Assessment of Cell Viability in Electrically Excitable Muscle Cells Through Intact Twitch Stimulation.

Muscle cells (i.e. skeletal muscle fibers) are fully viable and functional when their excitation-contraction (EC) coupling machinery is intact. This involves intact membrane integrity with polarized membrane, functional ion channels for action potential generation and conduction, an intact electro-chemical interface at the level of the fiber's triad, followed by sarcoplasmic reticulum Ca2+ release, and subsequent activation of the chemico-mechanical interface at the level of the contractile apparatus. The ultimate end result is then a visible twitch contraction upon a brief electrical pulse stimulation. For many biomedical studies involving single muscle cells, intact and viable myofibers are of utmost importance. Thus, a simple global screening method that involves a brief electrical stimulus applied to single muscle fibers and assessment of visible contraction would be of high value. In this chapter, we describe step-by-step protocols to (i) obtain intact single muscle fibers from freshly dissected muscle tissue using an enzymatic digestion procedure and (ii) provide a workflow for the assessment of twitch response of single fibers that can be ultimately classified as viable. For this, we have prepared a unique stimulation pen for which we provide the fabrication guide for do-it-yourself rapid prototyping to eliminate the need for expensive specialized commercial equipment.

Six weeks of N-acetylcysteine antioxidant in drinking water decreases pathological fiber branching in MDX mouse dystrophic fast-twitch skeletal muscle.

Introduction: It has been proposed that an increased susceptivity to oxidative stress caused by the absence of the protein dystrophin from the inner surface of the sarcolemma is a trigger of skeletal muscle necrosis in the destructive dystrophin deficient muscular dystrophies. Here we use the mdx mouse model of human Duchenne Muscular Dystrophy to test the hypothesis that adding the antioxidant NAC at 2% to drinking water for six weeks will treat the inflammatory phase of the dystrophic process and reduce pathological muscle fiber branching and splitting resulting in a reduction of mass in mdx fast-twitch EDL muscles. Methods: Animal weight and water intake was recorded during the six weeks when 2% NAC was added to the drinking water. Post NAC treatment animals were euthanised and the EDL muscles dissected out and placed in an organ bath where the muscle was attached to a force transducer to measure contractile properties and susceptibility to force loss from eccentric contractions. After the contractile measurements had been made the EDL muscle was blotted and weighed. In order to assess the degree of pathological fiber branching mdx EDL muscles were treated with collagenase to release single fibers. For counting and morphological analysis single EDL mdx skeletal muscle fibers were viewed under high magnification on an inverted microscope. Results: During the six-week treatment phase NAC reduced body weight gain in three- to nine-week-old mdx and littermate control mice without effecting fluid intake. NAC treatment also significantly reduced the mdx EDL muscle mass and abnormal fiber branching and splitting. Discussion: We propose chronic NAC treatment reduces the inflammatory response and degenerative cycles in the mdx dystrophic EDL muscles resulting in a reduction in the number of complexed branched fibers reported to be responsible for the dystrophic EDL muscle hypertrophy.

Calcium and strontium contractile activation properties of single skinned skeletal muscle fibres from elderly women 66-90 years of age.

The single freshly skinned muscle fibre technique was used to investigate Ca2+- and Sr2+-activation properties of skeletal muscle fibres from elderly women (66-90 years). Muscle biopsies were obtained from the vastus lateralis muscle. Three populations of muscle fibres were identified according to their specific Sr2+-activation properties: slow-twitch (type I), fast-twitch (type II) and hybrid (type I/II) fibres. All three fibre types were sampled from the biopsies of 66 to 72 years old women, but the muscle biopsies of women older than 80 years yielded only slow-twitch (type I) fibres. The proportion of hybrid fibres in the vastus lateralis muscle of women of circa 70 years of age (24%) was several-fold greater than in the same muscle of adults (< 10%), suggesting that muscle remodelling occurs around this age. There were no differences between the Ca2+- and Sr2+-activation properties of slow-twitch fibres from the two groups of elderly women, but there were differences compared with muscle fibres from young adults with respect to sensitivity to Ca2+, steepness of the activation curves, and characteristics of the fibre-type dependent phenomenon of spontaneous oscillatory contractions (SPOC) (or force oscillations) occurring at submaximal levels of activation. The maximal Ca2+ activated specific force from all the fibres collected from the seven old women use in the present study was significantly lower by 20% than in the same muscle of adults. Taken together these results show there are qualitative and quantitative changes in the activation properties of the contractile apparatus of muscle fibres from the vastus lateralis muscle of women with advancing age, and that these changes need to be considered when explaining observed changes in women's mobility with aging.

