Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility.

UNLABELLED: Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. METHODS: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-ß-cyclodextrin (MßCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. RESULTS: Treatment with MßCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MßCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. CONCLUSIONS: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility.

Adenosine receptor-mediated cardioprotection: are all 4 subtypes required or redundant?

Adenosine is a purine nucleoside, which is produced primarily through the metabolism of adenosine triphosphate (ATP), therefore its levels increase during stressful situations when ATP utilization increases. Adenosine exerts potent cardioprotective effects on the ischemic/reperfused heart, reducing reversible and irreversible myocardial injury. Adenosine receptors (ARs) are G-protein-coupled receptors, and 4 subtypes exist--A(1), A(2A), A(2B), and A(3), all of which have been shown to be cardioprotective. Adenosine receptors are expressed on multiple cardiac cells, including fibroblasts, endothelial cells, smooth muscle cells, and myocytes. Activation of both A(1) and A(3) receptors prior to ischemia has been shown in multiple experimental models to reduce ischemia/reperfusion-induced cardiac injury. Additionally, activation of the A(2A) receptor at the onset of reperfusion has been shown to reduce injury. Most recently, there is evidence that the A(2B) receptor has cardioprotective effects upon its activation. However, controversy remains regarding the precise timing of activation of these receptors required to induce cardioprotection, as well as their involvement in ischemic preconditioning and postconditioning. Adenosine receptors have been suggested to reduce cell death through actions at the mitochondrial ATP-dependent potassium (K(ATP)) channel, as well as protein kinase C and mitogen-activated protein kinase (MAPK) signaling. Additionally, the ability of ARs to interact has been documented, and several recent reports suggest that these interactions play a role in AR-mediated cardioprotection. This review summarizes the current knowledge of the cardioprotective effects of each AR subtype, as well as the proposed mechanisms of AR cardioprotection. Additionally, the role of AR interactions in cardioprotection is discussed.

Adenosine A2A and A2B receptors are both required for adenosine A1 receptor-mediated cardioprotection.

All four adenosine receptor subtypes have been shown to play a role in cardioprotection, and there is evidence that all four subtypes may be expressed in cardiomyocytes. There is also increasing evidence that optimal adenosine cardioprotection requires the activation of more than one receptor subtype. The purpose of this study was to determine whether adenosine A(2A) and/or A(2B) receptors modulate adenosine A(1) receptor-mediated cardioprotection. Isolated perfused hearts of wild-type (WT), A(2A) knockout (KO), and A(2B)KO mice, perfused at constant pressure and constant heart rate, underwent 30 min of global ischemia and 60 min of reperfusion. The adenosine A(1) receptor agonist N(6)-cyclohexyladenosine (CHA; 200 nM) was administrated 10 min before ischemia and for the first 10 min of reperfusion. Treatment with CHA significantly improved postischemic left ventricular developed pressure (74 ± 4% vs. 44 ± 4% of preischemic left ventricular developed pressure at 60 min of reperfusion) and reduced infarct size (30 ± 2% with CHA vs. 52 ± 5% in control) in WT hearts, effects that were blocked by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (100 nM). Treatments with the A(2A) receptor agonist CGS-21680 (200 nM) and the A(2B) agonist BAY 60-6583 (200 nM) did not exert any beneficial effects. Deletion of adenosine A(2A) or A(2B) receptor subtypes did not alter ischemia-reperfusion injury, but CHA failed to exert a cardioprotective effect in hearts of mice from either KO group. These findings indicate that both adenosine A(2A) and A(2B) receptors are required for adenosine A(1) receptor-mediated cardioprotection, implicating a role for interactions among receptor subtypes.

Sex differences and the effects of ovariectomy on the ß-adrenergic contractile response.

The presence of sex differences in myocardial ß-adrenergic responsiveness is controversial, and limited studies have addressed the mechanism underlying these differences. Studies were performed using isolated perfused hearts from male, intact female and ovariectomized female mice to investigate sex differences and the effects of ovarian hormone withdrawal on ß-adrenergic receptor function. Female hearts exhibited blunted contractile responses to the ß-adrenergic receptor agonist isoproterenol (ISO) compared with males but not ovariectomized females. There were no sex differences in ß(1)-adrenergic receptor gene or protein expression. To investigate the role of adenylyl cyclase, phosphodiesterase, and the cAMP-signaling cascade in generating sex differences in the ß-adrenergic contractile response, dose-response studies were performed in isolated perfused male and female hearts using forskolin, 3-isobutyl-1-methylxanthine (IBMX), and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Males showed a modestly enhanced contractile response to forskolin at 300 nM and 5 µM compared with females, but there were no sex differences in the response to IBMX or CPT-cAMP. The role of the A(1) adenosine receptor (A(1)AR) in antagonizing the ß-adrenergic contractile response was investigated using both the A(1)AR agonist 2-chloro-N(6)-cyclopentyl-adenosine and A(1)AR knockout (KO) mice. Intact females showed an enhanced A(1)AR anti-adrenergic effect compared with males and ovariectomized females. The ß-adrenergic contractile response was potentiated in both male and female A(1)ARKO hearts, with sex differences no longer present above 1 nM ISO. The ß-adrenergic contractile response is greater in male hearts than females, and minor differences in the action of adenylyl cyclase or the A(1)AR may contribute to these sex differences.

Differential effects of adenosine A2a and A2b receptors on cardiac contractility.

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or ß-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter ß-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.