Hydrogen Peroxide Elicits Constriction of Skeletal Muscle Arterioles by Activating the Arachidonic Acid Pathway

Aims

The molecular mechanisms of the vasoconstrictor responses evoked by hydrogen peroxide (H₂O₂) have not been clearly elucidated in skeletal muscle arterioles.

Methods and Results

Changes in diameter of isolated, cannulated and pressurized gracilis muscle arterioles (GAs) of Wistar-Kyoto rats were determined under various test conditions. H₂O₂ (10–100 µM) evoked concentration-dependent constrictions in the GAs, which were inhibited by endothelium removal, or by antagonists of phospholipase A (PLA; 100 µM 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid), protein kinase C (PKC; 10 µM chelerythrine), phospholipase C (PLC; 10 µM U-73122), or Src family tyrosine kinase (Src kinase; 1 µM Src Inhibitor-1). Antagonists of thromboxane A2 (TXA2; 1 µM SQ-29548) or the non-specific cyclooxygenase (COX) inhibitor indomethacin (10 µM) converted constrictions to dilations. The COX-1 inhibitor (SC-560, 1 µM) demonstrated a greater reduction in constriction and conversion to dilation than that of COX-2 (celecoxib, 3 µM). H₂O₂ did not elicit significant changes in arteriolar Ca²⁺ levels measured with Fura-2.

Conclusions

These data suggest that H₂O₂ activates the endothelial Src kinase/PLC/PKC/PLA pathway, ultimately leading to the synthesis and release of TXA2 by COX-1, thereby increasing the Ca²⁺ sensitivity of the vascular smooth muscle cells and eliciting constriction in rat skeletal muscle arterioles.     

Excitation–Contraction Coupling: Acute exposure to extracellular BTP2 does not inhibit Ca2+ release during EC coupling in intact skeletal muscle fibers

The inhibitor of store-operated Ca²⁺ entry (SOCE) BTP2 was reported to inhibit ryanodine receptor Ca²⁺ leak and electrically evoked Ca²⁺ release from the sarcoplasmic reticulum when introduced into mechanically skinned muscle fibers. However, it is unclear how effects of intracellular application of a highly lipophilic drug like BTP2 on Ca²⁺ release during excitation–contraction (EC) coupling compare with extracellular exposure in intact muscle fibers. Here, we address this question by quantifying the effect of short- and long-term exposure to 10 and 20 µM BTP2 on the magnitude and kinetics of electrically evoked Ca²⁺ release in intact mouse flexor digitorum brevis muscle fibers. Our results demonstrate that neither the magnitude nor the kinetics of electrically evoked Ca²⁺ release evoked during repetitive electrical stimulation were altered by brief exposure (2 min) to either BTP2 concentration. However, BTP2 did reduce the magnitude of electrically evoked Ca²⁺ release in intact fibers when applied extracellularly for a prolonged period of time (30 min at 10 µM or 10 min at 20 µM), consistent with slow diffusion of the lipophilic drug across the plasma membrane. Together, these results indicate that the time course and impact of BTP2 on Ca²⁺ release during EC coupling in skeletal muscle depends strongly on whether the drug is applied intracellularly or extracellularly. Further, these results demonstrate that electrically evoked Ca²⁺ release in intact muscle fibers is unaltered by extracellular application of 10 µM BTP2 for <25 min, validating this use to assess the role of SOCE in the absence of an effect on EC coupling.     

Altered Ca2+ signaling in skeletal muscle fibers of the R6/2 mouse,a model of Huntington’s disease

