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Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis
© The Author(s). 2016
- Received: 1 October 2015
- Accepted: 3 June 2016
- Published: 23 June 2016
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3–5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca2+ channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.
We recorded L-type currents, charge movements, and myoplasmic Ca2+ transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1G93A).
In FDB fibers obtained from "symptomatic" global SOD1G93A mice, we observed a substantial reduction of SR Ca2+ release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1G93A fibers when compared to control fibers. Ca2+ transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1G93A mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca2+ release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1G93A expression was restricted to skeletal muscle.
We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1G93A mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1G93A and skeletal muscle autonomous MLC/SOD1G93A mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.
- Amyotrophic lateral sclerosis
- CaV1.1
- Excitation-contraction coupling
- L-type
- Skeletal muscle
- Neuromuscular disease
Amyotrophic lateral sclerosis (ALS), known commonly as Lou Gehrig's disease in the USA or motoneurone disease in the UK, is an adult-onset neurodegenerative disorder that is usually lethal within 3 to 5 years of initial diagnosis [1, 2]. As a consequence of disrupted nerve-muscle communication, afflicted individuals progressively lose control of voluntary muscle function and die most frequently from respiratory failure. Nearly a quarter of familial ALS cases have been linked to point mutations in the type 1 Cu/Zn superoxide dismutase (SOD1) enzyme, which functions to mitigate cellular oxidative stress [3]. Since these mutations are autosomal dominant, transgenic mice overexpressing ALS-linked SOD1 mutations have proven to be useful models for investigation of ALS pathogenesis [4, 5].
Two independent groups have found that skeletal muscle-targeted, transgenic overexpression of SOD1G93A causes profound muscle atrophy [6, 7] and initiates motoneuron death [7]. These congruent reports reinforce earlier challenges to the dogma that ALS is solely a neurological disorder and support the "dying-back" phenomenon by which motor unit loss and associated muscle function precede the death of motor neurons (reviewed in refs. [8–11]). Additional support for this view is provided by the observations that destruction of neuromuscular junctions has been linked to oxidative stress induced by tissue-specific breakdown of muscle mitochondria [12] and trophic factors secreted from the skeletal muscle promote motoneuron survival in an ALS model by stabilizing neuromuscular junctions (e.g., IGF-1, GDNF) [13–15].
In the skeletal muscle, CaV1.1 functions both as the voltage-sensor for excitation-contraction (EC) coupling and as an L-type Ca2+ channel [16–18]. CaV1.1's role as voltage-sensor is firmly established [19], but recent work has also revealed that L-type Ca2+ entry maintains myoplasmic Ca2+ levels during repetitive activity [20, 21] augments muscle contraction [22], engages excitation-transcription coupling [23], regulates energy expenditure [24], and promotes development of neuromuscular junctions [25]. Thus, aberrant CaV1.1 activity may profoundly affect skeletal muscle biology.
The question of whether CaV1.1 function is disrupted during the progression of ALS was originally posed over 20 years ago [26–28], but the experimental approach used to investigate this premise left much ambiguity. In these earlier studies, serum of ALS patients was acutely applied to normal cut wild-type rat extensor digitorum longus (EDL) fibers and the Vaseline-gap voltage-clamp technique was used to record L-type currents and gating charge movements [26, 27]; L-type currents and intramembrane gating charge movements were both found to be reduced. Similarly, acute IgG application also produced inhibitory effects on single CaV1.1 channels in planar lipid bilayers [28]. In the time that has passed since the experiments that employed acute application of ALS IgG were performed, it has become evident that that muscle dysfunction in ALS manifests early and slowly over time. Fortunately, a number of ALS mouse models have been developed since then, including SOD1G93A transgenic lines [4, 29–31].
