10 min, P < 0.05) (Supplemental Fig. Recently, three independent lines of generalized PGC- 2B). Histologic analyses and electron microscopic stud- 1␤-deficient mice were generated. PGC-1␤−/− mice ex- ies of soleus muscles did not reveal any overt cellular or hibit stress-induced phenotypes that are generally milder ultrastructural abnormalities in the PGC-1␤−/− mice but very similar to PGC-1␣−/− mice (Lelliott et al. 2006; (data not shown). However, mean state 3 respiration Vianna et al. 2006; Sonoda et al. 2007). These results rates of mitochondria isolated from hindlimb muscle suggest the possibility that PGC-1␣ and PGC-1␤ control were modestly but significantly decreased in PGC-1␤−/− a subset of overlapping targets and are, therefore, capable mice compared with PGC-1␤+/+ controls (73.93 ± 6.12 of compensating for the loss of the other factor in the vs. 102.58 ± 3.22 nmol O2 per minute per milligram of PGC-1 loss-of-function mice. To address this question protein, P < 0.05) (Supplemental Fig. 2C). This finding of and to learn more about the role of PGC-1 coactivators in reduced muscle state 3 respiratory rates in the PGC- vivo, we generated mice that are doubly deficient in 1␤−/− mice is consistent with the results of recent studies PGC-1␣ and PGC-1␤ (PGC-1␣␤−/−) by targeting all four demonstrating that the expression of genes involved in alleles. As described by others, we found that our inde- mitochondrial oxidative phosphorylation (OXPHOS) are pendent line of PGC-1␤-deficient mice exhibits a mild down-regulated in skeletal muscle (Lelliott et al. 2006; basal phenotype similar to that of PGC-1␣-deficient Vianna et al. 2006; Sonoda et al. 2007) and BAT (Lelliott mice. In striking contrast, PGC-1␣␤−/− mice die shortly et al. 2006; Sonoda et al. 2007) in PGC-1␤-deficient after birth as a result of heart failure related to a perinatal mouse lines. Taken together, these results demonstrate

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Lai et al. that the muscle and BAT phenotypes of the PGC-1␤−/− PGC-1␤−/− tissues. Under basal conditions, PGC-1␣ mice are similar to that of PGC-1␣-deficient mice (Lin et mRNA levels in PGC-1␤−/− heart were not significantly al. 2004; Leone et al. 2005) indicating that both PGC-1␣ different from that of littermate PGC-1␤+/+ controls (Fig. and PGC-1␤ are necessary for adaptive thermogenesis to 1A). However, after short-term starvation, a condition cold and exertional exercise. that increases demands on mitochondrial oxidation of To determine the necessity of PGC-1␤ for cardiac fatty acids and ketones in heart, PGC-1␣ mRNA levels function, echocardiographic studies were performed on were induced to higher levels in PGC-1␤−/− hearts com- age-matched PGC-1␤−/− and PGC-1␤+/+ control mice at 2 pared with controls (Fig. 1A). In addition, mean levels of mo of age. Mean left ventricular (LV) mass (64.87 ± 3.52 BAT PGC-1␣ protein were higher in PGC-1␤−/− mice vs. 73.33 ± 4.13 mg) and cardiac systolic function (% LV compared with PGC-1␤+/+ mice under basal conditions fractional shortening, 64.96 ± 3.53 vs. 58.32 ± 0.99) were and following exposure to cold (Fig. 1B). Thus, PGC-1␣ not different between the groups. To assess the cardiac gene expression is induced in the heart and BAT of PGC- response to stress, exercise echocardiography was per- 1␤−/− mice, particularly in the context of physiological formed. The post-exercise heart rate response and LV stressors that increase requirements for mitochondrial fractional shortening was similar between the groups ATP production. (data not shown). Tissue histologic studies of the PGC- We next sought to determine whether the PGC-1 co- 1␤−/− ventricle did not reveal any significant fibrosis or activators share gene targets in cardiac myocytes given cellular abnormalities (data not shown). Electron micro- the minimal cardiac phenotype of the PGC-1␤−/− and scopic analyses of PGC-1␤−/− papillary muscle revealed PGC-1␣−/− mice. To this end, gene expression profiling normal sarcomeric architecture, mitochondrial mor- was conducted using RNA isolated from neonatal rat phology, and mitochondrial volume density (Supplemen- cardiac myocytes (NRCM) infected with adenovirus tal Fig. 3). Collectively, these results indicate that, as we overexpressing PGC-1␣, PGC-1␤, or GFP alone (adeno- reported previously for PGC-1␣ (Leone et al. 2005), PGC- viral backbone control). NRCM were used because, in 1␤ is dispensable for normal cardiac development, struc- culture conditions, the myocytes assume a late fetal ture, and function. metabolic phenotype including minimal expression of PGC-1 coactivators and targets (Lehman et al. 2000). Pathway analysis revealed that 38 pathways were up- PGC-1␣ compensates for the loss of PGC-1␤ regulated by PGC-1␤, 26 of which were also regulated by The lack of a cardiac phenotype in PGC-1␤−/− mice un- PGC-1␣ (Fig. 1C). As expected, mitochondrial-associated der nonstressed conditions strongly suggested that com- pathways were predominantly targeted by both coacti- pensatory mechanisms are activated, possibly through vators (Supplemental Table 2). Mitochondrial targets the actions of the related coactivator, PGC-1␣.Asan shared by the coactivators include genes involved in initial step to explore this possibility, levels of PGC-1␣ fatty acid oxidation (FAO), the TCA cycle, OXPHOS, gene expression were determined in several relevant and glucose oxidation (Supplemental Table 3). Notably,

Figure 1. PGC-1␣ and PGC-1␤ drive a significant 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).

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PGC-1␣/␤ deficiency causes cardiac death

