Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium.

Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, <5 % FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified >4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR < 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (-38); Adipoq (-17); Atgrl1/Aplnr (-14); H19 (-11) and Itga8 (-8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a 'cardio-depressant' profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia.

The adenosine A2A receptor - myocardial protectant and coronary target in endotoxemia.

BACKGROUND: Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A2A adenosine receptor (A2AAR) activity during lipopolysaccharide (LPS)-induced inflammation. METHODS: We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A2AARs (A2AAR KO). RESULTS: Cardiac injury was evident in LPS-treated WTs, with ~7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A2AARs increased LPS-provoked cTnI release at 24 h by 3-fold without additional demise of contraction function. Importantly, A2AAR deletion per se emulated detrimental effects of LPS on coronary function, and LPS was without effect in coronary vessels lacking A2AARs. Effects of A2AAR KO were independent of major shifts in circulating C-reactive protein (CRP) and haptoglobin. Cytokine responses were largely insensitive to A2AAR deletion; substantial LPS-induced elevations (up to 100-fold) in IFN-γ and IL-10 were unaltered in A2AAR KO mice, as were levels of IL-4 and TNF-α. However, late elevations in IL-2 and IL-5 were differentially modulated by A2AAR KO (IL-2 reduced, IL-5 increased). Data demonstrate that in the context of LPS-triggered cardiac and coronary injury, A2AAR activity protects myocardial viability without modifying contractile dysfunction, and selectively modulates cytokine (IL-2, IL-5) release. A2AARs also appear to be targeted by LPS in the coronary vasculature. CONCLUSIONS: These experimental data suggest that preservation of A2AAR functionality might provide therapeutic benefit in human sepsis.

Protection from doxorubicin-induced cardiomyopathy using the modified anthracycline N-benzyladriamycin-14-valerate (AD 198).

The anthracycline doxorubicin (Dox) is an effective antitumor agent. However, its use is limited because of its toxicity in the heart. N-Benzyladriamycin-14-valerate (AD 198) is a modified anthracycline with antitumor efficacy similar to that of Dox, but with significantly less cardiotoxicity and potentially cardioprotective elements. In the present study, we investigated the possibility of in vivo protective effects of low-dose AD 198 against Dox-induced cardiomyopathy. To do this, rats were divided into four groups: vehicle, Dox (20 mg/kg; single injection day 1), AD 198 (0.3 mg/kg per injection; injections on days 1, 2, and 3), or a combination treatment of Dox + AD 198. Seventy-two hours after beginning treatment, hearts from the Dox group had decreased phosphorylation of AMP kinase and troponin I and reduced poly(ADP-ribose) polymerase, ß-tubulin, and serum albumin expression. Dox also increased the phosphorylation of phospholamban and expression of inducible nitric-oxide synthase in hearts. Each of these Dox-induced molecular changes was attenuated in the Dox + AD 198 group. In addition, excised hearts from rats treated with Dox had a 25% decrease in left ventricular developed pressure (LVDP) and a higher than normal increase in LVDP when perfused with a high extracellular Ca(2+) solution. The Dox-induced decrease in baseline LVDP and hyper-responsiveness to [Ca(2+)] was not observed in hearts from the Dox + AD 198 group. Thus Dox, with well established and efficient antitumor protocols, in combination with low levels of AD 198, to counter anthracycline cardiotoxicity, may be a promising next step in chemotherapy.

A cardioprotective role for platelet-activating factor through NOS-dependent S-nitrosylation.

