Exercise during short-term exposure to hypoxia or hyperoxia - novel treatment strategies for type 2 diabetic patients?!

Both hypoxia (decreased oxygen availability) and hyperoxia (increased oxygen availability) have been shown to alter exercise adaptations in healthy subjects. This review aims to clarify the possible benefits of exercise during short-term exposure to hypoxia or hyperoxia for patients with type 2 diabetes mellitus (T2DM). There is evidence that exercise during short-term exposure to hypoxia can acutely increase skeletal muscle glucose uptake more than exercise in normoxia, and that post-exercise insulin sensitivity in T2DM patients is more increased when exercise is performed under hypoxic conditions. Furthermore, interventional studies show that glycemic control can be improved through regular physical exercise in short-term hypoxia at a lower workload than in normoxia, and that exercise training in short-term hypoxia can contribute to increased weight loss in overweight/obese (insulin-resistant) subjects. While numerous studies involving healthy subjects report that regular exercise in hypoxia can increase vascular health (skeletal muscle capillarization and vascular dilator function) to a higher extent than exercise training in normoxia, there is no convincing evidence yet that hypoxia has such additive effects in T2DM patients in the long term. Some studies indicate that the use of hyperoxia during exercise can decrease lactate concentrations and subjective ratings of perceived exertion. Thus, there are interesting starting points for future studies to further evaluate possible beneficial effects of exercise in short-term hypoxia or hyperoxia at different oxygen concentrations and exposure durations. In general, exposure to hypoxia/hyperoxia should be considered with caution. Possible health risks-especially for T2DM patients-are also analyzed in this review.

Muscular Oxygen Uptake Kinetics in Aged Adults.

Pulmonary oxygen uptake (V˙O2) kinetics and heart rate kinetics are influenced by age and fitness. Muscular V˙O2 kinetics can be estimated from heart rate and pulmonary V˙O2. In this study the applicability of a test using pseudo-random binary sequences in combination with a model to estimate muscular V˙O2 kinetics was tested. Muscular V˙O2 kinetics were expected to be faster than pulmonary V˙O2 kinetics, slowed in aged subjects and correlated with maximum V˙O2 and heart rate kinetics. 27 elderly subjects (73±3 years; 81.1±8.2 kg; 175±4.7 cm) participated. Cardiorespiratory kinetics were assessed using the maximum of cross-correlation functions, higher maxima implying faster kinetics. Muscular V˙O2 kinetics were faster than pulmonary V˙O2 kinetics (0.31±0.1 vs. 0.29±0.1 s; p=0.004). Heart rate kinetics were not correlated with muscular or pulmonary V˙O2 kinetics or maximum V˙O2. Muscular V˙O2 kinetics correlated with maximum V˙O2 (r=0.35; p=0.033). This suggests, that muscular V˙O2 kinetics are faster than estimates from pulmonary V˙O2 and related to maximum V˙O2 in aged subjects. In the future this experimental approach may help to characterize alterations in muscular V˙O2 under various conditions independent of motivation and maximal effort.

Effects of Wearing Compression Stockings on the Physical Performance of T2DM Men with MetS.

Metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) are associated with macro- and microcirculatory complications that reduce physical performance. Wearing compression garments to potentially optimize hemodynamics has been discussed. This study investigates the effects of wearing compression stockings on physical performance-related variables in type 2 diabetic men with metabolic syndrome (n=9, 57±12 years, BMI: 36±4 kg/m(2)). Participants served as their own controls in a randomized 3*3 crossover study wearing below-knee stockings with either compression (24 or 30 mmHg ankle pressure) or no compression. Venous pooling and lower limb oxygenation profiles were determined with near-infrared spectroscopy and arterial oxygen saturation was determined using a pulse oxymeter. Measurements were performed in the supine lying position, during standing, following 10 tiptoe exercises and after submaximal intensity cycling. In addition, lactate and erythrocyte deformability were analyzed in capillary blood pre- and post-exercise. Erythrocyte deformability was analyzed using a laser-assisted optical rotational red cell analyzer. No significant differences in any variables when wearing different compression or regular stockings were evident at any point of measurement. This study did not reveal any beneficial effects of wearing compression stockings at rest and during acute bouts of moderately intense exercise in this particular patient group.

Endurance training alters skeletal muscle MCT contents in T2DM men.