Table 1. Survival rates of the PGC-1␣␤−/− mice taken before death were normal, suggesting that the ab- (At weaning) normalities were secondary, possibly to congestive heart failure (Supplemental Fig. 6A). In addition, postmortem Genotype Expected Observed gross and histologic analyses of the PGC-1␣␤−/− mice did not reveal overt abnormalities in brain, liver, and kidney ␣−/− 27 (25%) 49 (45%) ␣ −/−, ␤+/−, ␤+/− 55 (50%) 60 (55%) (Supplemental Fig. 6B,C; data not shown). The PGC- ␣␤−/− ␣␤−/− 27 (25%) 0 (0%) P < 0.01 1 hearts were significantly smaller than the hearts from the other genotypes, but did not exhibit any gross (At birth) morphologic abnormalities; all four chambers were Genotype Expected Observed present, the great vessels were intact and in the proper orientation, and the ductus arteriosus was appropriately ␣−/− 78 (25%) 77 (25%) closed post-birth (data not shown). −/− +/− +/− ␣ , ␤ , ␤ 157 (50%) 157 (50%) PGC-1 loss-of-function studies conducted in isolated ␣␤−/− 78 (25%) 79 (25%) adipocytes have strongly suggested that the presence of Results are expressed as the total number of animals found with either PGC-1␣ or PGC-1␤ is necessary for the differen- either genotype and the percentage of the total. Percentage tiation and mitochondrial maturation of brown adipo- (rounded) is given in parentheses. cytes in vitro (Uldry et al. 2006). Accordingly, we char- acterized BAT structure and function in the doubly mutant mice. Lipid droplet size and density appeared greater 70.5% of genes involved in mitochondrial metabolism in brown adipocytes of PGC-1␣−/−, PGC-1␤−/−, and PGC- up-regulated by PGC-1␤ were also induced by PGC-1␣ 1␣␤−/− mice compared with the wild-type control, with (Fig. 1D). These results suggest that there is significant the PGC-1␣␤−/− group being slightly more abnormal overlap in the cardiac metabolic gene targets regulated (Supplemental Fig. 7A). Triglyceride content in BAT by PGC-1␣ and PGC-1␤. from all four genotypes paralleled the histologic results We next sought to evaluate the functional redundancy (Supplemental Fig. 7B). Electron microscopy coupled of the two coactivators by generating mice with targeted with quantitative morphometry demonstrated that deactivation of all four PGC-1␣ and PGC-1␤ alleles whereas BAT mitochondrial volume density was not dif- (PGC-1␣␤−/− mice). For these studies, PGC-1␤+/− mice on ferent in PGC-1␣−/− and PGC-1␤−/− BAT compared with a PGC-1␣-null background (PGC-1␣−/−␤+/−) were gener- wild-type control, it was significantly lower in PGC- ated and intercrossed to generate double-deficient off- 1␣␤−/− mice (Fig. 3A,B). The mitochondria of PGC-1␣␤−/− spring (PGC-1␣␤−/−). Of 109 pups generated from the BAT also exhibited reduced cristae density (Fig. 3A). PGC-1␣−/−␤+/− × PGC-1␣−/−␤+/− crosses, no double ho- Consistent with the ultrastructural findings, BAT ex- mozygous null mice (PGC-1␣␤−/−) were found at wean- pression of key PGC-1 mitochondrial gene targets (cyto- ing. Furthermore, the PGC-1␣−/−␤+/− mice that survived chrome c, somatic, Cycs; cytochrome oxidase 4, Cox4; until weaning were not found in the expected 2:1 ratio and ATP synthase, H+ transporting, mitochondrial F1 with PGC-1␣−/− mice (Table 1, top), indicating that the complex, ␤ polypeptide, Atp5b) was significantly re- PGC-1␣␤−/− genotype is lethal. To determine the age of duced in PGC-1␣␤−/− mice compared with the other death in the PGC-1␣␤−/− group, timed breedings were genotypes (Fig. 3C). To assess functional correlates of the performed. Inspection of birth sacs during the late fetal BAT histologic and gene expression results, cold expo- period did not reveal any evidence of embryonic lethal- sure studies were conducted with 5- to 6-wk-old PGC- ity. At birth, all of the PGC-1␣␤−/− mice were viable and 1␣−/−␤+/− mice. Triple allele mutants were used because genotyping revealed the expected Mendelian ratio (Table the PGC-1␣␤−/− mice did not survive. After 2 h, the core 1, bottom), although their birth weights were less than temperature of PGC-1␣−/−␤+/− mice dropped signifi- the other genotypes (Supplemental Fig. 4). However, the cantly lower than age- and sex-matched wild-type, PGC- majority (∼70%) of PGC-1␣␤−/− pups died within 24 h of 1␣−/−, or PGC-1␤−/− mice (Fig. 3D). Taken together, these birth and all died within 14 d (Fig. 2). These results dem- results suggest that all four alleles are necessary for a onstrate the importance of having at least one PGC-1␣ or normal adaptive thermogenic response, but that some 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 values were not significantly different among the four genotypes 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 Figure 2. Deficiency of both PGC-1␣ and PGC-1␤ is lethal. genotypes following a 4-h fast (Supplemental Fig. 5B). Mortality curve depicting the percent survival of male and fe- −/− Postmortem histologic analyses of the PGC-1␣␤ lungs male PGC-1␣−/− (diamonds, n = 55) and PGC-1␣␤-deficient revealed evidence of alveolar collapse, whereas sections (PGC-1␣␤−/−, squares, n = 31) pups 28 d after birth.

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Lai et al.

Figure 3. BAT phenotype in PGC-1␣␤-deficient 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 label denotes the vertical column below it. Bars: top row, 2 µm; bottom row, 500 nm. (B) Quantitative morphometric measurements 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, somatic (Cycs), cytochrome oxidase 4 (Cox4), ATP synthase, H+ transporting, mitochondrial F1 complex, ␤ polypeptide (Atp5b). The mRNA levels were normalized to 18s rRNA content, and expressed relative to PGC-1␣␤+/+ values. Bars represent 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 ␤−/−. functional overlap exists between the two PGC-1 coac- nents of diastolic LV filling revealed that they were not tivator proteins for mitochondrial biogenesis. always coupled to a systolic LV outflow jet, consistent with intermittent second-degree heart block, which occurred in a variety of A:V conduction patterns including PGC-1␣␤−/− mice die of heart failure 2:1, 3:1, 4:1, 6:1, and 8:1 (Supplemental Fig. 8). To assess cardiac structure and function in the PGC- Cardiac output measurements were made in the neo- 1␣␤−/− mice, echocardiographic and Doppler studies natal mice by measuring aortic diameter and blood flow were performed at ∼12 h after birth. Both absolute LV velocity via Doppler in the proximal portion of the de- mass (LVM) and LVM corrected for body weight (LVMI) scending aorta. Cardiac output was markedly reduced in were significantly reduced in PGC-1␣␤−/− mice com- the PGC-1␣␤−/− mice compared with the other geno- pared with wild-type, PGC-1␣−/−, and PGC-1␤−/− groups types (Fig. 4A,B), despite preservation of LV fractional consistent with a growth defect (Table 2). PGC-1␣␤−/− shortening (Table 2). To further analyze cardiac perfor- hearts also exhibited a significant reduction in mean LV mance, the Tei index, a noninvasively derived parameter internal diameter (LVID) during diastole consistent with of combined systolic and diastolic function, based on the reduced heart size (Table 2). Mean heart rate was signifi- timing of events during the cardiac cycle, was deter- cantly lower in the PGC-1␣␤−/− mice compared with the mined using the pulse wave Doppler spectra of the trans- other genotypes (Table 2). Careful inspection of the heart mitral and LV outflow tract velocities (Tei et al. 1995). rate and Doppler velocity waveforms representing the To account for the potential confounding effect of heart passive (E wave) and atrial contraction (A wave) compo- rate differences among the groups, the heart rate of wild-

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PGC-1␣/␤ deficiency causes cardiac death

Table 2. Echocardiographic parameters of the PGC-1␣␤−/− mice at birth Measure PGC-1␣␤+/+ PGC-1␣−/− PGC-1␤−/− PGC-1␣␤−/−