Here, we provide the first comprehensive voltage-clamp assessment of CaV1.1 function in the muscle of an engineered mammalian model of ALS. Specifically, we utilized both global SOD1G93A mice and mice expressing SOD1G93A only in skeletal muscle to systematically investigate whether CaV1.1 function is compromised during the progression of the disease. We found substantial reductions in myoplasmic Ca2+ transient amplitude, peak L-type current density, and maximal intramembrane charge movement in flexor digitorum brevis (FDB) fibers obtained from "symptomatic" congenic SOD1G93A mice. Consistent with a progressive muscle defect, we observed lesser reductions in L-type current amplitude and charge movement in FDB fibers obtained from "early-symptomatic" SOD1G93A mice in which widespread denervation had yet to occur. To investigate the possibility that events occurring in muscle contribute, at least in part, to the impairment of CaV1.1 function, we performed voltage-clamp experiments with FDB fibers of mice expressing SOD1G93A autonomously in skeletal muscle. In these experiments, we observed significant reductions in EC coupling, charge movement, and peak L-type current amplitude.
Ethical information/animals
All procedures involving mice were approved by the University of Colorado-Anschutz Medical Campus Institutional Animal Care and Use Committee (91813(05)1D). Male congenic SOD1G93A mice (B6.Cg-Tg(SOD1*G93A)1Gur/J; 32 transgene copies) and age-matched male C57BL/6J mice (both Jackson Laboratories, Bar Harbor, ME) were used at two time points based on the criteria for disease progression described by Hatzipetros et al. [31]: (1) "symptomatic" and (2) "early-symptomatic." Nine symptomatic mice presented classical ALS symptoms (i.e., tremor, abnormal gait, comprised ability to stand on hindquarters, etc.) when they were sacrificed at 149 ± 1 days. Six mice from the early-symptomatic cohort were largely devoid of overt symptoms when they were sacrificed at 105 ± 1 days. Age-matched C57BL/6J mice were used as background wild-type controls for both symptomatic and early-symptomatic global SOD1G93A mice (eight and seven mice, respectively). Seven MLC/SOD1G93A mice were sacrificed at (111 ± 1 days) [6]. In this case, six age-matched FVB/NJ mice were used as the background wild-type control strain. The dissimilar backgrounds precluded direct comparisons between global SOD1G93A and MLC/SOD1G93A strains.
Dissociation of FDB fibers
The FDB muscles were dissected in cold (~4 °C) Rodent Ringer's solution (in mM: 146 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, pH 7.4 with NaOH). Muscles were then digested in a mild collagenase solution (155 Cs-aspartate, 10 HEPES, 5 MgCl2, pH 7.4 with CsOH, supplemented with 1 mg/ml BSA, and 1 mg/ml collagenase type IA (Sigma-Aldrich, Saint Louis, MO) with agitation at 37 °C for ~1 h. Following digestion, the collagenase solution was replaced with dissociation solution (140 Cs-aspartate, 10 Cs2EGTA, 10 HEPES, 5 MgCl2, pH 7.4 with CsOH, supplemented with 1 mg/ml BSA) and muscles were triturated gently with a series of fire-polished glass Pasteur pipettes of descending bore. Dissociated FDB fibers destined for electrophysiological experiments were then plated onto ECL (Millipore, Billerica, MA)-coated 35-mm plastic culture dishes (Falcon, Tewksbury, MA). For Ca2+ imaging experiments, FDB fibers were plated onto laminin (Invitrogen, Eugene, OR)-coated 35-mm culture dishes with glass coverslip bottoms (MatTek, Ashland, MA). Experiments were performed with FDB fibers 1–6 h following dissociation.