Controversy exists as to whether platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, can actually protect the heart from postischemic injury. To determine whether endogenous activation of the PAF receptor is cardioprotective, we examined postischemic functional recovery in isolated hearts from wild-type and PAF receptor-knockout mice. Postischemic function was reduced in hearts with targeted deletion of the PAF receptor and in wild-type hearts treated with a PAF receptor antagonist. Furthermore, perfusion with picomolar concentrations of PAF improved postischemic function in hearts from wild-type mice. To elucidate the mechanism of a PAF-mediated cardioprotective effect, we employed a model of intracellular Ca2+ overload and loss of function in nonischemic ventricular myocytes. We found that PAF receptor activation attenuates the time-dependent loss of shortening and increases in intracellular Ca2+ transients in Ca2+ -overloaded myocytes. These protective effects of PAF depend on nitric oxide, but not activation of cGMP. In addition, we found that reversible S-nitrosylation of myocardial proteins must occur in order for PAF to moderate Ca2+ overload and loss of myocyte function. Thus our data are consistent with the hypothesis that low-level PAF receptor activation initiates nitric oxide-induced S-nitrosylation of Ca2+ -handling proteins, e.g., L-type Ca2+ channels, to attenuate Ca2+ overload during ischemia-reperfusion in the heart. Since inhibition of the PAF protective pathway reduces myocardial postischemic function, our results raise concern that clinical therapies for inflammatory diseases that lead to complete blockade of the PAF receptor may eliminate a significant, endogenous cardioprotective pathway.

N-Benzyladriamycin-14-valerate (AD 198): a non-cardiotoxic anthracycline that is cardioprotective through PKC-epsilon activation.

N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-epsilon, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD(50) doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198-mediated cardioprotection correlates with increased PKC-epsilon activation and is inhibited in hearts from PKC-epsilon knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-epsilon.

Targeted deletion of A2A adenosine receptors attenuates the protective effects of myocardial postconditioning.

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (POC), yet it is unclear which adenosine receptor subtype is primarily responsible. To evaluate the role of A(2A) adenosine receptors in POC-induced protection, global ischemia-reperfusion was performed with and without POC in isolated wild-type (WT) and A(2A) adenosine receptor knockout (A(2A)KO) mouse hearts. Injury was measured in terms of postischemic functional recovery and release of cardiac troponin I (cTnI). Activation of protective signaling with POC was assessed by Akt and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In WT hearts, POC improved recovery of postischemic developed pressure in early (81.6 +/- 6.4% of preischemic baseline vs. 37.5 +/- 5.6% for non-POC WT at 1 min) and late (62.2 +/- 4.2% of baseline vs. 45.5 +/- 5.3% for non-POC WT at 30 min) reperfusion, reduced cTnI release by 37%, and doubled the phosphorylation of both Akt and ERK1/2. These beneficial effects of POC were blocked by treatment with the selective A(2A) adenosine receptor antagonist ZM-241385 during reperfusion. Postischemic functional recovery, cTnI release, and phosphorylation of Akt and ERK1/2 were not different between non-POC WT and A(2A)KO hearts. In A(2A)KO hearts, POC did not improve functional recovery, reduce cTnI release, nor increase phosphorylation of Akt or ERK1/2. Thus the protective effects of POC are attenuated by both selective A(2A) receptor antagonism and targeted deletion of the gene encoding A(2A) adenosine receptors. These observations support the conclusion that endogenous activation of A(2A) adenosine receptors is an essential trigger leading to the protective effects of POC in isolated murine hearts.

H2O2 activation of HSP25/27 protects desmin from calpain proteolysis in rat ventricular myocytes.

