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

Deterioration of myocardial contractility is an obvious indicator for reduced oxygen supply. In coronary heart disease, when arteriosclerotic plaque formation reduces blood flow, the reduced coronary blood supply results in ischemia and damage to the myocardium. Myocardial respiration depends on Cytochrome c- Oxidase (CytOx) activity. It represents the rate limiting step for the function of the mitochondrial respiratory chain, also known as electron transmission chain (ETC). If cardiac failure is related to the compromised cellular respiration of the heart, remains unclear. But contractility requires abundant supply of adenosine triphosphate (ATP), and this kind of “energy currency” is produced in mitochondria (see Fig. 1) where oxygen consumption for water production at Cytochrome c- Oxidase (E.C. 1.9.3.1.) is a rate-limiting step. Decreased expression of COX 4 results in an impaired Cytochrome c oxidase activity [4, 5]. We hypothesize subsequent mitochondrial dysfunction associated with the formation of increased reactive oxygen species and inadequate maintenance of ATP levels when subunit 4 is reduced in the holoenzyme. This dysregulation is likely to contribute to the pathophysiology associated with myocardial insufficiency.

The extent of ROS formation herein, determines the pathophysiological consequences. Ischemic damage and reperfusion injury of myocardium proceed with an “Overspending ATP” and finally metabolic break down. The CytOx (complex IV) is directly involved and has the “center stage” [4, 5]. This enzyme is considered to be the rate limiting step of electron transfer chain (ETC) [6, 7]. The holoenzyme in mammals is always composed of 13 subunits [8] (Fig. 2) where subunit 4 is essential for the assembly and oxygen consumption of the enzyme [9].

Interestingly, Buchwald et al. [10] found in hearts of transplant recipients with dilated cardiomyopathy (DCM) reduced cytochrome content as well as decreased complex IV activity. The group claimed no differences in subunit composition, although only gel- electrophoresis was used and harvested hearts represented profound end stage status of terminal cardiac insufficiency. Opposite data were presented by Grossmann and co-workers [11] where they found in homozygous and heterozygous Cox7a1 knockout mice, although viable, the reduced enzymatic activity and development of DCM at 6 weeks of age. Surprisingly, the cardiomyopathy improves and stabilizes by 6 months of age. Cox7a1 knockout mice incorporated more of the “liver-type” isoform Cox7a2 into cardiac type of the enzyme.

Mitochondrial mtDNA mutations are known to be involved in the pathogenesis of DCM [12, 13]. Point mutations are found more frequently. Mutations involving the evolutionary conserved residues of CytOx subunit 1 and NADH dehydrogenase 5 have been identified [14]. Additionally, further mutations were located in highly conserved domains of the gene coding for the CytOx [15, 16]. After cardiac transplantation, Arbustini and collaborators [14] found in explanted recipients’ hearts suffering from DCM, significant lower CytOx- activity related to NADH dehydrogenase and succinic dehydrogenase activities. They correlated pathological mtDNA mutations to CytOx- deficiency and abnormal mitochondrial ultrastructure.

The prognosis of DCM is poor because of its progression to myocardial insufficiency. Progression results either in cardiac death or prolonged support with inotropic medication, implantation of left ventricular assist devices, or heart transplantation, respectively. Effective, early indicators are required for diagnosing heart failure and commencing justified interventions in due time. Diversely, a good prognostic parameter to start the weaning process from cardiac assist devices under intensive medical care are needed as well.

Reflecting the need of such new parameters, the present study addresses mitochondrial pathophysiology of myocardial respiration in case of myocardial insufficiency. The study addresses the question, if reduced transcription and protein translation rates of subunit 4 represents a good indicator for compromised myocardial oxygen consumption and utilization for ATP production. The mismatch between mitochondrial- and nuclear coded subunits of CytOx indicates compromised cardiac dysfunction. Subunit 2 binds to one of the copper ions (CuA) whilst subunit 1 is likely to bind the two hemes (a and a₃) and the other redox-active copper (CuB). Two cysteine and two histidine residues of subunit 2 are the likely ligands of CuA, forming a centre for electron transportation [17, 18]. Subunit 4 is known to be essential for the assembly and respiratory function of the enzyme complex [19, 20]. Accordingly, MT –CO₂ /COX 4 gene expression, CytOx subunit 2 and 4 protein content and enzymatic activity of the holoenzyme in biopsies of DCM patients were determined. The extent of myocardial insufficiency in patients was detected by heart catheterization and LV angiography. Hemodynamic data were correlated to the biochemical findings of CytOx subunit 2 and 4 measurements.