Assessment of SR Ca2+ store content
Freshly dissociated FDB fibers were washed with Ca2+/Mg2+-free Ringer's solution (in mM: 146 NaCl, 5 KCl, 10 HEPES, and 11 glucose, pH 7.4, with NaOH) twice and loaded with 5 μM Fluo 3-AM (Invitrogen) dissolved in Rodent Ringer's solution for 20–30 min. Fibers were then washed three times in Rodent Ringer's solution with gentle agitation. Fluo 3-AM-loaded fibers bathed in Rodent Ringer's solution were then placed on the stage of an LSM META scanning laser confocal microscope (Carl Zeiss, Inc., Jena, Germany) and viewed under ×10 magnification. N-benzyl-p-toluenesulfonamide (BTS; 100 μM; Sigma-Aldrich) was always present in the bath solution (∼25 °C) to prevent contractions. The Fluo-3 dye was excited with the 488 nm line of an argon laser (30 mW maximum output, operated at 50 % or 6.3 A, attenuated to 1–2 %). The emitted fluorescence was directed to a photomultiplier equipped with a 505-nm long-pass filter. Confocal fluorescence intensity data were digitized at 8 bits, with the photomultiplier gain and offset adjusted such that maximum pixel intensities were no more than ∼70 % saturated and cell-free areas had close to zero intensity. Application of 4-chloro-m-cresol (4-CmC; Pfaltz and Bauer, Waterbury, CT) via a manually operated, gravity-driven global perfusion system was used to deplete the SR of Ca2+. Fluorescence amplitude data are expressed as ΔF/F, where F represents the baseline fluorescence before the application of 4-CmC, and ΔF represents the change in fluorescence during the application of 4-CmC.
Electrophysiology and whole-cell recordings of myoplasmic Ca2+ transients
The hindlimb muscles from wild-type and SOD1G93A mice (tibialis anterior, gastrocnemius, and FDB) were promptly removed following sacrifice, flash-frozen in liquid nitrogen, and stored at −80 °C for later use. At a later time, muscle tissue was homogenized by centrifugation with aluminum beads (NextAdvance, Averill Park, NY) in lysis buffer (50 mM Tris, 1 % SDS, Bio-Rad, Hercules, CA). Sample protein concentration was determined using the Bradley method (BCA assay kit, ThermoFisher, Pittsburgh, PA); lysates were divided into 30–50 μg aliquots. After addition of Laemmli buffer (Bio-Rad), proteins were separated by standard SDS-PAGE (10 % gels; Bio-Rad), transferred to nitrocellulose and non-specific immunoreactivity was blocked with 5 % non-fat dry milk (Kroger, Cincinnati, OH). Primary antibodies used for immunoblotting were mouse anti-CaV1.1 (also referred to as mAB 1A; 1:1000; ThermoScientific, Rockford, IL), mouse α-actin (1:1000; Sigma-Aldrich), and rabbit histone H3 (1:1000; Cell Signaling, Danvers, MA). Goat anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were obtained from Southern Biotech (1:10000; Birmingham, AL). Blots were visualized with SuperSignal West Pico kit (ThermoFisher) viewed on a FluorChem HD2 scanner (Alpha Innotech, San Leandro, CA). Blots were stripped using Restore Western Blot Stripping Buffer (ThermoScientific). Cellular CaV1.1 protein levels measured using ImageJ densitometry (National Institutes of Health, Bethesda, MD) and were normalized to α-actin expression in the same sample (i.e., lane).
All data are presented as mean ± SEM. Statistical comparisons were made by two-tailed, unpaired t test with P < 0.05 considered significant. Figures were made using the software program SigmaPlot (version 11.0, SSPS Inc., San Jose, CA).
Results and discussion
EC coupling is impaired in symptomatic SOD1G93A FDB fibers
L-type currents and intramembrane charge movement are reduced in symptomatic SOD1G93A FDB fibers
Impaired CaV1.1 function in early-symptomatic SOD1G93A FDB fibers
EC coupling, L-type currents, and charge movement are reduced in FDB fibers expressing SOD1G93A autonomously in the skeletal muscle
Our experiments with early-symptomatic SOD1G93A mice raise the possibility that the reduction of CaV1.1 expression precedes overt neurological symptoms of ALS. Nonetheless, a degree of uncertainty exists concerning the contribution of denervation. On a more stringent level, the innervation profiles of the individual fibers used in our patch-clamp experiments were virtually impossible to assess accurately. To determine nerve-independent effects, we utilized a mouse model where transgenic expression of SOD1G93A is limited to skeletal muscle (MLC/SOD1G93A) [6]. This mouse line enabled us to examine whether events occurring locally in skeletal muscle can cause impaired CaV1.1 function because substantial neuromuscular junction defects in such models do not develop until at least 8 months of age ([6, 7]; A.M., unpublished results).