Huntington’s disease (HD) is caused by an expanded CAG trinucleotide repeat within the gene encoding the protein huntingtin. The resulting elongated glutamine (poly-Q) sequence of mutant huntingtin (mhtt) affects both central neurons and skeletal muscle. Recent reports suggest that ryanodine receptor–based Ca²⁺ signaling, which is crucial for skeletal muscle excitation–contraction coupling (ECC), is changed by mhtt in HD neurons. Consequently, we searched for alterations of ECC in muscle fibers of the R6/2 mouse, a mouse model of HD. We performed fluorometric recordings of action potentials (APs) and cellular Ca²⁺ transients on intact isolated toe muscle fibers (musculi interossei), and measured L-type Ca²⁺ inward currents on internally dialyzed fibers under voltage-clamp conditions. Both APs and AP-triggered Ca²⁺ transients showed slower kinetics in R6/2 fibers than in fibers from wild-type mice. Ca²⁺ removal from the myoplasm and Ca²⁺ release flux from the sarcoplasmic reticulum were characterized using a Ca²⁺ binding and transport model, which indicated a significant reduction in slow Ca²⁺ removal activity and Ca²⁺ release flux both after APs and under voltage-clamp conditions. In addition, the voltage-clamp experiments showed a highly significant decrease in L-type Ca²⁺ channel conductance. These results indicate profound changes of Ca²⁺ turnover in skeletal muscle of R6/2 mice and suggest that these changes may be associated with muscle pathology in HD.     

Intense Resistance Exercise Induces Early and Transient Increases in Ryanodine Receptor 1 Phosphorylation in Human Skeletal Muscle

Background

While ryanodine receptor 1 (RyR1) critically contributes to skeletal muscle contraction abilities by mediating Ca²⁺ion oscillation between sarcoplasmatic and myofibrillar compartments, AMP-activated protein kinase (AMPK) senses contraction-induced energetic stress by phosphorylation at Thr¹⁷². Phosphorylation of RyR1 at serine²⁸⁴³ (pRyR1Ser²⁸⁴³) results in leaky RyR1 channels and impaired Ca²⁺homeostasis. Because acute resistance exercise exerts decreased contraction performance in skeletal muscle, preceded by high rates of Ca²⁺-oscillation and energetic stress, intense myofiber contractions may induce increased RyR1 and AMPK phosphorylation. However, no data are available regarding the time-course and magnitude of early RyR1 and AMPK phosphorylation in human myofibers in response to acute resistance exercise.

Purpose

Determine the effects and early time-course of resistance exercise on pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² in type I and II myofibers.

Methods

7 male subjects (age 23±2 years, height: 185±7 cm, weight: 82±5 kg) performed 3 sets of 8 repetitions of maximum eccentric knee extensions. Muscle biopsies were taken at rest, 15, 30 and 60 min post exercise. pRyR1Ser²⁸⁴³ and pAMPKThr¹⁷² levels were determined by western blot and semi-quantitative immunohistochemistry techniques.

Results

While total RyR1 and total AMPK levels remained unchanged, RyR1 was significantly more abundant in type II than type I myofibers. pRyR1Ser²⁸⁴³ increased 15 min and peaked 30 min (p<0.01) post exercise in both myofiber types. Type I fibers showed relatively higher increases in pRyR1Ser²⁸⁴³ levels than type II myofibers and remained elevated up to 60 min post resistance exercise (p<0.05). pAMPKThr¹⁷² also increased 15 to 30 min post exercise (p<0.01) in type I and II myofibers and in whole skeletal muscle.

Conclusion

Resistance exercise induces acutely increased pRyR1Ser²⁸⁴³ and concomitantly pAMPKThr¹⁷² levels for up to 30 min in resistance exercised myofibers. This provides a time-course by which pRyR1Ser²⁸⁴³ can mechanistically impact Ca²⁺handling properties and consequently induce reduced myofiber contractility beyond immediate fatiguing mechanisms.     

Inhibitory control over Ca(2+) sparks via mechanosensitive channels is disrupted in dystrophin deficient muscle but restored by mini-dystrophin expression

Background

In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca²⁺ release events (ECRE) in high Ca²⁺ external environments. Such ‘uncontrolled’ Ca²⁺ sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition.

Methodology/Principal Findings

We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca²⁺ concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 µM), spider peptide GsMTx-4 (5 µM) and Gd³⁺ (20 µM) that block unspecific, specific cationic and Ca²⁺ selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation.

Conclusions/Significance

Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca²⁺ homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found.     