HR (bpm) 429 ± 14 413 ± 27 411 ± 20 316 ± 13*†# LVPWd (mm) 0.28 ± 0.005 0.27 ± 0.008 0.27 ± 0.006 0.26 ± 0.007 LVPWdi (mm/g) 0.189 ± 0.008 0.185 ± 0.006 0.196 ± 0.009 0.189 ± 0.006 LVIDd (mm) 1.59 ± 0.04 1.52 ± 0.06 1.59 ± 0.04 1.27 ± 0.03*†# LVPWs (mm) 0.49 ± 0.02 0.46 ± 0.2 0.53 ± 0.02† 0.44 ± 0.01# LVIDs (mm) 1.04 ± 0.06 1.01 ± 0.03 0.90 ± 0.03*† 0.82 ± 0.02*† LVM (mg) 6.19 ± 0.29 5.59 ± 0.52 5.94 ± 0.28 3.86 ± 0.25*†# LVMI (mg/g) 4.16 ± 0.21 3.79 ± 0.31 4.26 ± 0.25 2.80 ± 0.11*†# RWT 0.35 ± 0.01 0.36 ± 0.01 0.35 ± 0.02 0.41 ± 0.07*†# FS (%) 34.7 ± 2.6 33.5 ± 1.4 43.4 ± 1.8*† 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 shortening. type mice was lowered to that measured in the PGC- gene was deleted specifically in heart on a generalized 1␣␤−/− mice by the sinus node inhibitor, Zatebradine. PGC-1␣-deficient background (PGC-1␣−/−␤f/f/MHC-Cre) The mean Tei index was abnormal in the PGC-1␣␤−/− via Cre-recombinase-mediated excision of exons 4–6 us- mice compared with the other genotypes (Fig. 4A), re- ing the same parent targeting vector used to generate the flecting a significant abnormality in each of the three generalized PGC-1␤-null mice (strategy shown in components of the index (increased isovolumic contrac- Supplemental Fig. 9A). Cardiac specificity of the PGC-1␤ tion time and isovolumic relaxation time [IVRT], and gene deletion was achieved through the use of ␣MHC- decreased ejection time, depicted in Fig. 4B). Interest- Cre mice (Agah et al. 1997), which expresses Cre recom- ingly, the PGC-1␣-deficient heart exhibited a significant binase specifically in cardiac myocytes driven by the car- decrease in performance compared with the PGC-1␣␤+/+ diac ␣ myosin heavy chain promoter (Supplemental Fig. heart, albeit not as severe as that of the PGC-1␣␤−/− 9B; data not shown). Combined PGC-1␣−/− and cardiac- mice. Lastly, Doppler-derived parameters of diastolic specific PGC-1␤-deficient mice (PGC-1␣−/−␤f/f/MHC-Cre) filling showed decreased E/A ratio and prolonged IVRT were generated by breeding. Among the four geno- in PGC-1␣␤−/− mice, consistent with impaired ventricu- types expected in the offspring, three were viable and lar diastolic relaxation (Fig. 4A). Taken together, the car- produced with the expected 1:1:1 ratio (Supplemental diac function results indicate that the PGC-1␣␤−/− mice Table 4). Similar to the generalized PGC-1␣␤−/− mice, have markedly reduced postnatal cardiac output, likely PGC-1␣−/−␤f/f/MHC-Cre mice were born alive, 67% of the due to an inappropriately low heart rate combined with pups died within 24 h of birth, and all were dead within reduced contractile and diastolic function. 7 d (Supplemental Fig. 10). The mean echocardiographic- Despite our findings of a significant cardiac phenotype derived cardiac output measurements with the neonatal in the PGC-1␣␤−/− mice, it was possible that extracardiac PGC-1␣−/−␤f/f/MHC-Cre mice (controls, 920 ± 40 vs. PGC- effects including, but not limited to, abnormal thermo- 1␣−/−␤f/f/MHC-Cre, 479 ± 28 µL per minute) were strik- genesis contributed to the early postnatal lethality. To ingly similar to that of the PGC-1␣␤−/− mice (␣␤+/+, address this, mice were generated in which the PGC-1␤ 852 ± 52 vs. ␣␤−/−, 342 ± 47 µL per minute). These results

Figure 4. Evidence for cardiac failure in PGC-1␣␤-deficient mice. To evaluate cardiac function noninvasively in all four genotypes, high-resolution echocardiography 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 performance. (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.

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Lai et al. indicate that the PGC-1␣−/−␤f/f/MHC-Cre mice phenocopy tochondrial volume density of PGC-1␣−/− and PGC-1␤−/− the generalized PGC-1␣␤-deficient mice, providing fur- hearts was not reduced. This conclusion was also sup- ther support for the conclusion that the PGC-1␣␤−/− ported by the results of mitochondrial DNA quantifica- mice die of heart failure. tion (Supplemental Fig. 11). A variety of cellular ultrastructural abnormalities were also noted in the cardiac ventricles of PGC-1␣␤−/− Evidence for a mitochondrial maturational arrest mice. A subset of myocytes that were largely or partially in the hearts of PGC-1␣␤−/− mice devoid of mitochondria, sarcomeres, and other cellular We next sought to investigate the basis for the cardiac organelles was noted (Supplemental Fig. 12A,B). Simi- failure in the PGC-1␣␤−/− mice. Evidence for apoptosis or lar cellular and mitochondrial derangements, includ- fibrotic changes was absent based on TUNEL, caspase, ing a marked reduction in mitochondrial number and and trichrome staining (data not shown). However, elec- size, were also seen in the postnatal hearts of the tron microscopic studies demonstrated dramatic mito- PGC-1␣−/−␤f/f/MHC-Cre mice (data not shown). chondrial abnormalities in the hearts of PGC-1␣␤−/− The mitochondrial abnormalities in hearts of PGC- mice, most prominent of which was a significant dimi- 1␣␤−/− mice suggested a mitochondrial maturation ar- nution in mitochondrial number and size, consistent rest. A major surge in cardiac mitochondrial biogenesis with a defect in mitochondrial biogenesis (Fig. 5A). Car- occurs in heart during the late fetal period and continues diac mitochondria of the PGC-1␣␤−/− mice also exhib- through the early postnatal stages (Hallman 1971; Smol- ited a variety of ultrastructural abnormalities including ich et al. 1989; Marin-Garcia et al. 2000). To determine vacuoles and reduced cristae density, suggesting a defect whether this perinatal mitochondrial biogenic response in biogenesis or swelling (Fig. 5A). Quantitative mor- was defective in the PGC-1␣␤−/− mice, electron micros- phometry confirmed a significant reduction in mean cel- copy studies were conducted on heart samples from mice lular mitochondrial volume density despite normal myo- at embryonic days 16.5 and 17.5 (E16.5 and E17.5) com- fibrillar volume density in the cardiac myocytes of the pared with that of postnatal day 0.5 (PD0.5) across all PGC-1␣␤−/− mice (Fig. 5B). Importantly, the myocyte mi- four genotypes. At E16.5, mitochondria were small and

Figure 5. Abnormal mitochondrial density and structure in hearts of PGC-1␣␤−/− mice. (A) Representative electron micrographs of cardiac muscle (LV free wall) at PD0.5 from wild-type (␣␤+/+), PGC-1␣−/− (␣−/−), PGC-1␤−/− (␤−/−), and PGC-1␣␤−/− (␣␤−/−) mice at three different magnifications. Each genotype label denotes the vertical 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 measurements of the cellular volume density for the mitochondrial (left) and myofibrilar (right) fractions based on analysis of electron micrographs. Bars represent mean ± SEM. (*) P < 0.05 compared with ␣␤+/+.

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PGC-1␣/␤ deficiency causes cardiac death sparse compared with postnatal ventricular sections in transferase 1b [Cpt1b], carnitine palmitoyltransferase 2 all four genotypes (Fig. 6A). In striking contrast, at PD0.5 [Cpt2]), and OXPHOS (Cycs, Cox4, and Atp5b) were sig- a marked cardiac biogenic response occurred in PGC- nificantly reduced in the PGC-1␣␤−/− hearts compared 1␣␤+/+, PGC-1␣−/−, and PGC-1␤−/− mice, but not the with wild-type controls (Fig. 7). Glucose metabolic PGC-1␣␤−/− group (Fig. 6A). At E17.5, a modest increase markers were also analyzed. Hexokinase 2 (Hk2) was in- in mitochondrial density occurs in all genotypes except creased and pyruvate dehydrogenase kinase 4 (Pdk4) was the PGC-1␣␤−/− group, indicative of a block in this late significantly decreased in the PGC-1␣␤−/− hearts, sug- prenatal surge of mitochondrial biogenesis in the doubly gesting that the programs directing the switch from re- mutant mice. Mitochondrial DNA measurements also liance on glucose during the fetal period to fatty acids as revealed the same pattern (Supplemental Fig. 13). Expres- the preferred fuel after birth was blocked. In addition, sion of the PGC-1␣ and PGC-1␤ genes is coordinately expression of several fetal cardiac gene markers not di- induced in wild-type murine heart from E15.5 to PD0.5, rectly involved in cellular energy metabolism, including in parallel with the observed mitochondrial biogenic re- atrial natriuretic factor (Nppa) and brain natriuretic pep- sponse (Fig. 6B) providing additional evidence supporting tide (Nppb), remained elevated in the PGC-1␣␤−/− car- a role for these coactivators in the perinatal mitochon- diac ventricles (Fig. 7), whereas expression of ␣ myosin drial biogenic surge. heavy chain (Mhy6), an adult sarcomeric isoform, was The small heart size and mitochondrial biogenic arrest reduced in PGC-1␣␤−/− as well as PGC-1␣−/− hearts. noted in PGC-1␣␤−/− mice strongly suggested a general- These gene marker results are consistent with a general ized defect in cardiac maturation. To further explore this arrest in cardiac maturation and suggest a regulatory link possibility, gene regulatory signatures of postnatal car- between metabolic pathways and the broad program of diac myocyte maturation were assessed in the PGC- myocyte maturation. 1␣␤−/− mice and compared with the other genotypes. Known metabolic markers of terminal maturation in- Discussion clude PGC-1 target genes involved in mitochondrial oxidative pathways such as FAO and OXPHOS. Quantita- The mammalian heart functions as a constant pump tive RT–PCR analyses revealed that the expression of throughout the life of the organism. Following birth, the genes involved in FAO (acetyl-Coenzyme A dehydroge- myocardium burns tremendous amounts of ATP daily to nase, medium chain [Acadm], acyl-Coenzyme A dehy- meet the energy demands of postnatal life. During the drogenase, very long chain [Acadvl], carnitine palmitoyl- fetal period, the heart uses mainly glucose and lactate to

