Martin F. Schneider

Activity-dependent nuclear translocation and intranuclear distribution of NFATc in adult skeletal muscle fibers

TTranscription factor nuclear factor of activated T cells NFATc (NFATc1, NFAT2) may contribute to slow-twitch skeletal muscle fiber type–specific gene expression. Green fluorescence protein (GFP) or FLAG fusion proteins of either wild-type or constitutively active mutant NFATc [NFATc(S→A)] were expressed in cultured adult mouse skeletal muscle fibers from flexor digitorum brevis (predominantly fast-twitch). Unstimulated fibers expressing NFATc(S→A) exhibited a distinct intranuclear pattern of NFATc foci. In unstimulated fibers expressing NFATc–GFP, fluorescence was localized at the sarcomeric z-lines and absent from nuclei. Electrical stimulation using activity patterns typical of slow-twitch muscle, either continuously at 10 Hz or in 5-s trains at 10 Hz every 50 s, caused cyclosporin A–sensitive appearance of fluorescent foci of NFATc–GFP in all nuclei. Fluorescence of nuclear foci increased during the first hour of stimulation and then remained constant during a second hour of stimulation. Kinase inhibitors and ionomycin caused appearance of nuclear foci of NFATc–GFP without electrical stimulation. Nuclear translocation of NFATc–GFP did not occur with either continuous 1 Hz stimulation or with the fast-twitch fiber activity pattern of 0.1-s trains at 50 Hz every 50 s. The stimulation pattern–dependent nuclear translocation of NFATc demonstrated here could thus contribute to fast-twitch to slow-twitch fiber type transformation.

Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle.

1. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) in voltage clamped segments of frog skeletal muscle fibres was calculated from myoplasmic free calcium transients (delta[Ca2+]) measured with the calcium indicator dye Antipyrylazo III. 2. During a 100‐200 ms depolarizing pulse Rrel reached an early peak and then declined markedly. The time course and extent of decline of Rrel were independent of membrane potential over a range of potentials where release activation varied severalfold. 3. For test pulses applied shortly after relatively large or long conditioning pulses Rrel completely lacked the early peak. The peak gradually recovered as the interval between the conditioning and test pulses was increased to 1 s. 4. A latency was often observed before the start of recovery of the peak in Rrel. The latency appeared to be correlated with the time for delta[Ca2+] to fall below a certain level, indicating that recovery of the peak might represent reversal of a calcium‐dependent process. It was therefore proposed that the rapid decline in Rrel during a pulse was due to calcium‐dependent inactivation of the SR calcium release channels. 5. Inactivation continued to develop during the interval between a relatively large 20 ms conditioning pulse and a test pulse applied 20 ms later. This was as expected for calcium‐dependent inactivation of SR calcium release because of the elevated [Ca2+] between the conditioning and test pulses. It was not as expected for external membrane potential‐dependent inactivation. 6. Small steady elevations in [Ca2+] due to relatively small 200 ms conditioning pulses produced marked inactivation of Rrel, indicating an apparent dissociation constant for calcium‐dependent inactivation only slightly above resting [Ca2+]. 7. All observations could be well simulated by a two‐step model for inactivation in which myoplasmic free calcium equilibrates rapidly with a high‐affinity calcium receptor on the release channel and then the calcium‐receptor complex undergoes a slower conformational change to the inactivated state of the channel. 8. An alternative model in which calcium binds to a soluble receptor (e.g. free calmodulin) and then the calcium‐receptor complex binds to and directly inactivates the channel was shown to be formally identical to the preceding model. Either model could closely simulate all observations.

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)

Activity-dependent and -independent nuclear fluxes of HDAC4 mediated by different kinases in adult skeletal muscle.

Class II histone deacetylases (HDACs) may decrease slow muscle fiber gene expression by repressing myogenic transcription factor myocyte enhancer factor 2 (MEF2). Here, we show that repetitive slow fiber type electrical stimulation, but not fast fiber type stimulation, caused HDAC4-GFP, but not HDAC5-GFP, to translocate from the nucleus to the cytoplasm in cultured adult skeletal muscle fibers. HDAC4-GFP translocation was blocked by calmodulin-dependent protein kinase (CaMK) inhibitor KN-62. Slow fiber type stimulation increased MEF2 transcriptional activity, nuclear Ca2+ concentration, and nuclear levels of activated CaMKII, but not total nuclear CaMKII or CaM-YFP. Thus, calcium transients for slow, but not fast, fiber stimulation patterns appear to provide sufficient Ca2+-dependent activation of nuclear CaMKII to result in net nuclear efflux of HDAC4. Nucleocytoplasmic shuttling of HDAC4-GFP in unstimulated resting fibers was not altered by KN-62, but was blocked by staurosporine, indicating that different kinases underlie nuclear efflux of HDAC4 in resting and stimulated muscle fibers.

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.

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.

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice

The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.

Decay of calcium transients after electrical stimulation in rat fast‐ and slow‐twitch skeletal muscle fibres

1 Calcium transients were calculated from fura‐2 fluorescence signals (corrected for kinetic delays in the Ca2+–fura‐2 reaction) from single rat skeletal muscle fibres, either fully dissociated from the fast‐twitch flexor digitorum brevis (FDB) muscle or in small bundles from the slow‐twitch soleus muscle. Fibres or bundles were embedded in agarose gel to inhibit movement and stimulated by single or trains of 1–2 ms electrical pulses (100 Hz, 2–400 ms train duration). 2 The rate constant of decay of [Ca2+] determined from single‐exponential fits to the final decay phase of [Ca2+] after a single action potential was considerably faster in FDB fibres than in soleus fibres. As the stimulation duration increased, the rate constant of [Ca2+] decay decreased for both the FDB and soleus fibres, but the effect was greater in FDB than in soleus fibres. 3 Using the magnitude of the decline in the rate constant of [Ca2+] decay with increasing stimulation duration as an index of relative contribution of the saturable Ca2+ binding sites on parvalbumin, subpopulations termed ‘high’, ‘medium’ and ‘low’, referring to estimated parvalbumin content, were determined within each group of FDB and soleus fibres. In fibres assigned to the ‘high’ and ‘medium’ groups, parvalbumin was the major contributor (50.73%) to the [Ca2+] decay rate constant after a single action potential. In fibres in the ‘low’ group, parvalbumin contributed only 0–28% to the rate constant of [Ca2+] decay. 4 Fluorescence recordings using mag‐fura‐2, a lower‐affinity Ca2+ indicator expected to be in equilibrium with myoplasmic Ca2+, gave similar values for both the [Ca2+] decay rate constant after a single action potential and the decrease in this rate constant with increased stimulation duration, as found for the fura‐2 [Ca2+] transients from FDB and soleus fibres. Thus, the observed differences in decay rate of Ca2+ were not introduced by kinetic correction of the fura‐2 recordings, but are attributed to differences in the Ca2+ binding and transport properties of fast‐ and slow‐twitch mammalian fibres.

S100A1 and Calmodulin Compete for the Same Binding Site on Ryanodine Receptor.

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca2+ release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca2+-S100A1 competes with Ca2+-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca2+-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca2+-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca2+-CaM. A model for regulating muscle contraction is presented in which Ca2+-S100A1 and Ca2+-CaM compete directly for the same binding site on the ryanodine receptor.

S100A1 Binds to the Calmodulin-binding Site of Ryanodine Receptor and Modulates Skeletal Muscle Excitation-Contraction Coupling

S100A1, a 21-kDa dimeric Ca2+-binding protein, is an enhancer of cardiac Ca2+ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca2+ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca2+ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca2+ release in S100A1-/- muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca2+-dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.