Kalju Paju

Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice

The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the Km for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low Km for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.

Sarcoplasmic reticulum function in determining atrioventricular contractile differences in rat heart

The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+dT/d t) and relaxation (-dT/d t) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas +dT/d t and -dT/d t(normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca2+-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the α-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.The relationships between the contractile characteristics and the sarcoplasmic reticulum (SR) function of rat atrial and ventricular trabeculae were compared. The isometric developed tension (DT) and the rates of contraction (+ dT/dt) and relaxation (-dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower, respectively, in intact atrial strips compared with ventricular strips, whereas + dT/dt and -dT/dt (normalized to DT) were 2.3 and 2.8 times higher, respectively, in atria. Atria exhibited a maximal potentiation of DT after shorter rest periods than ventricles and a lower reversal for prolonged rest periods. Caffeine-induced tension transients in saponin-permeabilized fibers suggested that the Ca2+ concentration released in atrial myofibrils reached a lower maximum and decayed more slowly than in ventricular preparations. However, the tension-time integrals indicated an equivalent capacity of sequestrable Ca2+ in SR from both tissues. In atrial, as in ventricular myocardium, the SR Ca2+ uptake was more efficiently supported by ATP produced by the SR-bound MM form of creatine kinase (CK; MM-CK) than by externally added ATP, suggesting a tight functional coupling between the SR Ca2+ adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of oxalate-supported Ca2+ uptake was two times higher in atrial than in ventricular tissue homogenates. The SR Ca(2+)-ATPase 2a mRNA content normalized to 18S RNA was 38% higher in atria than in ventricles, whereas the amount of mRNA encoding the alpha-myosin heavy chain, calsequestrin, and the ryanodine receptor was similar in both tissues. Thus a lower amount of readily releasable Ca2+ together with a faster uptake rate may partly account for the shorter time course and lower tension development in intact atrial myocardium compared with ventricular myocardium.

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.

Compartmentation of energy metabolism in atrial myocardium of patients undergoing cardiac surgery.

The parameters of oxidative phosphorylation and its interaction with creatine kinase (CK)- and adenylate kinase (AK)-phosphotransfer networks in situ were studied in skinned atrial fibers from 59 patients undergoing coronary artery bypass surgery, valve replacement/correction and atrial septal defect correction. In atria, the mitochondrial CK and AK are effectively coupled to oxidative phosphorylation, the MM-CK is coupled to ATPases and there exists a direct transfer of adenine nucleotides between mitochondria and ATPases. Elimination of cytoplasmic ADP with exogenous pyruvate kinase was not associated with a blockade of the stimulatory effects of creatine and AMP on respiration, neither could it abolish the coupling of MM-CK to ATPases and direct transfer of adenine nucleotides. Thus, atrial energy metabolism is compartmentalized so that mitochondria form functional complexes with adjacent ATPases. These complexes isolate a part of cellular adenine nucleotides from their cytoplasmic pool for participating in energy transfer via CK- and AK-networks, and/or direct exchange. Compared to atria in sinus rhythm, the fibrillating atria were larger and exhibited increased succinate-dependent respiration relative to glutamate-dependent respiration and augmented proton leak. Thus, alterations in mitochondrial oxidative phosphorylation may contribute to pathogenesis of atrial fibrillation. (Mol Cell Biochem 270: 49–61, 2005)

Thyroid hormones increase the contractility but suppress the effects of β-adrenergic agonist by decreasing phospholamban expression in rat atria

OBJECTIVE The aim of the present study was to characterize the relationships between the thyroid-hormone-dependent changes in sarcoplasmic reticulum (SR) Ca2+ handling and contractile performance in atria. METHODS Hypothyroidism in rats was induced by adding 0.05% 6-n-propyl-2-thiouracil to their drinking water for 6 weeks. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (1 microgram/g body weight) to euthyroid rats for 1 week. Left atria from the hearts with different thyroid states were examined by means of contractile measurements, SR oxalate-supported Ca(2+)-uptake, and Western blot of SR proteins. RESULTS The tissue level of SR Ca(2+)-pump protein decreased in hypothyroid (46 +/- 6%) atria, but remained unchanged in hyperthyroid (110 +/- 8%) atria as compared with euthyroid atria. Hypothyroidism was associated with increased phospholamban expression (141 +/- 25%), whereas it was drastically downregulated under hyperthyroidism (21 +/- 4%). The rate of SR Ca(2+)-uptake, measured in the presence of the protein kinase A inhibitor, H-89, was higher in hyperthyroid atria and lower in hypothyroid atria than in euthyroid atria (397 +/- 40, 55 +/- 6 and 194 +/- 17 nmol Ca2+/g protein/min, respectively). However, the stimulation of SR Ca(2+)-uptake by the catalytic subunit of protein kinase A was relatively weaker in hyperthyroid (130 +/- 20% over control level without catalytic subunit) and stronger in hypothyroid (640 +/- 60%) than in euthyroid atria (280 +/- 40%). The rates of inotropic contraction (+dT/dt) were higher in the hyperthyroid atria (133 +/- 10 mN/s), but lower in hypothyroid atria (15 +/- 3 mN/s) than in their euthyroid counterparts (95 +/- 13 mN/s). Inversely, hypothyroid atria responded to isoproterenol with much larger increases in contractility (883 +/- 164% over the control values for the same muscle before addition of isoproterenol) and hyperthyroid with smaller increases (25 +/- 9%) than euthyroid preparations (207 +/- 17%) CONCLUSIONS Thyroid hormones increase the contractility, but decrease the inotropic response to isoproterenol through decreasing the phospholamban/SR Ca(2+)-pump ratio in rat atria.

