Prostate cancer is a bimodal disease with aggressive and indolent forms. Current prostate-specific-antigen testing and digital rectal examination screening provide ambiguous results leading to both under-and over-treatment. Accurate, consistent diagnosis is crucial to risk-stratify patients and facilitate clinical decision making as to treatment versus active surveillance. Diagnosis is currently achieved by needle biopsy, a painful procedure. Thus, there is a clinical need for a minimally-invasive test to determine prostate cancer aggressiveness. A blood sample to predict Gleason score, which is known to reflect aggressiveness of the cancer, could serve as such a test.

Calsequestrin (CS), the major Ca(2+)-binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation-contraction coupling: buffering free Ca(2+) increasing SR capacity, and modulating the activity of the Ca(2+) release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1-null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1-null muscle fibres are characterized by structural and functional changes, which are much more evident in fast-twitch muscles (EDL) in which most fibres express only CS1, than in slow-twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time-to-peak and half-relaxation time, probably related to impaired calcium release from and re-uptake by the SR. Ca(2+)-imaging studies show that the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca(2+)-release channels (confirmed also by (3)H-ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca(2+).

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

The medial nucleus of the trapezoid body (MNTB) in the superior olivary complex (SOC) is an inhibitory hub considered critical for binaural sound localization. We show that genetic ablation of MNTB neurons in mice only subtly affects this ability by prolonging the minimum time required to detect shifts in sound location. Furthermore, glycinergic innervation of the SOC is maintained without an MNTB, consistent with the existence of parallel inhibitory inputs. These findings redefine the role of MNTB in sound localization and suggest that the inhibitory network is more complex than previously thought.

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca(2+)]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+)]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+)]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+)]r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.

Developing skeletal myofibers in vertebrates are intrinsically 'pre-patterned' for motor nerve innervation. However, the intrinsic factors that regulate muscle pre-patterning remain unknown. We found that a functional skeletal muscle dihydropyridine receptor (DHPR, the L-type Ca(2+) channel in muscle) was required for muscle pre-patterning during the development of the neuromuscular junction (NMJ). Targeted deletion of the β1 subunit of DHPR (Cacnb1) in mice led to muscle pre-patterning defects, aberrant innervation and precocious maturation of the NMJ. Reintroducing Cacnb1 into Cacnb1(-/-) muscles reversed the pre-patterning defects and restored normal development of the NMJ. The mechanism by which DHPRs govern muscle pre-patterning is independent of their role in excitation-contraction coupling, but requires Ca(2+) influx through the L-type Ca(2+) channel. Our findings indicate that the skeletal muscle DHPR retrogradely regulates the patterning and formation of the NMJ.

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation-contraction coupling (voltage-gated Ca(2+) release from the SR) and increased amplitude of L-type Ca(2+) current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca(2+) release from the SR, membrane-bound charge movement, and/or L-type Ca(2+) current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca(2+) release from the SR was substantially reduced and shifted ( approximately 10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca(2+) release. The current-voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials ( approximately 7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist +/-Bay K 8644 (10 microM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to -50, -10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes.

Neuronal intracellular Ca2+ homeostasis is critical to the normal physiological functions of neurons and neuronal Ca2+ dyshomeostasis has been associated with the age-related decline of cognitive functions. Accumulated evidence indicates that the underlying mechanism for this is that abnormal intracellular Ca2+ levels stimulate the dysregulation of intracellular signaling, which subsequently induces neuronal cell death. We examined intracellular Ca2+ homeostasis in cortical (in vivo) and hippocampal (in vitro) neurons from young (3-months), middle-age (12-months), and aged (24-months) wild type C57BL6J mice. We found a progressive age-related elevation of intracellular resting calcium ([Ca2+]r) in cortical (in vivo) and hippocampal (in vitro) neurons associated with increased hippocampal neuronal calpain activity and reduced cell viability. In vitro, removal of extracellular Ca2+ or treatment with SAR7334 or dantrolene reduced [Ca2+]r in all age groups and dantrolene treatment lowered calpain activity and increased cell viability. In vivo, both middle-aged and aged mice showed cognitive deficits compared to young mice, which improved after dantrolene treatment. These findings support the hypothesis that intracellular Ca2+ dyshomeostasis is a major mechanism underlying the cognitive deficits seen in both normal aging and degenerative neurologic diseases.

