Peroxisome proliferator-activated receptor gamma coactivator-1 promotes cardiac mitochondrial biogenesis

Abstract

Cardiac mitochondrial function is altered in a variety of inherited and acquired cardiovascular diseases. Recent studies have identified the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) as a regulator of mitochondrial function in tissues specialized for thermogenesis, such as brown adipose. We sought to determine whether PGC-1 controlled mitochondrial biogenesis and energy-producing capacity in the heart, a tissue specialized for high-capacity ATP production. We found that PGC-1 gene expression is induced in the mouse heart after birth and in response to short-term fasting, conditions known to increase cardiac mitochondrial energy production. Forced expression of PGC-1 in cardiac myocytes in culture induced the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways, increased cellular mitochondrial number, and stimulated coupled respiration. Cardiac-specific overexpression of PGC-1 in transgenic mice resulted in uncontrolled mitochondrial proliferation in cardiac myocytes leading to loss of sarcomeric structure and a dilated cardiomyopathy. These results identify PGC-1 as a critical regulatory molecule in the control of cardiac mitochondrial number and function in response to energy demands.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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 (^AP < 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 (^AP < 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.

Figure 6

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.

Figure 7

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.