Figure 6. Perinatal mitochondrial biogenesis is blocked in PGC-1␣␤−/− hearts. (A) Representative electron micrographs of cardiac 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.

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Figure 7. Cardiac gene expression markers are consistent 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-cytochrome c, somatic (Cycs), cytochrome oxidase 4 (Cox4), ATP synthase, H+ transporting, mitochondrial F1 complex, ␤ polypeptide (Atp5b); fatty acid oxidation-acetyl-Coenzyme A dehydrogenase, medium chain (Acadm), acyl-Coenzyme A dehydrogenase, very long chain (Acadvl), carnitine palmitoyltransferase 1b (Cpt1b), carnitine palmitoyltransferase 2(Cpt2); Glycolysis/Glucose oxidation-hexokinase 2 (Hk2), phosphofructokinase (Pfk), pyruvate dehydrogenase kinase 4 (Pdk4); General adult cardiac gene markers-atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 (Serca2a), and ␣-myosin heavy chain (Myh6). The mRNA levels were normalized to ␤-actin mRNA content, and expressed relative to PGC-1␣␤+/+ values. Bars represent mean ± SEM. (*) P < 0.05 compared with ␣␤+/+;(†) P < 0.05 compared with ␣−/−; (#) P < 0.05 compared with ␤−/−. generate ATP (Fisher et al. 1980, 1981; Girard et al. cient for ERR␥, a known target of PGC-1-mediated co- 1992). The enormous energy demands of the adult heart activation, were recently shown to exhibit gene regula- are met, in large part, by the oxidation of fatty acids in tory signatures consistent with a block in the cardiac mitochondria (Bing 1955; Itoi and Lopaschuk 1993; Taha perinatal switch from relying on glucose to oxidative and Lopaschuk 2007). Accordingly, to meet the rigors of metabolism (Alaynick et al. 2007). Taken together, these postnatal life, the heart undergoes a perinatal metabolic results indicate that whereas PGC-1␣ and PGC-1␤ are maturation that involves a fuel “switch” concordant not required for early formation of mitochondria, they with a dramatic increase in mitochondrial functional ca- are necessary for programs directing late fetal and post- pacity (Marin-Garcia et al. 2000; Taha and Lopaschuk natal cardiac maturation. It is possible that the related 2007). Herein, we show that the collective actions of factor PRC is compensating for early fetal development PGC-1␣ and PGC-1␤ comprise a critical component of processes. In addition, given that our original PGC-1␣ the molecular circuitry that drives the perinatal mito- gene ablation strategy did not exclude the possible pro- chondrial biogenesis necessary for metabolic and func- duction of a smaller mutant PGC-1␣ protein (Leone et al. tional maturation of the murine heart and BAT. 2005), it is theoretically possible that a small amount of Our results indicate that key late fetal and perinatal residual PGC-1␣ activity compensates during early de- cardiac developmental events are not activated in the velopmental stages in the PGC-1␣␤−/− mice. PGC-1␣␤−/− mice. The heart chambers and great vessels Our results strongly suggest that PGC-1␣ and PGC-1␤ of the PGC-1␣␤−/− mice were overtly normal, suggesting share a subset of key gene targets and functions, at least that major early fetal developmental events were unim- in heart and BAT. This conclusion is supported by the paired. However, the hearts of the double mutant ani- following lines of evidence: (1) PGC-1␣ gene expression mals are small and the dramatic mitochondrial biogenic is induced in BAT and heart of PGC-1␤-deficient mice, response known to occur at the time of birth was found suggesting a compensatory response in this context; (2) to be completely absent in the PGC-1␣␤−/− mice. The the severity of the cold intolerance phenotype and BAT doubly deficient animals also exhibited evidence of an mitochondrial derangements increases in parallel with immature conduction system, including bradycardia and the number of deleted PGC-1␣ and PGC-1␤ alleles, re- heart block. Consistent with this conclusion, mice defi- sults that are consistent with the findings of a previous