Absence of the Z-disc protein α-actinin-3 impairs the mechanical stability of Actn3KO mouse fast-twitch muscle fibres without altering their contractile properties or twitch kinetics.

BACKGROUND: A common polymorphism (R577X) in the ACTN3 gene results in the complete absence of the Z-disc protein α-actinin-3 from fast-twitch muscle fibres in ~ 16% of the world's population. This single gene polymorphism has been subject to strong positive selection pressure during recent human evolution. Previously, using an Actn3KO mouse model, we have shown in fast-twitch muscles, eccentric contractions at L0 + 20% stretch did not cause eccentric damage. In contrast, L0 + 30% stretch produced a significant ~ 40% deficit in maximum force; here, we use isolated single fast-twitch skeletal muscle fibres from the Actn3KO mouse to investigate the mechanism underlying this. METHODS: Single fast-twitch fibres are separated from the intact muscle by a collagenase digest procedure. We use label-free second harmonic generation (SHG) imaging, ultra-fast video microscopy and skinned fibre measurements from our MyoRobot automated biomechatronics system to study the morphology, visco-elasticity, force production and mechanical strength of single fibres from the Actn3KO mouse. Data are presented as means ± SD and tested for significance using ANOVA. RESULTS: We show that the absence of α-actinin-3 does not affect the visco-elastic properties or myofibrillar force production. Eccentric contractions demonstrated that chemically skinned Actn3KO fibres are mechanically weaker being prone to breakage when eccentrically stretched. Furthermore, SHG images reveal disruptions in the myofibrillar alignment of Actn3KO fast-twitch fibres with an increase in Y-shaped myofibrillar branching. CONCLUSIONS: The absence of α-actinin-3 from the Z-disc in fast-twitch fibres disrupts the organisation of the myofibrillar proteins, leading to structural weakness. This provides a mechanistic explanation for our earlier findings that in vitro intact Actn3KO fast-twitch muscles are significantly damaged by L0 + 30%, but not L0 + 20%, eccentric contraction strains. Our study also provides a possible mechanistic explanation as to why α-actinin-3-deficient humans have been reported to have a faster decline in muscle function with increasing age, that is, as sarcopenia reduces muscle mass and force output, the eccentric stress on the remaining functional α-actinin-3 deficient fibres will be increased, resulting in fibre breakages.

Sarcoplasmic reticulum calcium handling in unbranched, immediately post-necrotic fast-twitch mdx fibres is similar to wild-type littermates.

NEW FINDINGS: What is the central question of this study? What are the early effects of dystrophin deficiency on sarcoplasmic reticulum Ca2+ handling in the mdx mouse? What is the main finding and its importance? In the mdx mouse, Ca2+ handling by the sarcoplasmic reticulum is little affected by the absence of dystrophin when looking at fibres without branches that have recently regenerated after massive myonecrosis. This has important implications for our understanding of Ca2+ pathology in the mdx mouse. ABSTRACT: There is a variety of results in the literature regarding the effects of dystrophin deficiency on the Ca2+ handling properties of the sarcoplasmic reticulum (SR) in the mdx mouse, an animal model of Duchenne muscular dystrophy. One possible source of variation is the presence of branched fibres. Fibre branching, a consequence of degenerative-regenerative processes such as muscular dystrophy, has in itself a significant influence on the function of the SR. In this study, we attempted to detect early effects of dystrophin deficiency on SR Ca2+ handling by using unbranched fibres from the immediate post-necrotic stage in mdx mice (recently regenerated after massive necrosis). Using kinetically corrected fura-2 fluorescence signals measured during twitch and tetanus, we analysed the amplitude, rise time and decay time of Δ[Ca2+ ]i in unfatigued and fatigued fibres. Decay was also resolved into SR pump and SR leak components. Fibres from mdx mice were similar in all respects to fibres from wild-type littermates apart from: (1) a smaller amplitude of the initial spike of Δ[Ca2+ ]i during a tetanus; and (2) a mitigation of the fall in Δ[Ca2+ ]i amplitude during the course of fatigue. Our findings suggest that the early effects of a loss of dystrophin on SR Ca2+ handling in mdx mice are subtle, and we emphasize the importance of distinguishing between Ca2+ pathology that is attributable to lack of dystrophin and Ca2+ pathology that is attributable to muscle degeneration.

