Pasteurized akkermansia muciniphila improves irritable bowel syndrome-like symptoms and related behavioral disorders in mice.

Gut - brain communications disorders in irritable bowel syndrome (IBS) are associated with intestinal microbiota composition, increased gut permeability, and psychosocial disturbances. Symptoms of IBS are difficult to medicate, and hence much research is being made into alternative approaches. This study assesses the potential of a treatment with pasteurized Akkermansia muciniphila for alleviating IBS-like symptoms in two mouse models of IBS with different etiologies. Two clinically relevant animal models were used to mimic IBS-like symptoms in C57BL6/J mice: the neonatal maternal separation (NMS) paradigm and the Citrobacter rodentium infection model. In both models, gut permeability, colonic sensitivity, fecal microbiota composition and colonic IL-22 expression were evaluated. The cognitive performance and emotional state of the animals were also assessed by several tests in the C. rodentium infection model. The neuromodulation ability of pasteurized A. muciniphila was assessed on primary neuronal cells from mice dorsal root ganglia using a ratiometric calcium imaging approach. The administration of pasteurized A. muciniphila significantly reduced colonic hypersensitivity in both IBS mouse models, accompanied by a reinforcement of the intestinal barrier function. Beneficial effects of pasteurized A. muciniphila treatment have also been observed on anxiety-like behavior and memory defects in the C. rodentium infection model. Finally, a neuroinhibitory effect exerted by pasteurized A. muciniphila was observed on neuronal cells stimulated with two algogenic substances such as capsaicin and inflammatory soup. Our findings demonstrate novel anti-hyperalgesic and neuroinhibitory properties of pasteurized A. muciniphila, which therefore may have beneficial effects in relieving pain and anxiety in subjects with IBS.

Insights into FcγR involvement in pain-like behavior induced by an RA-derived anti-modified protein autoantibody.

Joint pain is one of the most debilitating symptoms of rheumatoid arthritis (RA) and patients frequently rate improvements in pain management as their priority. RA is hallmarked by the presence of anti-modified protein autoantibodies (AMPA) against post-translationally modified citrullinated, carbamylated and acetylated proteins. It has been suggested that autoantibody-mediated processes represent distinct mechanisms contributing to pain in RA. In this study, we investigated the pronociceptive properties of monoclonal AMPA 1325:01B09 (B09 mAb) derived from the plasma cell of an RA patient. We found that B09 mAb induces pain-like behavior in mice that is not associated with any visual, histological or transcriptional signs of inflammation in the joints, and not alleviated by non-steroidal anti-inflammatory drugs (NSAIDs). Instead, we found that B09 mAb is retained in dorsal root ganglia (DRG) and alters the expression of several satellite glia cell (SGC), neuron and macrophage-related factors in DRGs. Using mice that lack activating FcγRs, we uncovered that FcγRs are critical for the development of B09-induced pain-like behavior, and partially drive the transcriptional changes in the DRGs. Finally, we observed that B09 mAb binds SGC in vitro and in combination with external stimuli like ATP enhances transcriptional changes and protein release of pronociceptive factors from SGCs. We propose that certain RA antibodies bind epitopes in the DRG, here on SGCs, form immune complexes and activate resident macrophages via FcγR cross-linking. Our work supports the growing notion that autoantibodies can alter nociceptor signaling via mechanisms that are at large independent of local inflammatory processes in the joint.

Involvement of toll-like receptor 5 in mouse model of colonic hypersensitivity induced by neonatal maternal separation.

BACKGROUND: Chronic abdominal pain is the most common cause for gastroenterology consultation and is frequently associated with functional gastrointestinal disorders including irritable bowel syndrome and inflammatory bowel disease. These disorders present similar brain/gut/microbiota trialogue alterations, associated with abnormal intestinal permeability, intestinal dysbiosis and colonic hypersensitivity (CHS). Intestinal dysbiosis can alter colon homeostasis leading to abnormal activation of the innate immunity that promotes CHS, perhaps involving the toll-like receptors (TLRs), which play a central role in innate immunity. AIM: To understand the mechanisms between early life event paradigm on intestinal permeability, fecal microbiota composition and CHS development in mice with TLRs expression in colonocytes. METHODS: Maternal separation model (NMS) CHS model, which mimics deleterious events in childhood that can induce a wide range of chronic disorders during adulthood were used. Colonic sensitivity of NMS mice was evaluated by colorectal distension (CRD) coupled with intracolonic pressure variation (IPV) measurement. Fecal microbiota composition was analyzed by 16S rRNA sequencing from weaning to CRD periods. TLR mRNA expression was evaluated in colonocytes. Additionally, the effect of acute intrarectal instillation of the TLR5 agonist flagellin (FliC) on CHS in adult naive wildtype mice was analyzed. RESULTS: Around 50% of NMS mice exhibited increased intestinal permeability and CHS associated with intestinal dysbiosis, characterized by a significant decrease of species richness, an alteration of the core fecal microbiota and a specific increased relative abundance of flagellated bacteria. Only TLR5 mRNA expression was increased in colonocytes of NMS mice with CHS. Acute intrarectal instillation of FliC induced transient increase of IPV, reflecting transient CHS appearance. CONCLUSION: Altogether, these data suggest a pathophysiological continuum between intestinal dysbiosis and CHS, with a role for TLR5.

