Cardiac ryanodine receptors (RyR2s) play a critical role in excitation-contraction coupling, mediating the efflux of Ca2+ from the sarcoplasmic reticulum. In left ventricular dysfunction (LVD) and in heart failure, where Ca2+ handling is disrupted, there is considerable controversy over whether RyR2 protein expression is selectively altered. Using a rabbit coronary artery ligation model of LVD, alterations in RyR2 mRNA and protein levels were studied.
Prior to experiments, rabbits were humanely killed with a lethal injection of sodium pentobarbital (1 ml kg-1) and hearts rapidly removed. Subendocardial and subepicardial regions of the left ventricle were analysed 8 weeks after coronary artery ligation and these were directly compared with the same regions in hearts from sham-operated animals. Real-time quantitative Taqman PCR revealed a subendocardial/subepicardial gradient for RyR2 mRNA in both experimental groups, with a decrease in the gradient in samples from animals with LVD.
The mean fold-difference (± S.E.M.) in mRNA for RyR2 normalised to GAPDH was 0.35 ± 0.02 (endo, n = 6) and 0.55 ± 0.04 (epi, n = 6) in the sham-operated group, compared with 0.26 ± 0.02 (endo, n = 10) and 0.56 ± 0.02 (epi, n = 10) in the LVD group (ANOVA, P = 0.013, where P < 0.05 is significant). The decrease in gradient observed is due to a down-regulation of RyR2 message in the subendocardial region. Quantitative analysis of RyR2 protein levels revealed similar findings. Two independent methods were used to assess regional RyR2 protein levels. Method 1 used quantitative densitometric analysis from Western blots probed with a specific monoclonal antibody against RyR2. Normalised data revealed down-regulation in the subendocardium in LVD preparations (2.2 ± 0.36 (sham, n = 7) vs. 1.2 ± 0.34 (LVD, n = 7)) (P = 0.009). As with mRNA data, there was no significant change in RyR2 protein levels in subepicardium (3.56 ± 0.53 (sham, n = 7) vs. 3.66 ± 0.51 (LVD, n = 7). Method 2 used [3H]ryanodine binding to assess the amount of RyR protein in subepicardial and subendocardial LV homogenates. Again the same observation was noted. Specific ryanodine binding (pmol (mg protein)-1) was reduced in subendocardial preparations from the LVD group (0.18 ± 0.03 (LVD, n = 7) and 0.3 ± 0.04 (sham, n = 4), P = 0.018) with a small but non-significant decrease in subepicardium (0.39 ± 0.03 (LVD, n = 7) and 0.45 ± 0.05 (sham, n = 4)). This gave results similar to those obtained with the quantitative immunoblotting.
Taken together, these results reveal the following in this rabbit model of LVD. (i) Subendocardial RyR2 mRNA expression is reduced by ~25 % whereas subepicardial message is unchanged. (ii) Subendocardial RyR2 protein is reduced by ~40 % with no significant change in subepicardium. Similar data was obtained using two independent methods. (iii) A subendocardial/ subepicardial gradient exists in both sham and LVD groups. This is decreased to a similar extent in LVD for both RyR2 message and protein expression (~20 % for mRNA and 20-30 % for protein).
This study highlights the importance of regional analysis in assessing cardiac protein dysfunction and could reconcile some of the controversy surrounding this area.
This work was sponsored by the British Heart Foundation.