Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

The dynamics of actin protrusions can be controlled by tip-localized myosin motors.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with non-syndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actin-activated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Essential nucleotide- and protein-dependent functions of Actb/ß-actin.

The highly similar cytoplasmic ß- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked ß-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, ß-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

Stereocilia morphogenesis and maintenance through regulation of actin stability.

Stereocilia are actin-based protrusions on auditory and vestibular sensory cells that are required for hearing and balance. They convert physical force from sound, head movement or gravity into an electrical signal, a process that is called mechanoelectrical transduction. This function depends on the ability of sensory cells to grow stereocilia of defined lengths. These protrusions form a bundle with a highly precise geometry that is required to detect nanoscale movements encountered in the inner ear. Congenital or progressive stereocilia degeneration causes hearing loss. Thus, understanding stereocilia hair bundle structure, development, and maintenance is pivotal to understanding the pathogenesis of deafness. Stereocilia cores are made from a tightly packed array of parallel, crosslinked actin filaments, the length and stability of which are regulated in part by myosin motors, actin crosslinkers and capping proteins. This review aims to describe stereocilia actin regulation in the context of an emerging "tip turnover" model where actin assembles and disassembles at stereocilia tips while the remainder of the core is exceptionally stable.

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia.

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a ß-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing ß-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of ß- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.

ß-Actin and fascin-2 cooperate to maintain stereocilia length.

Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both ß-actin and γ-actin as knock-out mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that ß-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking ß-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds ß-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on ß-actin for localization in vivo. Nevertheless, double-mutant mice lacking ß-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Because the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.

ß-actin and γ-actin are each dispensable for auditory hair cell development but required for Stereocilia maintenance.

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic ß-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since ß-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of ß-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither ß-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with ß-actin- or γ-actin-deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for ß-actin and γ-actin in maintaining hair cell stereocilia.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

Gamma-actin is required for cytoskeletal maintenance but not development.

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

Nucleotide- and Protein-Dependent Functions of Actg1.

Cytoplasmic ß- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb-/- mice are embryonic lethal and Actb-/- cells fail to proliferate, but editing the Actb gene to express γ-actin (Actbc-g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actbc-g and Actg1-/- mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actbc-g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1-/-, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions.

Calpain-mediated proteolysis of talin regulates adhesion dynamics.

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

Cell Biology: Function Guides Form of Auditory Sensory Cells.

Mechanosensory bundles on auditory sensory cells are composed of stereocilia that grow in rows of decreasing height. This pattern depends on the specification of the eventual tallest row, then the assignment of distinct molecular identities to the shorter rows. Mechanotransduction refines and maintains row identity, thus instructing the form of the bundle.

Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration.

Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.

The stable actin core of mechanosensory stereocilia features continuous turnover of actin cross-linkers.

Stereocilia are mechanosensitive protrusions on the surfaces of sensory hair cells in the inner ear that detect sound, gravity, and head movement. Their cores are composed of parallel actin filaments that are cross-linked and stabilized by several actin-binding proteins, including fascin-2, plastin-1, espin, and XIRP2. The actin filaments are the most stable known, with actin turnover primarily occurring at the stereocilia tips. While stereocilia actin dynamics has been well studied, little is known about the behavior of the actin cross-linking proteins, which are the most abundant type of protein in stereocilia after actin and are critical for stereocilia morphogenesis and maintenance. Here, we developed a novel transgenic mouse to monitor EGFP-fascin-2 incorporation . In contrast to actin, EGFP-fascin-2 readily enters the stereocilia core. We also compared the effect of EGFP-fascin-2 expression on developing and mature stereocilia. When it was induced during hair cell development, we observed increases in both stereocilia length and width. Interestingly, stereocilia size was not affected when EGFP-fascin-2 was induced in adult stereocilia. Regardless of the time of induction, EGFP-fascin-2 displaced both espin and plastin-1 from stereocilia. Altering the actin cross-linker composition, even as the actin filaments exhibit little to no turnover, provides a mechanism for ongoing remodeling and repair important for stereocilia homeostasis.

GRXCR2 Regulates Taperin Localization Critical for Stereocilia Morphology and Hearing.

Mutations in human GRXCR2, which encodes a protein of undetermined function, cause hearing loss by unknown mechanisms. We found that mouse GRXCR2 localizes to the base of the stereocilia, which are actin-based mechanosensing organelles in cochlear hair cells that convert sound-induced vibrations into electrical signals. The stereocilia base also contains taperin, another protein of unknown function required for human hearing. We show that taperin and GRXCR2 form a complex and that taperin is diffused throughout the stereocilia length in Grxcr2-deficient hair cells. Stereocilia lacking GRXCR2 are longer than normal and disorganized due to the mislocalization of taperin, which could modulate the actin cytoskeleton in stereocilia. Remarkably, reducing taperin expression levels could rescue the morphological defects of stereocilia and restore the hearing of Grxcr2-deficient mice. Thus, our findings suggest that GRXCR2 is critical for the morphogenesis of stereocilia and auditory perception by restricting taperin to the stereocilia base.

PNPase knockout results in mtDNA loss and an altered metabolic gene expression program.

Polynucleotide phosphorylase (PNPase) is an essential mitochondria-localized exoribonuclease implicated in multiple biological processes and human disorders. To reveal role(s) for PNPase in mitochondria, we established PNPase knockout (PKO) systems by first shifting culture conditions to enable cell growth with defective respiration. Interestingly, PKO established in mouse embryonic fibroblasts (MEFs) resulted in the loss of mitochondrial DNA (mtDNA). The transcriptional profile of PKO cells was similar to rho0 mtDNA deleted cells, with perturbations in cholesterol (FDR = 6.35 x 10-13), lipid (FDR = 3.21 x 10-11), and secondary alcohol (FDR = 1.04x10-12) metabolic pathway gene expression compared to wild type parental (TM6) MEFs. Transcriptome analysis indicates processes related to axonogenesis (FDR = 4.49 x 10-3), axon development (FDR = 4.74 x 10-3), and axonal guidance (FDR = 4.74 x 10-3) were overrepresented in PKO cells, consistent with previous studies detailing causative PNPase mutations in delayed myelination, hearing loss, encephalomyopathy, and chorioretinal defects in humans. Overrepresentation analysis revealed alterations in metabolic pathways in both PKO and rho0 cells. Therefore, we assessed the correlation of genes implicated in cell cycle progression and total metabolism and observed a strong positive correlation between PKO cells and rho0 MEFs compared to TM6 MEFs. We quantified the normalized biomass accumulation rate of PKO clones at 1.7% (SD ± 2.0%) and 2.4% (SD ± 1.6%) per hour, which was lower than TM6 cells at 3.3% (SD ± 3.5%) per hour. Furthermore, PKO in mouse inner ear hair cells caused progressive hearing loss that parallels human familial hearing loss previously linked to mutations in PNPase. Combined, our study reports that knockout of a mitochondrial nuclease results in mtDNA loss and suggests that mtDNA maintenance could provide a unifying connection for the large number of biological activities reported for PNPase.

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle.

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.

Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms.

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic ßcyto - or γcyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific ßcyto - and γcyto -actin KO mice. We found ßcyto - and γcyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking ßcyto - and/or γcyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific ßcyto - and γcyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old ßcyto - and γcyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.