Aging impairs regulation of ryanodine receptors from extensor digitorum longus but not soleus muscles

ABSTRACT: Introduction: Because impaired excitation‐contraction coupling and reduced sarcoplasmic reticulum (SR) Ca²⁺ release may contribute to the age‐associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. Methods: [³H]Ryanodine binding to membranes from 8‐ and 26‐month‐old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca²⁺ and calmodulin (CaM). Results: Aging reduced maximal Ca²⁺‐stimulated binding to EDL membranes. In 0.3 μM Ca²⁺, age reduced binding and CaM increased binding to EDL membranes. In 300 μM Ca²⁺, CaM reduced binding, but the age effect was not significant. Aging did not affect Ca²⁺ or CaM regulation of soleus RyR1. Discussion: In aged fast‐twitch muscle, impaired RyR1 Ca²⁺ regulation may contribute to lower SR Ca²⁺ release and reduced muscle function. Muscle Nerve 57 : 1022–1025, 2018     

Different mechanisms of Ca2+ regulation that influence synaptic transmission: Comparison between crayfish and Drosophila neuromuscular junctions

A brief historical background on synaptic transmission in relation to Ca²⁺ dynamics and short‐term facilitation is described. This study focuses on the mechanisms responsible for the regulation of intracellular calcium concentration ([Ca²⁺]_i) in high output terminals of larval Drosophila compared to a low‐output terminal of the crayfish neuromuscular junction (NMJ). Three processes; plasmalemmal Na⁺/Ca²⁺ exchanger [NCX], Ca²⁺‐ATPase (PMCA), and sarcoplasmic/endoplasmic Ca²⁺‐ATPase (SERCA) are important in regulating the [Ca²⁺]_i are examined. When the NCX is compromised by reduced [Na⁺]_o, no consistent effect occurred; but a NCX blocker KB‐R7943 decreased the excitatory postsynaptic potential (EPSP) amplitudes. Compromising the PMCA with pH 8.8 resulted in an increase in EPSP amplitude but treatment with a PMCA specific inhibitor carboxyeosin produced opposite results. Thapsigargin exposure to block the SERCA generally decreases EPSP amplitude. Compromising the activity of the above Ca²⁺ regulating proteins had no substantial effects on short‐term depression. The Kum^170TS strain (with dysfunctional SERCA), showed a decrease in EPSP amplitudes including the first EPSP within the train. Synaptic transmission is altered by reducing the function of the above three [Ca²⁺]_i regulators; but they are not consistent among different species as expected. Results in crayfish NMJ were more consistent with expected results as compared to the Drosophila NMJ. It is predicated that different mechanisms are used for regulating the [Ca²⁺]_i in high and low output synaptic terminals. Synapse 63:1100–1121, 2009. © 2009 Wiley‐Liss, Inc.     

Sarcoplasmic–endoplasmic–reticulum Ca2+‐ATPase and calsequestrin are overexpressed in spared intrinsic laryngeal muscles of dystrophin‐deficient mdx mice

In the mdx mouse model of Duchenne muscular dystrophy, the lack of dystrophin is associated with increased calcium levels and skeletal muscle myonecrosis. The intrinsic laryngeal muscles (ILM) are protected and do not undergo myonecrosis. We investigated whether this protection is related to an increased expression of calcium‐binding proteins, which may protect against the elevated calcium levels seen in dystrophic fibers. The expression of sarcoplasmic–endoplasmic–reticulum Ca²⁺‐ATPase and calsequestrin was examined in ILM and in nonspared limb muscles of control and mdx mice using immunofluorescence and immunoblotting. Dystrophic ILM presented a significant increase in the proteins studied when compared to controls. The increase of Ca²⁺‐handling proteins in dystrophic ILM may permit better maintenance of calcium homeostasis, with the consequent absence of myonecrosis. The results further support the concept that abnormal Ca²⁺‐handling is involved in dystrophinopathies. Muscle Nerve, 2009     

CaM kinase II in colonic smooth muscle contributes to dysmotility in murine DSS‐colitis

Background Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca²⁺ signalling contributes to the impaired contractility of colon circular smooth muscles. Methods Acute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days. Key Results Spontaneous and acetylcholine‐evoked contractions, caffeine‐evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca²⁺ waves were increased compared with controls. Caffeine abolished intracellular Ca²⁺ waves in control smooth muscle cells, but not in smooth muscle cells from DSS‐treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and IκBα levels were decreased in distal colon smooth muscles from DSS‐treated mice. Conclusions & Inferences These results suggest that disruptions in intracellular Ca²⁺ mobilization due to down‐regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF‐κB activity.     