In this study, we have investigated the mRNA expression of MT –CO₂ (mitochondrial encoded subunit) and COX 4 (nuclear encoded subunit) in endomyocardial biopsies (EMB) of patients with non- viral, non- inflammatory and non- familial idiopathic DCM and compared to controls. Hemodynamics of 54 patients were studied. Thirty patients were classified as DCM according to international criteria [21, 22]. In 24 individuals, neither infiltrating cells, cardiotropic virus, nor dilatation of the left or right ventricle and not even wall motion abnormalities were detected, although they had the clinical signs of infectious heart disease (recurrent fever, reported arrhythmia, dizziness etc.). These patients were considered as a control group (Table 1).

A significant decrease of COX 4 mRNA expression and protein content but no significant changes in correlating MT- CO₂ measurements, were found (1.1 ± 0.2 vs. 0.6 ± 0.3, p < 0.001; 7.7 ± 3.1% vs. 2.8 ± 1.4%, p < 0.0001) (Figs. 3 and 4.) COX 4 mRNA expression data correlated with ejection fractions, EF (r = 0.766, p = 0.00045,) and LVEDD (r = − 0.492, p = 0.0068) as well as shortening fractions (SF; r = 0.377, p = 0.032) (Fig. 5). Otherwise, MT- CO₂ did not correlate neither to EF and LVEDD nor shortening fractions (SF). The age of patients neither correlate to the MT- CO₂ mRNA nor with COX 4 mRNA data. For this reason the found alterations were age- independent. Even in the group of DCM the reduced SF correlated to the reduced COX 4 expression (r = 0.52, p = 0.017).

Interestingly, oxygen consumption testing provides no difference in both groups (3.1 ± 1.0 vs. 3.3 ± 0.6 nm/ml, p = 0.643, n.s.). However, enzymatic activity of CytOx in case of DCM was approximately 3fold increased (0.08 ± 0.06 vs. 0.26 ± 0.08 U/mg, p < 0.001, Fig. 6).

The question of myocardial oxygen supply, consumption and utilization remains to be one of the biggest problems in cardiovascular research [25]. Dilated Cardiomyopathy (DCM) is defined as a primary heart muscle disease with heart enlargement and impairment of the ventricular contractile function [26] and appears as a sufficient model to understand pathogenesis of myocardial insufficiency and cardiac failure, respectively. Mitochondrial dysfunction is a determinant of its pathogenesis [27]. In most cases, isolated CytOx- deficiencies are autosomal recessive disorders. Onset of the diseases starts at a very early age and is associated with fatal outcome (Leigh Syndrome and myopathies [28, 29]). Mutations in nuclear- encoded structural CytOx subunits have not been found in these phenotypes but still several assembly factors were affected. For example, SURF 1 (Leigh-syndrome), SCO2 (hypertrophic cardiopathy), SCO1 (hepatic failure) and COX 10 (sensorineural hearing loss, anemia, and hypertrophic cardiomyopathy) [16, 27, 30]. Saada and Coworkers [31] found a decreased mitochondrial thymidine kinase activity in association with mtDNA depletion responsible for certain kinds of myopathies. Reduced enzymatic activities of the electron transmission chain (ETC) in patients with dilated cardiomyopathy are found only in some cases [27]. The decrease of complex III activity was considered as a secondary phenomenon and not due to a mitochondrial defect for transcription and protein translation. In contrast, Hittel and coworkers found a differential expression of mitochondrial encoded genes in hibernation and specific increase of Complex IV- expression to prevent ETC alterations caused by cold and ischemia [32]. Evidence of a link between mutations in genes for respiratory chain components on one hand and human diseases on the other hand is reported with the emphasis on defects in respiratory complex IV and its assembly factors [27]. Early mutations in the mtDNA-encoded COX genes for CytOx subunits are relatively rare, but alterations in metabolism by thyroid hormones are known to influence the subunit composition at transcriptional and posttranscriptional level [33, 34]. Moreover increased mtDNA mutations are found with age in various human tissues as a result of oxidative stress. New aspect comes into consideration related to CytOx subunit composition in case of myocardial insufficiency and hypoxia with the emphasis on subunit 4 where the oxygen binding site of the holoenzyme is located [9].

