Original articleβ2-adrenergic receptors mediate cardioprotection through crosstalk with mitochondrial cell death pathways
Highlights
► β2-ARs protect against doxorubicin cardiotoxity via modulation of PKC and Akt. ► Doxorubicin decreases both εPKC and Akt activity in β2-/- mice but not in WT. ► β2-ARs attenuate mitochondrial dysfunction in doxorubicin cardiotoxicity. ► Complex I and II activities decrease only in doxorubicin treated β2-/- mice. ► Calcium blockade or MPT inhibition ameliorates doxorubicin toxicity in β2-/- mice.
Introduction
The two major cardiac β-adrenergic receptor (β-AR) subtypes, β1 and β2, modulate cardiac signaling through both parallel and opposing pathways. Over the past decade, our understanding of the function of these key receptors has increased beyond their classical role in regulating inotropy and chronotropy, and now includes the complex regulation of cardiac remodeling. Stimulation of β1-ARs can induce apoptosis through PKA-dependent [1] and independent (e.g. CaMKII) pathways [2]. In contrast, stimulation of β2-ARs can be anti-apoptotic, mediated through Gi, Gβγ, phosphatidylinositol 3′-kinase and Akt [3]. In addition, β2-ARs can also signal through ERK via a GRK5/6-β-arrestin-dependent pathway, independent of G protein coupling [4].
We have previously shown that β1-ARs play a cardiotoxic and that β2-ARs play a cardioprotective role in oxidative stress, using as a model the anthracycline anti-cancer drug doxorubicin (DOX) [5], [6]. β2-AR knockout (β2-/-) mice receiving a single therapeutic-level dose of DOX (which has no acute effect on WT or β1-/- mice) show markedly enhanced cardiotoxicity, with ECG, blood pressure and contractility changes within 2 min, and death within 30 min. Differential activation of MAPK isoforms was observed: p38 activity increased 20-fold only in the β2-/- and p38 inhibition partly rescued this phenotype. However, the exact mechanisms mediating the extremely rapid demise of the β2-/- mice and the additional pathways activated by β2-ARs to protect against anthracycline-induced oxidative stress are unknown.
In addition to the MAPKs, several other kinases play important roles in modulating cardiotoxicity/cardioprotection, including protein kinase C (PKC) and Akt. Of the 11 isozymes of PKC identified, opposing cardioprotective actions have been attributed to the novel subfamily members εPKC and δPKC. εPKC attenuates, whereas δPKC increases, ischemia/reperfusion injury [7]. Similar roles have been observed in both ethanol [8] and reactive oxygen species-induced cardioprotection [9], and may explain some of the cardioprotective effects of diazoxide [10]. Akt, a key regulator of cardiac growth and metabolism, also plays a central role in cardioprotection. Activation of Akt inhibits hypoxia-induced myocyte apoptosis [11] and plays a role in both ischemic preconditioning and postconditioning [12]. In DOX cardiotoxicity, Akt activation protects against myocyte apoptosis [13], attenuates heart failure [14] and may mediate the protective effects of dexrazoxane [15]. The interrelationship between these pro-survival kinases and β2-ARs in mediating protection against DOX cardiotoxicity is unknown.
A central intracellular target of DOX toxicity, and a point of convergence of signaling by the above kinases, is the mitochondria. The extremely rapid demise of the β2-/- mice when exposed to DOX makes apoptosis an unlikely mechanism, and we have found no evidence of either apoptosis or ischemia. Rapid cell death in this time frame can be mediated by opening of the mitochondrial permeability transition (MPT) pore in the inner mitochondrial membrane, causing reversal of the F0–F1 ATPase and collapse of the mitochondrial membrane potential [16]. Previous evidence implicates mitochondrial dysfunction in DOX toxicity. DOX is redox cycled through mitochondrial complex I NADH dehydrogenase [17], increasing the formation of free radical intermediates. Mitochondria are also targets for the [Ca2+]i dysregulation and bioenergetic failure that are hallmarks of DOX cardiotoxicity [17]. However, how signaling mediated through a cell surface G-protein coupled receptor (GPCR), such as the β2-AR, can so profoundly affect these mitochondrial cell death pathways is unknown.
In this study, we sought to determine the mechanisms by which a cell surface GPCR like the β2-AR can crosstalk with mitochondrial cell death pathways to mediate cardiotoxicity/cardioprotection. We used DOX cardiotoxicity as a well established and highly reproducible model of oxidative stress. We hypothesized that absence of β2-AR signaling would lead to subtle alterations in pro-survival kinase signaling and in [Ca2+]i regulation. These alterations would be below the threshold for altering baseline cardiac physiology. However, when exposed to an oxidative stressor such as DOX, these alterations would combine to predispose to opening of the MPT and subsequent cell demise.
Section snippets
Doxorubicin cardiotoxicity
DOX was used as a model of oxidative stress and has been well established in the mouse [6], [18], [19]. 3 month old male WT and β2-/- mice, both on a congenic FVB background, were injected with a single dose of DOX (NovaPlus, Bedford, OH) (200–300 μl) via the dorsal tail vein. Three different doses were administered in order to maximize the effect of the inhibitors tested: 8 mg/kg (LD50 dose), 10 mg/kg and 15 mg/kg. These doses are within the normal range of dosing for patients with malignancies,
β2-ARs mediate cardioprotection through differential regulation of PKC isozymes
To gain insight into whether β2-ARs mediate their protective effects trough crosstalk with specific PKC isozymes, levels of εPKC and δPKC and their translocation from soluble to particulate fractions were assessed at baseline and 20 min. after DOX (15 mg/kg) administration to WT and β2-/- mice.
At baseline there was no difference in εPKC levels in either soluble or particulate fraction between WT and β2-/-, however baseline δPKC was increased slightly (16.0 ± 4.6%) in the particulate fraction in
Discussion
Our results suggest that β2-ARs mediate their cardioprotective effects in oxidative stress in part through crosstalk with mitochondrial cell death pathways. Anthracycline cardiotoxicity was used as a model of oxidative stress to dissect the mechanisms by which a cell surface GPCR receptor such as the β2-AR can influence mitochondrial signaling. Although the exact mechanism of DOX cardiotoxicity is still incompletely defined, the mitochondria are a prime target for DOX action. Cardiomyocytes may
Funding
This work was supported by the National Institutes of Health Grant HL061535 to D.B.
Conflict of interest statement
None.
Acknowledgments
We are grateful to Dr. Joel Karliner, Dr. Gary Cecchini, Dr. Elena Maklashina and Dr. Conrad Alano at UCSF/San Francisco Veteran's Hospital and Dr. Hannes Vogel at Stanford for their advice.
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