Characterization of neuromuscular synapse function abnormalities in multiple Duchenne muscular dystrophy mouse models

Duchenne muscular dystrophy (DMD) is an X‐linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin‐associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn^+/? and mdx/utrn^?/? mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn^?/? mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation‐evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high‐rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn^?/? mice. Overall, we showed mild ‘myasthenia‐like’ neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration.     

Macrophage-Derived Vascular Endothelial Growth Factor-A Is Integral to Neuromuscular Junction Reinnervation after Nerve Injury

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (Vegf-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and Vegf-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes (Ccr2−/− mice), the absence of CD68+ cells (macrophages) in the muscle resulted in diminished muscle function. Using a Vegf-receptor 2 (VegfR2) inhibitor (cabozantinib; CBZ) via oral gavage in wild-type (WT) mice resulted in reduced tSC cytoplasmic process extension and decreased NMJ reinnervation compared with saline controls. Mice with Vegf-A conditionally knocked out in macrophages (Vegf-A^fl/fl; LysM^Cre mice) demonstrated a more prolonged detrimental effect on NMJ reinnervation and worse functional muscle recovery. Together, these results show that contributions of the immune system are integral for NMJ reinnervation and functional muscle recovery after nerve injury.SIGNIFICANCE STATEMENT This work demonstrates beneficial contributions of a macrophage-mediated response for neuromuscular junction (NMJ) reinnervation following nerve injury and repair. Macrophage recruitment occurred at the NMJ, distant from the nerve injury site, to support functional recovery at the muscle. We have shown hindered terminal Schwann cell (tSC) injury response and NMJ recovery with inhibition of: (1) macrophage recruitment after injury; (2) vascular endothelial growth factor receptor 2 (VegfR2) signaling; and (3) Vegf secretion from macrophages. We conclude that macrophage-derived Vegf is a key component of NMJ recovery after injury. Determining the mechanisms active at the end target muscle after motor nerve injury reveals new therapeutic targets that may translate to improve motor recovery following nerve injury.     

Mitochondrial DNA deletions and depletion within paraspinal muscles

G. R. Campbell, A. Reeve, I. Ziabreva, T. M. Polvikoski, R. W. Taylor, R. Reynolds, D. M. Turnbull and D. J. Mahad (2013) Neuropathology and Applied Neurobiology 39, 377–389 Mitochondrial DNA deletions and depletion within paraspinal muscles Aims: Although mitochondrial abnormalities have been reported within paraspinal muscles in patients with axial weakness and neuromuscular disease as well as with ageing, the basis of respiratory deficiency in paraspinal muscles is not known. This study aimed to determine the extent and basis of respiratory deficiency in paraspinal muscles from cases undergoing surgery for degenerative spinal disease and post mortem cases without a history of spinal disease, where age‐related histopathological changes were previously reported. Methods: Cervical and lumbar paraspinal muscles were obtained peri‐operatively from 13 patients and from six post mortem control cases (age range 18–82 years) without a neurological disease. Sequential COX/SDH (mitochondrial respiratory chain complex IV/complex II) histochemistry was performed to identify respiratory‐deficient muscle fibres (lacking complex IV with intact complex II activity). Real‐time polymerase chain reaction, long‐range polymerase chain reaction and sequencing were used to identify and characterize mitochondrial DNA (mtDNA) deletions and determine mtDNA copy number status. Mitochondrial respiratory chain complex subunits were detected by immunohistochemistry. Results: The density of respiratory‐deficient fibres increased with age. On average, 3.96% of fibres in paraspinal muscles were respiratory‐deficient (range 0–10.26). Respiratory deficiency in 36.8% of paraspinal muscle fibres was due to clonally expanded mtDNA deletions. MtDNA depletion accounted for further 13.5% of respiratory deficiency. The profile of immunohistochemically detected subunits of complexes was similar in respiratory‐deficient fibres with and without mtDNA deletions or mtDNA depletion. Conclusions: Paraspinal muscles appeared to be particularly susceptible to age‐related mitochondrial respiratory chain defects. Clonally expanded mtDNA deletions and focal mtDNA depletion may contribute towards the development of age‐related postural abnormalities.     