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching.

The common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin-deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this, we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model, which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse, we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%), and this correlates with a graded force loss over three eccentric contractions for dKO muscles (~36% after first contraction, ~66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (~75% after first contraction, ~89% after three contractions). In dKO, protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

Lifespan Analysis of Dystrophic mdx Fast-Twitch Muscle Morphology and Its Impact on Contractile Function.

Duchenne muscular dystrophy is caused by the absence of the protein dystrophin from skeletal muscle and is characterized by progressive cycles of necrosis/regeneration. Using the dystrophin deficient mdx mouse model, we studied the morphological and contractile chronology of dystrophic skeletal muscle pathology in fast-twitch Extensor Digitorum Longus muscles from animals 4-22 months of age containing 100% regenerated muscle fibers. Catastrophically, the older age groups lost ∼80% of their maximum force after one eccentric contraction (EC) of 20% strain with the greatest loss of ∼92% recorded in senescent 22-month-old mdx mice. In old age groups, there was minimal force recovery ∼24% after 120 min, correlated with a dramatic increase in the number and complexity of branched fibers. This data supports our two-phase model where a "tipping point" is reached when branched fibers rupture irrevocably on EC. These findings have important implications for pre-clinical drug studies and genetic rescue strategies.

Dystrophin-negative slow-twitch soleus muscles are not susceptible to eccentric contraction induced injury over the lifespan of the mdx mouse.

Duchenne muscular dystrophy (DMD) is the second most common fatal genetic disease in humans and is characterized by the absence of a functional copy of the protein dystrophin from skeletal muscle. In dystrophin-negative humans and rodents, regenerated skeletal muscle fibers show abnormal branching. The number of fibers with branches and the complexity of branching increases with each cycle of degeneration/regeneration. Previously, using the mdx mouse model of DMD, we have proposed that once the number and complexity of branched fibers present in dystrophic fast-twitch EDL muscle surpasses a stable level, we term the "tipping point," the branches, in and of themselves, mechanically weaken the muscle by rupturing when subjected to high forces during eccentric contractions. Here, we use the slow-twitch soleus muscle from the dystrophic mdx mouse to study prediseased "periambulatory" dystrophy at 2-3 wk, the peak regenerative "adult" phase at 6-9 wk, and "old" at 58-112 wk. Using isolated mdx soleus muscles, we examined contractile function and response to eccentric contraction correlated with the amount and complexity of regenerated branched fibers. The intact muscle was enzymatically dispersed into individual fibers in order to count fiber branching and some muscles were optically cleared to allow laser scanning confocal microscopy. We demonstrate throughout the lifespan of the mdx mouse that dystrophic slow-twitch soleus muscle is no more susceptible to eccentric contraction-induced injury than age-matched littermate controls and that this is correlated with a reduction in the number and complexity of branched fibers compared with fast-twitch dystrophic EDL muscles.

Minocycline Treatment Reduces Mass and Force Output From Fast-Twitch Mouse Muscles and Inhibits Myosin Production in C2C12 Myotubes.