Impairment of mitochondrial respiration and oxidative phosphorylation induces an increase in ROS production that causes mtDNA rearrangements, deletions and apoptosis [35]. A previous study found activities of complexes II and V of the ETC unchanged in dilated cardiomyopathy although cytochrome- containing complexes III and IV showed impaired activity, although subunit composition of CytOx remained the same as compared to the normal hearts in this study [10].

Opposite data addresses impaired cytochrome c oxidase- assembly in pathogenesis of myocardial insufficiency [36] and a heart-type cytochrome c oxidase subunit 7a1 was found to be associated with the development of DCM [37].

In our study, gene expression of mitochondrial encoded subunit 2 (MT-CO₂) was throughout the same, but COX 4 was down-regulated and protein content of subunit 4 was also reduced in cases of myocardial insufficiency. This effect is age- independent. The complex IV in the ETC is involved in the electron transfer to dioxygen via subunit 4 that determines the subsequent respiratory activity of the tissue [6, 8, 9]. Exchange of bound ADP through ATP at a high-affinity binding site in the matrix domain of subunit 4 has a regulatory function for the whole enzymatic activity and for the final respiratory performance [38, 39]. Interestingly, in concern of previous findings [10] the enzymatic activity of CytOx in our estimations was found to be increased, but myocardial oxygen consumption had the equal amount in both groups. The data opens the question if down- regulation of subunit 4 is a consequence of hypoxia [40] or it represents the malfunctioning of the molecular subunit assembly among multiple factors [41] beside the uncoupling status of respiratory control [42]. Nonetheless, reduced subunit 4 appears as an indicator for myocardial insufficiency. Additional information in this regard has also been presented by the further analysis of subunit 4 isoforms (4i1 and 4i2) and their role in cardiac diseases [43].

New phase in heart surgery has begun. Testing indications and improving the outcome of patients in heart surgery require an understanding of molecular medicine and knowledge to be transferred at the bed- side level. In early studies, assessment of myocardial blood flow suggested myocardial ischemia as a reason for cardiac insufficiency and dilated cardiomyopathy [44]. Later studies corrected this idea [45]. Myocardial hypoxia has to be discussed as a matter of supply and demand and the different status of workload, respectively [46]. A third, new and an interesting fact with clinical relevance comes into consideration. Present study appears coherent with myocardial respiratory data from patients with left ventricular hypertrophy [47] and perhaps gives an explanation for a universal pathomechanism. The myocardial oxygen consumption is unchanged but efficiency is reduced. Different states of respiration determine efficiency of ATP synthesis and extent of ROS formation [1‐3]. Quantity of ROS production results finally in myocardial damage and a progression of myocardial insufficiency, respectively.

In this study, patients suspected for inflammatory heart disease and dilated cardiomyopathy (DCM) underwent heart catheterization. Thirty patients suffering from reduced LV ejection fraction below 45% and an enlarged left ventricular enddiastolic diameter bigger than 56 mm were considered to be myocardial insufficient. In biopsies of all patients the activity of Cytochrome c-oxidase (Complex IV of the mitochondrial respiratory chain) was measured. Furthermore, the subunit mRNA expression and protein content of the mitochondrial encoded catalytic subunit II (MT-CO2) and nuclear encoded regulatory subunit 4 (COX 4) were detected. Patients with myocardial insufficiency showed a significant decrease in COX 4 but not in MT-CO2. Correlations were found between COX 4 expression and EF% and LVEDD. Therefore, it is concluded that reduced COX 4 leads to impaired activity of Cytochrome c oxidase, subsequent reduction in mitochondiral respiration and hence myocardial insufficiency.