ICC‐MY coordinate smooth muscle electrical and mechanical activity in the murine small intestine

Background Animals carrying genetic mutations have provided powerful insights into the role of interstitial cells of Cajal (ICC) in motility. One classic model is the W/W^V mouse which carries loss‐of‐function mutations in c‐kit alleles, but retains minimal function of the tyrosine kinase. Previous studies have documented loss of slow waves and aberrant motility in the small intestine of W/W^V mice where myenteric ICC (ICC‐MY) are significantly depleted. Methods Here, we used morphological and electrophysiological techniques to further assess the loss of ICC around the circumference of the small intestine and determine consequences of losing ICC‐MY on electrical activity, Ca²⁺ transients and contractions of the longitudinal muscle (LM). Key Results In wild‐type mice, there was coherent propagation of Ca²⁺ transients through the ICC‐MY network and spread of this activity to the LM. In short segments of small intestine in vitro and in exteriorized segments, slow waves coordiated smoothly propagating Ca²⁺ waves and contractions in the LM of wild‐type mice. In W/W^V mice, Ca²⁺ waves were initiated at variable sites along and around intestinal segments and propagated without constraint unless they collided with other Ca²⁺ waves. This activity resulted in abrupt, uncoordinated contractions. Conclusions & Inferences These results show how dominance of pacemaking by ICC‐MY coordinates propagating con‐tractions and regulates the spontaneous activity of smooth muscle.     

Calendar of events

Intramyofiber accumulation of β‐amyloid fragments (Aβ) is a pathologic hallmark of inclusion‐body myositis (IBM), a progressive skeletal muscle disorder. We investigated the temporal pattern of alterations in the resting cytoplasmic [Ca²⁺] ([Ca²⁺]_i) as well as the depolarization‐evoked Ca²⁺ release from the sarcoplasmic reticulum in skeletal muscle from transgenic mice expressing human βAPP (MCK‐βAPP). MCK‐βAPP mice show an age‐dependent increase in [Ca²⁺]_i along with a reduction in depolarization‐evoked Ca²⁺ release, which appear well before the other reported aspects of IBM, such as inclusion formation, inflammation, centralized nuclei, atrophy, and skeletal muscle weakness. In the young MCK‐βAPP animals the increase in resting [Ca²⁺]_i can be attributed largely to Ca²⁺ influx through nifedipine‐sensitive Ca²⁺ channels. In the adult MCK‐βAPP mice, in addition to the nifedipine‐sensitive pathway, there is also a substantial contribution by the intracellular compartments to the increase in [Ca²⁺]_i. These results suggest that β‐amyloid‐induced disuption of Ca²⁺ handling may represent an early event in the pathogenesis of IBM. Muscle Nerve, 2010     

Extracellular magnesium and calcium reduce myotonia in isolated ClC‐1 chloride channel‐inhibited human muscle

Introduction: Experimental myotonia induced in rat muscle by ClC‐1 chloride channel‐inhibited has been shown to be related inversely to extracellular concentrations of Mg²⁺ and Ca²⁺ ([Mg²⁺]_o and [Ca²⁺]_o) within physiological ranges. Because this implicates a role for [Mg²⁺]_o and [Ca²⁺]_o in the variability of symptoms among myotonia congenita patients, we searched for similar effects of [Mg²⁺]_o and [Ca²⁺]_o on myotonia in human muscle. Methods: Bundles of muscle fibers were isolated from abdominal rectus in patients undergoing abdominal surgery. Myotonia was induced by ClC‐1 inhibition using 9‐anthracene carboxylic acid (9‐AC) and was assessed from integrals of force induced by 5‐Hz stimulation for 2 seconds. Results: Myotonia disappeared gradually when [Mg²⁺]_o or [Ca²⁺]_o were elevated throughout their physiological ranges. These effects of [Mg²⁺]_o and [Ca²⁺]_o were additive and interchangeable. Conclusions: These findings suggest that variations in symptoms in myotonia congenita patients may arise from physiological variations in serum Mg²⁺ and Ca²⁺. Muscle Nerve 51 : 65–71, 2015     