Minocycline, a tetracycline-class of antibiotic, has been tested with mixed effectiveness on neuromuscular disorders such as amyotrophic lateral sclerosis, autoimmune neuritis and muscular dystrophy. The independent effect of minocycline on skeletal muscle force production and signalling remain poorly understood. Our aim here is to investigate the effects of minocycline on muscle mass, force production, myosin heavy chain abundance and protein synthesis. Mice were injected with minocycline (40 mg/kg i.p.) daily for 5 days and sacrificed at day six. Fast-twitch EDL, TA muscles and slow-twitch soleus muscles were dissected out, the TA muscle was snap-frozen and the remaining muscles were attached to force transducer whilst maintained in an organ bath. In C2C12 myotubes, minocycline was applied to the media at a final concentration of 10 µg/mL for 48 h. In minocycline treated mice absolute maximal force was lower in fast-twitch EDL while in slow-twitch soleus there was an increase in the time to peak and relaxation of the twitch. There was no effect of minocycline treatment on the other contractile parameters measured in isolated fast- and slow-twitch muscles. In C2C12 cultured cells, minocycline treatment significantly reduced both myosin heavy chain content and protein synthesis without visible changes to myotube morphology. In the TA muscle there was no significant changes in myosin heavy chain content. These results indicate that high dose minocycline treatment can cause a reduction in maximal isometric force production and mass in fast-twitch EDL and impair protein synthesis during myogenesis in C2C12 cultured cells. These findings have important implications for future studies investigating the efficacy of minocycline treatment in neuromuscular or other muscle-atrophy inducing conditions.

Single muscle fibre biomechanics and biomechatronics - The challenges, the pitfalls and the future.

Interest in muscle biomechanics is growing with availabilities of patient biopsies and animal models related to muscle diseases, muscle wasting (sarcopenia, cachexia), exercise and drug effects. However, development of technologies or facilitated systems required to measure biomechanical and contractile properties of single fibres has not kept pace with this demand. Most studies use manual mechatronics systems that have not changed in decades and are confined to a few labs worldwide. Available commercial systems are expensive and limited in versatility, throughput and user-friendliness. We review major standard systems available from research labs and commercial sources, and benchmark those to our recently developed automated MyoRobot biomechatronics platform that provides versatility to cover multiple organ scales, is flexible in programming for active/passive muscle biomechanics using custom-made graphics user interfaces, employs on-the-fly data analyses and does not rely on external research microscopes. With higher throughput, this system blends Industry 4.0 automation principles into myology.

The Effect of ACTN3 Gene Doping on Skeletal Muscle Performance.

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

Branched fibers from old fast-twitch dystrophic muscles are the sites of terminal damage in muscular dystrophy.

A striking pathological feature of dystrophinopathies is the presence of morphologically abnormal branched skeletal muscle fibers. The deterioration of muscle contractile function in Duchenne muscular dystrophy is accompanied by both an increase in number and complexity of these branched fibers. We propose that when number and complexity of branched fibers reaches a critical threshold, or "tipping point," the branches in and of themselves are the site of contraction-induced rupture. In the present study, we use the dystrophic mdx mouse and littermate controls to study the prediseased dystrophic fast-twitch extensor digitorum longus (EDL) muscle at 2-3 wk, the peak myonecrotic phase at 6-9 wk, and finally, "old," at 58-112 wk. Using a combination of isolated muscle function contractile measurements coupled with single-fiber imaging and confocal microscope imaging of cleared whole muscles, we identified a distinct pathophysiology, acute fiber rupture at branch nodes, which occurs in "old" fast-twitch EDL muscle approaching the end stage of the dystrophinopathy muscle disease, where the EDL muscles are entirely composed of complexed branched fibers. This evidence supports our concept of "tipping point" where the number and extent of fiber branching reach a level where the branching itself terminally compromises muscle function, irrespective of the absence of dystrophin.

Quantitative Ratiometric Ca2+ Imaging to Assess Cell Viability.

Viability of cells is strongly related to their Ca2+ homeostasis. Ca2+ signal fluctuations can be on a slow time scale, e.g., in non-excitable cells, but also in the range of tens of milliseconds for excitable cells, such as nerve and muscle. Muscle fibers respond to electrical stimulation with Ca2+ transients that exceed their resting basal level about 100 times. Fluorescent Ca2+ dyes have become an indispensable means to monitor Ca2+ fluctuations in living cells online. Fluorescence intensity of such "environmental dyes" relies on a buffer-ligand interaction which is not only governed by laws of mass action but also by binding and unbinding kinetics that have to be considered for proper Ca2+ kinetics and amplitude validation. The concept of Ca2+ dyes including the different approaches using ratiometric and non-ratiometric dyes, the way to correctly choose dyes according to their low-/high-affinity properties and kinetics as well as staining techniques, and in situ calibration are reviewed and explained. We provide detailed protocols to apply ratiometric Fura-2 imaging of resting Ca2+ and Ca2+ fluctuations during field-stimulation in single isolated skeletal muscle cells and how to translate fluorescence intensities into absolute Ca2+ concentrations using appropriate calibration techniques.

