Myocardial insufficiency is related to reduced subunit 4 content of cytochrome c oxidase

Abstract

Background: Treatment of heart failure remains one of the most challenging task for intensive care medicine, cardiology and cardiac surgery. New options and better indicators are always required. Understanding the basic mechanisms underlying heart failure promote the development of adjusted therapy e.g. assist devices and monitoring of recovery. If cardiac failure is related to compromised cellular respiration of the heart, remains unclear. Myocardial respiration depends on Cytochrome c- Oxidase (CytOx) activity representing the rate limiting step for the mitochondrial respiratory chain. The enzymatic activity as well as mRNA expression of enzyme's mitochondrial encoded catalytic subunit 2, nuclear encoded regulatory subunit 4 and protein contents were studied in biopsies of cardiac patients suffering from myocardial insufficiency and dilated cardiomyopathy (DCM).

Methods: Fifty-four patients were enrolled in the study and underwent coronary angiography. Thirty male patients (mean age: 45 +/- 15 yrs.) had a reduced ejection fraction (EF) 35 ± 12% below 45% and a left ventricular end diastolic diameter (LVEDD) of 71 ± 10 mm bigger than 56 mm. They were diagnosed as having idiopathic dilated cardiomyopathy (DCM) without coronary heart disease and NYHA-class 3 and 4. Additionally, 24 male patients (mean age: 52 +/- 11 yrs.) after exclusion of secondary cardiomyopathies, coronary artery or valve disease, served as control (EF: 68 ± 7, LVEDD: 51 ± 7 mm). Total RNA was extracted from two biopsies of each person. Real-time PCR analysis was performed with specific primers followed by a melt curve analysis. Corresponding protein expression in the tissue was studied with immune-histochemistry while enzymatic activity was evaluated by spectroscopy.

Results: Gene and protein expression analysis of patients showed a significant decrease of subunit 4 (1.1 vs. 0.6, p < 0.001; 7.7 ± 3.1% vs. 2.8 ± 1.4%, p < 0.0001) but no differences in subunit 2. Correlations were found between reduced subunit 2 expression, low EF (r = 0.766, p < 0.00045) and increased LVEDD (r = 0.492, p < 0.0068). In case of DCM less subunit 4 expression and reduced shortening fraction (r = 0.524, p < 0.017) was found, but enzymatic activity was higher (0.08 ± 0.06 vs. 0.26 ± 0.08 U/mg, p < 0.001) although myocardial oxygen consumption continued to the same extent.

Conclusion: In case of myocardial insufficiency and DCM, decreased expression of COX 4 results in an impaired CytOx activity. Higher enzymatic activity but equal oxygen consumption contribute to the pathophysiology of the myocardial insufficiency and appears as an indicator of oxidative stress. This kind of dysregulation should be in the focus for the development of diagnostic and therapy procedures.

Keywords: Cytochrome c oxidase; MT –CO2 and COX 4 expression; Myocardial insufficiency.

Fig. 1

Mitochondrial electron transport chain (ETC) is composed of four multisubunit respiratory complexes and the carrier proteins that together perform mitochondrial respiration. Electrons are delivered from NADH and FADH₂ to complex I and complex II, respectively, and subsequently passed through the electron transport chain to the final acceptor i.e. molecular oxygen to form water. This transfer of electrons through the ETC is associate with the translocation of protons at complexes I, III and IV across the mitochondrial membrane into the intermembrane space, thus trapping the energy in the form of electrochemical gradient., This energy stored in the gradient is used to perform oxidative phosphorylation i.e. formation of ATP by ATP synthase (complex V). Energy supply is closely related to the energy demand of the cell. Higher myocardial workload results in increased contractility spending more ATP and consuming more oxygen. Depending on the H⁺/e⁻ -stoichiometry and different efficiency of ATP synthesis, two different states of respiration (relaxed/active) may be considered [–3]. A schematic representation of the enzymes involved in mitochondrial respiration and oxidative phosphorylation. Models of protein crystal structures were taken from the Protein Data Bank (https://www.rcsb.org/). The corresponding PDB IDs’ for complex I to V were: 2FUG, 1YQ3, INTZ, 10CC and 101

Fig. 2

The subunit composition of the human ‘heart-type’ cytochrome c oxidase in 2D- gelelectrophoresis according to the Kadenbach- classification. Present study is focused on subunit 2 and 4 (arrows). CytOx from mammals and birds is composed of 13 subunits. The three catalytic subunits 1–3 are encoded by mitochondrial DNA, while 10 are nuclear-encoded. The latter ones are essentially involved in the regulation of oxygen consumption and proton translocation, since their removal or modification changes the activity and while their mutations may lead to mitochondrial diseases. Respiration is differently regulated in organs and species by expression of tissue-, developmental-, and species-specific isoforms for COX subunits 4, 6a, 6b, 7a, 7b, and 8, but the holoenzyme in mammals is always composed of 13 subunits [8]. Essential for the assembly and the oxygen consumption of the enzyme is the subunit 4 [9]. In the text, the subunits are termed in arabic numbers while mitochondrial enzyme complexes of the ETC are termed in latin numbers

Fig. 3

Relative mRNA expression of MT-CO₂ and COX 4 in endomyocardial biopsies of patients with dilated cardiomyopathy (DCM) compared to controls

Fig. 4

Immune histomorphometric detection of CytOx subunit 2 and 4 protein expressions in biopsies from DCM patients and controls. Evaluation shows clear reduction of CytOx subunit 4 protein expression in DCM tissue (p < 0.0001)

Fig. 5

Correlations of normalized COX 4 expression vs. ejection fraction (EF) as well as left ventricular enddiastolic diameter (LVEDD) and shortening fraction (SF). Tests for significance of correlations are shown in the figure. In case of DCM less subunit 4 expression and reduced shortening fraction (r = 0.524, p < 0.017) was found

Fig. 6

Protein content related to measurements of myocardial oxygen consumption in DCM group and control group (p = 0.643, n.s.) and simultaneous detection of CytOx activity in the presence of 20 μM reduced Cytochrome c (p < 0.001). Although the oxygen consumption rate is equal, the enzymatic activity is higher in the DCM group. The data indicates reduced efficiency in oxygen utilization as pathogenic factor for myocardial insufficiency and DCM, respectively