Preferent Diaphragmatic Involvement in TK2 Deficiency: An Autopsy Case Study

Abstract

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular β-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.

Keywords: diaphragm; mitochondrial diseases; respiratory failure; thymidine kinase 2.

Figure 1

mtDNA content and mtDNA deletions. (A) mtDNA/nDNA copy-number ratio analyzed by real-time PCR in different tissues of the patient expressed as a percentage of the corresponding control tissue. Data are mean ± SD of three independent determinations. (B) Agarose gel showing mtDNA amplified by long-range PCR in different tissues of the patient and in control diaphragms. Normal sized amplicon of 16.5 kb is indicated. Lower molecular weight bands are suggestive of mtDNA deletions. A lower-size band of presumably non-specific amplification can be observed in B, K and controls. Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain; L, liver; K, kidney; I, small intestine; C+, skeletal muscle positive control for mtDNA deletions; C−, skeletal muscle negative control for mtDNA deletions; Mw, Roche DNA Molecular Weight Marker II (Merck, Sigma-Aldrich, Darmstadt, Germany); Cv₁, ventilated control diaphragm 1; C₃, non-ventilated control diaphragm 3.

Figure 2

OXPHOS system subunits. (A) Representative Western blotting of OXPHOS system subunits in tissue homogenates of a control (C) (n = 3 for muscle, n = 1 for other tissues) and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of control values. Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain; L, liver; K, kidney; I, intestine. Two samples of control intestine are shown; large (left) and small (right). ATP5a, ATP synthase subunit alpha; UQCRC2, ubiquinol-cytochrome C reductase core protein 2; SDHB, succinate dehydrogenase complex iron sulfur subunit B; COXII, cytochrome c oxidase subunit II; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8.

Figure 3

Constitutive proteins. (A) Representative western blotting of proteins with constitutive expression in tissue homogenates of a control (C) (n = 3 for muscle, n = 1 for other tissues) and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of control values. Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MHC-β/slow, myosin heavy chain beta/slow.

Figure 4

OXPHOS system subunits in patient and ventilated control diaphragms. (A) Representative Western blotting of OXPHOS system subunits in diaphragm homogenates of non-ventilated controls (C, n = 3), ventilated controls (Cv, n = 2), and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of non-ventilated control values. Abbreviations: ATP synthase subunit alpha; UQCRC2, ubiquinol-cytochrome C reductase core protein 2; SDHB, succinate dehydrogenase complex iron sulfur subunit B; COXII, cytochrome c oxidase subunit II; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Actin isoforms and TK2 in diaphragm. (A) Representative Western blotting of skeletal muscle α-actin, microtubule β-actin and TK2 in diaphragm homogenates of non-ventilated controls (C) (n = 3), ventilated controls (Cv, n = 2), and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of non-ventilated control values. Abbreviations: TK2, thymidine kinase 2.

Figure 6

Antioxidant enzymes. (A) Representative Western blotting of antioxidant enzymes in tissue homogenates of a control (C) (n = 1) and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of control values. Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain; L, liver; K, kidney; I, small intestine. PRDX6, peroxiredoxin 6; cSOD, cytosolic superoxide dismutase.

Figure 7

Autophagy and ubiquitin-proteasome system markers. (A) Representative Western blotting of autophagy and ubiquitin proteasome markers in tissue homogenates of a control (C, n = 1) and the patient (P). Shown below is a representative Coomassie blue-stained gel. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of control values. Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain; L, liver; K, kidney; I, small intestine. LC3, microtubule-associated proteins 1A/1B light chain 3A; Ubi-prots, ubiquitinated proteins; α + β Proteas, α and β catalytic subunits of the 26S proteasome.

Figure 8

TK1 levels. (A) Western blotting of TK1 in tissue homogenate samples treated with dithiothreitol from a control (C) (n = 1) and the patient (P). Coomassie straining of the membranes is shown as total protein loading control. (B) Densitometry analysis: protein levels were normalized to total protein loading according to Coomassie blue staining. Data are expressed as percentage of control tissue Abbreviations: M, skeletal muscle; H, heart; D, diaphragm; B, brain; L, liver; K, kidney; I, small intestine. TK1, thymidine kinase 1.