A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient mouse hearts

Abstract

We performed global gene expression analyses in mouse hearts with progressive respiratory chain deficiency and found a metabolic switch at an early disease stage. The tissue-specific mitochondrial transcription factor A (Tfam) knockout mice of this study displayed a progressive heart phenotype with depletion of mtDNA and an accompanying severe decline of respiratory chain enzyme activities along with a decreased mitochondrial ATP production rate. These characteristics were observed after 2 weeks of age and became gradually more severe until the terminal stage occurred at 10-12 weeks of age. Global gene expression analyses with microarrays showed that a metabolic switch occurred early in the progression of cardiac mitochondrial dysfunction. A large number of genes encoding critical enzymes in fatty acid oxidation showed decreased expression whereas several genes encoding glycolytic enzymes showed increased expression. These alterations are consistent with activation of a fetal gene expression program, a well-documented phenomenon in cardiac disease. An increase in mitochondrial mass was not observed until the disease had reached an advanced stage. In contrast to what we have earlier observed in respiratory chain-deficient skeletal muscle, the increased mitochondrial biogenesis in respiratory chain-deficient heart muscle did not increase the overall mitochondrial ATP production rate. The observed switch in metabolism is unlikely to benefit energy homeostasis in the respiratory chain-deficient hearts and therefore likely aggravates the disease. It can thus be concluded that at least some of the secondary gene expression alterations in mitochondrial cardiomyopathy do not compensate but rather directly contribute to heart failure progression.

Fig. 1.

Hearts of Tfam knockouts (Tfam^loxP/Tfam^loxP,+/Ckmm-NLS-cre) show increased size, accumulation of mitochondria, and reduced amounts of mtDNA and mtDNA-encoded cytochrome c oxidase (COX) I mtRNA. (A) Heart weight/body weight (hw/bw) ratio in knockout and control (Tfam^loxP/Tfam^loxP) mice at different ages. (B) Southern and Northern blot analysis showing levels of mtDNA and mtRNA in knockout (K) and control (C) mouse hearts at 2, 4, and 8 weeks of age. Membranes were first hybridized with a Cox1 DNA probe and then reprobed with an 18S rRNA DNA probe as a loading control. (C) Transmission electron microscopy performed on heart sections from knockout and control mice at 8 weeks of age.

Fig. 2.

Biochemical analysis of respiratory chain function in knockout (filled bars) and control (open bars) mouse hearts at 2 (2w), 4 (4w), and 8 (8w) weeks of age. Bars present mean levels ± SEM. Asterisks indicate level of statistical significance: ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. (A) Citrate synthase (CS) activity in heart muscle. (B) Relative enzyme activities of respiratory chain enzymes. NADH cytochrome c reductase (NCR), corresponding to complex I + III; NADH coenzyme Q reductase (NQR), complex I; succinate:cytochrome c reductase (SCR), complex II + III; cytochrome c oxidase (COX), complex IV. The relative enzyme activities presented as 100% in the figure correspond to the following absolute ratios of enzyme activity per unit of CS activity at 2, 4, and 8 weeks of age, respectively: NCR, 1.32, 0.74 and 0.75; NQR, 0.35, 0.22, and 0.23; SCR, 0.77, 0.66, and 0.78; COX, 2.88, 2.45, and 2.36. (C) Measurements of MAPR, per unit of CS activity, by using substrates that enter the respiratory chain at different points. The relative MAPR/CS presented as 100% in the figure corresponds to the following absolute ratios of MAPR/unit of CS activity at 2, 4, and 8 weeks of age, respectively: glutamate plus succinate (G + S), 0.27, 0.26, and 0.27; TMPD plus ascorbate (T + A), 0.25, 0.26, and 0.26; palmitoyl-l-carnitine plus malate (PC + M), 0.22, 0.15, and 0.14; succinate plus rotenone (S + R), 0.05, 0.05 and 0.05. (D) Measurements of MAPR per kg of muscle. The relative MAPR/kg presented as 100% in the figure corresponds to the following absolute ratios of MAPR/kg of heart muscle (mmol/ATP/min/kg heart muscle) at 2, 4, and 8 weeks of age, respectively: G + S, 71, 86, and 97; T + A, 66, 86, and 94; PC + M, 57, 50, and 52; S + R, 13, 17, and 19.

Fig. 3.

Dendrogram showing results from cluster analysis of gene expression patterns. Pools of RNA from knockout (K) and control (C) mouse hearts at 2, 4, 5, 7, and 9 weeks of age were hybridized to gene microarrays. Some samples were performed in duplicate. The RNA pools with the most similar gene expression profile will cluster together within the same branch of the tree.

Fig. 4.

Biochemical analysis of glycolytic enzymatic activities. Relative enzymatic activities assayed from heart of knockout and control mice at 2, 4, and 8 weeks of age. Bars present the mean ± SEM. Asterisks indicate level of statistical significance: ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001. The relative enzymatic activities presented as 100% in the figure correspond to the following absolute enzymatic activities (mmol/min per kg of heart tissue) at 2, 4, and 8 weeks of age, respectively: hexokinase (HXK) 6.6, 8.1, and 9.3; PFK 41.6, 63.9, and 50.3; phosphoglucose isomerase (PGI) 229, 267, and 279; PGK 121, 171, and 190.