β‐Catenin regulates membrane potential in muscle cells by regulating the α2 subunit of Na,K‐ATPase

Muscle β‐catenin has been shown to play a role in the formation of the neuromuscular junction (NMJ). Our previous studies showed that muscle‐specific conditional knockout of β‐catenin (HSA‐β‐cat^?/?) results in early postnatal death in mice. To understand the underlying mechanisms, we investigated the electrophysiological properties of muscle cells from HSA‐β‐cat^?/? and control mice, and found that, in the absence of muscle β‐catenin, the resting membrane potential (RMP) depolarised in muscle cells from the diaphragm, gastrocnemius and extensor digitorum longus muscles. Furthermore, in a primary line of mouse myoblasts (C2C12 cells) transfected with small‐interfering RNAs targeting β‐catenin, the RMP was depolarised as well. Finally, the expression levels of the α2 subunit of sodium/potassium adenosine triphosphatase were reduced by β‐catenin knockdown in vitro or deletion in vivo. These results suggest a possible mechanism underlying the depolarised RMP in the absence of muscle β‐catenin, and provide additional evidence supporting a role for β‐catenin in the development of NMJs.     

Dopamine elevates and lowers astroglial Ca2+ through distinct pathways depending on local synaptic circuitry

Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca²⁺ rises, the dopamine sensitivity of astroglia in situ and its physiological roles remain unknown. To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca²⁺ in cells connected via gap junctions to astrocytes loaded whole‐cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca²⁺] measurements, we also employed life‐time imaging of the Ca²⁺ indicator Oregon Green BAPTA‐1. We found that dopamine triggered a dose‐dependent, bidirectional Ca²⁺ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below‐baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca²⁺ storage and removal whereas the dopamine‐induced [Ca²⁺] decrease involved D2 receptors only and was sensitive to Ca²⁺ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher‐threshold dopamine‐induced Ca²⁺ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter—dopamine—could either elevate or decrease astrocyte [Ca²⁺] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca²⁺] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447–459     

Impaired propulsive motility in the distal but not proximal colon of BK channel β1‐subunit knockout mice

Background Large‐conductance Ca²⁺‐activated K⁺ (BK) channels regulate smooth muscle tone. The BK channel β1‐subunit increases Ca²⁺ sensitivity of the α‐subunit in smooth muscle. We studied β1‐subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. Methods Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. Key Results Bethanechol‐induced contractions were larger in the distal colon of β1‐subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsβ1‐subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of β1‐subunit KO compared to WT mice. Gastrointestinal transit was unaffected by β1‐subunit KO. Fecal output was decreased and glass bead expulsion times were increased in β1‐subunit KO mice. Membrane potential of distal colon smooth muscle cells from β1‐subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. Conclusions & Inferences BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.     

Role for Toll-like receptor 3 in muscle regeneration after cardiotoxin injury

Introduction: Sterile tissue injury induces an inflammatory response involving cytokines that have crucial roles in the tissue repair that follows. Methods: MyH3 and type 1 interferon (IFN) were assessed by qPCR after cardiotoxin (CTX)‐induced muscle injury. Results: CTX‐induced injury increased expression of IFN‐regulated genes, IFIT1 and MX‐2, which was blocked in type 1 IFN receptor (IFNR)‐deficient mice. However, IFNR‐deficient mice showed no significant differences in muscle regeneration as assessed by MyH3 expression. MyH3 was significantly reduced in TLR3‐deficient but not MyD88‐deficient mice. TLR3‐deficient mice also showed altered expression of proinflammatory cytokines, IL‐6, IL‐1β, and TNF‐α. Conclusions: CTX‐induced muscle injury increased markers of innate immune activation, but blocking type 1 IFN signaling had no effect on muscle regeneration. Taken together, these results suggest a role for TLR3, and perhaps other innate immune signals, in the inflammatory response to CTX‐induced muscle injury and consequent muscle regeneration. Muscle Nerve, 2011     

