Peroxisome proliferator–activated receptor γ coactivator-1 promotes cardiac mitochondrial biogenesis (original) (raw)
PGC-1 gene expression parallels known changes in mitochondrial energy-producing capacity during perinatal development and in response to fasting. As an initial step to determine whether the inducible transcriptional coactivator PGC-1 is involved in the physiologic control of mitochondrial function in the heart, the expression of its gene was delineated in a context in which cardiac fatty acid-utilization rates, cellular mitochondrial number, and respiratory functional capacity is increased: the perinatal developmental transition (32). Mouse heart PGC-1 mRNA levels were coordinately induced on the first day after birth and during later postnatal stages in parallel with the expression of its interacting partner PPARα; two PPARα target genes encoding mitochondrial FAO enzymes, M-CPT I and medium-chain acyl-CoA dehydrogenase (MCAD); and the mitochondrial ATP synthase β gene (Figure 1). As has been described (14), two PGC-1 transcripts of approximately 5 and 8 kb were observed. The nature of the two PGC-1 transcripts is unknown, although mRNAs of similar sizes are expressed by the human PGC-1 gene as a result of differential polyadenylation (33). The expression pattern of the gene encoding the glycolytic enzyme, GAPDH, was distinct from the other genes studied, exhibiting greatest expression during the prenatal period.
Induction of PGC-1 gene expression during perinatal cardiac development. The expression of the genes encoding PGC-1, PPARα, and mitochondrial energy production–pathway enzymes during cardiac postnatal development. Representative autoradiograph of Northern blot analyses performed with total RNA (15 μg/lane) isolated from mouse hearts at several late embryonic (e), postnatal, and adult (A, 2 months old) time points using the cDNA probes denoted on the left. The ethidium bromide–stained 28S ribosomal subunit (28S) was used as a control for loading.
PGC-1 gene expression was also evaluated in response to short-term fasting, a physiologic condition known to rapidly increase cardiac mitochondrial fatty acid utilization rates (34) and FAO enzyme gene expression by PPARα-mediated transcriptional activation (23). PGC-1 mRNA levels were induced within 24 hours of the onset of fasting (Figure 2). As expected, PPARα target genes encoding mitochondrial (MCAD, M-CPT I) and peroxisomal (acyl-CoA oxidase [ACO]) FAO enzymes were also induced in the mouse heart during the fast (Figure 2). In contrast to the results observed in the heart, fasting did not induce PGC-1 gene expression in brown adipose, a mitochondrial-enriched tissue specialized for thermogenesis through uncoupled respiration (data not shown).
Induction of cardiac PGC-1 gene expression during short-term starvation. Representative autoradiograph of Northern blot analyses performed with total RNA isolated from sex-matched, adult littermate 129SvJ mice after a 24-hour fast (F) or controls fed ad libitum (C). The expression of nuclear genes encoding mitochondrial (M-CPT I, MCAD) and peroxisomal (acyl-CoA oxidase [ACO]) FAO enzymes was also assessed. The results shown are representative of three independent experiments.
Forced expression of PGC-1 in neonatal cardiac myocytes induces the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways. Gain-of-function studies were performed to determine whether PGC-1 could regulate cardiac mitochondrial oxidative capacity. Primary rat neonatal ventricular myocytes in culture were chosen for these studies because they exhibit a fetal energy metabolic phenotype, including low mitochondrial number, in contrast to the adult mammalian heart that has abundant mitochondria and a high capacity for oxidative energy production. In addition, although cultured neonatal cardiac myocytes express functional PPARα (13), PGC-1 transcripts and protein were not detectable by Northern and Western blot analyses, respectively (Figure 3a). Infection of the cardiac myocytes with an adenoviral vector designed to express a myc epitope-tagged PGC-1 (Ad-PGC-1) resulted in high-level expression of the _myc_-PGC-1 transcript and protein (Figure 3a). Expression of the endogenous PGC-1 gene was also increased in the Ad-PGC-1–infected cells (Figure 3a), suggesting the existence of an autoregulatory mechanism. Forced expression of PGC-1 markedly induced the expression of PPARα target genes encoding the mitochondrial FAO enzymes, MCAD and M-CPT I (top panels, Figure 3a). Immunodetectable MCAD protein levels were also significantly greater in the Ad-PGC-1–infected myocytes compared with control cells (bottom panels, Figure 3a).