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+) imaging and Ca(2+) selective microelectrodes we found that changes in e-c coupling, SR Ca(2+)content and resting [Ca(2+)] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+) regulation than Jct/CASQ association.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

Contractile activation in striated muscles requires a Ca(2+) reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca(2+)] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca(2+) signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca(2+) release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca(2+) permeability and inversely to its Ca(2+) buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca(2+) depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca(2+) depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca(2+)], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca(2+), but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca(2+) as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca(2+) storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.

Bidirectional signaling between the sarcolemmal L-type Ca(2+) channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca(2+) release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation-contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K(+) depolarization. The new findings are: (a) For all three genotypes, Ca(2+) transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca(2+) or the addition of Ca(2+) entry blockers (nifedipine, SKF96365, and Ni(2+)) enhanced the rate of decay in all genotypes. (d) When Ca(2+) entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K(+) depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor.

Aging causes skeletal muscles to become atrophied, weak, and easily fatigued. Here, we have tested the hypothesis that normal aging in skeletal muscle cells is associated with Ca2+ intracellular dyshomeostasis and oxidative stress. Intracellular Ca2+ concentration ([Ca2+]i), resting intracellular Na+ concentration ([Na+]i) and reactive oxygen species (ROS) production were measured in vivo (superficial gastrocnemius fibers) using double-barreled ion-selective microelectrodes, and in vitro [isolated single flexor digitorum brevis fibers] using fluorescent ROS sensor CM-H2DCFDA in young (3 months of age), middle-aged (12 months of age), and aged (24 months of age) mice. We found an age-related increase in [Ca2+]i from 121 ± 4 nM in young muscle cells which rose to 255 ± 36 nM in middle-aged and to 409 ± 25 nM in aged cells. [Na+]i also showed an age-dependent elevation, increasing from 8 ± 0.5 mM in young muscle fibers, to 12 ± 1 mM in middle-aged and to 17 ± 1 mM in old muscle fibers. Using the fluorescent ROS sensor CM-H2DCFDA we found that these increases in intracellular cation concentrations were associated with significantly increased basal ROS production as demonstrated by age related increases in the rate of dichlorodihydrofluorescein fluorescence. To determine is this could be modified by reducing ROS and/or blocking sarcolemmal Ca2+ influx we administered flufenamic acid (FFA), a non-steroidal anti-inflammatory drug which is also a non-selective blocker of the transient receptor potential canonical channels (TRPCs), for 4 weeks to determine if this would have a beneficial effect. FFA treatment reduced both basal ROS production and muscle [Ca2+]i and [Na+]i in middle-aged and aged muscle fibers compared to fibers and muscles of untreated 12 and 24-months old mice. [Ca2+]i was reduced to 134 ± 8 nM in middle-aged muscle and to 246 ± 40 nM in muscle from aged mice. Likewise [Na+]i was reduced to 9 ± 0.7 mM in middle-aged muscles and to 13 ± 1 mM in muscle from aged mice. FFA treatment also reduced age associated increases in plasma interleukin 6 and tumor necrosis factor-alpha (TNF-α) concentrations which were elevated in 12 and 24-months old mice compared to young mice and decreased age-related muscle damage as indicated by a reduction in serum creatine kinase (CK) activity. Our data provides a direct demonstration that normal aging is associated with a significant elevation [Ca2+]i, [Na+]i, and intracellular ROS production in skeletal muscle fibers. Furthermore, the fact that FFA reduced the intracellular [Ca2+], [Na+], and ROS production as well as the elevated IL6, TNF-α, and CK levels, led us to suggest that its pharmacological effect may be related to its action both as a TRPC channel blocker and as an anti-inflammatory.

Topical sinonasal rinse therapies may alter the local microbiome and improve disease control in chronic rhinosinusitis (CRS). The objective of this study was to examine microbiome changes in post-surgical CRS patients when rinsing with commercially available products containing xylitol or Lactococcus lactis.