Patients suffering from type 2 diabetes mellitus (T2DM) often exhibit chronic elevated lactate levels which can promote peripheral insulin resistance by disturbing skeletal muscle insulin-signaling. Monocarboxylate transporter (MCT) proteins transfer lactate molecules through cellular membranes. MCT-1 and MCT-4 are the main protein isoforms expressed in human skeletal muscle, with MCT-1 showing a higher affinity (lower Km) for lactate than MCT-4. T2DM patients have reduced membranous MCT-1 proteins. Consequently, the lactate transport between muscle cells and the circulation as well as within an intracellular lactate shuttle, involving mitochondria (where lactate can be further metabolized), can be negatively affected. This study investigates whether moderate cycling endurance training (3 times per week for 3 months) can change skele-tal muscle MCT contents in T2DM men (n=8, years=56±9, body mass index (BMI)=32±4 kg/m(2)). Protein content analyses (immuno-histochemical stainings) were performed in bio-psies taken from the vastus lateralis muscle. Intracellular MCT-1 proteins were up-regulated (relative increase+89%), while intracellular MCT-4 contents were down-regulated (relative decrease - 41%) following endurance training. Sarcolemmal MCT-1 and MCT-4 did not change. The question of whether the training-induced up-regulation of intracellular MCT-1 leads to an improved lactate transport (and clearance) in T2DM patients requires further research.

Training alters the skeletal muscle antioxidative capacity in non-insulin-dependent type 2 diabetic men.

The present study analyzes the oxidative stress situation in the skeletal muscle of overweight/obese men suffering from non-insulin-dependent type 2 diabetes mellitus [T2DM, n=16, years=61±7, body mass index (BMI)=31±4 kg/m(2) ] and BMI-matched non-diabetic male control subjects (CON, n=7, years=53±6, BMI=30±4 kg/m(2) ). Furthermore, it investigates whether physical training can alter the skeletal muscle antioxidative capacity of T2DM patients at rest. Molecule content analyses (immunohistochemical stainings) of 8-iso-prostaglandin-F2α (8-Iso-PGF), superoxide dismutase-2 (SOD2), glutathione peroxidase-1 (GPX1), peroxiredoxin isoforms (PRDX 1-6) and heat-shock-protein-70 (HSP70) were performed in biopsies taken from the vastus lateralis muscle. Under basal conditions, 8-Iso-PGF was significantly decreased in T2DM patients (-35.7%), whereas PRDX2 and PRDX6 were significantly increased relative to CON (+82.6%; +82.3%). Differences were neither observed in SOD2 nor in GPX1 or PRDX1, 3, 4, 5 density. Regular physical activity (moderate endurance or resistance training twice a week for 3 months) did not alter PRDX1, 2, 3, 4, 6 in the skeletal muscle of T2DM patients, but significantly increased SOD2 (+65.9%), GPX1 (+62.4%), PRDX5 (+37.5%), and HSP70 (+48.5%). Overweight/obese men with non-insulin-dependent T2DM exhibit up-regulated cytosolic peroxiredoxin contents relative to BMI-matched controls. Regular training further up-regulates cytosolic and mitochondrial antioxidative enzymes in T2DM patients and improves their cellular protection systems. This may contribute to a retardation of the disease's progression.

Sport and oxidative stress in oncological patients.

Oxidative stress is thought to be an important factor in the onset, progression and recurrence of cancer. In order to investigate how it is influenced by physical activity, we measured oxidative stress and antioxidative capacity (aoC) in 12 women with breast cancer and 6 men with prostate cancer, before and after long hiking trips. Before the hike, the men had a ROS-concentration of 1.8±0.6 mM H2O2 and an aoC of 0.7±0.6 mM Trolox-equivalent (Tro), while the women had a ROS-concentration of 3.1±0.7 mM H2O2 and an aoC of 1.2±0.2 mM Tro. After the hike, women showed no significant change in ROS and a significant increase in aoC (1.3±0.2 mM Tro), while the ROS concentration in men increased significantly (2.1±0.3 mM H2O2) and their aoC decreased (0.25±0.1 mM Tro). After a regenerative phase, the ROS concentration of the men decreased to 1.7±0.4 mM H2O2 and their aoC recovered significantly (1.2±0.4 mM Tro), while the women presented no significant change in the concentration of H2O2 but showed an ulterior increase in antioxidant capacity (2.05±0.43 mM Tro). From this data we conclude that physical training programs as for example long distance hiking trips can improve the aoC in the blood of oncological patients.

eNOS phosphorylation and translocation are altered in male but not female mice by increased activation of the Gαq protein.