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PGC-1␣/␤ deficiency causes cardiac death study conducted in isolated brown adipocytes (Uldry et this latter conclusion, inborn errors in mitochondrial al. 2006); (3) gene expression profiling in cardiac myo- FAO enzymes are an important cause of inherited car- cytes demonstrated a high degree of overlap in PGC-1␣ diomyopathy in children (Kelly and Strauss 1994). In ad- and PGC-1␤ target pathways, especially those involved dition, mice with targeted ablation of the gene encoding in mitochondrial metabolism; and (4) whereas the PGC- ERR␥, a known target of PGC-1␣ and ␤ (Huss et al. 2002; 1␤-deficient mice and PGC-1␣-deficient mice survive Kamei et al. 2003; Schreiber et al. 2003), die early after with a minimal cardiac phenotype, combined deficiency birth with a small heart and reduced expression of results in 100% postnatal lethality due to heart failure. genes involved in myocardial FAO (Alaynick et al. 2007). Further analysis of our gene expression array results also Lastly, mice with cardiac-specific deficiency of PGC-1␤ provided some idea about which of the downstream tran- in a generalized PGC-1␣-deficient background (PGC- scription factors may be involved in the shared PGC-1 1␣−/−␤f/f/MHC-Cre mice) phenocopy the generalized PGC- functions. A significant subset of genes activated by both 1␣␤−/− mice, providing additional support for the conclu- coactivators (Supplemental Tables 2, 3) are also direct sion that the lethal phenotype is caused by cardiac de- targets for ERR␣ and ERR␥, known PGC-1 coactivating rangements. However, given the severe abnormalities targets in cardiac myocytes based on gene expression found in the BAT of the PGC-1␣␤−/− mice, it is possible profiling and ChIP-chip promoter occupation assays (Du- that this phenotype is also incompatible with postnatal four et al. 2007). This comparative analysis reveals that a survival. significant number of ERR target genes involved in mitochondrial FAO, respiration, and ATP synthesis were also shown to be activated by PGC-1␣ and PGC-1␤ in Materials and methods this study. These results suggest that the profound cardiac mitochondrial phenotype that occurs in the PGC- Generation of generalized PGC-1␤-null and cardiac-specific 1␣␤−/− mice is related to deactivation of the ERR gene PGC-1␤-null mice regulatory pathway. Sv129 genomic DNA was used as a template to create three Although these results indicate significant gene target amplicons using PCR, which were subsequently inserted into and functional redundancy, evidence for complementary pGKNeo-p1339 (GenBank Accession #AF335420). The 3Ј am- PGC-1 coactivator-specific roles also exist. For example, plicon also contained an engineered LoxP site that was used to the stress-induced phenotypes of the single PGC-1-null excise exons 4, 5, and 6 via Cre recombinase. The construct was mice indicate that both coactivators are necessary to linearized with XhoI and electroporated into SCC10 ES cells meet the full range of physiological demands imposed on (derived from RW4) using G418 selection. The clones were screened by Southern blotting and PCR. One clone out of 216 postnatal life. Cold exposure, treadmill exercise, or fast- screened was positive for the recombination event. This clone ing precipitate phenotypes in the single PGC-1 gene de- was injected into a C57BL6/J blastocyst. Germ-line transmis- letion mice (Lin et al. 2004; Leone et al. 2005; Lelliott et sion was confirmed by coat color as well as PCR analysis of tail al. 2006; Vianna et al. 2006; Sonoda et al. 2007). In addi- DNA. The female mice containing the targeted allele were bred tion, the results of studies focused on noncardiac tissues with the male EIIa-Cre mice to generate both complete and have suggested that PGC-1␤ drives a subset of programs conditional knockout mice. The offspring were screened by distinct from that of PGC-1␣, including hepatic lipogen- Southern blotting and PCR. Mice that contained the recombi- esis and cholesterol metabolism (Lin et al. 2005) and nation allele with the exon 4–6 cassette as well as the neomycin skeletal muscle IIx fiber type determination (Mortensen cassette removed were chosen for generation of the generalized ␤−/− et al. 2006; Arany et al. 2007). Single and combined PGC- “knockout” (PGC-1 ). Mice that had only the neomycin cassette removed were chosen to create the conditional knockout. 1␣ and PGC-1␤ loss-of-function studies conducted in Mice were maintained in the hybrid background, C57BL6/ brown adipocytes in culture have also revealed comple- J × sv129. Littermates were used whenever possible (as indi- mentary effects on mitochondrial function (Uldry et al. cated in the text) to control for strain effects. 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 Generation of PGC-1a␤ double-deficient mice coactivator-specific patterns based on the specific physi- Mice with a single gene deletion of either PGC-1␣ (Leone et al. ological stimulus among tissues or cell types. 2005) or PGC-1␤ were bred to generate compound heterozygous Extensive phenotyping revealed that the early postna- (PGC-1␣+/−␤+/−) mice, which in turn were crossed to generate tal death of the PGC-1␣␤−/− mice is due to severe cardiac mice deficient for PGC-1␣ and heterozygous for PGC-1␤ (PGC- −/− +/− dysfunction. The PGC-1␣␤−/− hearts exhibited intermit- 1␣ ␤ ). These mice were used as breeders to generate the tent second-degree AV block and generated a very low offspring used in this study. Mice were maintained in a hybrid background (C57BL6/J × sv129) and littermates for PGC-1␣−/− cardiac output. The basis for the low cardiac output and PGC-1␣␤−/− were used for comparative analysis to control likely relates to the relatively small size of the heart, for genetic background effects. PGC-1␤−/− and PGC-1␣␤+/+ mice bradycardia, and reduced contractile function. The con- were generated with separate breeding pairs. tractile dysfunction is likely due, at least in part, to reduced capacity for mitochondrial ATP production related to immature mitochondria and a block in the post- Animal phenotyping studies natal induction of genes involved in FAO, the chief All animal experiments and euthanasia protocols were con- source of energy in the postnatal period. In support of ducted in strict accordance with the National Institutes of

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Health guidelines for humane treatment of animals and were reverse transcribed with Taqman reverse transcription reagents reviewed and approved by the Institutional Animal Care and (Applied Biosystems). PCR reactions were performed in tripli- Use Committee of Washington University School of Medicine. cate in a 96-well format using a Prism 7500 Sequence Detector Animals were weighed at different time points between 2 and (Applied Biosystems). The mouse-specific primer-probe sets 8 wk of age and compared directly to their sex-matched litter- used to detect specific gene expression can be found in Supple- mates. For cold exposure experiments, male and female PGC- mental Table 5. Either ␤-actin (Applied Biosystems), 36B4, or 1␤+/+ and PGC-1␤−/− mice were singly housed and placed for 3–4 18s primer-probe sets (Supplemental Table 5) was included in a hat4°C without food. Core body temperatures were monitored separate well (in triplicate) and used to normalize the gene ex- by rectal probe at baseline and every hour thereafter. Mice were pression data as noted in the figure legends. monitored at least every 30 min to check for lethargy. At the Genomic/mitochondrial DNA was isolated using RNAzol, end of 4 h, mice were sacrificed and tissues harvested for RNA followed by back extraction with 4 M guanidine thiocyanate, 50 and protein extraction. For fasting studies, animals were singly mM sodium citrate, and 1 M tris, and an alcohol precipitation. housed and given water ad libitum. Food was removed from Mitochondrial DNA content was determined by SYBR green cages in the morning and tissues harvested at 36 h for RNA and analysis (Applied Biosystems). To this end, the levels of NADH histology. For prenatal and mortality curve studies, timed dehydrogenase subunit 1 (mitochondrial DNA) were normal- breedings were performed, and pregnancy was determined by ized to the levels of lipoprotein lipase (genomic DNA). The detection of a vaginal plug (E0.5). The time of birth was closely primer sequences are noted in Supplemental Table 5. monitored, and newborns were counted and genotyped within For Southern blot analysis, 5 µg of genomic DNA was di- 12 h after birth for ␹2 analysis. Postnatal survival was followed gested with SpeI, electrophoresed on a 0.8% TAE gel, and trans- daily for 28 d. Blood glucose in newborns was measured with a ferred to Gene Screen (Perkin Elmer) membrane for hybridiza- One-Touch Ultra glucometer (LifeScan, Inc.). Blood lactate in tion. Western blotting was performed as described (Cresci et al. newborns was measured with a Lactate Pro blood lactate test 1996) using Super Signal West Dura Extended Duration Sub- meter (ARKRAY, Inc.). strate (Pierce) for detection. The polyclonal PGC-1␤ antibody For the low-intensity exercise studies, 9-wk-old female PGC- was a generous gift provided by Dr Anastasia Kralli. The PGC- 1␤+/+ (n = 6) and PGC-1␤−/− (n = 6) mice were run to exhaustion 1␣ antibody has been previously described (Lehman et al. 2000). using a motorized, speed-controlled modular treadmill system Total tissue triglyceride analysis was performed by the Ani- (Columbus Instruments). The treadmill was equipped with an mal Model Research Core at Washington University School of electric shock stimulus and an adjustable inclination angle. Medicine (supported by the CNRU) from frozen tissue using a Running velocity was set at 10 m/min for an hour, and in- modified Bligh and Dyer technique as described previously creased by 2 m/min increments every 15 min until exhaustion (Bligh and Dyer 1959). was achieved. For echocardiographic studies performed on adult mice, Mitochondrial respiration studies 2-mo-old female PGC-1␤+/+ (n = 5) and PGC-1␤−/− (n = 5) mice were lightly anesthetized with an intraperitoneal injection of Mitochondrial respiration was assessed in isolated mitochon- 3% avertin (tribromoethanol, 0.01 mL/g). Cardiac ultrasound dria from the hindlimb muscle with pyruvate as substrate as studies were performed as described previously (Rogers et al. described previously (Bhattacharya et al. 1991). In brief, 3-mo-