Single Subcutaneous Injection of Lysophosphatidyl-Choline Evokes ASIC3-Dependent Increases of Spinal Dorsal Horn Neuron Activity.

Lysophosphatidyl-choline (LPC), a member of the phospholipid family, is an emerging player in pain. It is known to modulate different pain-related ion channels, including Acid-Sensing Ion Channel 3 (ASIC3), a cationic channel mainly expressed in peripheral sensory neurons. LPC potentiates ASIC3 current evoked by mild acidifications, but can also activate the channel at physiological pH. Very recently, LPC has been associated to chronic pain in patients suffering from fibromyalgia or osteoarthritis. Accordingly, repetitive injections of LPC within mouse muscle or joint generate both persistent pain-like and anxiety-like behaviors in an ASIC3-dependent manner. LPC has also been reported to generate acute pain behaviors when injected intraplantarly in rodents. Here, we explore the mechanism of action of a single cutaneous injection of LPC by studying its effects on spinal dorsal horn neurons. We combine pharmacological, molecular and functional approaches including in vitro patch clamp recordings and in vivo recordings of spinal neuronal activity. We show that a single cutaneous injection of LPC exclusively affects the nociceptive pathway, inducing an ASIC3-dependent sensitization of nociceptive fibers that leads to hyperexcitabilities of both high threshold (HT) and wide dynamic range (WDR) spinal neurons. ASIC3 is involved in LPC-induced increase of WDR neuron's windup as well as in WDR and HT neuron's mechanical hypersensitivity, and it participates, together with TRPV1, to HT neuron's thermal hypersensitivity. The nociceptive input induced by a single LPC cutaneous rather induces short-term sensitization, contrary to previously described injections in muscle and joint. If the effects of peripheral LPC on nociceptive pathways appear to mainly depend on peripheral ASIC3 channels, their consequences on pain may also depend on the tissue injected. Our findings contribute to a better understanding of the nociceptive signaling pathway activated by peripheral LPC via ASIC3 channels, which is an important step regarding the ASIC3-dependent roles of this phospholipid in acute and chronic pain conditions.

Lysophosphatidylcholine 16:0 mediates chronic joint pain associated to rheumatic diseases through acid-sensing ion channel 3.

ABSTRACT: Rheumatic diseases are often associated to debilitating chronic pain, which remains difficult to treat and requires new therapeutic strategies. We had previously identified lysophosphatidylcholine (LPC) in the synovial fluids from few patients and shown its effect as a positive modulator of acid-sensing ion channel 3 (ASIC3) able to induce acute cutaneous pain in rodents. However, the possible involvement of LPC in chronic joint pain remained completely unknown. Here, we show, from 2 independent cohorts of patients with painful rheumatic diseases, that the synovial fluid levels of LPC are significantly elevated, especially the LPC16:0 species, compared with postmortem control subjects. Moreover, LPC16:0 levels correlated with pain outcomes in a cohort of osteoarthritis patients. However, LPC16:0 do not appear to be the hallmark of a particular joint disease because similar levels are found in the synovial fluids of a second cohort of patients with various rheumatic diseases. The mechanism of action was next explored by developing a pathology-derived rodent model. Intra-articular injections of LPC16:0 is a triggering factor of chronic joint pain in both male and female mice, ultimately leading to persistent pain and anxiety-like behaviors. All these effects are dependent on ASIC3 channels, which drive sufficient peripheral inputs to generate spinal sensitization processes. This study brings evidences from mouse and human supporting a role for LPC16:0 via ASIC3 channels in chronic pain arising from joints, with potential implications for pain management in osteoarthritis and possibly across other rheumatic diseases.

Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis.

ABSTRACT: Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

Antibody-induced pain-like behavior and bone erosion: links to subclinical inflammation, osteoclast activity, and acid-sensing ion channel 3-dependent sensitization.