In this study, we found that FDB fibers harvested from symptomatic global SOD1G93A mice had substantially reduced EC coupling relative to fibers of age-matched wild-type mice (Fig. 1). The impairment of EC coupling was not a consequence of a depleted SR Ca2+ store (Fig. 2), but appeared to be the result of reduced L-type Ca2+ channel membrane expression (though we cannot discount the presence of electrically silent channels). In particular, L-type Ca2+ current (Fig. 3a–c) and intramembrane charge movement (Fig. 3d–f) were both decreased by ~40 % in symptomatic SOD1G93A FDB fibers. The observed reductions in CaV1.1 function in symptomatic SOD1G93A fibers resembled the effect of application of ALS patient serum to normal rat extensor digitorum longus muscle [26, 27], but our results imply that these impairments develop more slowly over time.
Our data indicating that EC coupling and L-type current were compromised in symptomatic SOD1G93A muscle fibers contrast with those of another group who found that depolarization-dependent Ca2+ transients were slightly augmented in SOD1G93A mice (B6SJL-Tg(SOD1*G93A)1Gur/J) as a consequence of insufficient local mitochondrial Ca2+ buffering near the neuromuscular junction [40, 41]. Our findings also differ with those of another study which also examined Ca2+ handling in FDB fibers obtained from symptomatic SOD1G93A mice (B6.Cg-Tg(SOD1*G93A)1Gur/J). In this latter study [42], the authors observed virtually no difference in the amplitude of Ca2+ transients evoked by maximal field stimulation frequencies (100 Hz) using ratiometric Ca2+-sensitive dyes. It was concluded that the ability of CaV1.1 to engage EC coupling was normal in symptomatic SOD1G93A superficial gastrocnemius muscle because western blots showed no change in CaV1.1 expression [42]. By contrast, our western blot analysis of whole-cell lysates obtained from symptomatic SOD1G93A tibialis anterior muscles indicated a clear reduction in total CaV1.1 expression (Fig. 4a). We did not observe significant differences for either gastrocnemius or FDB (Fig. 4b, c), though the band intensity difference in gastrocnemius was marginal (P = 0.074). The ambiguous nature of our biochemical results underscores the critical importance of our voltage-clamp approach to determine the relative number of functional CaV1.1 channels in the plasma membrane.
One reasonable explanation for the observed decreases in Ca2+ transient amplitude, peak L-type current density, and maximal charge movement is that SOD1G93A-expressing muscle undergoes fast-twitch (type IIb or IIx) to slow or intermediate (type I or type Ia, respectively) fiber-type switching [6, 30]. In an earlier study, slow-twitch rat soleus fibers were found to support voltage-dependent SR Ca2+ release only about half as well as fast-twitch fibers dissected from EDL of the same animals [43, 44]. Likewise, radioactive 1,4-dihydropyridine binding of the soleus was reduced ~50 % compared to that of EDL in the same study. In view of this difference in CaV1.1 expression between general fiber types, it is possible that we recorded from a population more heavily weighted in type I or type IIa fibers and that our data reflect fiber-type switching. Nevertheless, the dramatic alteration in the expression profile of CaV1.1 that accompanies fiber-type switching appears to be a downstream consequence of mutant SOD1 expression. Future experiments with more complex models that enable the distinction amongst fiber types in live cells are required to investigate this idea further.
