Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy

IntroductionGrowth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD).MethodsHere, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.).ResultsIn vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-β1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD.DiscussionOur results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling

Blockers of Skeletal Muscle Nav1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back.

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure–activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activit

Blockers of Skeletal Muscle Na_v1.4 Channels: From Therapy of Myotonic Syndrome to Molecular Determinants of Pharmacological Action and Back

The voltage-gated sodium channels represent an important target for drug discovery since a large number of physiological processes are regulated by these channels. In several excitability disorders, including epilepsy, cardiac arrhythmias, chronic pain, and non-dystrophic myotonia, blockers of voltage-gated sodium channels are clinically used. Myotonia is a skeletal muscle condition characterized by the over-excitability of the sarcolemma, resulting in delayed relaxation after contraction and muscle stiffness. The therapeutic management of this disorder relies on mexiletine and other sodium channel blockers, which are not selective for the Nav1.4 skeletal muscle sodium channel isoform. Hence, the importance of deepening the knowledge of molecular requirements for developing more potent and use-dependent drugs acting on Nav1.4. Here, we review the available treatment options for non-dystrophic myotonia and the structure–activity relationship studies performed in our laboratory with a focus on new compounds with potential antimyotonic activity

Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy

Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X-linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild-type (WT) H2K-2B4 and the dystrophic (DYS) H2K-SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day-11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium-sensitive outward current compared to WT. Furthermore, day-11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.Ion channels have an active crosstalk with the dystrophin complex, which is disrupted in Duchenne muscular dystrophy, but little is known about their involvement in myogenesis, especially in dystrophic settings. We performed an electrophysiological characterization of wild-type and dystrophic myoblasts/myocytes to shed light on the relationship between dystrophin loss and ion channel activity during myogenesis with the final aim to identify new biomarkers and drug targets in regenerative medicine approaches. # imag

LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model

: The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies

DataSheet_1_Growth hormone secretagogues modulate inflammation and fibrosis in mdx mouse model of Duchenne muscular dystrophy.pdf

IntroductionGrowth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD).MethodsHere, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.).ResultsIn vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-β1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD.DiscussionOur results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.</p

Incidence, Risk Factors and Outcome of Pre-engraftment Gram-Negative Bacteremia after Allogeneic and Autologous Hematopoietic Stem Cell Transplantation: An Italian Prospective Multicenter Survey

Abstract BACKGROUND: Gram-negative bacteremia (GNB) is a major cause of illness and death after hematopoietic stem cell transplantation (HSCT), and updated epidemiological investigation is advisable. METHODS: We prospectively evaluated the epidemiology of pre-engraftment GNB in 1118 allogeneic HSCTs (allo-HSCTs) and 1625 autologous HSCTs (auto-HSCTs) among 54 transplant centers during 2014 (SIGNB-GITMO-AMCLI study). Using logistic regression methods. we identified risk factors for GNB and evaluated the impact of GNB on the 4-month overall-survival after transplant. RESULTS: The cumulative incidence of pre-engraftment GNB was 17.3% in allo-HSCT and 9% in auto-HSCT. Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were the most common isolates. By multivariate analysis, variables associated with GNB were a diagnosis of acute leukemia, a transplant from a HLA-mismatched donor and from cord blood, older age, and duration of severe neutropenia in allo-HSCT, and a diagnosis of lymphoma, older age, and no antibacterial prophylaxis in auto-HSCT. A pretransplant infection by a resistant pathogen was significantly associated with an increased risk of posttransplant infection by the same microorganism in allo-HSCT. Colonization by resistant gram-negative bacteria was significantly associated with an increased rate of infection by the same pathogen in both transplant procedures. GNB was independently associated with increased mortality at 4 months both in allo-HSCT (hazard ratio, 2.13; 95% confidence interval, 1.45-3.13; P <.001) and auto-HSCT (2.43; 1.22-4.84; P = .01). CONCLUSIONS: Pre-engraftment GNB is an independent factor associated with increased mortality rate at 4 months after auto-HSCT and allo-HSCT. Previous infectious history and colonization monitoring represent major indicators of GNB. CLINICAL TRIALS REGISTRATION: NCT02088840