A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts Anna Hansson*, Nicole Hance*, Eric Dufour*, Anja Rantanen*, Kjell Hultenby†, David A. Clayton‡, Rolf Wibom§, and Nils-Go¨ ran Larsson*¶ Departments of *Medical Nutrition and Biosciences and §Laboratory Medicine and †Clinical Research Center, Karolinska Institutet, Novum, Karolinska University Hospital, S-141 86 Stockholm, Sweden; and ‡Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD 20815-6789 Communicated by Rolf Luft, Karolinska Institutet, Stockholm, Sweden, December 30, 2003 (received for review November 15, 2003) We performed global gene expression analyses in mouse hearts plified by increased levels of transcripts encoding the glycolytic with progressive respiratory chain deficiency and found a meta- enzyme GAPDH in respiratory chain-deficient heart muscle bolic switch at an early disease stage. The tissue-specific mitochon- (14) and normal GAPDH transcript levels in respiratory chain- drial transcription factor A (Tfam) knockout mice of this study deficient cerebral cortex (9). Our studies have also demonstrated displayed a progressive heart phenotype with depletion of mtDNA that the mitochondrial mass increases in respiratory chain- and an accompanying severe decline of respiratory chain enzyme deficient embryos and differentiated mouse tissues, suggesting activities along with a decreased mitochondrial ATP production that increased mitochondrial biogenesis represents a cellular rate. These characteristics were observed after 2 weeks of age and compensatory mechanism. Measurement of the MAPR has became gradually more severe until the terminal stage occurred at demonstrated that an increase of mitochondrial mass increases 10–12 weeks of age. Global gene expression analyses with mi- overall ATP production to near normal levels in mitochondrial croarrays showed that a metabolic switch occurred early in the myopathy mice (11). MAPR may thus not be as critical for the progression of cardiac mitochondrial dysfunction. A large number pathophysiology of respiratory chain deficiency as previously of genes encoding critical enzymes in fatty acid oxidation showed thought, and other mechanisms, such as changes in reduction͞ decreased expression whereas several genes encoding glycolytic oxidation status and secondary metabolic alterations, may be enzymes showed increased expression. These alterations are con- important. In this article, we have characterized temporal sistent with activation of a fetal gene expression program, a changes in gene expression patterns and induction of mitochon- well-documented phenomenon in cardiac disease. An increase in drial biogenesis in Tfam knockout mouse hearts with progressive mitochondrial mass was not observed until the disease had respiratory chain deficiency. Surprisingly, we found that an early reached an advanced stage. In contrast to what we have earlier switch in metabolism may worsen the situation and contribute to observed in respiratory chain-deficient skeletal muscle, the in- the disease progression in respiratory chain-deficient hearts. creased mitochondrial biogenesis in respiratory chain-deficient heart muscle did not increase the overall mitochondrial ATP pro- Materials and Methods duction rate. The observed switch in metabolism is unlikely to Mating and Genotyping of Transgenic Animals. TfamloxP͞TfamloxP benefit energy homeostasis in the respiratory chain-deficient mice of mixed genetic background (10, 12) were mated to hearts and therefore likely aggravates the disease. It can thus be heterozygous transgenic mice (ϩ͞Ckmm-NLS-cre) (15). Double concluded that at least some of the secondary gene expression heterozygotes (ϩ͞TfamloxP,ϩ͞Ckmm-NLS-cre) were obtained alterations in mitochondrial cardiomyopathy do not compensate and backcrossed to the TfamloxP͞TfamloxP strain to generate but rather directly contribute to heart failure progression. tissue-specific knockouts (TfamloxP͞TfamloxP,ϩ͞Ckmm-NLS- cre). We analyzed a total of 416 offspring from this cross and espiratory chain dysfunction can generate symptoms in observed Mendelian distribution of the four obtained genotypes: loxP͞ loxP ϩ͞ ϭ loxP͞ almost any organ with varying ages of onset (1, 2). Many Tfam Tfam , Ckmm-NLS-cre (n 93), Tfam R TfamloxP (n ϭ 105), ϩ͞TfamloxP (n ϭ 116), and ϩ͞TfamloxP,ϩ͞ distinct genetic mitochondrial disease syndromes caused by ϭ mutations in nuclear genes or mtDNA have been extensively Ckmm-NLS-cre (n 102). studied (3). Furthermore, mitochondrial dysfunction is also RNA Isolation, Target Preparation, and Microarray Hybridization. implicated in common age-associated diseases as well as in the ͞ normally occurring aging process (4, 5). Respiratory chain Total RNA was isolated