1999). Exercise echocardiography was performed on a motorized old male mice were euthanized by CO2 inhalation. The entire treadmill as described previously at a duration tolerated by the hindlimb was dissected from the bone and minced well, fol- PGC-1␤−/− mice (1–1.5 min) (Leone et al. 2005). lowed by a 5-min incubation in Ionic Medium (IM) plus Nagarse For neonatal echocardiography, male and female pups were (100 mM sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46 mM Kcl imaged within 12 h after birth using a Vevo 770 ultrasound at pH 7.4, 10 mg nagarse). Samples were homogenized using an system (Visual Sonics, Inc.). Unanesthetized mice were placed Eberbach homogenizer, centrifuged at 500g for 10 min at 4°C, on an imaging table under a heating lamp, and were lightly and supernatant transferred to a clean tube. The supernatant restrained in a left lateral decubitus position. Parasternal long- was centrifuged at 12,000g and the pellet was resuspended in and short-axis images of the heart were obtained from standard IM + 0.5% BSA. The sample was spun again and the pellet re- echocardiographic views. Semi-apical long-axis views of the LV suspended in Suspension Buffer (230 mM mannitol, 70 mM su- were used to interrogate the combined trans-mitral and LV out- crose, 0.02 mM EDTA, 20 mM Tris-HCl, 5 mM K2HPO4 at pH flow tract blood flow velocities. Basal short-axis views were 7.4). Total protein was quantified by a BCA assay (Pierce) and used to image the pulmonary artery and the proximal portion of respiration was performed at 25°C using an optical probe (Oxy- the descending thoracic aorta. Pulse wave Doppler sample vol- gen FOXY Probe, Ocean Optics). Following measurement of ume was placed parallel with the direction of blood flow, and basal respiration, maximal (ADP-stimulated) respiration was aortic diameter was measured at the same level. Cardiac output determined by exposing the mitochondria to 1 mM ADP. Un- was measured as the product of aortic area and velocity time coupled respiration was evaluated following addition of oligo- integral of the Doppler tracing. The Tei index was calculated as mycin (1 µg/mL). The solubility of oxygen in the respiration

(IVCT + IVRT)/LVET, where IVCT is isovolumic contraction buffer at 25°C was taken as 246.87 nmol O2 per milliliter. Res- и −1 и time (period between mitral valve closure and aortic valve open- piration rates were expressed as “nmol O2 min mg pro- ing), IVRT is isovolumic relaxation time (period between aortic tein−1.” valve closure and mitral valve opening), and LVET is the LV ejection period (time between aortic valve opening and closure). Histology and electron microscopy Adult mice were anesthetized and perfused with Karnovsky’s RNA, DNA, protein, and tissue triglyceride analyses fixative (2% glutaraldehyde, 1% paraformaldehyde, and 0.08% Total RNA was isolated from various mouse tissues using the sodium cacodylate) to avoid artifact. Neonates were euthanized RNAzol method (Tel-Test). Northern blotting (Kelly et al. 1989) and hearts were fixed in Karnovsky’s fixative. Cardiac papillary and quantitative real-time RT–PCR were performed as de- muscle (adult), LV free wall (neonates), BAT, soleus, and EDL scribed (Huss et al. 2004). In brief, total RNA was isolated and muscle were dissected and postfixed in 1% osmium tetroxide,

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PGC-1␣/␤ deficiency causes cardiac death dehydrated in graded ethanol, embedded in Poly Bed plastic Crouch, Dr. Feng Chen, and Dr. Robert Schmidt for careful resin, and sectioned for electron microscopy. Mitochondrial and analysis of the histopathology; Dr. Jeffrey Saffitz (Beth Israel myofibrillar volume densities were determined from electron Deaconess Medical Center) for consultative advice on electron micrographs as described previously (Russell et al. 2004). For microscopy; Dr. Patrick Jay for help with assessing cardiac mor- each animal, three different fields at the magnification of 7500× phology; and Mary Wingate for assistance with manuscript were quantified in blinded fashion. Data were expressed as preparation. L.L. is supported by the AHA Fellowship award mean volume density of mitochondria or myofibrils in each (0525743Z). C.Z. is a recipient of the Deutsche Forschungsge- field. meinschaft research Fellowship ZE 796/2-1. This work was sup- H&E staining was performed by the Morphology Core at ported by NIH grants RO1 DK045416, RO1 HL058427, Diges- Washington University School of Medicine (DDRCC). The tis- tive Diseases Research Core Center (P30 DK052574), Diabetes sues were fixed with 10% buffered formalin overnight, dehy- Research and Training Center (P60 DK020579), Alvin J. Siteman drated in graded concentrations of alcohol, and embedded in Cancer Center Bioinformatics Core and Embryonic Stem Cell paraffin from which 5-µm sections were prepared. Core (P30 CA91842), and Clinical Nutrition Research Unit Core Center (P30 DK56341). Gene expression profiling References Total RNA from cultured NRCM post adenoviral infection with either Ad-GFP, Ad-PGC-1␣, or Ad-PGC-1␤ was reverse tran- Agah, R., Frenkel, P.A., French, B.A., Michael, L.H., Overbeek, scribed using Superscript II (Invitrogen Corp.) and primed with P.A., and Schneider, M.D. 1997. Gene recombination in T7 promoter-polyA primer (T7T24), followed by second-strand postmitotic cells. Targeted expression of cre recombinase synthesis according to the manufacturer’s protocol. Biotin-la- provokes cardiac-restricted, site-specific rearrangement in beled cRNA was synthesized using T7-coupled ENZO BioArray adult ventricular muscle in vivo. J. Clin. Invest. 100: 169– High Yield RNA Transcript Labeling Kit (ENZO Diagnostics, 179. Inc.). The Alvin J. Siteman Cancer Center’s Bioinformatics Core Alaynick, W.A., Kondo, R.P., Xie, W., He, W., Dufour, C.R., at Washington University School of Medicine performed hy- Downes, M., Jonker, J.W., Giles, W., Naviaux, R.K., Giguère, bridization to Affymetrix Rat Expression Set 230 chip. Af- V., et al. 2007. ERR␥ directs and maintains the transition to fymetrix MAS 5.0 software was used for initial analysis and oxidative metabolism in the postnatal heart. Cell Metab. 6: background normalization, and Z score calculation and subse- 13–24. quent data analysis were performed using Spotfire DecisionSite Andersson, U. and Scarpulla, R.C. 2001. PGC-1-related coacti- for Functional Genomics 9.0. Probe sets called “absent” by vator, a novel, serum-inducible coactivator of nuclear respi- MAS 5.0 in all Ad-GFP control, Ad-PGC-1␣, and Ad-PGC-1␤ ratory factor-1-dependent transcription in mammalian cells. were excluded. Two independent trials were performed. Stu- Mol. Cell. Biol. 21: 3738–3749. dent’s t-test was performed and a P-value <0.05 was used to Arany, Z., Novikov, M., Chin, S., Ma, Y., Rosenzweig, A., and determine genes significantly up-regulated. Signal intensity ra- Spiegelman, B.M. 2006. Transverse aortic constriction leads tios were averaged from both trials and were calculated as ei- to accelerated heart failure in mice lacking PPAR␥ coactiva- ther Ad-PGC-1␣/Ad-GFP or Ad-PGC-1␤/Ad-GFP to determine tor 1␣. Proc. Natl. Acad. Sci. 103: 10086–10091. changes due to exogenous expression of either PGC-1␣ or PGC- Arany, Z., Lebrasseur, N., Morris, C., Smith, E., Yang, W., Ma, 1␤. A gene with a calculated fold change Ն1.5 was considered an Y., Chin, S., and Spiegelman, B.M. 2007. The transcriptional up-regulated gene target in cultured NRCM. For pathway analy- coactivator PGC-1␤ drives the formation of oxidative type sis, the filtered data sets were uploaded into GenMAPP software IIX fibers in skeletal muscle. Cell Metab. 5: 35–46. to review the biopathways using the Gene Ontology database. Bhattacharya, S.K., Thakar, J.H., Johnson, P.L., and Shanklin, GenMAPP produced a ranked list of Gene Ontology biological D.R. 1991. Isolation of skeletal muscle mitochonria using an categories based on the following criteria: (1) at least five regu- ionic medium containing ethylenediaminetetraacetic acid lated genes in selected GO terms; (2) >50% of genes regulated in and nagarse. Anal. Biochem. 192: 344–349. selected pathways (>50 “percent changed”). The “percent Bing, R.J. 1955. The metabolism of the heart. Harvey Lect. 50: changed” was calculated by “the genes meeting the criteria/ 27–70. genes measured * 100.” The Z score was calculated by subtract- Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid ing the expected number of genes meeting the criteria from the extraction and purification. Can. J. Biochem. Physiol. 37: observed number, and then dividing by the standard deviation of 911–917. the observed number of genes. Buroker, N.E., Ning, X.-H., and Portman, M. 2008. Cardiac PPAR␣ protein expression is constant as alternate nuclear receptors and PGC-1 coordinately increase during the post- Statistical analysis natal metabolic transition. PPAR Research 2008: 279531. Data were analyzed using t-tests or ANOVA where appropriate. doi: 10.1155/2008/279531. The level of significance was set at P < 0.05 in all cases. Data are Cresci, S., Wright, L.D., Spratt, J.A., Briggs, F.N., and Kelly, D.P. reported as mean values ± the standard error of the mean, unless 1996. Activation of a novel metabolic gene regulatory path- otherwise noted. way by chronic stimulation of skeletal muscle. Am. J. Phys- iol. 270: C1413–C1420. Dufour, C.R., Wilson, B.J., Huss, J.M., Kelly, D.P., Alaynick, Acknowledgments W.A., Downes, M., Evans, R.M., Blanchette, M., and Giguère, V. 2007. Genome-wide orchestration of cardiac We thank Dr. Anastasia Kralli (Scripps) for providing the PGC- functions by orphan nuclear receptors ERR␣ and ␥. Cell Me- 1␤ antibody; Juliet Fong, Michelle Trusgnich, and Alicia Wallis tabolism 5: 345–356. for help with mouse husbandry; William Kraft for expert tech- Finck, B.N. and Kelly, D.P. 2006. PGC-1 coactivators: Inducible nical assistance with electron microscopy; Mike Courtois for regulators of energy metabolism in health and disease. J. assistance with echocardiography; Dr. Suellen Greco, Dr. Erica Clin. Invest. 116: 615–622.