Thyroid hormones differentially affect sarcoplasmic reticulum function in rat atria and ventricles

The present study was undertaken to compare the effects of hypothyroidism and hyperthyroidism on sarcoplasmic reticulum (SR) Ca2+-pump activity, together with assessment of the functional role of SR in providing activator Ca2+ under these altered thyroid states. In response to a shift from hypothyroid to hyperthyroid state, a 10 fold and 2 fold increase in SR Ca2+-pump activity in atria and ventricles, respectively, were observed. This was associated with the 8-9 fold increases in atrial contractility (+dT/dt) and relaxation (-dT/dt), but only with a 3-4 fold increase in their ventricular counterparts. Also, the recirculation fraction of activator Ca2+ (RFA) increased to a far greater extent in atria (4 fold) than in papillary muscles, and the relative increment in inhibition of developed tension by ryanodine became 3 times larger in atria than in papillary muscles. A positive force-frequency relationship (FFR) was observed in hypothyroid atria, whereas the hyperthyroid atria, hypothyroid and hyperthyroid papillary muscles showed a negative FFR. These results suggest the greater role of transsarcolemmal (SL) Ca2+ and smaller role of SR Ca2+ in activating contraction in hypothyroid atria compared to other preparations. Thyroid hormones decrease the contribution of SL and increase that of SR in providing activator Ca2+ to the greater extent in atria than in ventricles. This effect of thyroid hormones is based on larger stimulation of SR Ca2+-pump in atria compared to ventricles.

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.

Proliferation of Human Primary Myoblasts Is Associated with Altered Energy Metabolism in Dependence on Ageing In Vivo and In Vitro

Background. Ageing is associated with suppressed regenerative potential of muscle precursor cells due to decrease of satellite cells and suppressive intramuscular milieu on their activation, associated with ageing-related low-grade inflammation. The aim of the study was to characterize the function of oxidative phosphorylation (OXPHOS), glycolysis, adenylate kinase (AK), and creatine kinase (CK) mediated systems in young and older individuals. Materials and Methods. Myoblasts were cultivated from biopsies taken by transcutaneous conchotomy from vastus lateralis muscle in young (20–29 yrs, n = 7) and older (70–79 yrs, n = 7) subjects. Energy metabolism was assessed in passages 2 to 6 by oxygraphy and enzyme analysis. Results. In myoblasts of young and older subjects the rate of OXPHOS decreased during proliferation from passages 2 to 6. The total activities of CK and AK decreased. Myoblasts of passage 2 cultivated from young muscle showed higher rate of OXPHOS and activities of CK and AK compared to myoblasts from older subjects while hexokinase and pyruvate kinase were not affected by ageing. Conclusions. Proliferation of myoblasts in vitro is associated with downregulation of OXPHOS and energy storage and transfer systems. Ageing in vivo exerts an impact on satellite cells which results in altered metabolic profile in favour of the prevalence of glycolytic pathways over mitochondrial OXPHOS of myoblasts.

Mechanisms of thyroid hormone control over sensitivity and maximal contractile responsiveness to β-adrenergic agonists in atria

This paper discusses the mechanisms of two basic effects of thyroid hormones on atrial responses to β-adrenergic agonists, i.e. increased inotropic sensitivity and decreased maximal contractile responsiveness. The increased sensitivity of atria to β; adrenergic agonists under thyroid hormones appears to be related to increases in β-adrenoceptor density and Gs/Gi. protein ratio, leading to activation of Gs-mediated pathway, but suppression of Gi.-mediated pathway of adenylate cyclase regulation. Therefore, the i/c concentrations of cAMP and corresponding inotropic responses achieve their maximums at lower doses of β-adrenergic agonist. Thyroid hormones also decrease the expression of phospholamban, but increase the expression of sarcoplasmic reticulum Ca2+-pump. As a result, the basal activity of sarcoplasmic reticulum Ca2+-pump increases, but its β-adrenergic activation through phosphorylation of phospholamban decreases. It is suggested that these changes are causal for decreased maximal inotropic and lusitropic responses of atria to β-adrenergic agonists.(Mol Cell Biochem 184: 419–426, 1998)

Deep Quantitative Proteomics Reveals Extensive Metabolic Reprogramming and Cancer-Like Changes of Ectopic Endometriotic Stromal Cells

Endometriosis is a prevalent health condition in women of reproductive age characterized by ectopic growth of endometrial-like tissue in the extrauterine environment. Thorough understanding of the molecular mechanisms underlying the disease is still incomplete. We dissected eutopic and ectopic endometrial primary stromal cell proteomes to a depth of nearly 6900 proteins using quantitative mass spectrometry with a spike-in SILAC standard. Acquired data revealed metabolic reprogramming of ectopic stromal cells with extensive upregulation of glycolysis and downregulation of oxidative respiration, a widespread metabolic phenotype known as the Warburg effect and previously described in many cancers. These changes in metabolism are additionally accompanied by attenuated aerobic respiration of ectopic endometrial stromal cells as measured by live-cell oximetry and by altered mRNA levels of respective enzyme complexes. Our results additionally highlight other molecular changes of ectopic endometriotic stromal cells indicating reduced apoptotic potential, increased cellular invasiveness and adhesiveness, and altered immune function. Altogether, these comprehensive proteomics data refine the current understanding of endometriosis pathogenesis and present new avenues for therapies.