Na+/Ca2+ exchanger 2‐heterozygote knockout mice display decreased acetylcholine release and altered colonic motility in vivo

Background The Na⁺/Ca²⁺ exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca²⁺ concentrations. NCX is critical for Ca²⁺ regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. However, little is known about the physiological role of NCX in the myenteric neurons and smooth muscles of the gastrointestinal tract. Methods To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)‐induced responses in the longitudinal smooth muscle of the distal colon in NCX1‐ and NCX2‐heterozygote knockout mice. Key Results We found that the amplitudes of EFS‐induced relaxation that persisted during EFS were greater in NCX2 heterozygous mice (HET) than in wild‐type mice (WT). Under the nonadrenergic, noncholinergic (NANC) condition, EFS‐induced relaxation in NCX2 HET was similar in amplitude to that of WT. In addition, an NCX inhibitor, YM‐244769 enhanced EFS‐induced relaxation but did not affect EFS‐induced relaxation under the NANC condition, as in NCX2 HET. Unlike NCX2 HET, NCX1 HET displayed no marked changes in colonic motility. These results indicate that cholinergic function in the colon is altered in NCX2 HET. The magnitude of acetylcholine (ACh)‐induced contraction in NCX2 HET was similar to that in WT. In contrast, EFS‐induced ACh release was reduced in NCX2 HET compared with that in WT. Conclusions & Inferences In this study, we demonstrate that NCX2 regulates colonic motility by altering ACh release onto the myenteric neurons of the distal colon.     

Calcium accumulation precedes the degenerative effects ofl-glutamate on locust muscle fibres

Agonist-induced degeneration of locust muscle occurs only when desensitization of the excitatory glutamate receptors present on this tissue is inhibited. It has been suggested that an increase in intracellular Ca²⁺ is responsible for this degeneration. To test this proposal the accumulation of⁴⁵Ca by locusts muscle has been studied under various conditions, including those under which receptor desensitization was inhibited. Retractor unguis muscles from the metathoracic leg of locusts (Schistocerca gregaria) were used in these studies. All muscles exposed tol-glutamate exhibited an early increase in intracellular radioactivity but this was 2–3 times greater in muscle pretreated with concanavalin A (Con A) to block receptor desensitization. In the desensitizing system the increase in muscle radioactivity was not maintained, intracellular Ca²⁺-levels declining to control values after 30 min in⁴⁵Ca-saline-containing glutamate. In Con A-treated muscles intracellular Ca²⁺-levels plateaued well above control levels within 5 min of exposure to glutamate and were maintained at these levels throughout the period of glutamate treatment. These results support the contention that agonist-induced degeneration of locust muscle is triggered by entry of Ca²⁺ and a rise in intracellular concentration of this cation to a toxic level.     

Canonical transient receptor potential channel subtype 3‐mediated hair cell Ca2+ entry regulates sound transduction and auditory neurotransmission

The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca²⁺ entry pathway, with dual activation via G protein‐coupled receptor (GPCR)–phospholipase C–diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca²⁺ releases Ca²⁺–calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca²⁺ homeostasis. Confocal microfluorimetry with the Ca²⁺ indicator Fluo‐4 acetoxymethylester showed that, when cytosolic Ca²⁺ was depleted, Ca²⁺ re‐entry was significantly impaired in mature TRPC3^?/? inner and outer hair cells. The impact of this disrupted Ca²⁺ homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3^?/? mice showed significantly stronger DPOAE (2f₁ ? f₂) growth functions than wild‐type (WT) littermates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3^?/? and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca²⁺ set‐point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback‐regulated Ca²⁺ entry pathway. This TPRC3‐regulated Ca²⁺ homeostasis shapes the sound transduction input–output function and auditory neurotransmission.