ABSTRACT: Several bone conditions, eg, bone cancer, osteoporosis, and rheumatoid arthritis (RA), are associated with a risk of developing persistent pain. Increased osteoclast activity is often the hallmark of these bony pathologies and not only leads to bone remodeling but is also a source of pronociceptive factors that sensitize the bone-innervating nociceptors. Although historically bone loss in RA has been believed to be a consequence of inflammation, both bone erosion and pain can occur years before the symptom onset. Here, we have addressed the disconnection between inflammation, pain, and bone erosion by using a combination of 2 monoclonal antibodies isolated from B cells of patients with RA. We have found that mice injected with B02/B09 monoclonal antibodies (mAbs) developed a long-lasting mechanical hypersensitivity that was accompanied by bone erosion in the absence of joint edema or synovitis. Intriguingly, we have noted a lack of analgesic effect of naproxen and a moderate elevation of few inflammatory factors in the ankle joints suggesting that B02/B09-induced pain-like behavior does not depend on inflammatory processes. By contrast, we found that inhibiting osteoclast activity and acid-sensing ion channel 3 signaling prevented the development of B02/B09-mediated mechanical hypersensitivity. Moreover, we have identified secretory phospholipase A2 and lysophosphatidylcholine 16:0 as critical components of B02/B09-induced pain-like behavior and shown that treatment with a secretory phospholipase A2 inhibitor reversed B02/B09-induced mechanical hypersensitivity and bone erosion. Taken together, our study suggests a potential link between bone erosion and pain in a state of subclinical inflammation and offers a step forward in understanding the mechanisms of bone pain in diseases such as RA.

The Class I HDAC Inhibitor, MS-275, Prevents Oxaliplatin-Induced Chronic Neuropathy and Potentiates Its Antiproliferative Activity in Mice.

Oxaliplatin, the first-line chemotherapeutic agent against colorectal cancer (CRC), induces peripheral neuropathies, which can lead to dose limitation and treatment discontinuation. Downregulation of potassium channels, which involves histone deacetylase (HDAC) activity, has been identified as an important tuner of acute oxaliplatin-induced hypersensitivity. MS-275, a class I histone deacetylase inhibitor (HDACi), prevents acute oxaliplatin-induced peripheral neuropathy (OIPN). Moreover, MS-275 exerts anti-tumor activity in several types of cancers, including CRC. We thus hypothesized that MS-275 could exert both a preventive effect against OIPN and potentially a synergistic effect combined with oxaliplatin against CRC development. We first used RNAseq to assess transcriptional changes occurring in DRG neurons from mice treated by repeated injection of oxaliplatin. Moreover, we assessed the effects of MS-275 on chronic oxaliplatin-induced peripheral neuropathy development in vivo on APCMin/+ mice and on cancer progression when combined with oxaliplatin, both in vivo on APCMin/+ mice and in a mouse model of an orthotopic allograft of the CT26 cell line as well as in vitro in T84 and HT29 human CRC cell lines. We found 741 differentially expressed genes (DEGs) between oxaliplatin- and vehicle-treated animals. While acute OIPN is known as a channelopathy involving HDAC activity, chronic OIPN exerts weak ion channel transcriptional changes and no HDAC expression changes in peripheral neurons from OIPN mice. However, MS-275 prevents the development of sensory neuropathic symptoms induced by repeated oxaliplatin administration in APCMin/+ mice. Moreover, combined with oxaliplatin, MS-275 also exerts synergistic antiproliferative and increased survival effects in CT26-bearing mice. Consistently, combined drug associations exert synergic apoptotic and cell death effects in both T84 and HT29 human CRC cell lines. Our results strongly suggest combining oxaliplatin and MS-275 administration in CRC patients in order to potentiate the antiproliferative action of chemotherapy, while preventing its neurotoxic effect.

A physicochemical model of reaction kinetics supports peroxyl radical recombination as the main determinant of the FLASH effect.