Ischemia-reperfusion-induced Ca(2+) overload results in activation of calpain-1 in the heart. Calpain-dependent proteolysis contributes to myocardial dysfunction and cell death. Previously, preischemic treatment with low doses of H(2)O(2) was shown to improve postischemic function and reduce myocardial infarct size. Our aim was to determine the mechanism by which H(2)O(2) protects the heart. We hypothesized that H(2)O(2) causes the activation of p38 MAPK which initiates translocation of heat shock protein 25/27 (HSP25/27) to the myofilament Z disk. We further hypothesized that HSP25/27 shields structural proteins, particularly desmin, from calpain-induced proteolysis. To address this hypothesis, we first determined that an ischemia-reperfusion-induced decrease in desmin content could be blocked by H(2)O(2) pretreatment of hearts from rats. We next determined that ventricular myocytes that underwent Ca(2+) overload also demonstrated a calpain-dependent disruption of desmin that could be reduced by H(2)O(2)/p38 MAPK activation. Furthermore, myocytes acutely treated with H(2)O(2) exhibited a decrease in cleavage of desmin upon exposure to exogenous calpain-1 compared with myocytes not pretreated with H(2)O(2). The H(2)O(2)-induced attenuation of desmin degradation by calpain-1 was blocked by inhibition of p38 MAPK. In a final series of experiments, we demonstrated that cardiac myofilaments exposed to recombinant phosphorylated HSP27, but not nonphosphorylated HSP27, had a significant reduction in the calpain-induced degradation of desmin compared with non-HSP27-treated myofilaments. These findings are consistent with the hypothesis that H(2)O(2)-induced activation of p38 MAPK and subsequent HSP25/27 translocation attenuates desmin degradation brought about by calpain-1 activation in ischemia-reperfused hearts.

Acute modulation of PP2a and troponin I phosphorylation in ventricular myocytes: studies with a novel PP2a peptide inhibitor.

The present study demonstrates that acute activation with either beta-adrenergic receptor agonists or H(2)O(2) treatment increases protein phosphatase 2a (PP2a) activity in ventricular myocytes. PP2a activation occurs concomitant with an increase in methylation of PP2a, changes in localization of a PP2a targeting subunit PP2aB56alpha, and a decrease in phosphorylation of PP2a substrates, such as troponin I (TnI) and ERK in ventricular myocytes. Okadaic acid, a well-established pharmacological inhibitor of PP2a, and the peptide Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL) were used to block PP2a methylation, localization, and phosphorylations. TPDYFL is a highly conserved sequence of the PP2a catalytic subunit COOH-terminus. Specifically, both okadaic acid and the peptide increased beta-adrenergic-cAMP-dependent phosphorylation of TnI and blocked the beta-adrenergic-cAMP-dependent translocation of PP2aB56alpha. TPDYFL, but not a scrambled version of this sequence, blocked H(2)O(2)-induced changes in PP2a methylation and TnI dephosphorylation. Okadaic acid produces similar inhibition of H(2)O(2) effects. Thus we propose that the novel peptide TPDYFL acts as an inhibitor of PP2a activity and may be a useful tool to increase our understanding of how PP2a is regulated and the role of PP2a in a variety of physiological and pathological processes. In addition, the present study is consistent with acute beta-adrenergic receptor activation and H(2)O(2) exposure, simultaneously activating kinases and PP2a to work on common substrates, such as TnI. We hypothesize that dual activation of opposing enzymes provides for a tighter regulation of substrate phosphorylations in ventricular myocytes.

Modulation of myosin phosphatase targeting subunit and protein phosphatase 1 in the heart.

Myosin light chain 2 (LC2) phosphorylation is of both physiological and pathological importance to myocardial function. The phosphatase that directly dephosphorylates LC2 is a type 1 protein phosphatase (PP1) that contains a catalytic subunit that complexes with a myosin-binding phosphatase targeting subunit (MYPT). The goal of the present study was to examine the role of MYPT in the regulation of PP1 in ventricular myocytes. In the first part of the study, regional distribution of MYPT expression and phosphorylation were determined in unstimulated hearts. The pattern of MYPT phosphorylation was inversely related to the LC2 phosphorylation spatial gradient as described by Epstein and colleagues (Davis JS, Hassanzadeh S, Winitsky S, Lin H, Satorius C, Vemuri R, Aletras AH, Wen H, and Epstein ND. Cell 107: 631-641, 2001). In the second part of the study, adult rat isolated ventricular myocytes were exposed to an alpha-adrenergic receptor agonist, and properties of MYPT, PP1, and LC2 were studied. We found MYPT associates with cardiac myofilaments, and this association increases upon alpha-adrenergic receptor stimulation. Activation of alpha-adrenergic receptors also led to a decrease in the PP1-myofilament association. Furthermore, alpha-adrenergic receptor stimulation results in phosphorylation of MYPT and LC2 and an increase in myocyte Ca(2+) sensitivity of tension that all depend on Rho kinase activation. These data support the hypothesis that alpha-adrenergic receptor activation works through Rho kinase to phosphorylate MYPT, and phosphorylated MYPT dissociates from PP1 so that PP1 is no longer physically associated with LC2. Hence, we propose a pathway for the dynamic modulation of LC2 phosphorylation through receptor-dependent phosphorylation of MYPT, and a spatial gradient of LC2 phosphorylation under basal conditions that occurs due to varied levels of phosphorylation of MYPT in ventricles.