Little is known about sex-dependent physiological and pathophysiological differences in cardiac endothelial nitric oxide synthase (eNOS) expression and activation. Therefore, we investigated cardiac morphology and eNOS protein expression, including its translocation-dependent activation and phosphorylation, in cardiac tissue of male and female wild-type mice and transgenic heart-failure mice having a cardiac-specific, 5-fold overexpression of the Galphaq protein. In addition, we measured calcineurin protein expression. Heart-to-body weight ratio was increased in Galphaq mice. Female wild-type mice showed higher eNOS protein expression and activation (translocation and phosphorylation) than did wild-type males. In cardiac tissue of Galphaq mice, these sex-dependent differences remained or were enhanced. Protein expression of the catalytic subunit calcineurin A, which has been shown to dephosphorylate eNOS, was higher in wild-type males than in wild-type females. These differences were increased in the Galphaq mice model. We conclude that sex differences exist in cardiac eNOS protein expression and phosphorylation. Increased activation of the Galphaq protein appears to alter eNOS protein expression and phosphorylation only in males.

Regulation of extracellular matrix compounds involved in angiogenic processes in short- and long-track elite runners.

Exercise induces alterations of the extracellular matrix (ECM), e.g. by an increased release of endostatin or by regulation of matrix metalloproteases (MMP)-2/-9, and cathepsin L. To investigate the influence of training status on exercise-induced ECM-processing of angiogenic molecules, alterations of endostatin-, MMP-2, and MMP-9 plasma concentrations during incremental running step tests in male elite short-track (n=6) and male elite long-track runners (n=7) were studied. Three blood samples (pre-exercise, 0, and 1 h post-exercise) were taken from each subject at each running test. In both groups, the basal endostatin plasma concentration was significantly decreased at the second running test, i.e. after the training season. Exercise-related acute alterations of the parameters were also observed only during the second test. In the long-track group, there was a significant increase in endostatin at 0 h and of MMP-2 at 1 h post-exercise. In the short-track group, only MMP-9 was significantly increased at 0 h post-exercise. Cathepsin L was increased at 0 h post-exercise. In conclusion, regular exercise performance decreases the basal endostatin plasma concentration, facilitates ECM-processing of angiogenic molecules by regular performance, and seems to be dependent on the kind of training.

Total beta-adrenoceptor deficiency results in cardiac hypotrophy and negative inotropy.

The present study investigated cardiac function in hearts of mice with total deficiency of the beta1-, beta2- and beta3-adrenoceptors (TKO) in comparison to wildtype mice (WT). We investigated cardiac morphology and echocardiographic function, measured protein expression of Ca2+-regulatory proteins, SERCA 2a activity, myofibrillar function, and performed running wheel tests. Heart weight and heart-to-body weight ratio were significantly smaller in TKO as compared to WT. This was accompanied by a decrease in the size of the cardiomyocytes in TKO. Heart rate and ejection fraction were significantly diminished in TKO as compared to WT. Protein expressions of SERCA 2a, ryanodine receptor and Na+/Ca2)-exchanger were similar in TKO and WT mice, but phospholamban protein expression was increased. PKA-dependent phosphorylation of phospholamban at serine 16 was absent and CaMKII-dependent phosphorylation at threonine 17 was decreased in TKO. All alterations were paralleled by a decrease in SERCA 2a-activity. A similar maximal calcium-dependent tension but an increased myofibrillar calcium-sensitivity was measured in TKO as compared to WT. We did not observe relevant functional impairments of TKO in running wheel tests. In the absence of beta-agonistic stimulation, SERCA 2a activity is mainly regulated by alterations of phospholamban expression and phosphorylation. The decreased SERCA 2a activity following beta-adrenoceptor deficiency may be partly compensated by an increased myofibrillar calcium-sensitivity.

Regulation of endothelial nitric oxide synthase (eNOS) in myocardium subjected to cardioplegic arrest.

