What is cardiolipin in mitochondria

Abstract:The heart has an extremely high energy requirement, and mitochondria are the main place for energy production in heart muscle cells (cardiomyocytes). In mitochondria, NADH and FADH2 are produced in the citric acid cycle, which donate electrons to complexes I and II of the respiratory chain, the transfer of which along the respiratory chain complexes I-IV produces a proton gradient, which is the driving force for ATP production at the F1Fo-ATP synthase. In order to adapt the ATP production to the constantly changing demand, ADP and Ca2 + regulate the flow of electrons along the respiratory chain. ADP activates F1Fo-ATP synthase, which speeds up the flow of electrons and oxidizes NADH and FADH2 to NAD + and FAD. An increase in workload is induced under physiological conditions by β-adrenergic stimulation, which increases the amplitude and frequency of cytosolic Ca2 + transients in cardiomyocytes. Ca2 + is absorbed into mitochondria via the mitochondrial Ca2 + uniporter (MCU), where it stimulates dehydrogenases of the citric acid cycle. This reduces the redox status of NADH / NAD + and FADH2 / FAD again. NADH is in equilibrium with NADPH via mitochondrial transhydrogenase, and NADPH is essential for the detoxification of H2O2. Thus the mitochondrial Ca2 + uptake plays an important role for the energetic adaptation and the regeneration of the anti-oxidative capacity. Mitochondrial dysfunction with energetic deficit and oxidative stress has been observed in various forms of heart failure. Genetic mutations are rare causes of heart failure. One such form of cardiomyopathy is found in Barth syndrome, an X-linked recessive disease that only affects boys and causes not only cardiac insufficiency but also skeletal muscle myopathy and immune defects. The genetic defect affects the gene (Taz) that encodes Tafazzin (TAZ). TAZ is a transacylase that is required for the last step in the biosynthesis of cardiolipin, a phospholipid that occurs particularly in the inner mitochondrial membrane. The aim of the present work was to investigate the mechanisms for the development of oxidative stress in mice with tafazzin knockdown (Taz-KD), as this is ascribed an important role both in the pathophysiology of cardiac diseases and in Barth syndrome. In particular, we focused on a possibly disturbed mitochondrial Ca2 + transmission, which could lead to a redox shift. To achieve this goal, experiments were carried out on isolated cardiomyocytes and mitochondria from Taz-KD and wild-type (WT) control mice. Isolated cardiomyocytes were examined for their contractility and their redox status based on the determination of NADH, NADPH and FAD. Furthermore, the cytosolic Ca2 + concentration and the production of reactive oxygen species (ROS) were detected. It was shown that the Taz-KD cardiomyocytes, especially under workload, have a shorter diastolic length, which indicates diastolic dysfunction. There was no systolic dysfunction. With regard to the redox status, we were able to show that Taz-KD cardiomyocytes experienced a significantly stronger oxidation of NAD (P) H and FADH2 in the course of the experiment, especially during the increased stimulation frequency. This suggests an energetic “mismatch” in which Taz-KD cardiomyocytes may not be able to reproduce enough NADH and FADH2 in the citric acid cycle. In contrast to WT cardiomyocytes, in which the cytosolic Ca2 + amplitudes increased under β-adrenergic stimulation, this was not the case in Taz-KD cardiomyocytes. The emission of H2O2 did not differ in both groups, especially in additional experiments on isolated mitochondria, despite the increased NADPH oxidation. Looking at the results together, we concluded that the Taz-KD cardiomyocytes have a diastolic dysfunction, which is possibly due to a lack of increase in mitochondrial Ca2 + concentration when the workload increases. In fact, in further experiments that were not carried out by myself, a pronounced defect in mitochondrial Ca2 + uptake could be observed, which was due to a strong downregulation of the mitochondrial Ca2 + uniporter. The lack of mitochondrial Ca2 + uptake explains the severe deficit of bioenergetic adaptation, as it leads to pronounced oxidation of NAD (P) H and FADH2 during β-adrenergic stimulation due to a lack of activation of various key enzymes of the citric acid cycle. This results in a reduced regeneration of NAD (P) H, which is crucial for the elimination of H2O2. Previous work, which saw the sole reason for ROS production in a disturbed function of the respiratory chain due to an impaired super complex formation through aberrant cardiolipin forms, could thus be refuted.