Forced expression of PGC-1 in cultured rat neonatal cardiac myocytes induces the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways. (a) The expression of mitochondrial FAO enzyme genes. The autoradiographs represent Northern (top panels) and Western (bottom panels) analyses, respectively. The panels depict the expression of the _myc_-tagged PGC-1 and endogenous FAO enzyme genes (MCAD and M-CPT I) in rat neonatal cardiac myocytes grown in serum-free culture conditions. Total RNA (15 μg/lane) or protein (10 μg/lane) was isolated from uninfected cells (U), or 48 hours after infection with control adenovirus expressing GFP alone (C), or an adenoviral vector expressing both GFP and PGC-1 (P). As determined by GFP fluorescence, 95–100% of the total cells plated were infected (data not shown). Adenoviral epitope-tagged PGC-1 mRNA is denoted _myc_-PGC-1 and endogenous PGC-1 transcripts are labeled endogenous PGC-1. The α-actin signal is shown as a control. PGC-1 protein was not detected in extracts from control adenovirus-infected cells by Western blot analysis (bottom panels), even with prolonged exposures. (b) The expression of genes and proteins involved in the TCA cycle, electron transport, and oxidative phosphorylation. The autoradiographs shown at the top represent Northern blot studies performed with total RNA isolated from cardiac myocytes under the conditions described in a. Western blot studies (bottom) were performed with cellular protein extracts prepared from myocytes 4 days after adenoviral infection. Bov. COX, bovine COX protein standards; Cyt C, cytochrome c. All data shown are representative of at least three independent experiments.
We next sought to explore a broader role for PGC-1 in the transcriptional control of genes involved in mitochondrial pathways critical for cardiac myocyte energy production. PGC-1 induced the expression of the nuclear genes encoding a TCA cycle enzyme (citrate synthase) and components of the oxidative phosphorylation complex (β and c subunits of F1–F0 ATP synthase; Figure 3b). PGC-1–expressing myocytes also exhibited increased protein levels of nuclear-encoded (COX subunits IV, Va, Vb, and cytochrome c) and mitochondrial-encoded (COX subunit I) components of the electron transport chain (Figure 3b). Taken together, these results indicate that PGC-1 coordinately activates expression of nuclear and mitochondrial genes encoding proteins involved in multiple mitochondrial energy-transducing/energy-producing pathways.
PGC-1 induces mitochondrial biogenesis in cardiac myocytes in culture. The results of the gene and protein expression studies shown in Figure 3 are indicative of a mitochondrial biogenic response to PGC-1 overexpression. To determine whether PGC-1 promotes mitochondrial biogenesis in cardiac myocytes, a fluorescent, mitochondrion-selective dye (MitoTracker; see Methods) was used. MitoTracker staining was significantly greater in cells expressing PGC-1 compared with control cells infected with the viral backbone, consistent with increased mitochondrial capacity (Figure 4a). Electron microscopy was performed on histologic sections of pellets prepared from cardiac myocytes expressing PGC-1. Cells infected with Ad-PGC-1 exhibited increased cellular mitochondrial number compared with control myocytes (Figure 4b). Marked mitochondrial size heterogeneity was also noted in the Ad-PGC-1–infected myocytes, including the presence of very large mitochondria (Figure 4b, far right panel). Quantitative morphometric analysis demonstrated that the mean mitochondrial volume density (total mitochondrial area/total cytoplasmic area) in cardiac myocytes infected with Ad-PGC-1 was 57% higher relative to control virus-infected cells (0.36 ± 0.04, n = 15 cells, vs. 0.23 ± 0.04, n = 13 cells, μm3 per μm3 of cell cytoplasm; P < 0.05).
PGC-1 promotes mitochondrial biogenesis in cardiac myocytes. (a) Fluorescence micrograph panels representing the use of the mitochondrion-selective dye MitoTracker to estimate mitochondrial capacity in cardiac myocytes 5 days after infection with either a GFP-expressing control adenovirus (Ad-GFP) or an adenovirus expressing both GFP and PGC-1 (Ad-PGC-1). MitoTracker, which is oxidized, sequestered, and conjugated in mitochondria, is identified by the orange-red color. GFP, which confirms adenoviral infection, is seen as a nuclear-localized green color. (b) Electron micrographs obtained from sections of cultured rat neonatal cardiac myocyte pellets representing cells infected with either Ad-GFP (Control) or Ad-PGC-1 (PGC-1). The far right panel is representative of regions containing markedly enlarged mitochondria observed in the PGC-1–expressing myocytes. Bar represents 1 μm, the size standard for all three panels. M, mitochondria; N, nucleus.
