Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.     

Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle

Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered--localization of diffusion restrictions--is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.     

Study of possible interactions of tubulin, microtubular network, and STOP protein with mitochondria in muscle cells

We studied possible connections of tubulin, microtubular system, and microtubular network stabilizing STOP protein with mitochondria in rat and mouse cardiac and skeletal muscles by confocal microscopy and oxygraphy. Intracellular localization and content of tubulin was found to be muscle type-specific, with high amounts in oxidative muscles, and much lower in glycolytic skeletal muscle. STOP protein localization and content in muscle cells was also muscle type-specific. In isolated heart mitochondria, addition of 1 μM tubulin heterodimer increased apparent K _m for ADP significantly. Dissociation of microtubular system into free tubulin by colchicine treatment only slightly decreased initially high apparent K _m for ADP in permeabilized cells, and diffusely distributed free tubulin stayed inside the cells, obviously connected to the intracellular structures. To identify the genes that are specific for oxidative muscle, we developed and applied a method of kindred DNA. The results of sequencing and bioinformatic analysis of isolated cDNA pool common for heart and m. soleus showed that in adult mice the β-tubulin gene is expressed predominantly in oxidative muscle cells. It is concluded that whereas dimeric tubulin may play a significant role in regulation of mitochondrial outer membrane permeability in the cells in vivo, its organization into microtubular network has a minor significance on that process.     

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes

Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.     

Characteristics of the frequency dependence of the contraction force in the hyperthyroid rat myocardium

We have studied the effect of increased contraction frequency (from 0.2 to 1.5 Hz) on developed tension (delta T) in thin papillary muscles of eu- and hyperthyroid rats. The results show that while increasing the contraction frequency, the delta T of euthyroid papillary muscles decreased at lower frequencies than in hyperthyroid group. Also, at the contraction frequencies above 1.0 Hz the absolute and relative levels of delta T of hyperthyroid myocardium were less decreased than in euthyroid preparations. In conclusion, the myocardium of hyperthyroid rat is characterized by a decreased sensitivity to negative inotropic effect of enhanced contraction frequency. In is probably due to the acceleration of the processes of intracellular Ca2+ recycling during diastole under the influence of hyperthyroidism.     

Synthesis of phosphocreatine in heart mitochondria of rats with hyperthyroidism

The mechanisms of the phosphocreatine/creatine ratio decrease in female Wistar rats with hyperthyroidism were studied. L-Thyroxin was injected to animals in doses of 50 and 100 micrograms/100 g of body weight, daily for 1 and 2 weeks. Oxidative phosphorylation and the rate of phosphocreatine synthesis were studied in isolated rat heart mitochondria. It was found that hyperthyroidism caused an increase in the ADP-activated mitochondrial respiration, whereas the coupling between electron transport and ADP phosphorylated remained at a constant level. Besides oxidative phosphorylation, activation, hyperthyroidism increased the rate of phosphocreatine synthesis at high values of the phosphocreatine/oxygen ratio. Thus, hyperthyroidism is unaccompanied by and significant changes in the coupling of mitochondrial creatine kinase with oxidative phosphorylation.     

Impaired regulation of brain mitochondria by extramitochondrial Ca2+ in transgenic Huntington disease rats

Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.     

Hormone regulation of cardiac energy metabolism. I. Creatine transport across cell membranes of euthyroid and hyperthyroid rat heart

Hyperthyroid rat heart was studied with the purpose of identifying the mechanism for the significant decrease in total creatine (free creatine plus phosphocreatine) observed in this pathology and its consequences on heart function. Administration of L-thyroxine in doses of 50-100 micrograms/100 g of body weight during a week resulted in a reversible decrease of the total creatine by 40-50%. Simultaneously, remarkable changes in the creatine transport system across the cardiac cell membranes were observed: both the maximal rate of its active uptake and its passive movement along its concentration gradient were enhanced. In euthyroid hearts, the parameters of creatine uptake (Km approximately or equal to 0.05 mM, Vmax = 20 nmole/min/g dry weight) were similar to those for skeletal muscle and the passive movement of creatine was negligible. In hyperthyroid hearts the latter rate was enhanced to 0.4 mumole min/g dry weight, this showing reversible damages in the cell membrane structure induced by L-thyroxine. This conclusion is consistent with observed penetration of colloidal lanthanum into the cells of hyperthyroid hearts. Perfusion of hyperthyroid rat hearts with 50 mM creatine significantly restored creatine content in the cells, Hyperthyroid hearts with decreased creatine content were found to develop ischemic contracture more rapidly and in higher extent than the euthyroid hearts. Increased sensitivity to ischemic damage may be related to decreased efficiency of energy channeling via phosphocreatine pathway.