Evidence for ACTN3 as a genetic modifier of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10 m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that α-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that α-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients.

Analysis of the ACTN3 heterozygous genotype suggests that α-actinin-3 controls sarcomeric composition and muscle function in a dose-dependent fashion.

A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in ∼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.

Altered Ca2+ kinetics associated with α-actinin-3 deficiency may explain positive selection for ACTN3 null allele in human evolution.

Over 1.5 billion people lack the skeletal muscle fast-twitch fibre protein α-actinin-3 due to homozygosity for a common null polymorphism (R577X) in the ACTN3 gene. α-Actinin-3 deficiency is detrimental to sprint performance in elite athletes and beneficial to endurance activities. In the human genome, it is very difficult to find single-gene loss-of-function variants that bear signatures of positive selection, yet intriguingly, the ACTN3 null variant has undergone strong positive selection during recent evolution, appearing to provide a survival advantage where food resources are scarce and climate is cold. We have previously demonstrated that α-actinin-3 deficiency in the Actn3 KO mouse results in a shift in fast-twitch fibres towards oxidative metabolism, which would be more "energy efficient" in famine, and beneficial to endurance performance. Prolonged exposure to cold can also induce changes in skeletal muscle similar to those observed with endurance training, and changes in Ca2+ handling by the sarcoplasmic reticulum (SR) are a key factor underlying these adaptations. On this basis, we explored the effects of α-actinin-3 deficiency on Ca2+ kinetics in single flexor digitorum brevis muscle fibres from Actn3 KO mice, using the Ca2+-sensitive dye fura-2. Compared to wild-type, fibres of Actn3 KO mice showed: (i) an increased rate of decay of the twitch transient; (ii) a fourfold increase in the rate of SR Ca2+ leak; (iii) a threefold increase in the rate of SR Ca2+ pumping; and (iv) enhanced maintenance of tetanic Ca2+ during fatigue. The SR Ca2+ pump, SERCA1, and the Ca2+-binding proteins, calsequestrin and sarcalumenin, showed markedly increased expression in muscles of KO mice. Together, these changes in Ca2+ handling in the absence of α-actinin-3 are consistent with cold acclimatisation and thermogenesis, and offer an additional explanation for the positive selection of the ACTN3 577X null allele in populations living in cold environments during recent evolution.

Membrane Injury and Repair in the Muscular Dystrophies.

Muscle cells have an elaborate plasma membrane and t-tubule system that has been evolutionarily refined to maximize electrical conductivity for synchronous muscle contraction. However, this elaborate plasma membrane network has intrinsic vulnerabilities to stretch-induced membrane injury, and thus requires ongoing maintenance and repair. Herein we discuss the types of membrane injuries encountered by myofibers in healthy muscle and in muscular dystrophy. We review the different mechanisms by which muscle fibers in patients with muscular dystrophy are rendered more susceptible to injury, and we summarize the latest developments in our understanding of how the muscular dystrophy protein dysferlin mediates satellite-cell independent membrane repair.

RhoA/ROCK signaling and pleiotropic α1A-adrenergic receptor regulation of cardiac contractility.

AIMS: To determine the mechanisms by which the α1A-adrenergic receptor (AR) regulates cardiac contractility. BACKGROUND: We reported previously that transgenic mice with cardiac-restricted α1A-AR overexpression (α1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gαq/11-coupled receptor in hypertrophy. METHODS: Contractility, calcium (Ca(2+)) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition. RESULTS: Hypercontractility without hypertrophy with α1A-AR overexpression is shown to result from increased intracellular Ca(2+) release in response to agonist, augmenting the systolic amplitude of the intracellular Ca(2+) concentration [Ca(2+)]i transient without changing resting [Ca(2+)]i. In the absence of agonist, however, α1A-AR overexpression reduced contractility despite unchanged [Ca(2+)]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gαq/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca(2+) sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility. CONCLUSIONS: We report for the first time pleiotropic α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.

Aged skeletal muscle retains the ability to fully regenerate functional architecture.

While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.