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca²⁺ signaling is a fundamental physiological process in living cells. Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in the striated muscle fibers that appear as highly localized Ca²⁺ release events mediated by ryanodine receptor (RyR) Ca²⁺ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca²⁺ sparks could provide information on the intracellular Ca²⁺ handling properties of healthy and diseased striated muscles. Although Ca²⁺ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.Detailed here is an experimental protocol for measuring Ca²⁺ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca²⁺ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca²⁺ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca²⁺ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP₃R) and RyR contributes to Ca²⁺ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca²⁺ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).     

Amiodarone Inhibits Apamin-Sensitive Potassium Currents

Background

Apamin sensitive potassium current (I _KAS), carried by the type 2 small conductance Ca²⁺-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.

Objective

To test the hypothesis that amiodarone inhibits I _KAS in human embryonic kidney 293 (HEK-293) cells.

Methods

We used the patch-clamp technique to study I _KAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.

Results

Amiodarone inhibited I_KAS in a dose-dependent manner (IC₅₀, 2.67±0.25 µM with 1 µM intrapipette Ca²⁺). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of I_KAS induced with 1 µM intrapipette Ca²⁺ (n = 3). I_KAS inhibition by amiodarone was not voltage-dependent, but was Ca²⁺-dependent: 30 µM amiodarone inhibited 81.5±1.9% of I _KAS induced with 1 µM Ca²⁺ (n = 4), and 16.4±4.9% with 250 nM Ca²⁺ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca²⁺ dependent inhibition of I _KAS.

Conclusion

Both amiodarone and desethylamiodarone inhibit I _KAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca²⁺ concentration. SK2 current inhibition may in part underlie amiodarone''s effects in preventing electrical storm in failing ventricles.     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit,Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca²⁺ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca²⁺ release and Ca²⁺ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca²⁺ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca²⁺ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.     

Beat-to-Beat Variation in Periodicity of Local Calcium Releases Contributes to Intrinsic Variations of Spontaneous Cycle Length in Isolated Single Sinoatrial Node Cells

Spontaneous, submembrane local Ca²⁺ releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward Na⁺/Ca²⁺-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca²⁺ release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence.

Aim

The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells.

Methods

We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp.

Results

LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca²⁺ transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca²⁺ transient into a rise, resulting in a late, whole-cell diastolic Ca²⁺ elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca²⁺ transient peak to an LCR’s subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length.

Conclusion

Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle.     

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

Background

There is limited knowledge about atrial myocyte Ca²⁺ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca²⁺ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.

Methods and results

Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca²⁺ amplitude, decreased SR Ca²⁺ content, and slower Ca²⁺ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca²⁺ ATPase activity, increased Na⁺/Ca²⁺-exchanger activity and increased diastolic Ca²⁺ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca²⁺ handling in HF.

Conclusion

Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca²⁺ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.     

A Non Invasive Estimate of Dead Space Ventilation from Exercise Measurements

Rationale

During exercise, heart failure patients (HF) show an out-of-proportion ventilation increase, which in patients with COPD is blunted. When HF and COPD coexist, the ventilatory response to exercise is unpredictable.

Objectives

We evaluated a human model of respiratory impairment in 10 COPD-free HF patients and in 10 healthy subjects, tested with a progressive workload exercise with different added dead space. We hypothesized that increased serial dead space upshifts the VE vs. VCO₂ relationship and that the VE-axis intercept might be an index of dead space ventilation.

Measurements

All participants performed a cardiopulmonary exercise test with 0, 250 and 500 mL of additional dead space. Since DS does not contribute to gas exchange, ventilation relative to dead space is ventilation at VCO₂ = 0, i.e. VE-axis intercept. We compared dead space volume, estimated dividing VE-axis intercept by the intercept on respiratory rate axis of the respiratory rate vs. VCO₂ relationship with standard method measured DS.

Main results

In HF, adding dead space increased VE-axis intercept (+0 mL = 4.98±1.63 L; +250 mL = 9.69±2.91 L; +500 mL = 13.26±3.18 L; p<0.001) and upshifted the VE vs.VCO₂ relationship, with a minor slope rise (+0 mL = 27±4 L; +250 = 28±5; +500 = 29±4; p<0.05). In healthy, adding dead space increased VE-axis intercept (+0 mL = 4.9±1.4 L; +250 = 9.3±2.4; +500 = 13.1±3.04; p<0.001) without slope changes. Measured and estimated dead space volumes were similar both in HF and healthy subjects.