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 μM H2O2 increased NF-κB activity; after administration of 100 and 200 μM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 μM and 200 μM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

Chronic Chagas' disease cardiomyopathy is a leading cause of congestive heart failure in Latin America, affecting more than 3 million people. Chagas' cardiomyopathy is more aggressive than other cardiomyopathies, but little is known of the molecular mechanisms responsible for its severity. We characterized gene expression profiles of human Chagas' cardiomyopathy and dilated cardiomyopathy to identify selective disease pathways and potential therapeutic targets. Both our customized cDNA microarray (Cardiochip) and real-time reverse transcriptase-polymerase chain reaction analysis showed that immune response, lipid metabolism, and mitochondrial oxidative phosphorylation genes were selectively up-regulated in myocardial tissue of the tested Chagas' cardiomyopathy patients. Interferon (IFN)-gamma-inducible genes represented 15% of genes specifically up-regulated in Chagas' cardiomyopathy myocardial tissue, indicating the importance of IFN-gamma signaling. To assess whether IFN-gamma can directly modulate cardio-myocyte gene expression, we exposed fetal murine cardiomyocytes to IFN-gamma and the IFN-gamma-inducible chemokine monocyte chemoattractant protein-1. Atrial natriuretic factor expression increased 15-fold in response to IFN-gamma whereas combined IFN-gamma and monocyte chemoattractant protein-1 increased atrial natriuretic factor expression 400-fold. Our results suggest IFN-gamma and chemokine signaling may directly up-regulate cardiomyocyte expression of genes involved in pathological hypertrophy, which may lead to heart failure. IFN-gamma and other cytokine pathways may thus be novel therapeutic targets in Chagas' cardiomyopathy.

Approximately 15% of American adults report some degree of difficulty hearing in a noisy environment or have auditory filtering difficulties. There are objective clinical tests of auditory filtering, yet few tests exist for mouse models that do not rely on extensive training. We have used reflex modification audiometry (RMA) and developed exclusion criteria for the mouse model. This RMA based test makes use of the acoustic startle response (ASR) and the ability of prepulses to inhibit the ASR [i.e., prepulse inhibition (PPI)] to assess the mouse's ability to detect prepulse signals presented in quiet or embedded in masking noise. We have studied PPI behavior across four inbred mouse strains with normal cochlear function and developed pre-testing exclusion criteria and test/retest reliability measures. Moreover, because both the medial (MOC) and the lateral (LOC) olivocochlear efferent feedback systems have been proposed to improve auditory behavior performance, especially in noisy backgrounds, we have examined PPI abilities in mice (with their littermate controls) either lacking the MOC receptor subunit α9 nicotinic acetylcholine receptor [α9 nAChR (-/-)] or expressing an overactive receptor [Ld'T mutation in α9 nAChR KI], or lacking an LOC efferent neuropeptide, alpha calcitonin gene-related peptide [αCGRP (-/-)] only in the CNS. Because CGRP receptor formation has been shown to mature from juvenile to adult ages, we also studied if this maturation would be reflected in PPI behavioral responses in juvenile and adult (+/+) controls and in adult αCGRP (-/-) animals. We show that 50% PPI response thresholds (sound level with 50% correct responses) in quiet are decreased in the (-/-) α9 nAChR animals, and 50% PPI responses are increased for mice with an overactive receptor (α9 nAChR KI) and are increased in adult mice lacking αCGRP (-/-). However, in background noise, only mice lacking αCGRP exhibited increased 50% PPI response thresholds, as there were no significant differences between α9 nAChR adult mouse lines and their littermate controls. These findings suggest that MOC and LOC olivocochlear neurotransmission work in tandem to improve behavioral responses to sound. These experiments further pave the way for rapid behavioral hearing assessments in other mouse models.

Muscle contraction depends on tightly regulated Ca2+ release. Aberrant Ca2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca2+ leak initiates a cascade of events that cause precise redistribution of Ca2+ among the SR, cytoplasm, and mitochondria through altering the Ca2+ permeability of the transverse tubular system membrane. This redistribution of Ca2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.