Importantly, we also found that the impairment of CaV1.1 function in SOD1G93A mice began to develop prior the presentation of classical ALS symptoms. Though the reduction in depolarization-induced Ca2+ transients were not significantly reduced in early-symptomatic SOD1G93A muscle fibers (Fig. 5), decreased L-type current amplitude and intramembrane charge movement were clearly evident (Fig. 6). A number of explanations could account for this apparently discordant result. For example, the early-symptomatic SOD1G93A mice may have had a dissimilar RyR (type 1 or type 3) complement than the symptomatic mice or there have been a compensatory reversion to a juvenile RyR1 splice variant (ASI+) that supports greater SR Ca2+ release in response to depolarization [45, 46]. In any event, the results of our experiments with early-symptomatic SOD1G93A fibers suggested that the impairment of CaV1.1 function manifested in symptomatic SOD1G93A fibers arises, at least in part, from signals that originate in muscle (Figs. 5, 6, and 7). To test this idea, we assessed CaV1.1 function in fibers obtained from 4-month-old MLC/SOD1G93A mice in which expression of the mutant SOD1 protein is restricted to skeletal muscle. In these experiments, we observed ~35, ~20, and ~15 % reductions in Ca2+ transient amplitude, peak L-type current density, and maximal charge movement, respectively (Figs. 8 and 9). The reductions in L-type current amplitude and charge movement resembled the reductions in the parameters observed in early-symptomatic global SOD1G93A fibers. Interestingly, the impact of muscle-specific expression of SOD1G93A on EC coupling was greater than that observed in early-symptomatic global fibers. Previous ultrastructural analysis of the MLC/SOD1G93A strain suggests that concurrent uncoupling of triad junctions with transverse tubules may amplify the EC coupling impairment [6]. Together, the ultrastructural and electrophysiological changes induced by muscle-specific expression of SOD1G93A can reasonably account for marked reductions in specific force observed in both fast- and slow-twitch hindlimb muscles of MLC/SOD1G93A mice [6]. Thus, the results of our experiments with MLC/SOD1G93A fibers support the idea that muscle-specific events also make a significant contribution to ALS pathology, similar to what has been observed previously with myotonic dystrophy [47] and Huntington's disease [48].
We did not observe a decrease in total membrane capacitance in SOD1G93A-expressing fibers, indicating that there was not substantial atrophy and/or loss of tubular surface area in the fibers that we examined electrophysiologically (Additional file 1: Figure S1). Based on the indisputable fact that atrophy is characteristic of both global and muscle-targeted SOD1G93A muscles, we think that our data are representative of a subpopulation of fibers healthy enough to survive the dissociation process and to meet the set visual criteria for voltage-clamp experiments (see Methods). If this is indeed the case, the reductions in voltage-dependent SR Ca2+ release, charge movement, and L-type current in the entire population of SOD1G93A fibers are likely more severe (and earlier onset) than we report.
Until recently, the involvement of CaV1.1 in the maintenance of muscle mass and composition was impossible to investigate because mice null for CaV1.1 (dysgenic) die immediately following parturition [49]. To circumvent the perinatal lethality of CaV1.1 deletion, Piétri-Rouxel and colleagues delivered anti-CaV1.1 siRNA to the mouse hindlimb muscle with a serotype 1 Adeno-Associated Virus (AAV1) vector [36]. Using this approach, they demonstrated that targeted, long-term suppression of L-type channel expression via induced exon-skipping produced prominent atrophy and extensive fibrosis. This atrophic effect of experimental downregulation of CaV1.1 expression suggested that pathological decreases in L-type channel activity, such as those we have observed in the two SOD1G93A models utilized in this study, may also lead to muscle degeneration and, possibly, contribute to the destabilization of neuromuscular junctions early in the progression of ALS. Our current work provides initial support for this idea, but further work is needed to reveal the precise mechanism. The first step will be to determine whether the downregulation of the channel and/or its auxiliary subunits occurs at the message level or post-translationally. For the former, quantification of CaV1.1 subunit message levels by qRT-PCR should be revealing. In regard to the latter, the chronic upregulation of expression the RGK family small GTP binding protein Rad (Ras-like Associated with Diabetes) — a potent constitutively active inhibitor of CaV1.1 [50, 51]—has been demonstrated in muscle of sporadic ALS patients of unknown aetiology and two established familial ALS mouse models (SOD1G93A and SOD1G86R) [52, 53]. The increases in muscle Rad expression were attributed to elevated cellular oxidative stress levels and coincided with the onset of spinal motoneuron death [53]. Without being overspeculative, these observations raise the possibility that such chronic enhancement of muscle Rad expression may contribute to both atrophy and the dissolution of neuromuscular junctions in human ALS [53].