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Lai et al.

Fisher, D.J., Heymann, M.A., and Rudolph, A.M. 1980. Myocar- N., Bernal-Mizrachi, C., et al. 2005. PGC-1␣ deficient mice dial oxygen and carbohydrate consumption in fetal lambs in exhibit multi-system energy metabolic derangements: Mus- utero and in adult sheep. Am. J. Phys. 238: H399–H405. cle dysfunction, abnormal weight control, and hepatic stea- Fisher, D.J., Heymann, M.A., and Rudolph, A.M. 1981. Myocar- tosis. PLoS Biol. 3: 672–687. dial consumption of oxygen and carbohydrates in newborn Lin, J., Puigserver, P., Donovan, J., Tarr, P., and Spiegelman, sheep. Pediatr. Res. 15: 843–846. B.M. 2002a. Peroxisome proliferator-activated receptor ␥ co- Girard, J., Ferré, P., Pégorier, J.P., and Duée, P.H. 1992. Adapta- activator 1␤ (PGC-1␤), a novel PGC-1-related transcription tions of glucose and fatty acid metabolism during perinatal coactivator associated with host cell factor. J. Biol. Chem. period and suckling–weaning transition. Physiol. Rev. 72: 277: 1645–1648. 507–562. Lin, J., Wu, H., Tarr, P.T., Zhang, C.Y., Wu, Z., Boss, O., Mi- Hallman, M. 1971. Changes in mitochondrial respiratory chain chael, L.F., Puigserver, P., Isotanni, E., Olson, E.N., et al. proteins during perinatal development. Evidence of the im- 2002b. Transcriptional co-activator PGC-1␣ drives the for- portance of environmental oxygen tension. Biochim. Bio- mation of slow-twitch muscle fibers. Nature 418: 797–801. phys. Acta 253: 360–372. Lin, J., Wu, P.-H., Tarr, P.T., Lindenberg, K.S., St-Pierre, J., Handschin, C. and Spiegelman, B.M. 2006. Peroxisome prolif- Zhang, C.-Y., Mootha, V.K., Jãger, S., Vianna, C.R., Reznick, erator-activated receptor ␥ coactivator 1 coactivators, energy R.M., et al. 2004. Defects in adaptive energy metabolism homeostasis, and metabolism. Endocr. Rev. 27: 728–735. with CNS-linked hyperactivity in PGC-1␣ null mice. Cell Herzig, S., Long, F., Jhala, U.S., Hedrick, S., Quinn, R., Bauer, A., 119: 121–135. Rudolph, D., Schutz, G., Yoon, C., Puigserver, P., et al. 2001. Lin, J., Yang, R., Tarr, P.T., Wu, P.-H., Handschin, C., Li, S., CREB regulates hepatic gluconeogenesis through the coacti- Yang, W., Pei, L., Uldry, M., Tontonoz, P., et al. 2005. Hy- vator PGC-1. Nature 413: 179–183. perlipidemic effects of dietary saturated fats mediated Huss, J.M., Kopp, R.P., and Kelly, D.P. 2002. PGC-1␣ coacti- through PGC-1␤ coactivation of SREBP. Cell 120: 261–273. vates the cardiac-enriched nuclear receptors estrogen-related Marin-Garcia, J., Ananthakrishnan, R., and Goldenthal, M.J. receptor-␣ and -␥. J. Biol. Chem. 277: 40265–40274. 2000. Heart mitochondrial DNA and enzyme changes during Huss, J.M., Pinéda Torra, I., Staels, B., Giguère, V., and Kelly, early human development. Mol. Cell. Biochem. 210: 47–52. D.P. 2004. ERR␣ directs PPAR␣ signaling in the transcrip- Michael, L.F., Wu, Z., Cheatham, R.B., Puigserver, P., Adel- tional control of energy metabolism in cardiac and skeletal mant, G., Lehman, J.J., Kelly, D.P., and Spiegelman, B.M. muscle. Mol. Cell. Biol. 24: 9079–9091. 2001. Restoration of insulin-sensitive glucose transporter Itoi, T. and Lopaschuk, G.D. 1993. The contribution of glycol- (GLUT4) gene expression in muscle cells by the transcrip- ysis, glucose oxidation, lactate oxidation, and fatty acid oxi- tional coactivator PGC-1. Proc. Natl. Acad. Sci. 98: 3820– dation to ATP production in isolated biventricular working 3825. hearts from 2-week-old rabbits. Pediatr. Res. 34: 735–741. Mortensen, O.H., Frandsen, L., Schjerling, P., Nishimura, E., Kamei, Y., Ohizumi, H., Fujitani, Y., Nemoto, T., Tanaka, T., and Grunnet, N. 2006. PGC-1␣ and PGC-1␤ have both simi- Takahashi, N., Kawada, T., Miyoshi, M., Ezaki, O., and Kaki- lar and distinct effects on myofiber switching toward an oxi- zuka, A. 2003. PPAR␥ coactivator 1␤/ERR ligand 1 is an ERR dative phenotype. Am. J. Phys. Endocrinol. Metab. 4: E807– protein ligand, whose expression induces a high-energy ex- E816. doi: 10.1152/ajpendo.00591.2005. penditure and antagonizes obesity. Proc. Natl. Acad. Sci. Puigserver, P. and Spiegelman, B.M. 2003. Peroxisome prolifera- 100: 12378–12383. tor-activated receptor-␥ coactivator 1␣ (PGC-1␣): Transcrip- Kelly, D.P. and Strauss, A.W. 1994. Inherited cardiomyopathies. tional coactivator and metabolic regulator. Endocr. Rev. 24: N. Engl. J. Med. 330: 913–919. 78–90. Kelly, D.P., Gordon, J.I., Alpers, R., and Strauss, A.W. 1989. The Puigserver, P., Wu, Z., Park, C.W., Graves, R., Wright, M., and tissue-specific expression and developmental regulation of Spiegelman, B.M. 1998. A cold-inducible coactivator of the two nuclear genes encoding rat mitochondrial proteins: nuclear receptors linked to adaptive thermogenesis. Cell 92: Medium-chain acyl-CoA dehydrogenase and mitochondrial 829–839. malate dehydrogenase. J. Biol. Chem. 264: 18921–18925. Puigserver, P., Adelmant, G., Wu, Z., Fan, M., Xu, J., O’Malley, Koo, S.H., Satoh, H., Herzig, S., Lee, C.H., Hedrick, S., Kulkarni, B., and Spiegelman, B.M. 1999. Activation of PPAR␥ coacti- R., Evans, R.M., Olefsky, J., and Montminy, M. 2004. PGC-1 vator-1 through transcription factor docking. Science 286: promotes insulin resistance in liver through PPAR␣-depen- 1368–1371. dent induction of TRB-3. Nat. Med. 10: 530–534. Puigserver, P., Rhee, J., Donovan, J., Walkey, C.J., Yoon, J.C., Kressler, D., Schreiber, S.N., Knutti, D., and Kralli, A. 2002. The Oriente, F., Kitamura, Y., Altomonte, J., Dong, H., Accili, D., PGC-1-related protein PERC is a selective coactivator of es- et al. 2003. Insulin-regulated hepatic gluconeogenesis trogen receptor ␣. J. Biol. Chem. 277: 13918–13925. through FOXO1-PGC-1␣ interaction. Nature 423: 550–555. Lehman, J.J. and Kelly, D.P. 2002. Transcriptional activation of Rogers, J.H., Tamirisa, P., Kovacs, A., Weinheimer, C., Cour- energy metabolic switches in the developing and hypertro- tois, M., Blumer, K.J., Kelly, D.P., and Muslin, A.J. 1999. phied heart. Clin. Exp. Pharmacol. Physiol. 29: 339–345. RGS4 causes increased mortality and reduced cardiac hyper- Lehman, J.J., Barger, P.M., Kovacs, A., Saffitz, J.E., Medeiros, D., trophy in response to pressure overload. J. Clin. Invest. 104: and Kelly, D.P. 2000. PPAR␥ coactivator-1 (PGC-1) pro- 567–576. motes cardiac mitochondrial biogenesis. J. Clin. Invest. 106: Russell, L.K., Mansfield, C.M., Lehman, J.J., Kovacs, A., Cour- 847–856. tois, M., Saffitz, J.E., Medeiros, D.M., Valencik, M.L., Mc- Lelliott, C.J., Medina-Gomez, G., Petrovic, N., Kis, A., Feld- Donald, J.A., and Kelly, D.P. 2004. Cardiac-specific induc- mann, H.M., Bjursell, M., Parker, N., Curtis, K., Campbell, tion of the transcriptional coactivator peroxisome prolifera- M., Hu, P., et al. 2006. Ablation of PGC-1␤ results in defec- tor-activated receptor ␥ coactivator-1␣ promotes mito- tive mitochondrial activity, thermogenesis, hepatic func- chondrial biogenesis and reversible cardiomyopathy in a de- tion, and cardiac performance. PLoS Biol. 4: 2042–2056. velopmental stage-dependent manner. Circ. Res. 94: 525– Leone, T.C., Lehman, J.J., Finck, B.N., Schaeffer, P.J., Wende, 533. A.R., Boudina, S., Courtois, M., Wozniak, D.F., Sambandam, Sano, M., Tokudome, S., Shimizu, N., Yoshikawa, N., Ogawa,