BACKGROUND: Nitric oxide (NO) production by both coronary endothelial cells and cardiomyocytes is thought to play a significant role in myocardial pathophysiology following ischemia/reperfusion (I/R). METHODS: In thirteen pigs subjected to 1 hour cardioplegic arrest (CA) on CPB, left ventricular (LV) biopsies were collected prior to CPB (baseline), at 60 min CPA, at 15 and 30 min reperfusion on CPB, and at 120 min post CPB. LV specimens were immunocytochemically stained against phospho-eNOS (Ser1177), phospho-eNOS (Thr495), phosphorylated ERK1/2, and AKT/PKB. Four additional pigs without CA served as controls. Cardiomyocytes were quantitatively investigated using TV densitometry (gray units: U). RESULTS: After 60 min CA phosphorylation of eNOS (Ser1177) increased significantly and remained elevated until 30 min of reperfusion. In contrast, eNOS (Thr495) phosphorylation remained unchanged during CA and throughout reperfusion. In control animals, eNOS phosphorylation remained unchanged. Akt/PKB activity significantly increased after 60 min CA and decreased thereafter. ERK1/2 activity remained unchanged during ischemia but increased during reperfusion. CONCLUSIONS: ENOS activation during ischemia occurs through phosphorylation at Ser1177 mediated by Akt/PKB. ERK1/2 does not seem to be involved in myocardial eNOS regulation especially not via phosphorylation at eNOS (Thr495).

Long-term endurance exercise decreases antiangiogenic endostatin signalling in overweight men aged 50-60 years.

BACKGROUND: Endurance training may decrease the risk of coronary artery disease. It has been speculated that these effects may be due to an exercise-induced stimulation of angiogenesis. The underlying mechanisms are not yet clear. Therefore, using ELISA, we investigated the plasma level of vascular endothelial growth factor (VEGF, angiogenic factor) and endostatin (antiangiogenic factor) in a group of untrained men aged 50-60 years with obesity. METHODS: All men were randomised into a "running" group (training 3 times/week, 60 min each, n = 7), a "cycling" group ( training 3 times/week, 90 min each, n = 7) and a sedentary control group ( n = 7). Both training groups worked at moderate intensity (2-4 mmol/l lactate). The intervention had a duration of 6 months. Before and after this period, blood samples were taken from the participants at rest and they underwent a medical investigation. RESULTS: Body mass index (BMI), systolic and diastolic blood pressure, and plasma levels of VEGF and endostatin were comparable in all three groups. Endurance training significantly reduced BMI in both exercise groups (mean (SEM) before v after 29.7 (0.7) v 29.1 (0.6) kg/m2 and 31.1 (0.7) v 30.1 (0.9) kg/m2 for the running and cycling groups respectively) but not in the control group (30.0 (1.0) v 30.2 (0.8) kg/m2). Endurance training did not influence VEGF plasma level (before v after 1.3 (0.4) v 1.5 (0.2) ng/ml for the running group; 1.6 (0.3) v 1.5 (0.2) ng/ml for the cycling group; and 2.5 (0.6) v 2.1 (0.7) ng/ml for the control group). Plasma level of endostatin was significantly reduced in both exercise groups (mean (SEM) before v after: 20.9 (1.6 v 17.5 (1.0) ng/ml and 21.3 (1.4 v 18.0 (1.6) ng/ml for the running and cycling groups respectively) but not in controls (19.7 (1.3 v 17.7 (1.1 ng/ml). CONCLUSIONS: Endurance training may reduce the antiangiogenic mechanisms in men aged 50-60 years by reducing endostatin plasma level and this may subsequently decrease the risk of cardiovascular disease.

Second-harmonic generation as a probe for structural and electronic properties of buried GaP/Si(0 0 1) interfaces.

Optical second-harmonic generation is demonstrated to be a sensitive probe of the buried interface between the lattice-matched semiconductors gallium phosphide and silicon with (0 0 1) orientation. Ex situ rotational anisotropy measurements on GaP/Si heterostructures show a strong isotropic component of the second-harmonic response not present for pure Si(0 0 1) or GaP(0 0 1). The strength of the overlaying anisotropic response directly correlates with the quality of the interface as determined by atomically resolved scanning transmission electron microscopy. Systematic comparison of samples fabricated under different growth conditions in metal-organic vapor phase epitaxy reveals that the anisotropy for different polarization combinations can be used as a selective fingerprint for the occurrence of anti-phase domains and twins. This all-optical technique can be applied as an in situ and non-invasive monitor even during growth.

