Introduction: Catecholamine hypo-responsiveness is a hallmark of sepsis, but its pathogenesis remains elusive. The adrenergic pathway, from catecholamine binding to the adrenergic receptor (AR) to the final physiological effect is a multistep process. Knowledge about protein expression and localisation of different constituents of the adrenergic signalling in sepsis, including adrenergic receptors themselves, is sparse.
Objectives: Using our well-characterised 72 h fluid-resuscitated rat model of faecal peritonitis (1), where accurate prognostication can be made as early as 6 h, we analysed expression and membrane localisation of the b2-AR protein in heart tissue at early (6 h) and late (24 h) timepoints.
Methods: Sepsis was induced in male Wistar rats by i.p. injection of faecal slurry under general anaesthesia. The animals were then recovered and i.v. fluid resuscitation was started 2 h after sepsis induction. Sham-operated rats (SH) were treated identically but did not receive the slurry. At 6 h, an echo-measured heart rate cut-off of 460 bpm was used to classify animals into predicted survivors (SR) and non-survivors (NSR). Rats were culled at either 6 h or 24 h with heart tissue collected and snap frozen in liquid nitrogen. To study b2-AR expression, either protein lysates of whole heart tissue or membrane fractions were prepared and analysed by Western Blot with antibodies against b2-AR and PFK or Caveolin 3. Specific signals were analysed in Image Studio Lite 5.2. Results are presented as mean ± SE and considered significant at p < 0.05 (Student’s t-test).
Results: The level of expression of b2-AR myocardial protein expression did not change in survivors but declined in non-survivors being significantly lower than survivors at 24 h (Table 1). Representative results of membrane localisation of b2-AR within different membrane fractions are presented in Figure 1. Lipid rafts were observed in only two of the 12 gradient fractions, namely fractions 4 and 5. This was confirmed by cholesterol distribution and caveolin 3 staining. In sham operated animals and in predicted survivors at 24h, b2-AR appeared strongly in fraction 5, associated with the lipid rafts. In predicted non-survivors, b2-AR was not detected in fractions 4 and 5 either at 6h or 24h post-sepsis.
Conclusion: More severe sepsis, with corresponding poor prognosis, is accompanied by increasing catecholamine hyporesponsiveness. One cause could be a decrease in adrenergic receptor protein expression. Localisation of the b2-AR within lipid rafts is also a prerequisite for their normal functioning. In predicted non-survivors myocardial b2-AR was dissociated from the lipid rafts. Further studies are necessary to identify strategies that restore adrenergic signalling in sepsis.
