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Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
Published on: July 3, 2013
Effects of A1 adenosine receptor stimulation on the expression of genes involved in calcium homeostasis
Sandra Ghelardoni1, Vittoria Carnicelli, Sabina Frascarelli
1Dipartimento di Scienze dell'Uomo e dell'Ambiente, Sezione di Biochimica, University of Pisa, via Roma 55, I-56126 Pisa, Italy.
Abstract:
We investigated whether A(1) adenosine receptor stimulation affects expression of genes involved in calcium homeostasis, including sarcolemmal L-type Ca(2+) channel, Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, phospholamban, or ryanodine receptor. Three models of A(1) stimulation were used: i) an acute model, i.e. isolated perfused rat hearts treated for 120 min with 15 nM R-phenylisopropyladenosine (R-PIA), an A(1) receptor agonist; ii) a subacute model, i.e. rats treated with 1.5 mg/kg R-PIA e.v. and sacrificed after 24 h; iii) a transgenic model, i.e. mice overexpressing A(1) adenosine receptors. In all models gene expression was determined by RT-PCR, and oxalate-supported Ca(2+) uptake, representing SR Ca(2+) uptake, was measured in the crude homogenate. Significant increase in the expression of the phospholamban gene was observed in each model of A(1) stimulation, while the expression of the other four genes was not significantly modified. In the acute model, SR Ca(2+) uptake was unaffected, however in the subacute and transgenic models uptake rate was significantly reduced. In parallel experiments, hearts obtained from the subacute model demonstrated a significant reduction in irreversible tissue injury from 30 min of ischemia and 120 min of reperfusion. Increased resistance to ischemia has already been reported also in our transgenic model. In conclusion, A(1) adenosine receptor stimulation up-regulates phospholamban gene expression, which leads within 24 h to a reduced rate of SR Ca(2+) uptake. Changes in Ca(2+) homeostasis might contribute to the delayed cardioprotective effect of adenosine.
Insights
Adenosine receptor A(1) stimulation increases phospholamban gene expression. This leads to reduced calcium uptake in the sarcoplasmic reticulum and offers cardioprotection against ischemia and reperfusion injury.
Area of Science:
- Cardiovascular Physiology
- Molecular Biology
- Pharmacology
Background:
- Adenosine receptors play crucial roles in cardiovascular function.
- Calcium homeostasis is vital for cardiac contractility and survival.
- Understanding adenosine's effects on cardiac calcium handling is key to developing cardioprotective strategies.
Purpose of the Study:
- To investigate the impact of A(1) adenosine receptor stimulation on genes regulating cardiac calcium homeostasis.
- To determine if A(1) receptor activation influences sarcoplasmic reticulum (SR) Ca(2+) uptake.
- To explore the link between altered calcium handling and adenosine-mediated cardioprotection.
Main Methods:
- Utilized three models of A(1) adenosine receptor stimulation: acute (R-PIA in isolated hearts), subacute (R-PIA in rats), and transgenic (overexpressing A(1) receptors in mice).
- Assessed gene expression of key calcium-handling proteins (L-type Ca(2+) channel, Na(+)/Ca(2+) exchanger, SR Ca(2+)-ATPase, phospholamban, ryanodine receptor) via RT-PCR.
- Measured SR Ca(2+) uptake using oxalate-supported Ca(2+) uptake assays.
- Evaluated myocardial infarct size following ischemia-reperfusion injury.
Main Results:
- A(1) adenosine receptor stimulation consistently upregulated phospholamban gene expression across all models.
- No significant changes were observed in the expression of other investigated calcium-handling genes.
- SR Ca(2+) uptake was reduced in subacute and transgenic models, but not in the acute model.
- Hearts from the subacute model exhibited significantly reduced infarct size after ischemia-reperfusion.
- Transgenic hearts also showed increased resistance to ischemia.
Conclusions:
- A(1) adenosine receptor stimulation upregulates phospholamban gene expression.
- This upregulation leads to a decreased rate of SR Ca(2+) uptake within 24 hours.
- Altered calcium homeostasis likely contributes to the observed cardioprotective effects of adenosine, particularly against ischemia-reperfusion injury.
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