Oxygen uptake kinetics following six weeks of interval and continuous endurance exercise training - An explorative pilot study.

PURPOSE: The aim of the study was to compare the responses of pulmonary (V˙O2pulm) and muscle (V˙O2musc) oxygen uptake kinetics before (PRE) and after (POST) six weeks of endurance exercise training. METHODS: Nine untrained individuals performed pseudo-random binary sequences work rate changes between 30W and 80W at PRE and POST training intervention. Heart rate (HR) and V˙O2pulm were measured beat-to-beat and breath-by-breath, respectively. V˙O2musc was estimated applying the approach of Hoffmann et al. (Eur J Appl Physiol 113: 1745-1754, 2013). RESULTS: Maximal oxygen uptake showed significant increases from PRE (3.2±0.3Lmin-1) to POST (3.7±0.2Lmin-1; p<0.05). For HR, V˙O2pulm and V˙O2musc kinetics no significant changes from PRE to POST training intervention were observed (p>0.05). CONCLUSIONS: Discrepancies in the adaptations of the involved exercise induced physiological systems seem to be responsible for the observed significant alterations in maximal V˙O2 after six weeks of the training intervention in contrast to no changes in the kinetics responses.

Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.

Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.

Nebivolol induces eNOS activation and NO-liberation in murine corpus cavernosum.

Erectile function is critically dependent upon the activation of the endothelial nitric oxide synthase (eNOS) in the smooth muscle cells of penile corpus cavernosum tissue. Nebivolol is a beta(1)-selective beta-adrenoceptor blocker (beta-ARB) with additional vasodilating properties, which have been attributed to eNOS-activation. Our study investigated whether nebivolol is able to increase eNOS activity in erectile tissue. Murine penile tissue was incubated in an organ bath under control conditions and in the presence of nebivolol or metoprolol. Immunofluorescence staining was performed using specific antibodies against eNOS-activation or eNOS-serine 1177 phosphorylation. Corpus cavernosum smooth muscle tissue was identified using a smooth muscle actin antibody. In addition, slices of murine erectile tissue were incubated with diaminofluorescein (DAF), a specific fluorescence marker for NO-liberation. Under control conditions and after application of metoprolol, we observed a small eNOS-activation and serine 1177-phosphorylation in murine corpus cavernosum tissue. A significant increase in eNOS-activation and serine 1177-phosphorylation of eNOS was observed only in the presence of nebivolol (10 muM). These alterations of the eNOS protein induced after application of nebivolol were associated with a time-dependent increase in DAF fluorescence in murine erectile tissue. We conclude that beta-adrenoceptor blockers differentially influence erectile tissue. Since cardiovascular diseases are often associated with the development of erectile dysfunction, the nebivolol-induced eNOS-activation in corpus cavernosum may be beneficial when treating patients suffering from cardiovascular disease.

Beta3-adrenergic stimulation in the human heart: signal transduction, functional implications and therapeutic perspectives.

Next to beta1- and beta2-adrenoceptors, a third beta-adrenoceptor population is expressed in the human heart, the beta3-adrenoceptor. In mammalian ventricular myocytes, beta3-adrenergic stimulation leads to a decrease in contractility via a release of nitric oxide (NO). Recently, different molecular mechanisms of beta3-adrenergic activation of endothelial nitric oxide synthase (eNOS) have been uncovered in cardiac myocytes. In the non-failing and especially the failing heart, beta3-adrenergic stimulation may offer protection against excessive catecholaminergic beta1-adrenoceptor stimulation. In this context, the beta3-adrenoceptor is discussed as a novel target for the pharmacological therapy of heart failure.

Endurance training alters enzymatic and rheological properties of red blood cells (RBC) in type 2 diabetic men during in vivo RBC aging.