BACKGROUND AND PURPOSE: FLASH radiotherapy, a technique based on delivering large doses in a single fraction at the micro/millisecond timescale, spares normal tissues from late radiation-induced toxicity, in an oxygen-dependent process, whilst keeping full anti-tumor efficiency. We present a theoretical model taking into account the kinetics of formation and decay of reactive oxygen species, in particular of organic peroxyl radicals ROO. formed by addition of O2 to primary carbon-centred radicals R. and known to play a major role at the origin radio-induced complications. MATERIALS AND METHODS: The model focuses on the time-dependent evolution of radiolytic products in living matter exposed to continuous irradiation at dose-rates in the range 10-3-107Gy·s-1. The 9 differential rate equations resulting from the radiolytic and enzymatic reactions network were solved using the published values of these reactions rate constants in a cellular environment. RESULTS: The model suggests a correlation between the area-under-the-curve of time-evolving [ROO.] and the probability of normal tissue complications. The model does not lend weight to the hypothesis of transient oxygen depletion as a main determinant of FLASH but rather suggests a major role of radical-radical recombination. CONCLUSION: The model gives support to the reduction of ROO. lifetime as the main root of FLASH and compares favorably with published experimental results. We conclude that any process - in this case radical recombination - that shortens the lifetime or limits the radiolytic yield of ROO. is likely to protect normoxic tissues against the deleterious effects of radiation.

Fecal dysbiosis associated with colonic hypersensitivity and behavioral alterations in chronically Blastocystis-infected rats.

BACKGROUND: Infectious gastroenteritis is a risk factor for the development of post-infectious Irritable Bowel Syndrome (PI-IBS). Recent clinical studies reported a higher prevalence of the intestinal parasite Blastocystis in IBS patients. Using a rat model, we investigated the possible association between Blastocystis infection, colonic hypersensitivity (CHS), behavioral disturbances and gut microbiota changes. METHODS: Rats were orally infected with Blastocystis subtype 4 (ST4) cysts, isolated from human stool samples. Colonic sensitivity was assessed by colorectal distension and animal behavior with an automatic behavior recognition system (PhenoTyper), the Elevated Plus Maze test and the Forced Swimming tests. Feces were collected at different time points after infection to study microbiota composition by 16 S rRNA amplicon sequencing and for short-chain fatty acid (SFCA) analysis. RESULTS: Blastocystis-infected animals had non-inflammatory CHS with increased serine protease activity. Infection was also associated with anxiety- and depressive-like behaviors. Analysis of fecal microbiota composition showed an increase in bacterial richness associated with altered microbiota composition. These changes included an increase in the relative abundance of Oscillospira and a decrease in Clostridium, which seem to be associated with lower levels of SCFAs in the feces from infected rats. CONCLUSIONS: Our findings suggest that experimental infection of rats with Blastocystis mimics IBS symptoms with the establishment of CHS related to microbiota and metabolic shifts.

c-Jun/p38MAPK/ASIC3 pathways specifically activated by nerve growth factor through TrkA are crucial for mechanical allodynia development.

Mechanical allodynia is a cardinal sign of several inflammatory pain disorders where nerve growth factor, a prototypic neurotrophin, plays a crucial role by binding to TrkA receptors. Here, we took the advantage of our generated knock-in mouse model expressing a chimeric TrkA/TrkC receptor that seems to not specifically develop mechanical allodynia after inflammation, to identify the TrkA downstream pathways involved in this phenomenon. We confirmed and extended that disrupting TrkA-specific pathways leads to a specific deficit in mechanical hypersensitivity development after somatic (systemic nerve growth factor administration and paw incision) and, to a lesser extent, visceral injuries. Despite a deficit in thin, mainly peptidergic, fibre innervation in TrkAC mice, thermal hyperalgesia development was not different from WT mice. Inflammatory reaction (oedema, IL-6 content), pain behaviours after intraplantar capsaicin, as well as TRPV1 calcium imaging response of dorsal root ganglion neurons were similar between TrkAC and WT mice. This deficiency in mechanical allodynia development in TrkAC mice is likely due to the alteration of the expression of different TrkA transduction pathways (ie, Akt, p38 MAPK, and c-Jun) especially p38 MAPK, in the dorsal root ganglion cell bodies, ultimately leading to an alteration of at least, ASIC3 channel overexpression, known to participate in nociceptor mechanosensory function.

Structural and functional alterations in the retrosplenial cortex following neuropathic pain.

Human and animal imaging studies demonstrated that chronic pain profoundly alters the structure and the functionality of several brain regions. In this article, we conducted a longitudinal and multimodal study to assess how chronic pain affects the brain. Using the spared nerve injury model which promotes both long-lasting mechanical and thermal allodynia/hyperalgesia but also pain-associated comorbidities, we showed that neuropathic pain deeply modified the intrinsic organization of the brain functional network 1 and 2 months after injury. We found that both functional metrics and connectivity of the part A of the retrosplenial granular cortex (RSgA) were significantly correlated with the development of neuropathic pain behaviours. In addition, we found that the functional RSgA connectivity to the subiculum and the prelimbic system are significantly increased in spared nerve injury animals and correlated with peripheral pain thresholds. These brain regions were previously linked to the development of comorbidities associated with neuropathic pain. Using a voxel-based morphometry approach, we showed that neuropathic pain induced a significant increase of the gray matter concentration within the RSgA, associated with a significant activation of both astrocytes and microglial cells. Together, functional and morphological imaging metrics of the RSgA could be used as a predictive biomarker of neuropathic pain.

