Physiological Modification of the Contractile Force of Isolated Right Ventricular Papillary Muscles.

<p><b>(A) Frequency-dependent activation</b>; Isometric developed force values are expressed as a fraction of its corresponding value at the basal frequency of 4 Hz and presented as mean ± SEM, and (<b>B</b>) <b>β–adrenergic stimulation</b>; Isometric developed force values are expressed as a fraction of its corresponding value at the basal frequency of 4 Hz before isoproterenol addition and presented as mean ± SEM. Control; n = 12, Thyroxin (T4); n = 15, Dimethyl sulfoxide (DMSO); N = 10, Sorafenib; n = 9, Tadalafil_IP (intraperitoneal, 1 mg/kg); n = 10, Tadalafil_Or (oral, 4 mg/kg); n = 8, carboxymethylcellulose (CMC); n = 8, Macitentan_LD (Low dose: 30 mg/kg); n = 8, Macitentan_HD (High dose: 100 mg/kg); n = 7. Note: in the β–adrenergic stimulation curve (<b>B</b>), all isometric developed force values at which the muscles exhibited an arrhythmic behavior were excluded from the analysis. For example, the Macitentan_HD group has no representative point at isoproterenol concentration of 1 μM, because all muscles became arrhythmic at this concentration [i.e. 7 out of 7 (100%)]. (<b>C</b>) <b>Development of Arrhythmia</b>: % of arrhythmic muscles at different isoproterenol (Iso) concentrations. The absence of the representative bar of any group at any Iso concentration on the curve means the absence of arrhythmia at this concentration. *: indicates a significant change as revealed by one-way ANOVA followed by Dunnett Multiple Comparisons post-hoc test, comparing all groups to T4. +: indicates a significant change as revealed by two-way ANOVA.</p

The Effect of Sorafenib, Tadalafil and Macitentan Treatments on Thyroxin-Induced Hemodynamic Changes and Cardiac Abnormalities

<div><p>Multikinase inhibitors (e.g. Sorafenib), phosphodiesterase-5 inhibitors (e.g. Tadalafil), and endothelin-1 receptor blockers (e.g. Macitentan) exert influential protection in a variety of animal models of cardiomyopathy; however, their effects on thyroxin-induced cardiomyopathy have never been investigated. The goal of the present study was to assess the functional impact of these drugs on thyroxin-induced hemodynamic changes, cardiac hypertrophy and associated altered responses of the contractile myocardium both <i>in-vivo</i> at the whole heart level and <i>ex-vivo</i> at the cardiac tissue level. Control and thyroxin (500 μg/kg/day)-treated mice with or without 2-week treatments of sorafenib (10 mg/kg/day; I.P), tadalafil (1 mg/kg/day; I.P or 4 mg/kg/day; oral), macitentan (30 and 100 mg/kg/day; oral), and their vehicles were studied. Blood pressure, echocardiography and electrocardiogram were non-invasively evaluated, followed by <i>ex-vivo</i> assessments of isolated multicellular cardiac preparations. Thyroxin increased blood pressure, resulted in cardiac hypertrophy and left ventricular dysfunction <i>in-vivo</i>. Also, it caused contractile abnormalities in right ventricular papillary muscles <i>ex-vivo</i>. None of the drug treatments were able to significantly attenuate theses hemodynamic changes or cardiac abnormalities in thyroxin-treated mice. We show here for the first time that multikinase (raf1/b, VEGFR, PDGFR), phosphodiesterase-5, and endothelin-1 pathways have no major role in thyroxin-induced hemodynamic changes and cardiac abnormalities. In particular, our data show that the involvement of endothelin-1 pathway in thyroxine-induced cardiac hypertrophy/dysfunction seems to be model-dependent and should be carefully interpreted.</p></div

Contractile Profile of Isolated Right Ventricular Papillary Muscles.

<p>Contractile Profile of Isolated Right Ventricular Papillary Muscles.</p

Physiological Modification of the Contractile Force of Isolated Right Ventricular Papillary Muscles.

<p><b>(A) Frequency-dependent activation</b>; Isometric developed force values are expressed as a fraction of its corresponding value at the basal frequency of 4 Hz and presented as mean ± SEM, and (<b>B</b>) <b>β–adrenergic stimulation</b>; Isometric developed force values are expressed as a fraction of its corresponding value at the basal frequency of 4 Hz before isoproterenol addition and presented as mean ± SEM. Control; n = 12, Thyroxin (T4); n = 15, Dimethyl sulfoxide (DMSO); N = 10, Sorafenib; n = 9, Tadalafil_IP (intraperitoneal, 1 mg/kg); n = 10, Tadalafil_Or (oral, 4 mg/kg); n = 8, carboxymethylcellulose (CMC); n = 8, Macitentan_LD (Low dose: 30 mg/kg); n = 8, Macitentan_HD (High dose: 100 mg/kg); n = 7. Note: in the β–adrenergic stimulation curve (<b>B</b>), all isometric developed force values at which the muscles exhibited an arrhythmic behavior were excluded from the analysis. For example, the Macitentan_HD group has no representative point at isoproterenol concentration of 1 μM, because all muscles became arrhythmic at this concentration [i.e. 7 out of 7 (100%)]. (<b>C</b>) <b>Development of Arrhythmia</b>: % of arrhythmic muscles at different isoproterenol (Iso) concentrations. The absence of the representative bar of any group at any Iso concentration on the curve means the absence of arrhythmia at this concentration. *: indicates a significant change as revealed by one-way ANOVA followed by Dunnett Multiple Comparisons post-hoc test, comparing all groups to T4. +: indicates a significant change as revealed by two-way ANOVA.</p

Blood Pressure of Mice.

<p>Blood Pressure of Mice.</p

Morphological Data.

<p>Morphological Data.</p

Electrocardiogram Analysis of Mice.

<p>Electrocardiogram Analysis of Mice.</p

Additional file 3: Figure S2. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

H&E-stained histological sections of the gastrocnemius and quadriceps from 8-month-old mice of the indicated genotypes. Scale bar = 50 μm. The number of mice is 6–8 for each group

Additional file 4: Figure S3. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Masson’s trichrome-stained histological sections of the gastrocnemius and quadriceps from 8- to 18-month-old mice of the indicated genotypes. Scale bar = 50 μm. The number of mice is 6–8 for each group

Additional file 2: Figure S1. of Genetic disruption of Ano5 in mice does not recapitulate human ANO5-deficient muscular dystrophy

Test of anti-Ano5 antibodies by Western blotting. a The anti-Ano5 antibody (sc-169626) from Santa Cruz Biotechnology did not detect any positive signal in human Ano5-expressing cell lysate, which was correctly detected by the anti-GFP antibody at the predicted size. b The anti-Ano5 antibody (AP8580B) from Abgent produced a prominent band at about 100 kDa in both negative and positive samples; however, this band is not Ano5 because the Ano5-YFP fusion protein should be around 135 kDa as correctly detected by the anti-GFP antibody. The arrows point to the correct Ano5-YFP fusion protein. These experiments were repeated at least three times