We report that myoplasmic Ca2+ transient amplitude, L-type current density, and intramembrane charge movement are progressively downregulated in flexor digitorum brevis (FDB) fibers obtained from SOD1G93A mice, the most extensively utilized mouse model of familial ALS. We also observed impairments of EC coupling, L-type current density, and charge movement in fibers of mice expressing SOD1G93A only in the skeletal muscle, signaling a muscle-specific contribution to SOD1G93A-induced downregulation of CaV1.1 expression and/or function. Since CaV1.1 is a positive regulator of muscle mass, our results suggest that altered CaV1.1 activity is a contributor to the muscle remodeling that occurs in ALS patients prior to the presentation of overt neurological symptoms.
4-CmC, 4-chloro-m-cresol; AAV1, serotype 1 adeno-associated virus; ALS, amyotrophic lateral sclerosis; EC, excitation-contraction; EDL, extensor digitorum longus; FDB, flexor digitorum brevis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF-1, insulin-like growth factor 1; RyR, ryanodine-sensitive intracellular Ca2+ release channel; SOD1, Cu/Zn superoxide dismutase 1; SR, sarcoplasmic reticulum; TA, tibialis anterior
We thank Drs. U. Meza and S. Papadopoulos for comments on an early draft of the manuscript. We are grateful to Drs. K.G. Beam and D. Oskar for the continued support. Confocal images were acquired in the University of Colorado-AMC Advanced Light Microscopy Core (funded in part by NIH/NCRR Colorado CTSI Grant Number UL1 RR025780).
This work was supported by grants from the Boettcher Foundation, the Judith and Jean Pape Adams Charitable Foundation (to R.A.B.), Fondazione Telethon and Fondazione Roma (to A.M.). D.B. received a stipend from 2T32 AG000279-11 (to R.S. Schwartz, University of Colorado School of Medicine, Department of Medicine-Geriatrics Division).
Authors' contributions
DB and RAB designed research, performed research, analyzed data, and wrote the paper. CFR performed experiments and analyzed data. AAV designed research and wrote the paper. GD and MM contributed unique materials. AM contributed unique materials, designed research, and wrote the paper. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Authors' information
D.B. is a post-doctoral scientist in the Department of Medicine of the University of Colorado School of Medicine. C.F.R. is a production laboratory associate at GE Healthcare Dharmacon. G.D. is a post-doctoral scientist at the Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology at La Sapienza University. M.M. is Ph.D. student at the Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology at La Sapienza University. A.A.V. is an assistant professor in the Department of Biological Sciences at Wright State University. A.M. is an associate professor at the Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology at La Sapienza University. R.A.B. is an assistant professor in the Department of Medicine of the University of Colorado School of Medicine.
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Membership and Service Costs
Burn In
$250 for 6 X 2hr classes of instruction with certified coaches and workouts in a small class setting of 4 participants, maximum.
$300 for 3 X 90 min sessions. The Fundamentals program is intended for individuals who have a need for an accelerated or individual program. Integration into regular group classes is at the coach's discretion.
Drop-in Packages
$250 for 10 class sessions, $30 for individual drop ins. Discounted packages are available to Chrysalis members.
Monthly Memberships
*Contract and payment arrangements made at time of signup. Drop in classes can be added on an as-needed basis to supplement membership during membership commitment timeframe. Membership allows you to sign up for classes and manage your account online with MindBody Online.
Unlimited members may not attend more than one class per day.
Chrysalis Physiques Personal Training & Counselling Sessions
*Fees are based on per person, per hour. 1.5 and 2 hr sessions are also available. All PT is provided by certified coaches on staff at pre-determined dates and times. The scheduled is based on coach/client availability. PT classes enable a group to get specialized training in an area of interest to those attending.
Note: All membership and coaching prices shown above exclude applicable taxes. Cheques are payable to "Chrysalis Physiques". Chrysalis Physiques accepts three forms of payment: Cash, cheque or electronic transfer.