PGC-1 increases cardiac myocyte capacity for mitochondrial respiration. To investigate whether forced expression of PGC-1 increased the functional capacity for mitochondrial respiration in cardiac myocytes in culture, oxygen consumption rates were determined. For these studies, saponin-permeabilized, cultured rat neonatal cardiac myocytes were studied under conditions in which mitochondrial substrate and ADP were not limiting, to mimic state 3 respiration. Relative to control cells, PGC-1–expressing cardiac myocytes consumed oxygen at a 2.8-fold greater rate, consistent with an increase in mitochondrial oxidative capacity and the observed increase in mitochondrial number (Figure 5a).
Forced expression of PGC-1 in neonatal cardiac myocytes increases oxygen consumption and coupled respiration. (a) Oxygen-consumption rates assessed in saponin-permeabilized cultured rat neonatal cardiac myocytes under conditions in which mitochondrial substrate and ADP were not limiting. The bars represent mean (± SE) oxygen consumption rates (nanomole of oxygen per minute per milligram of protein) in cells infected with control adenovirus expressing GFP alone (C) or adenovirus expressing GFP and PGC-1 (P), corrected for total cellular protein content (A_P_ < 0.05). The values represent four samples in two independent experiments. (b) Myocyte oxygen-consumption rates in serum-free growth medium (without saponin) at base line and after exposure to the ATP synthase inhibitor, oligomycin. The values represent the mean of at least three samples in two independent experiments (A_P_ < 0.001). (c) Representative autoradiograph of Northern blot analyses performed with total RNA isolated from cultured rat neonatal cardiac myocytes after a 48-hour exposure to three different conditions: uninfected cells (U), cells infected with control adenovirus expressing GFP alone (C), and cells infected with adenovirus expressing both GFP and PGC-1 (P). The blot was hybridized to cDNA probes encoding UCP-2, UCP-3, and the ATP synthase β subunit. The ethidium bromide–stained 28S ribosomal subunit (28S) was used as a control for lane loading.
A recent report demonstrated that PGC-1 increased uncoupled mitochondrial respiration and the expression of UCPs in adipocyte and C2C12 cell lines in culture (15). We hypothesized that in contrast to these cell types, PGC-1 would induce coupled mitochondrial respiration in the primary cardiac myocyte, given the obligate high level of ATP production in the heart. To determine whether PGC-1 induced coupled or uncoupled respiration in cardiac myocytes, the ATP synthase inhibitor, oligomycin, was employed to inhibit oxidative phosphorylation before the measurement of oxygen consumption rates. If PGC-1 overexpression induces predominantly coupled respiration in the cardiac myocytes, oligomycin would reduce oxygen consumption rates to a similar degree in cells infected with Ad-PGC and control cells. If, however, PGC-1 induces uncoupled respiration, oligomycin would reduce oxygen-consumption rates to a greater degree in control cells compared with PGC-1–expressing cells. The percentage of reduction in oxygen consumption after oligomycin treatment was similar in cardiac myocytes infected with either control or Ad-PGC-1 vector (Figure 5b), indicating that the PGC-1–induced increase in mitochondrial oxygen consumption mainly was due to an augmentation in coupled respiration through oxidative phosphorylation. Inhibition of the adenine nucleotide translocase by atractyloside also resulted in a similar degree of reduction in total oxygen consumption in control and PGC-1 adenovirus–infected cardiac myocytes (data not shown).
The effect of PGC-1 on cardiac myocyte UCP expression was delineated. UCP-1 is a brown adipose-specific inner mitochondrial membrane protein that generates heat by dissipating the inner mitochondrial membrane proton gradient (35). Two homologous proteins, UCP-2 and UCP-3, are expressed in multiple tissues, including skeletal muscle and heart (19). PGC-1 overexpression decreased cardiac myocyte UCP-2 mRNA levels by approximately 60%, while increasing ATP synthase-β subunit mRNA levels threefold (Figure 5c). Although detectable in adult heart total RNA (data not shown), UCP-3 mRNA was not detected in RNA isolated from neonatal rat cardiac myocytes in culture under either condition. Thus, in contrast to the effect of PGC-1 in several noncardiac cell types (15), PGC-1 overexpression decreased rather than increased mitochondrial UCP-2 gene expression in the cardiac myocyte, coincident with an observed increase in mitochondrial number and function. Taken together with the results of the oxygen consumption studies, these data indicate that PGC-1 is capable of increasing the capacity for cardiac myocyte energy production through coupled mitochondrial respiration.