Modest actomyosin energy conservation increases myocardial postischemic function.

We have proposed that pharmacological preconditioning, leading to PKC-epsilon activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in beta-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 +/- 6.2 vs. 32.6 +/- 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 +/- 5.5 and 62.5 +/- 14.5% compared with 20.0 +/- 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.

Protein phosphatase 2A-mediated cross-talk between p38 MAPK and ERK in apoptosis of cardiac myocytes.

Mitogen-activated protein kinases (MAPKs) play different regulatory roles in signaling oxidative stress-induced apoptosis in cardiac ventricular myocytes. The regulation and functional role of cross-talk between p38 MAPK and extracellular signal-regulated kinase (ERK) pathways were investigated in cardiac ventricular myocytes in the present study. We demonstrated that inhibition of p38 MAPK with SB-203580 and SB-239063 enhanced H(2)O(2)-stimulated ERK phosphorylation, whereas preactivation of p38 MAPK with sodium arsenite reduced H(2)O(2)-stimulated ERK phosphorylation. In addition, pretreatment of cells with the protein phosphatase 2A (PP2A) inhibitors okadaic acid and fostriecin increased basal and H(2)O(2)-stimulated ERK phosphorylation. We also found that PP2A coimmunoprecipitated with ERK and MAPK/ERK (MEK) in cardiac ventricular myocytes, and H(2)O(2) increased the ERK-associated PP2A activity that was blocked by inhibition of p38 MAPK. Finally, H(2)O(2)-induced apoptosis was attenuated by p38 MAPK or PP2A inhibition, whereas it was enhanced by MEK inhibition. Thus the present study demonstrated that p38 MAPK activation decreases H(2)O(2)-induced ERK activation through a PP2A-dependent mechanism in cardiac ventricular myocytes. This represents a novel cellular mechanism that allows for interaction of two opposing MAPK pathways and fine modulation of apoptosis during oxidative stress.

Cardioprotection through a PKC-dependent decrease in myofilament ATPase.

Activation of myocardial kappa-opioid receptor-protein kinase C (PKC) pathways may improve postischemic contractile function through a myofilament reduction in ATP utilization. To test this, we first examined the effects of PKC inhibitors on kappa-opioid receptor-dependent cardioprotection. The kappa-opioid receptor agonist U50,488H (U50) increased postischemic left ventricular developed pressure and reduced postischemic end-diastolic pressure compared with controls. PKC inhibitors abolished the cardioprotective effects of U50. To determine whether kappa-opioid-PKC-dependent decreases in Ca2+-dependent actomyosin Mg2+-ATPase could account for cardioprotection, we subjected hearts to three separate actomyosin ATPase-lowering protocols. We observed that moderate decreases in myofibrillar ATPase were equally cardioprotective as kappa-opioid receptor stimulation. Immunoblot analysis and confocal microscopy revealed a kappa-opioid-induced increase in myofilament-associated PKC-epsilon, and myofibrillar Ca2+-independent PKC activity was increased after kappa-opioid stimulation. This PKC-myofilament association led to an increase in troponin I and C-protein phosphorylation. Thus we propose PKC-epsilon activation and translocation to the myofilaments causes a decrease in actomyosin ATPase, which contributes to the kappa-opioid receptor-dependent cardioprotective mechanism.