subset: commoncrawl


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Somewhere inside, you know the angels can guide you to a happier, fulfilling and more balanced lifestyle. Perhaps you aren't certain whether you can learn to hear them…or learn to trust them, but you remain open to the possibility. You hold fast to the belief they can help you, so you're optimistic.
Or perhaps you're one who knows with certainty the angels are guiding you, but you're resistant to listening. You're wondering… if you had formal training, the gears would all kick in and your life would be so much easier.
Maybe you have a passion to help people or want to change careers. You've been trying really hard, but you just can't seem to get the manifestation process to work consistently for you. You're pondering…if I could just follow the angels' guidance more closely…
We work with people who are motivated to learn how to hear their angels and to follow their guidance to create the changes they desire.
Many of our students feel challenged with abundance, relationships, or health. They may understand the basics of "Law of Attraction" but they don't understand how to apply it to their own life. They are motivated to make change, but just don't know how.
Many of our students are closet healers. Some already have practices that need focus. And some dream about it during their day job. They want to take the jump from where they are now to feeling success in a healing business they love. But they feel trapped by financial obstacles and don't see a path to paying the bills while doing a job they love. Or, they lack clarity. They wonder, "what exactly would I do if I were a healer?"
She has felt challenged with family, relationships or her career. She believes there must be a higher power that can help her, because she wants to feel alive again.
Our students are ready for well-established, comprehensive training with practical, proven techniques for learning how to access their intuition and the power of the angels. Some of our students have already experienced this higher power, but are not sure whether to trust it or not. Some of them want to learn angel communication or sacred manifestation techniques for their own personal growth. Some of them want to pursue a new career.
Our clients are ready for change. They have sometimes lost their sense of direction and are looking for a beacon to show them the path of healing. They want to know how to feel better about life and they are motivated and ready to take positive steps forward to make it happen.
Our students desperately want to make a difference in the world. They just don't always know how.
Our students and clients know there is an ever-present loving force in the Universe that is there to help. And they want to harness that power…and trust it.
Our clients want to feel better. They know life has its challenges and can be better than it is. They are looking for answers that feel good and are lovingly supportive.
You want to create positive change for you, for your family and for your world. You long to feel gently supported in making changes in your life that are necessary for you to be truly happy. It may be on the surface, or it may be presently hidden, but somewhere within…you are courageous, strong and vibrant.
You believe in the potential of your abilities and are determined to keep trying no matter what. You understand that deep trust in divine guidance is not something that develops overnight. Somewhere inside, however, you know that as you do learn to trust this guidance, it can only lead to goodness.
Integrity and balance are important to you. You understand that when life becomes stressful and overwhelming, change is in the air. It may not always be possible in the moment, but your goal is to stand in the power of your truth…to live the life that God has intended for you.
You are open minded and willing to explore new ideas. You aren't afraid to ask questions. You love to learn, evolve and share space with like-minded friends.


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Lady and the Tramp
Lady is an elegant pooch who falls in love with the most unlikely hound!
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Sheriff's sales now on Web site
Andre B. Laird - Thursday, October 15, 2009 9:11 AM
Effective immediately, postings of upcoming Sheriff sales within Huntington County, will now be posted on the county's Web site, according to Commissioner Tom Wall.
"I'm happy that we have a county employee, Capt. Steve McIntyre, who saw the need to get us up-to-speed with something like this and took the initiative to do so," Wall states.
McIntyre says after getting the go-ahead from Major Tom Carney, he modeled the look of the site after the one similar to Tippecanoe County's, with one exception.
"Our site is much more detailed and offers the visitor more information about the properties," he states.
The site lists properties that will be sold in Sheriff's sales and includes a link to the County's Geographic Information System (GIS).
"The GIS gives detailed information on the property in question, including any improve-ments, liens and tax information," McIntyre says. "People can also view the property from different angles, including a bird eye's view."
He adds that a common misconception is that the Sheriff's Department has access to keys to the property and information on the interior.