Cardiac overexpression of PGC-1 in vivo in transgenic mice induces uncontrolled mitochondrial biogenesis. To evaluate the effect of forced PGC-1 expression in the intact postnatal heart in vivo, the cardiac α-MHC gene promoter (28) was used to produce transgenic mice with high-level postnatal, cardiac-specific expression of PGC-1 (MHC-PGC-1 mice). MHC-PGC-1 transgenic mice from three independent lines (ages 1–5 weeks) exhibited high-level cardiac-specific transgene expression and increased mitochondrial DNA content compared with nontransgenic littermate controls (data not shown). The transgenic mice of all three independent lines exhibited massive edema, increased heart size, and four-chamber enlargement consistent with a dilated cardiomyopathy (Figures 6, a and b). Echocardiographic analysis of MHC-PGC-1 mice demonstrated marked four-chamber cardiac enlargement and severely decreased global contractile function (Figure 6c). All of the MHC-PGC-1 mice died by 6 weeks of age. The postnatal phenotype of the MHC-PGC-1 mice is consistent with the temporal pattern of activation of the cardiac α-MHC gene promoter used to drive expression of the transgene.
MHC-PGC-1 mice develop a severe dilated cardiomyopathy. (a) Representative photographs of a nontransgenic mouse (Control) and a littermate MHC-PGC-1–transgenic mouse (MHC-PGC-1) at 6 weeks of age. (b) Midventricular transverse histologic sections of a nontransgenic control mouse (Control) and littermate MHC-PGC-1–transgenic mouse heart (MHC-PGC-1) at 4 weeks of age. The tissue was fixed in 10% neutral-buffered formalin and stained with Masson’s trichrome. (c) M-mode echocardiograms at the midventricular level of nontransgenic (Control) and littermate MHC-PGC-1–transgenic (MHC-PGC-1) mice at 6 weeks of age.
Histologic studies performed on left-ventricular tissue from the MHC-PGC-1 mice before death revealed marked structural abnormalities, including numerous regions of sarcomeric disruption (Figure 7a). Trichrome staining of the histologic sections revealed a granular appearance in areas where the sarcomeric assembly was altered or absent (Figure 7a). Patchy areas of mild fibrosis were also seen. Electron microscopic analysis of the transgenic hearts revealed that the regions of granular densities represented massive expansion of enlarged mitochondria (Figure 7b). The mitochondria in the cardiac ventricles of transgenic mice were more numerous and often significantly larger than the littermate nontransgenic control mouse hearts. In some myocytes, the marked mitochondrial proliferation appeared to have replaced the sarcomeric assembly (lower-left panel, Figure 7b). Thus, as predicted by the cell-culture studies, overexpression of PGC-1 resulted in a striking mitochondrial proliferation in the hearts of the transgenic mice.
Induction of uncontrolled mitochondrial proliferation in the cardiac ventricle of MHC-PGC-1 mice. (a) Representative histologic sections of cardiac ventricles from a nontransgenic littermate control (Control) and a MHC-PGC-1–transgenic mouse heart (MHC-PGC-1) obtained at 3.5 weeks of age. The tissue was fixed in formalin and stained with Gomori’s trichrome. The red blood cells within a vessel present in the lower-left region of the Control panel serve as a relative size standard. Note the granular appearance of the myocytes and loss of sarcomeric structure in the section shown in the lower panel. (b) Low-power (left panels) and high-power (right panels) electron micrographs of histologic sections prepared from 3.5-week-old MHC-PGC-1 transgenic mouse ventricle (MHC-PGC-1) and littermate non-transgenic control (Control). Bar represents 1 μM.
To determine whether the cardiac phenotype of the MHC-PGC-1 mice was determined by the level of forced PGC-1 expression, additional lines with lower levels of transgene expression were characterized. Two independent MHC-PGC-1 lines with approximately fivefold lower PGC-1 mRNA expression compared with the three high-expressing lines described above did not develop cardiomyopathy (data not shown). However, a small number of homozygous offspring resulting from crosses of hemizygotes of the low-expressing MHC-PGC-1 lines developed cardiomyopathy associated with marked cardiac mitochondrial proliferation at 5–7 weeks of age. These data indicate that the level of transgene expression predicts the cardiac mitochondrial proliferative phenotype in the MHC-PGC-1 mice.