The heart has an enormous demand for energy. Mitochondria are the organelles of energy production within the heart muscle cells (cardiomyocytes). NADH and FADH2 are produced in mitochondria via the Krebs cycle, afterwards both molecules are oxidized by complex I and II of the mitochondrial respiratory chain. The transfer of those electrons along the respiratory chain complexes I-IV establishes a proton gradient, which serves as the driving force for ATP production via the F1F0-ATP-synthase. The demand of ATP production constantly varies, therefore the electron transfer of the respiratory chain is regulated by the intracellular ADP- and Ca2 + concentrations. ADP activates the F1Fo-ATP synthase leading to the oxidation of NADH to NAD + as well as FADH2 to FAD. Under physiological conditions, however, an increased ATP production is induced by β-adrenergic stimulation, which leads to an increased cytosolic Ca2 + level in cardiomyocytes. Subsequently, the mitochondrial Ca2 + uniporter (MCU) ensures the uptake of Ca2 + into mitochondria where it stimulates the dehydrogenases of the citric acid cycle. Thereby, NAD + and FAD are reduced to NADH and FADH2 respectively. NADPH is essential for the detoxification of H2O2. Due to the equilibrium of NADH and NADPH, which is maintained by the mitochondrial transhydrogenase, the mitochondrial Ca2 + uptake mediates energetic adaptation as well as the regeneration of the oxidative capacity. In various forms of heart failure, mitochondrial dysfunction with energetic defects and oxidative stress have been observed. Genetic mutations are only rare causes for cardiac insufficiencies. One form of such a cardiomyopathy is the Barth syndrome, an X-linked recessive inherited disease, which only affects men and also causes skeletal muscular myopathy or immune deficiencies. The genetic defect affects the TAZ gene, which encodes for Tafazzin (TAZ). TAZ is a transacylase, which is essential for the biosynthesis of cardiolipin, a phospholipid of the inner mitochondrial membrane. The aim of this thesis was to examine the mechanisms that lead to the development of oxidative stress in mice with Tafazzin knockdown (Taz-KD), as Taz-KD is supposed to play a key role with the pathophysiology of cardiac disorders as well as the Barth syndrome. Focus was put on a potentially disturbed mitochondrial Ca2 + transport, which may lead to a redox shift. Isolated mitochondria from cardiomyocytes of Taz-KD mice were compared to those of wild type (WT) mice. The isolated cardiomyocytes were examined with respect to contractility and redox state. Furthermore, the cytosolic Ca2 + concentration and the formation of reactive oxygen species (ROS) were detected. The results show a shorter diastolic sarcomere length in Taz-KD cardiomyocytes under an increased workload, which indicates a diastolic dysfunction. A systolic dysfunction could not be verified. Taz-KD cardiomyocytes show a significantly stronger oxidation of NAD (P) H and FADH2 over the course of the experiment, especially during times of increased stimulation frequencies. This indicates an energetic mismatch, where Taz-KD cardiomyocytes may provide a decreased amount of NADH and FADH2 in the citric acid cycle. In contrast to Taz-KD cardiomyocytes, a rising cytosolic Ca2 + concentration during β-adrenergic stimulation could only be shown for WT cardiomyocytes. Although isolated mitochondria showed increased NADPH oxidation, the emission of H2O2 did not differ between both groups. Based on the results, it can be concluded that Taz-KD cardiomyocytes have a diastolic dysfunction, and that during workload transitions, oxidation of pyridine nucleotides may be the result of decreased mitochondrial Ca2 + uptake. Consecutive experiments of our working group indeed identified a distinctive defect of mitochondrial Ca2 + uptake, which is caused by a strong downregulation of the mitochondrial Ca2 + uniporter. The lack of mitochondrial Ca2 + uptake therefore explains the strong deficiency of bioenergetic adaptation and leads to the oxidation of NAD (P) H and FADH2 during β-adrenergic stimulation due to inactive Ca2 + dependent key enzymes of the citric acid cycle, hence hindering the regeneration of NAD (P) H, which is crucial for the H2O2 elimination. These data argue against the currently prevailing concept that in Barth syndrome, mitochondrial ROS formation is the result of defects in the respiratory chain secondary to cardiolipin deficiency, but rather point towards defective mitochondrial Ca2 + uptake that hampers the adaptation of the antioxidative capacity during workload transitions.