Conclusions

VE-axis intercept is related to dead space ventilation and dead space volume can be non-invasively estimated.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.     

Receptor Activated Ca2+ Release Is Inhibited by Boric Acid in Prostate Cancer Cells

Background

The global disparity in cancer incidence remains a major public health problem. We focused on prostate cancer since microscopic disease in men is common, but the incidence of clinical disease varies more than 100 fold worldwide. Ca²⁺ signaling is a central regulator of cell proliferation, but has received little attention in cancer prevention. We and others have reported a strong dose-dependent reduction in the incidence of prostate and lung cancer within populations exposed to boron (B) in drinking water and food; and in tumor and cell proliferation in animal and cell culture models.

Methods/Principal Findings

We examined the impact of B on Ca²⁺ stores using cancer and non-cancer human prostate cell lines, Ca²⁺ indicators Rhod-2 AM and Indo-1 AM and confocal microscopy. In DU-145 cells, inhibition of Ca²⁺ release was apparent following treatment with Ringers containing RyR agonists cADPR, 4CmC or caffeine and respective levels of BA (50 µM), (1, 10 µM) or (10, 20, 50,150 µM). Less aggressive LNCaP cancer cells required 20 µM BA and the non-tumor cell line PWR1E required 150 µM BA to significantly inhibit caffeine stimulated Ca²⁺ release. BA (10 µM) and the RyR antagonist dantroline (10 µM) were equivalent in their ability to inhibit ER Ca²⁺ loss. Flow cytometry and confocal microscopy analysis showed exposure of DU-145 cells to 50 µM BA for 1 hr decreased stored [Ca²⁺] by 32%.

Conclusion/Significance

We show B causes a dose dependent decrease of Ca²⁺ release from ryanodine receptor sensitive stores. This occurred at BA concentrations present in blood of geographically disparate populations. Our results suggest higher BA blood levels lower the risk of prostate cancer by reducing intracellular Ca²⁺ signals and storage.     

Co-Expression of SERCA Isoforms,Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs) by reducing their apparent affinity for Ca²⁺. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca²⁺ affinity of SERCA (K _Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.     

Similar Ca dependences of [H]ryanodine binding to α- and β-ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium

To understand the functions of the two ryanodine receptor isoforms (α- and β-RyRs) in nonmammalian skeletal muscles, we determined [³H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M-NaCl medium, both isoforms demonstrated similar Ca²⁺ dependent ryanodine binding activities, while the Ca²⁺ sensitivity for activation of β-RyR was increased in 1 M-NaCl medium. This enhancement in Ca²⁺ sensitivity depended on the kind of salts used. These results imply that α- and β-RyRs may have similar properties as Ca²⁺-induced Ca²⁺ release channels in bullfrog skeletal muscle.     

NT-ProBNP Levels in Saliva and Its Clinical Relevance to Heart Failure

Background

Current blood based diagnostic assays to detect heart failure (HF) have large intra-individual and inter-individual variations which have made it difficult to determine whether the changes in the analyte levels reflect an actual change in disease activity. Human saliva mirrors the body’s health and well being and ∼20% of proteins that are present in blood are also found in saliva. Saliva has numerous advantages over blood as a diagnostic fluid which allows for a non-invasive, simple, and safe sample collection. The aim of our study was to develop an immunoassay to detect NT-proBNP in saliva and to determine if there is a correlation with blood levels.

Methods

Saliva samples were collected from healthy volunteers (n = 40) who had no underlying heart conditions and HF patients (n = 45) at rest. Samples were stored at −80°C until analysis. A customised homogeneous sandwich AlphaLISA^(R) immunoassay was used to quantify NT-proBNP levels in saliva.