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PGC-1␣/␤ deficiency causes cardiac death

C., Shirakawa, K., Endo, J., Katayama, T., Yuasa, S., Ieda, M., et al. 2007. Intramolecular control of protein stability, sub- nuclear compartmentalization, and coactivator function of peroxisome proliferator-activated receptor ␥ coactivator 1␣. J. Biol. Chem. 282: 25970–25980. Schreiber, S.N., Knutti, D., Brogli, K., Uhlmann, T., and Kralli, A. 2003. The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor estrogen-related receptor ␣ (ERR␣). J. Biol. Chem. 278: 9013– 9018. Smolich, J.J., Walker, A.M., Campbell, G.R., and Adamson, T.M. 1989. Left and right ventricular myocardial morphometry in fetal, neonatal, and adult sheep. Am. J. Phys. 257: 1–9. Sonoda, J., Mehl, I.R., Chong, L.W., Nofsinger, R.R., and Evans, R.M. 2007. PGC-1␤ controls mitochondrial metabolism to modulate circadian activity, adaptive thermogenesis, and hepatic steatosis. Proc. Natl. Acad. Sci. 104: 5223–5228. St-Pierre, J., Lin, J., Krauss, S., Tarr, P.T., Yang, R., Newgard, C.B., and Spiegelman, B.M. 2003. Bioenergetic analysis of peroxisome proliferator-activated receptor ␥ coactivators 1␣ and 1␤ (PGC-1␣ and PGC-1␤) in muscle cells. J. Biol. Chem. 278: 26597–26603. Taha, M. and Lopaschuk, G.D. 2007. Alterations in energy metabolism in cardiomyopathies. Ann. Med. 39: 594–607. Tei, C., Ling, L.H., Hodge, D.O., Bailey, K.R., Oh, J.K., Rodehef- fer, R.J., Tajik, A.J., and Seward, J.B. 1995. New index of combined systolic and diastolic myocardial performance: A simple and reproducible measure of cardiac function-a study in normals and dilated cardiomyopathy. J. Cardiol. 26: 357– 366. Uldry, M., Yang, W., St-Pierre, J., Lin, J., Seale, P., and Spiegel- man, B.M. 2006. Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differen- tiation. Cell Metab. 3: 333–341. Vega, R.B., Huss, J.M., and Kelly, D.P. 2000. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor ␣ in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol. Cell. Biol. 20: 1868–1876. Vianna, C.R., Huntgeburth, M., Coppari, R., Choi, C.S., Lin, J., Krauss, S., Barbatelli, G., Tzameli, I., Kim, Y.B., Cinti, S., et al. 2006. Hypomorphic mutation of PGC-1␤ causes mitochondrial dysfunction and liver insulin resistance. Cell Metab. 4: 453–464. Wallberg, A.E., Yamamura, S., Malik, S., Spiegelman, B.M., and Roeder, R.G. 2003. Coordination of p300-mediated chromatin remodeling and TRAP/mediator function through coactivator PGC-1␣. Mol. Cell 12: 1137–1149. Wende, A.R., Schaeffer, P.J., Parker, G.J., Zechner, C., Han, D.H., Chen, M.M., Hancock, C.R., Lehman, J.J., Huss, J.M., McClain, D.A., et al. 2007. A role for the transcriptional coactivator PGC-1␣ in muscle refueling. J. Biol. Chem. 282: 36642–36651. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, R.C., et al. 1999. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115–124. Yoon, J.C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn, C.R., Granner, D.K., et al. 2001. Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413: 131–138.

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Transcriptional coactivators PGC-1α and PGC-lβ control overlapping programs required for perinatal maturation of the heart

Ling Lai, Teresa C. Leone, Christoph Zechner, et al.

Genes Dev. 2008, 22: Access the most recent version at doi:10.1101/gad.1661708

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