Molecular basis of arrhythmic substrate in ageing murine peroxisome proliferator-activated receptor γ co-activator deficient hearts modelling mitochondrial dysfunction

Figures

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  Figure 1. WB analysis of atrial NaV1.5, Cx40 and Cx43 expression (A) Representative Western blots of NaV1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. (B) Expression levels of atrial NaV1.5 obtained by densitometric analysis. (C) Expression levels of atrial Cx40 obtained by densitometric analysis. (D) Expression levels of atrial Cx43 expression obtained by densitometric analysis. (E) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. In (B–D), red boxes indicate young mice and blue boxes indicate aged mice. Primary monoclonal rabbit anti-NaV1.5 antibodies used at dilution 1:500; polyclonal goat anti-Cx40 used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used in blots staining for Cx40 at dilution 1:15000. Donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for NaV1.5, Cx43 and GAPDH. Significant P-values obtained by post-hoc testing with Tukey’s HSD test are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental…

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  Figure 2. IF analysis of atrial Cx43 expression (A) Representative micrographs of Cx43 signal in stained atrial sections visualised at 40×. (B) Expression levels of atrial Cx43 as obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P-values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

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  Figure 3. WB analysis of ventricular NaV1.5 and Cx43 expression (A) Representative Western blots of NaV1.5, Cx40, Cx43 and the housekeeping protein GAPDH, used as loading control. (B) Expression levels of ventricular NaV1.5 obtained by densitometric analysis. (C) Expression levels of ventricular Cx43 obtained by densitometric analysis. In (B,C), red boxes indicate young mice and blue boxes indicate aged mice. (D) Control blots using lysed WT young atria or erythrocytes (RBC) together with the indicated primary and secondary antibodies. Primary monoclonal rabbit anti-NaV1.5 antibody used at dilution 1:500; polyclonal goat anti-Cx40 antibody used at dilution 1:500; polyclonal rabbit anti-Cx43 used at dilution 1:1000; polyclonal rabbit anti-GAPDH used at dilution 1:1000. Secondary donkey anti-goat IgG antibody used at dilution 1:15000 in blots staining for Cx40, and donkey anti-rabbit IgG antibody used at dilution 1:10000 in blots staining for NaV1.5, Cx43 and GAPDH. Significant P-values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Abbreviations: A, aged; N, number of biological replicates per experimental group; RBC, erythrocyte; WT-Y-V, wild type young ventricular.

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  Figure 4. IF analysis of ventricular Cx43 expression (A) Representative micrographs of Cx43 signal in stained ventricular sections visualised at 40×. (B) Expression levels of ventricular Cx43 obtained by histomorphometric grid analysis. In (B), red boxes indicate young mice and blue boxes indicate aged mice. Primary polyclonal rabbit anti-Cx43 antibody used at dilution 1:1000; secondary goat anti-rabbit IgG antibody used at dilution 1:250. Significant P-values obtained by post-hoc testing with Tukey’s HSD tests are indicated. Note that brightness and contrast have been adjusted to make pictures more legible in print.

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  Figure 5. Proposed mechanistic links among NaV1.5 channels, gap junctions and conduction velocity (A) Circuit diagram schematic of the classical cable theory model of longitudinal conduction across cardiomyocytes. INa refers to the inward Na+ current that is carried by NaV1.5 channels, the conductance of which is represented by the term gNa. ra refers to the longitudinal intercellular conductance along cardiomyocytes, which acts in the direction of AP propagation (dotted arrow). cm and rm refer to the capacitance and resistance of a quiescent region of adjacent membrane. In boxes, possible mechanisms that may reduce conduction velocity connected by arrow to the term of the cable equation which they affect. (B) The ra term can be further broken down into two resistance values lying in series: rj, representing the resistance at gap junctions, and rcyt, the resistance within a cardiomyocyte arising due to the cytoplasm.