High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function

Abstract

Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

Fig 1. Representative high-resolution respirometry recordings of the human vastus lateralis muscle and the murine prefrontal cortex and heart.

Representative measurements of high-resolution respirometry recordings are shown for the human vastus lateralis muscle (A), the murine prefrontal cortex (B) and the murine heart (C). The blue line represents the oxygen concentration in the chamber, while the red line indicates the oxygen flux. (A) For experiments with human vastus lateralis muscle samples 10 mM pyruvate were added before the recording started. After administration of 5 mM malate (M), 10 mM glutamate (G) and 5 mM ADP complex I activity (CI) was recorded and the leak respiration was calculated (respiration after MG/respiration after ADP). 10 μM cytochrome c was added to survey the integrity of the outer mitochondrial membrane. 1 mM octanoyl carnitine was administered to measure the fatty acid induced respiration. Further addition of 10 mM succinate, which is the complex II substrate, enables the determination of the maximum OxPhos capacity. The ATP synthase inhibitor oligomycin (O) (5 μM), which was not used for the murine heart (C) and soleus muscle was administered. After adding the uncoupling agent carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (0.5, 0.25, 0.25 μM FCCP) the maximum uncoupled capacity was measured. Finally complex I was inhibited by rotenone (R) (0.5 μM) allowing the determination of complex II activity (CII). In the end complex III was inhibited by administration of antimycin A (5 μM). (B) The protocol for murine cortex and liver matched the human muscle protocol with the following exceptions: cytochrome c was added after pyruvate before the recording started and octanoyl carnitine was not used. (C) The protocol for murine heart and soleus muscle is equivalent to the one used for murine cortex (B) except for the oligomycin administration.

Fig 2. High-resolution respirometry of human muscle biopsies show no difference between mutation carriers and healthy controls.

High-resolution respirometry analysis of human vastus lateralis muscle muscle biopsies. The white boxes represent human controls (n = 9) versus HD expansion mutation carriers (HDEMC) (gray boxes) (n = 7). (A) Respiratory function of complex I activity (CI), complex II activity (CII), the maximum OxPhos capacity (mO), the maximum uncoupled capacity (mU) and the O2ATP (calculated oxygen consumption linked to ATP production) are shown. The leak respiration is illustrated in (B). The respiratory control ratio (RCR) is indicated in (C). The mitochondrial mass, determined using a citrate synthase (CS) activity assay, is shown in (D) (n = 7 controls, n = 4 for HDEMCs). Mann-Whitney test comparing controls vs. HDEMCs, ns = P>0.05, * = P≤0.05.

Fig 3. Respiration of cortex, liver, soleus muscle and heart of Hdh^Q20 and Hdh^Q111 mice.

High-resolution respirometry in the murine tissues prefrontal cortex, liver, soleus muscle and heart. The white boxes represent Hdh^Q20 control mice versus the gray boxes, which represent the HD mouse model Hdh^Q111. (A) Complex I activity (C_I) is shown in a coupled state determined after addition of ADP. (B) Complex II activity (C_II) was measured in an uncoupled state after addition of rotenone. (C) The maximum OxPhos capacity was measured and (D) the maximum uncoupled capacity was determined after application of FCCP in all four tissues. (E) For murine cortex and liver the oxygen consumption linked to ATP production (O₂ATP) was calculated. n = 6, student’s t-test comparing genotypes, ns = p>0.05, * = p≤0.05, ** = p≤0.01. 95% Confidence interval limits: (B) liver: 14.72–65.71; (D) cortex: 1.20–28.16; liver: 4.99–73.43.; (E) cortex: 0.96–13.67. The Mann-Whitney Rank-Test was used for the following samples: (A): cortex; (B): cortex, m. soleus, heart; (D): m. soleus.

Fig 4. Mitochondrial mass in HdhQ mice in brain, soleus muscle, liver and heart.

(A) The mtDNA copy number was determined by qPCR for the murine cortex, liver, soleus and heart muscle, comparing Hdh^Q20 control mice (white boxes) to Hdh^Q111 HD mice (gray boxes). n = 6–7. (B) The citrate synthase (CS) activity was measured by a spectrophotometric assay. n = 5, student’s t-test comparing genotypes of one tissue, ns = p>0.05. (C) Correlation of mtDNA copy number to the citrate synthase activity, which was determined in triplicates (±SD). Pearson value p≤0.001, R² = 0.5371. The Mann-Whitney Rank-Test was used for the following samples: (B): cortex, liver.

Fig 5. mRNA levels of mitochondrial energy metabolism related target genes and genes involved in ROS detoxification.

mRNA analysis of mitochondrial targets in the murine tissues (A) cortex, (B) liver, (C) gastrocnemius muscle and (D) heart. mRNA levels of target genes are normalized to the reference genes (hypoxanthine guanine phosphoribosyl transferase (Hprt), polymerase (RNA) II (DNA directed) polypeptide A (Polr2a), methionyl aminopeptidase 1 (Metap1) for the cortex, liver, heart and Polr2a, Metap1 for the gastrocnemius muscle) and the results are shown relative to the mean of Hdh^Q20 values in box plots. n = 5–6, student’s t-test comparing genotypes of one tissue, ns = p>0.05, * = p≤0.05, ** = p≤0.01. 95% confidence interval limits: (A) Sod2: 6.24–32.35; Ucp2: 3.21–60.09. The Mann-Whitney Rank-Test was used for the following samples: (B) Ppargc1a, Ucp2; (D): Ppargc1a.