Additional file 1: Figure S1. of Impaired Ca2+ release contributes to muscle weakness in a rat model of critical illness myopathy

Coomassie protein staining. Membranes used for western blot analysis of Na+ channels, DHPR (left panel), RyR1and SERCA1 (right panel). Lanes are marked as SHAM (S) or ICU (I); the value 110 kDa refers to the protein marker size in the ladder lane (PDF 5931 kb

Additional file 2: Figure S2. of Impaired Ca2+ release contributes to muscle weakness in a rat model of critical illness myopathy

Typical examples of the negative controls obtained for Na+ channels, DHPR, RyR and SERCA1 immunohistochemical staining (PDF 871 kb

The antioxidant NAC has no effect on the β-adrenergic stimulated SR Ca²⁺ release and contractility in cardiomyocytes from mice with the metabolic syndrome.

<p><b>(A)</b> Total body weight of mice after 8–10 weeks on control diet (Ctrl, n = 34) or high fat diet (HFD, n = 40). <b>(B)</b> Total body fat measured with DXA whole-body scan (n = 9–10). <b>(C)</b> Typical example of cross-sectional ORO staining showing fat accumulation (stained red) in left ventricles and mean data of ORO staining; eight sections per left ventricle from each group were analyzed (n = 3). Representative [Ca²⁺]_i transients from ctrl <b>(D)</b>, HFD <b>(E)</b> and <i>ob/ob</i> <b>(F)</b> cardiomyocytes obtained in the absence (full lines) and presence (dashed lines) of ISO (100 nM), and without (black lines) and with (red lines) NAC (5 mM). Average amplitude of Ca²⁺ transients <b>(G)</b>, fractional cell shortening (FS, <b><i>H</i></b>) and [Ca²⁺]_i transient decay time constant (tau) <b>(I)</b> with ISO and/or NAC as indicated from control (white bars), HFD (black bars) and <i>ob/ob</i> (grey bars) cardiomyocytes (n>16 cells from at least three mice). Representative Western blots <b>(J)</b> and mean data (n = 6) of ISO-induced PLB phosphorylation normalized to total PLB expression in left ventricles from control and HFD mice. Data are mean ± SEM; **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

Cardiomyocytes from mice with the metabolic syndrome show normal β₁-AR, β₂-AR expression and distribution.

<p><b>(A)</b> Representative Western blots and mean data ± SEM (<b><i>B</i></b>; n = 6) of total expression of β₁- and β₂-ARs in left ventricles from control mice and HFD mice. Immunofluorescence staining of β₁-AR <b>(C)</b> and β₂-AR <b>(D)</b> co-stained with RyR2 in control and HFD cardiomyocytes. Merged (yellow) show the intensity overlap between β-ARs and RyR2 in the dyads. <b><i>E</i></b> and <b><i>F</i></b> show plotted intensity profiles of along the dashed lines in <i>C</i> and <i>D</i> with β-AR (red) and RyR2 (green).</p

The ISO-induced increases in L-type Ca²⁺ current density is ROS-independent in cardiomyocytes with the metabolic syndrome.

<p>Mean data (±SEM; n = 6–8 in each group) of <i>I-V</i> curves of peak current density in the absence and presence of ISO (100nM) and NAC (20 mM) as indicated from control <b>(A, D)</b>, HFD <b>(B, E)</b> and <i>ob/ob</i> <b>(C, F)</b> cardiomyocytes. The <i>I-V</i> relationships were obtained by giving test pulses varying from -80 mV to +50 mV from a holding potential of -80 mV. The mean basal current density (i.e. in the absence of NAC and ISO) was -6.1±0.5 pA/pF and for comparisons between groups each group were normalized its basal current to give a relative current density (relative pA/pF).</p