Modulation of protein phosphatase 2a by adenosine A1 receptors in cardiomyocytes: role for p38 MAPK.

Adenosine A1 receptor activation causes protein phosphatase 2a (PP2a) activation in ventricular myocytes. This attenuates beta-adrenergic functional effects in the heart (Liu Q and Hofmann PA. Am J Physiol Heart Circ Physiol 283: H1314-H1321, 2002). The purpose of the present study was to identify the signaling pathway involved in the translocation/activation of PP2a by adenosine A1 receptors in ventricular myocytes. We found that N6-cyclopentyladenosine (CPA; an adenosine A1 receptor agonist)-induced PP2a translocation was blocked by p38 MAPK inhibition but not by JNK inhibition. CPA increased phosphorylation of p38 MAPK, and this effect was abolished by pertussis toxin and inhibitors of the cGMP pathway. Moreover, CPA-induced PP2a translocation was blocked by inhibition of the cGMP pathway. Guanylyl cyclase activation mimicked the effects of CPA and caused p38 MAPK phosphorylation and PP2a translocation. Finally, CPA-induced dephosphorylations of troponin I and phospholamban were blocked by pertussis toxin and attenuated by p38 MAPK inhibition. These results suggest that adenosine A1 receptor-mediated PP2a activation uses a pertussis toxin-sensitive Gi protein-guanylyl cyclase-p38 MAPK pathway. This proposed, novel pathway may play a role in acute modulation of cardiac function.

Age-related functional effects linked to phosphatase activity in ventricular myocytes.

Conflicting reports exist regarding the influence of beta-adrenergic stimulation on the maximum velocity of shortening (Vmax) in ventricular myocytes. This may be due to an unrecognized effect of maturation. In the present study, the effects of beta-adrenergic receptor stimulation on myocytes from hearts of juvenile nonbred and young adult retired breeder female rats were compared. Ventricular myocytes from young adults had a beta-adrenergic-dependent increase in Vmax and Ca2+-dependent actomyosin ATPase that was not observed in myocytes from juveniles. Myocytes from young adults had both an increase in beta-myosin heavy chain (MHC) and higher basal serine/threonine phosphatase activity compared with juvenile rats. Additional studies established moderate increases in beta-MHC induced by hypothyroidism do not confer myocardial beta-adrenergic responsiveness, whereas inhibition of the higher phosphatase activity in myocytes from young adults blocks the age-dependent, beta-adrenergic-induced increase in cross-bridge cycling rates. We propose that the higher phosphatase activity of myocytes from young adults compared with juveniles allows for a greater functional response of the myocardium to beta-adrenergic stimulation.

Antiadrenergic effects of adenosine A(1) receptor-mediated protein phosphatase 2a activation in the heart.

The ability of adenosine A(1) receptors to activate type 2a protein phosphatase (PP2a) and account for antiadrenergic effects was investigated in rat myocardial preparations. We observed that the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) significantly reduces the isoproterenol-induced increase in left ventricular developed pressure of isolated heats, and this effect is blocked by pretreatment of hearts with the PP2a inhibitor cantharidin. CPA alone or given in conjunction with isoproterenol stimulation decreases phosphorylation of phospholamban and troponin I in ventricular myocytes. These dephosphorylations are blocked by an adenosine A(1) receptor antagonist and by PP2a inhibition with okadaic acid. Adenosine A(1) receptor activation was also shown to increase carboxymethylation of the PP2a catalytic subunit (PP2a-C) and cause translocation of PP2a-C to the particulate fraction in ventricular myocytes. These results support the hypothesis that adenosine A(1) receptor activation leads to methylation of PP2a-C and subsequent translocation of the PP2a holoenzyme. Increases in localized PP2a activity lead to dephosphorylation of key cardiac proteins responsible for the positive inotropic effects of beta-adrenergic stimulation.