Eduardo Ríos

Amplitude distribution of calcium sparks in confocal images: theory and studies with an automatic detection method.

Determination of the calcium spark amplitude distribution is of critical importance for understanding the nature of elementary calcium release events in striated muscle. In the present study we show, on general theoretical grounds, that calcium sparks, as observed in confocal line scan images, should have a nonmodal, monotonic decreasing amplitude distribution, regardless of whether the underlying events are stereotyped. To test this prediction we developed, implemented, and verified an automated computer algorithm for objective detection and measurement of calcium sparks in raw image data. When the sensitivity and reliability of the algorithm were set appropriately, we observed highly left-skewed or monotonic decreasing amplitude distributions in skeletal muscle cells and cardiomyocytes, confirming the theoretical predictions. The previously reported modal or Gaussian distributions of sparks detected by eye must therefore be the result of subjective detection bias against small amplitude events. In addition, we discuss possible situations when a modal distribution might be observed.

β-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase

Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether β-adrenergic receptor (β-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca]i) and SR Ca load ([Ca]SRT) were assessed using fluo-4. β-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca]i decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2–related inhibitory peptide II or β-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the β-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca]i decline rate. The results suggest that β-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca]i.

Local calcium release in mammalian skeletal muscle

1 Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline‐gap chamber. 2 Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice. 3 Depolarization‐induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at −50 mV (n= 7). In rats, R had an essentially voltage‐independent value of 2.3 (n= 5). 4 Ca2+‐induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse ‘small event’ release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.

Regulation of Skeletal Muscle Ca2+ Release Channel (Ryanodine Receptor) by Ca2+ and Monovalent Cations and Anions

The effects of ionic composition and strength on rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) activity were investigated in vesicle-45Ca2+ flux, single channel and [3H]ryanodine binding measurements. In <0.01 μM Ca2+ media, the highest 45Ca2+ efflux rate was measured in 0.25 M choline-Cl medium followed by 0.25 M KCl, choline 4-morpholineethanesulfonic acid (Mes), potassium 1,4-piperazinediethanesulfonic acid (Pipes), and K-Mes medium. In all five media, the 45Ca2+ efflux rates were increased when the free [Ca2+] was raised from <0.01 μM to 20 μM and decreased as the free [Ca2+] was further increased to 1 mM. An increase in [KCl] augmented Ca2+-gated single channel activity and [3H]ryanodine binding. In [3H]ryanodine binding measurements, bell-shaped Ca2+ activation/inactivation curves were obtained in media containing different monovalent cations (Li+, Na+, K+, Cs+, and choline+) and anions (Cl−, Mes−, and Pipes−). In choline-Cl medium, substantial levels of [3H]ryanodine binding were observed at [Ca2+] <0.01 μM. Replacement of Cl− by Mes− or Pipes− reduced [3H]ryanodine binding levels at all [Ca2+]. In all media, the Ca2+-dependence of [3H]ryanodine binding could be well described assuming that the skeletal muscle ryanodine receptor possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inactivation sites. AMP primarily affected [3H]ryanodine binding by decreasing the apparent affinity of the Ca2+ inactivation site(s) for Ca2+, while caffeine increased the apparent affinity of the Ca2+ activation site for Ca2+. Competition studies indicated that ionic composition affected Ca2+-dependent receptor activity by at least three different mechanisms: (i) competitive binding of Mg2+ and monovalent cations to the Ca2+ activation sites, (ii) binding of divalent cations to the Ca2+ inactivation sites, and (iii) binding of anions to specific anion regulatory sites.

Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye.