Adherent-Invasive E. coli enhances colonic hypersensitivity and P2X receptors expression during post-infectious period.

Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.

Relationships between sports-specific characteristics of athlete's heart and maximal oxygen uptake.

BACKGROUND: Improvement to maximal oxygen uptake is mainly due to myocardial adaptations brought about by physical training. As a consequence, the athlete's heart echocardiographic modifications associated with these adaptations are already well-known. We studied the relationships between maximal oxygen uptake (ml/min) and resting echocardiographic patterns in three athlete groups. METHODS: Tumbling (n=16), canoeing (n=12), cycling (n=12) and untrained (n=19) participants performed clinical examination and an echocardiogram. Trained groups performed a maximal graded exercise test on a cycle ergometer with gas exchange analysis. RESULTS: Sport-specific cardiac hypertrophy was observed. No significant echocardiographic difference was noted between untrained and tumbling participants. Canoeists showed higher end-diastolic thickness of the interventricular septum (P<0.001) and left ventricle mass (P<0.05) than untrained and higher posterior wall thickness (P<0.001) and than untrained and tumbling participants. In comparison between untrained, tumbling and cycling participants, left ventricular end-diastolic diameter (P<0.001) and left ventricular mass (P<0.001) was higher in cyclists. In trained subjects studied as a global group, the main linear correlation with maximal oxygen uptake concerned left ventricular end-diastolic diameter (r=0.92; P<0.001), left ventricular mass (r=0.60; P<0.001) and to a lesser extent aortic (r=0.39; P<0.01) and left atrium (r=0.36; P<0.05) diameters and E (r=0.38; P<0.05) and A (r=-0.33; P<0.05) Doppler peak velocities. Each trained group showed specific correlations between echocardiographic parameters and absolute maximal oxygen uptake. No further correlation was noted with left ventricular end-diastolic diameter or left ventricle mass when each group was studied individually. CONCLUSIONS: In athletes, maximal oxygen uptake is partly linked to some resting echocardiographic parameters. Specific relationships between maximal oxygen uptake and some echocardiographic parameters in relation to the sport practised are also observed.

The effects of exercise training on myocardial adrenergic and muscarinic receptors.

We investigated the effects of exercise training on heart rate variability (HRV) and myocardial adrenergic and muscarinic receptors in rats. Exercise training induced a decrease in body mass while ventricular size remained unchanged, a development we considered as a relative cardiac hypertrophy. In addition, there was a reduction in the density of myocardial beta(1)-adrenergic receptors. These structural changes were associated with functional adaptations, as illustrated by the increased response of the sinus node to sympathetic blockade.

Effect of training on beta1 beta2 beta3 adrenergic and M2 muscarinic receptors in rat heart.

OBJECTIVE: Physical training is known to alter several cardiovascular parameters. These adaptations are for a great part linked to an alteration of the myocardial responses to its autonomic nervous regulation. To further explain the parasympathetic and catecholamine effects, we hypothesized that endurance training could modify rat myocardial beta1, beta2, beta3 adrenoreceptors (AR) and M2 muscarinic cholinergic receptor (AchR) densities. METHODS: Two groups of adults female Wistar rats were studied: controls (C) (N = 7) and trained (T) (N = 9). An 8-wk treadmill training protocol was performed, 5 d x wk and of 1 h x d. At the end of the training session, left ventricle and atria muscle were isolated and weighed. Then, quantification of beta1, beta2, beta3 AR and M2 AchR was performed using Western blot analysis. RESULTS: M2 AchR densities were not modified in left ventricle or in atria by training (respectively, 100 +/- 22%, C vs 101 +/- 14%, T and 100 +/- 23%, C vs 119 +/- 30%, T). Concerning the left ventricle beta AR isoforms, beta1AR density was decreased in T (80 +/- 10% T vs 100 +/- 14% C, P = 0.01), beta2AR was unaltered (102 +/- 12%, T vs 100 +/- 17%, C), and beta3 AR density was increased in T (139 +/- 38% T vs 100 +/- 15% C; P < 0.05). CONCLUSIONS: Our results show for the first time that in female rats an 8-wk treadmill training protocol alters specifically the left ventricle beta AR isoforms densities but not the M2 AchR one. These results could explain some of the beneficial cardiovascular adaptations of the physically trained heart.