Sex differences in sympathetic gene expression and cardiac neurochemistry in Wistar Kyoto rats

Fig 1. Female WKY rat hearts have higher neurotransmitter content than male hearts. A) Tissue collection schematic. Atria (shaded) were separated from the ventricles. The ventricles were cut into 4–5 mm sections highlighted by the dashed lines. Adapted from [28]. B) NE concentrations were higher in females than males in all areas of both the left and right ventricle. Mean±SEM, n = 6, *p<0.05, **p<0.01 female vs. male, 2-way ANOVA. Difference between regions of the ventricle, p<0.0001. C) Both NE and ACh were higher in the right atria of female rats compared to males, while the ratio of NE to ACh did not change. Mean±SEM, n = 6; (except Female RA n = 5) *p<0.05, **p<0.01 female vs male, unpaired t-test.

Fig 2. Multidimensional scaling plot of log-CPM values. MDS plot over 2 dimensions highlighting clear separation of sexes. F, Female Stellate (Red); M, Male Stellate (Blue). Samples were sequenced across 2 lanes (circles and squares). The distances on the plot approximate the typical log2 fold changes (Normalised CPM, counts per million) between the samples.

Fig 3. qPCR quantification of selected genes of interest in the stellate ganglion. Relative gene expression levels expressed as a ratio to gapdh expression in right stellate ganglion (Mean ± SEM, n = 6M vs 6F). There was strong agreement between RNA-seq and qPCR results for select genes of interest. *p<0.05, **p<0.01, ***p<0.001.

Fig 4. qPCR quantification of selected genes of interest in the SCG. Gene expression was assessed in the SCG as an alternative sympathetic ganglion. Relative gene expression levels expressed as a ratio to gapdh expression in right SCG (Mean ± SEM, n = 6M vs 6F). The gene expression levels of most genes were much higher relative to gapdh in the SCG compared to the stellate. The sex differences for Penk and Htr3b only were present in both the stellate and SCG. *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Tyrosine hydroxylase protein in left ventricle. (A) TH and GAPDH proteins were identified in the base (left) and apex (right) of the left ventricle by western blot and quantified via densitometry. Samples shown are base and apex from the same hearts. TH was normalized to GAPDH in the same sample. No significant differences were identified in either base or apex. (B) Average TH:GAPDH values for each heart (Mean ± SEM, n = 6/group).