Myoplasmic free calcium transients were monitored with the metallochromic indicator dye Antipyrylazo III (AP III) in single frog skeletal muscle fibres cut at both ends, stretched so as to minimize or eliminate contractile filament overlap and voltage clamped using a double‐Vaseline‐gap system (approximately 6 degrees C). The dye entered the central fibre segment by diffusion from the solution applied to the two cut ends. The diffusion coefficient of AP III was about 20 times lower in the fibre than in solution. This very slow diffusion was not due to binding of dye since the ratio of bound to free dye obtained from analysis of the diffusion was only about 0.45. For a given depolarizing pulse, the ratio of dye‐related absorbance changes delta A at 720 and 550 nm was the same as that produced on adding calcium to dye in calibrating solution, indicating that these signals were due to changes in myoplasmic calcium. The delta A signals at 700 or 720 nm were used to monitor transient changes in concentration of calcium‐dye complex [CaD2] and of free calcium [Ca] in the myofilament space. By applying the same pulse at different times during dye entry, it was observed that increasing dye concentrations [D]T produced the following effects: (a) [CaD2] was increased; (b) [Ca] was decreased at early times during a pulse; (c) a declining phase of [Ca] observed at late times during pulses was decreased and finally reversed to a slow rising phase at high [D]T; (d) the decay of [Ca] after the pulse was slowed. Analyses of the effects of [D]T on (a) the magnitude of [CaD2] at a given early time during the calcium release produced by pulses to a given voltage and on (b) the time constant for [Ca] decay after a pulse were both consistent with a calcium: dye stoichiometry of 1:2 in the fibre as found in calibrating solution. Analysis of the effect of [D]T on the [Ca] decay time constants also revealed the presence of intrinsic rapidly equilibrating myoplasmic calcium binding sites and provided the basis for obtaining estimates of the combined concentration [Ca] of free calcium plus calcium bound to such sites. Unlike the estimates of [Ca], these estimates of [Ca] are independent of the value of the calcium‐dye dissociation constant.(ABSTRACT TRUNCATED AT 400 WORDS)

The mechanical hypothesis of excitation—contraction (EC) coupling in skeletal muscle

SummaryThe mechanism of transmission in skeletal muscle EC coupling is still an open question. There is some indirect evidence in favour of the mechanical coupling hypothesis, deriving mostly from consideration of the structure of the Ca2+ release channel protein. A new functional approach is proposed, that consists in comparing the properties of the complete system — EC coupling in a skeletal muscle fibre — with those of the EC coupling molecules in bilayers. In this approach, those properties of the whole system that are not traceable to its constitutive molecules, are ascribed to the physiological interaction, and are expected to yield new information on the nature of this interaction.

Time course of calcium release and removal in skeletal muscle fibers.

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fiber is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We present a procedure for empirically characterizing the calcium removal processes in voltage-clamped fibers and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse. Our results reveal a decline of the SR calcium release rate during depolarization that was not anticipated from simple inspection of the calcium transients.

Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis

Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pHi in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na+/H+ antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pHi decreased sharply, and recovery required both Na+/H+ antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn2+ resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn2+. Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.

Hyperactive Intracellular Calcium Signaling Associated with Localized Mitochondrial Defects in Skeletal Muscle of an Animal Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1G93A mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca2+ release activity, which can include propagating Ca2+ waves. These Ca2+ waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca2+ uptake by Ru360 lead to cell-wide propagation of such Ca2+ release events. Our data reveal that mitochondria regulate Ca2+ signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.

Store-operated Ca2+ entry during intracellular Ca2+ release in mammalian skeletal muscle.

Store‐operated Ca2+ entry (SOCE) is activated following the depletion of internal Ca2+ stores in virtually all eukaryotic cells. Shifted excitation and emission ratioing of fluorescence (SEER) was used to image mag‐indo‐1 trapped in the tubular (t) system of mechanically skinned rat skeletal muscle fibres to measure SOCE during intracellular Ca2+ release. Cytosolic Ca2+ transients were simultaneously imaged using the fluorescence of rhod‐2. Spatially and temporally resolved images of t system [Ca2+] ([Ca2+]t‐sys) allowed estimation of Ca2+ entry flux from the rate of decay of [Ca2+]t‐sys. Ca2+ release was induced pharmacologically to activate SOCE without voltage‐dependent contributions to Ca2+ flux. Inward Ca2+ flux was monotonically dependent on the [Ca2+] gradient, and strongly dependent on the transmembrane potential. The activation of SOCE was controlled locally. It could occur without full Ca2+ store depletion and in less than a second after initiation of store depletion. These results indicate that the molecular agonists of SOCE must be evenly distributed throughout the junctional membranes and can activate rapidly. Termination of SOCE required a net increase in [Ca2+]SR. Activation and termination of SOCE are also demonstrated, for the first time, during a single event of Ca2+ release. At the physiological [Ca2+]t‐sys, near 2 mm (relative to t system volume), SOCE flux relative to accessible cytoplasmic volume was at least 18.6 μm s−1, consistent with times of SR refilling of 1–2 min measured in intact muscle fibres.