This study examines the effects of endurance training on red blood cells (RBC) in seventeen non-insulin-dependent type 2 diabetic men with a special focus on in vivo RBC aging. Venous blood was collected pre- and post-training at rest. RBC from whole blood and RBC separated according to cell age by density-gradient centrifugation were analyzed. RBC deformability was measured by ektacytometry. Immunohistochemical staining was performed to quantify the RBC-nitric oxide (NO) synthase activation (RBC-NOSSer1177) because RBC-NOS-produced NO can contribute to increased RBC deformability. The proportion of "young" RBC was significantly higher post-training. RBC deformability of all RBC (RBC of all ages) remained unaltered post-training. During RBC aging, RBC deformability decreased in both pre- and post-training. However, the training significantly increased RBC deformability in "young" and reduced their deformability in aging RBC. RBC-NOS activation remained unaltered in all RBC post-training. It tendentially increased in aging RBC pre-training, but did not change during aging post-training. The training significantly reduced RBC-NOS activation in "old" RBC. Endurance training may improve the RBC system (higher amount of "young" RBC which are more deformable). It remains speculative whether changes in older RBC (reduced RBC-NOS activation and deformability) could lead to more rapid elimination of aged RBC.

Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na+,K+-ATPase activity but unchanged Na+-Ca2+ exchanger protein levels in human heart failure.

BACKGROUND: Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+, K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. METHODS AND RESULTS: Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic alpha1-, alpha2-, alpha3-, and glycoprotein beta1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain-binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase alpha1- (0.62+/-0.06 of control), alpha3- (0.70+/-0.09), and beta1- (0.61+/-0.04) but not alpha2-isoforms were significantly reduced (P<0.01), whereas levels of NCE (0.92+/-0.13 of control) and calsequestrin (0.98+/-0.06) remained unchanged. Both Na+,K+-ATPase activity (NF: 1.9+/-0.29; NYHA class IV: 1.1+/-0.17 micromol ATP/min per milligram of protein) and the 3H-ouabain binding sites (Bmax NF: 15.9+/-1.9 pmol/mg protein; NYHA class IV: 9.7+/-1.5) were reduced in NYHA class IV and correlated significantly to each other (r2=0. 73; P<0.0001), as did beta1-subunit expression. In left ventricular papillary muscle strips from NYHA class IV compared with nonfailing tissue the Na+-channel modulator BDF 9198 exerted an increase in force of contraction with unchanged effectiveness but enhanced potency. CONCLUSIONS: The enhanced sensitivity of failing human myocardium toward cardiac glycosides may be, at least in part, attributed to a reduced protein expression and activity of the sarcolemmal Na+,K+-ATPase without a change in Na+-Ca2+ exchanger protein expression.

Increased expression of isoform 1 of the sarcoplasmic reticulum Ca(2+)-release channel in failing human heart.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca(2+)-release channel plays a key role in the excitation-contraction coupling of cardiac myocytes. Because respective alterations have been reported in human heart failure, we investigated isoform expression of the SR Ca(2+)-release channel in human hearts from patients with terminal heart failure (dilated cardiomyopathy [DCM], n=8) and nonfailing organ donors (NF, n=8). METHODS AND RESULTS: Expression of mRNA of SR Ca(2+)-release channel isoforms in isolated human cardiomyocytes and myocardial tissue was analyzed by reverse-transcription polymerase chain reaction. Protein expression was quantified in myocardial tissue with [(3)H]-ryanodine binding and with Western blots, expressed as densitometric units per microgram of protein (DU), and cellular localization was visualized with immunohistochemistry. We found mRNA expression of isoforms 1, 2, and 3 in cardiomyocytes and myocardial tissue both in NF and DCM. Total SR Ca(2+)-release channel protein expression in NF (B(max) 2.16+/-0.43 pmol/mg protein) and in DCM (B(max) 2.33+/-0.22 pmol/mg protein) myocardium was unchanged. Expression of isoform 1 of the SR Ca(2+)-release channel was significantly (P=0.0037) increased in DCM myocardium (NF 1.97+/-0.25 versus DCM 3.37+/-0.31 DU), whereas protein expression of isoform 2 (NF 14.62+/-0.87 versus DCM 13.52+/-0.43 DU) and isoform 3 (NF 1.39+/-0.13 versus DCM 1.35+/-0.19 DU) was unchanged. All 3 isoforms of the protein could be localized in human ventricular myocytes with fluorescence immunohistochemistry. CONCLUSIONS: All 3 isoforms of the SR Ca(2+)-release channel were determined in human ventricular cardiomyocytes. Increased expression of isoform 1 of the SR Ca(2+)-release channel could contribute to impaired excitation-contraction coupling in human heart failure.

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium.

The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.