Results

Our NT-proBNP immunoassay was validated against a commercial Roche assay on plasma samples collected from HF patients (n = 37) and the correlation was r² = 0.78 (p<0.01, y = 1.705× +1910.8). The median salivary NT-proBNP levels in the healthy and HF participants were <16 pg/mL and 76.8 pg/mL, respectively. The salivary NT-proBNP immunoassay showed a clinical sensitivity of 82.2% and specificity of 100%, positive predictive value of 100% and negative predictive value of 83.3%, with an overall diagnostic accuracy of 90.6%.

Conclusion

We have firstly demonstrated that NT-proBNP can be detected in saliva and that the levels were higher in heart failure patients compared with healthy control subjects. Further studies will be needed to demonstrate the clinical relevance of salivary NT-proBNP in unselected, previously undiagnosed populations.     

Molecular Imaging of Cell Death in Tumors. Increasing Annexin A5 Size Reduces Contribution of Phosphatidylserine-Targeting Function to Tumor Uptake

Objective

Annexin A5 is a phosphatidylserine binding protein that binds dying cells in vivo. Annexin A5 is a potential molecular imaging agent to determine efficacy of anti-cancer therapy in patients. Its rapid clearance from circulation limits tumor uptake and, hence, its sensitivity. The aim of this study is to determine if non-invasive imaging of cell death in tumors will benefit from increasing circulation time of annexin A5 by increasing its size.

Procedures

Annexin A5 size was increased by complexation of biotinylated annexin A5 with Alexa-Fluor680-labeled streptavidin. The non-binding variant of annexin A5, M1234, was used as negative control. The HT29 colon carcinoma xenograft model in NMRI nude mice was used to measure tumor uptake in vivo. Tumor uptake of fluorescent annexin A5-variants was measured using non-invasive optical imaging.

Results

The annexin A5-streptavidin complex (4∶1, moles:moles, M_w ∼200 kDa) binds phosphatidylserine-expressing membranes with a Hill-coefficient of 5.7±0.5 for Ca²⁺-binding and an EC50 of 0.9±0.1 mM Ca²⁺ (EC50 is the Ca²⁺ concentration required for half maximal binding)(annexin A5: Hill-coefficient 3.9±0.2, EC50 1.5±0.2 mM Ca²⁺). Circulation half-life of annexin A5-streptavidin is ±21 minutes (circulation half-life of annexin A5 is ±4 min.). Tumor uptake of annexin A5-streptavidin was higher and persisted longer than annexin A5-uptake but depended less on phosphatidylserine binding.

Conclusion

Increasing annexin A5 size prolongs circulation times and increases tumor uptake, but decreases contribution of PS-targeting to tumor uptake and abolishes power to report efficacy of therapy.     

High-Resolution Echo-Planar Spectroscopic Imaging of the Human Calf

Background

This study exploits the speed benefits of echo-planar spectroscopic imaging (EPSI) to acquire lipid spectra of skeletal muscle. The main purpose was to develop a high-resolution EPSI technique for clinical MR scanner, to visualise the bulk magnetic susceptibility (BMS) shifts of extra-myocellular lipid (EMCL) spectral lines, and to investigate the feasibility of this method for the assessment of intra-myocellular (IMCL) lipids.

Methods

The study group consisted of six healthy volunteers. A two dimensional EPSI sequence with point-resolved spectroscopy (PRESS) spatial localization was implemented on a 3T clinical MR scanner. Measurements were performed by means of 64×64 spatial matrix and nominal voxel size 3×3×15 mm³. The total net measurement time was 3 min 12 sec for non-water-suppressed (1 acquisition) and 12 min 48 sec for water-suppressed scans (4 acquisitions).

Results

Spectra of the human calf had a very good signal-to-noise ratio and linewidths sufficient to differentiate IMCL resonances from EMCL. The use of a large spatial matrix reduces inter-voxel signal contamination of the strong EMCL signals. Small voxels enabled visualisation of the methylene EMCL spectral line splitting and their BMS shifts up to 0.5 ppm relative to the correspondent IMCL line. The mean soleus muscle IMCL content of our six volunteers was 0.30±0.10 vol% (range 0.18–0.46) or 3.6±1.2 mmol/kg wet weight (range: 2.1–5.4).

Conclusion

This study demonstrates that high-spatial resolution PRESS EPSI of the muscle lipids is feasible on standard clinical scanners.