"We do not have pictures of the inside of the properties, because we ourselves don't know what inside looks like," he states. "In a sheriff's sale, the property is turned over to us to sell at the bank or the mortgage company's request."
McIntyre says that guidelines and all relevant information needed before coming to the sale and after the sale is also included on the Web site.
Wall says the move to posting the sales online is in the right direction.
"It has always been the commissioner's office desire to make things more efficient and speed things up wherever we can," Wall states. "In the future, we're looking to add a link to tax sales properties as well."
Prior to the link, people interested in purchasing properties at the Sheriff sales would visit the Huntington County Jail, where they could acquire a list of all the properties. Wall says the list will still be made available for those without Internet access.
McIntyre adds that future plans also include listing all outstanding warrants as well as active inmates at the jail.
"The whole aim behind that is to give people more information," he states. "We have people who call all the time inquiring about warrants or wanting to check on family members in lock up."
In the meanwhile, access to the link for Sheriff sales in now available. Visit www.huntington.in.us and click on the "County" tab. Navigate to the "Sheriff" link and click on "Sheriff Sales."


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Your first birthday should be something special and Eliya's sure topped that special scale on her big day. She even dressed to match her cupcake.
Does she not look like she became one with her birthday cupcake? Brother Sam was right there ensuring that the requisite sugar high and big mess were perfectly executed.
The inspiration for Eliya's layout came from the May 1st Stuck?! sketch designed by Laura Whitaker. I love that I got to play with thread and do some stitching on the page. This sketch lends itself to a quick fun layout.
Stuck?! will have another sketch on May 15th.
Check it out and get your creative on. Link up for a chance to win one of the monthly prizes.


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If you are a health professional in Wide Bay and would like to have access to the Wide Bay HealthPathways website, please contact the Wide Bay HealthPathways Team who will provide you with a username and password. The Team can be contacted via email or telephone (07) 4131-0802 (Monday to Friday).
HealthPathways is web-based information portal supporting primary care clinicians to plan patient care through primary, community and secondary health care systems within Wide Bay. It is like a 'care map', so that all members of a health care team – whether they work in a hospital or the community - can be on the same page when it comes to looking after a particular person.
HealthPathways are designed to be used at the point of care, primarily for General Practitioners but is also available to Hospital Specialists, Nurses, Allied Health and other Health Professionals within Wide Bay.
Please read
this disclaimer before using the information on this site.
These pathways have been developed by primary care clinicians, or jointly by primary and secondary care clinicians, for the care and management of patients within Wide Bay.
They provide guidance particularly for general practice teams but are read by other registered health professionals to diagnose and manage patients suffering from a number of different conditions, and contain patient information resources. They are not for access or use by non registered health professionals or patients.
The pathways are local agreements with services accessed by referral within the region. Although the pathways are strongly evidenced based, they are not full clinical guidelines.
As the pathways are suggested guidance only, while using them you must exercise your own clinical judgement and use pertinent clinical data when treating your patient. This includes sourcing additional prescribing information as HealthPathways only includes some treatment recommendations and advice on selected issues.
This site is intended to be flexible and frequently updated. While every effort has been made to ensure accuracy, all information should be verified.
The inclusion of links to websites or health providers in HealthPathways is not a recommendation or endorsement of any health provider, health professional or their services. Use of services featured in these websites is undertaken at the user's own risk.
Pathways that have been developed in New Zealand by the Canterbury Initiative, and are not yet localised for the Wide Bay region, are indicated by an orange background and a warning icon.


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- Error
DOVE received its 501(c)3 nonprofit status in May 2000, thanks to the hard work and dedication of our five founders:
- Jo Damiano
- Kristie Lukowicz
- Michele (Mattio) McCarthy
- Elaine (Woody) Navratil
- Jennifer Pfau
In honor of DOVE's 14th anniversary in May 2014, the founders reconvened and created a vlog describing DOVE's four core values. These core values are the reason DOVE was established and the reason DOVE continues to exist.
English captions are available by clicking on "CC" in the bottom right corner.


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