by using the TRIzol Reagent (GIBCO dysfunction thus plays an important role in human pathology, BRL) and equal amounts of RNA from at least four animals and it has generally been assumed that the pathogenesis is were pooled for each experiment. Target preparation and hy- directly related to deficient mitochondrial ATP production rate bridization to Affymetrix Mu11K Probe Arrays were performed (MAPR). However, there is very little direct experimental according to the protocol supplied by Affymetrix (Santa support for this hypothesis, and other mechanisms may have Clara, CA). been overlooked. Gene expression studies of respiratory chain- deficient budding yeast cells have demonstrated a variety of Microarray Data Analysis. Raw data were analyzed with the Af- responses, including major reconfiguration of the metabolic fymetrix software MICROARRAY SUITE 4.0. We used global scaling pathways and induction of peroxisomal biogenesis (6). We have to correct for differences in hybridization performance between generated mouse models for studies of the pathogenesis of the arrays. Only values scored as ‘‘Present’’ by the software were respiratory chain deficiency in vivo by using a conditional knockout strategy to disrupt the mitochondrial transcription Abbreviations: Tfam, mitochondrial transcription factor A; MAPR, mitochondrial ATP factor A (Tfam) gene in selected cell types (7–12). Increased cell production rate; PPAR␣, peroxisome proliferator activated receptor ␣; CS, citrate synthase; death is a common consequence of prolonged respiratory chain PFK, phosphofructokinase; LS, late-stage; MS, mid-stage; Hk1, hexokinase I; PGK, phos- deficiency, with the mechanism for cell death induction differing phoglycerate kinase. between affected tissues (8, 9, 13, 14). We have found evidence ¶To whom correspondence should be addressed. E-mail: [email protected]. for tissue-specific secondary alterations of metabolism as exem- © 2004 by The National Academy of Sciences of the USA 3136–3141 ͉ PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308710100 Downloaded by guest on September 29, 2021 used for analysis. Genes with no ‘‘Present’’ values for any of the effects on the other enzyme activities. The homogenate was experiments were eliminated, resulting in a data set of 7,706 stored at Ϫ80°C for subsequent analyses of the following enzyme genes. The intensities for each probe set, designated average activities: hexokinase (HXK, EC 2.7.1.1) (22), glucose-6- differences (AvgDiff) by the software, were averaged for all phosphate isomerase (GPI, EC 5.3.1.9) (23), PFK (EC 2.7.1.11) experiments and used as the denominator to calculate a fold (24), and phosphoglycerate kinase (PGK, EC 2.7.2.3) (25). change ratio for each gene and experiment. The ratios were log All enzyme activities were determined spectrophotometrically transformed, and the data were analyzed by the CLUSTER at 35°C. program (16). We filtered the data to keep genes with data available for at least 90% of the samples. We then performed Statistical Analyses. A two-way ANOVA was used to analyze the hierarchical clustering of the arrays, by using average linkage statistical significance of observed difference in enzyme activi- clustering. The results were displayed with the TREEVIEW pro- ties and MAPR. gram (16). Each branch of the resulting array tree contained a number of experiments that were used as a group for further Results analyses. The mean intensity (mean AvgDiff) for each gene and Progressive Cardiomyopathy and Increased Mitochondrial Mass in group was calculated and compared with the other groups to Knockout Mice. We have previously characterized a mouse strain obtain the fold change of their respective intensities. The Student with tissue-specific disruption of Tfam in the heart and skeletal t test was used to compare the different groups. The signal muscle (TfamloxP͞TfamloxP,ϩ͞Ckmm-cre) causing dilated cardio- intensities for a gene represented by two or more probe sets were myopathy, atrioventricular heart conduction blocks, and death at normalized to each other and then used to calculate the fold 2–4 weeks of age (10). This strain does not develop a muscle change and the P value. Annotations and gene ontology infor- phenotype during their limited life span (10), consistent with our mation were obtained from the NetAffx Analysis Center, avail- recent observation that it takes Ϸ4 months before TFAM able on the Affymetrix website (17). We further categorized the protein depletion in skeletal muscle causes severely reduced genes manually according to function as defined by public MAPR (11). The usefulness of this knockout strain was limited databases and literature. because of short postnatal survival, and the