Bruce G. Allen

The biochemical basis of the regulation of smooth-muscle contraction

The primary signal for smooth-muscle contraction is an increase in sarcoplasmic free Ca2+ concentration ([Ca2+]i). This triggers activation of calmodulin-dependent myosin light-chain kinase, which catalyses myosin phosphorylation, thereby activating crossbridge cycling and the development of force or contraction of the muscle cell. Restoration of resting [Ca2+]i deactivates the kinase; myosin is dephosphorylated by myosin light-chain phosphatase and the muscle relaxes. Recent evidence suggests that other signal-transduction pathways can modulate the contractile state of a smooth-muscle cell by affecting specific steps in the myosin phosphorylation-dephosphorylation mechanism.

Protein Kinase C–Induced Changes in the Stoichiometry of ATP Binding Activate Cardiac ATP-Sensitive K+ Channels A Possible Mechanistic Link to Ischemic Preconditioning

Activation of both ATP-sensitive K+ (KATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (< or = 50 mumol/L), PKC inhibits KATP channel activity. This finding is surprising, as both KATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximately 2 to approximately 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATP channels at millimolar levels of ATP. The effects of PKC on single KATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on KATP channel properties. KATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKCs action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in KATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates KATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.

Sildenafil and cardiomyocyte-specific cGMP signaling prevent cardiomyopathic changes associated with dystrophin deficiency

We recently demonstrated early metabolic alterations in the dystrophin-deficient mdx heart that precede overt cardiomyopathy and may represent an early “subclinical” signature of a defective nitric oxide (NO)/cGMP pathway. In this study, we used genetic and pharmacological approaches to test the hypothesis that enhancing cGMP, downstream of NO formation, improves the contractile function, energy metabolism, and sarcolemmal integrity of the mdx heart. We first generated mdx mice overexpressing, in a cardiomyocyte-specific manner, guanylyl cyclase (GC) (mdx/GC+/0). When perfused ex vivo in the working mode, 12- and 20-week-old hearts maintained their contractile performance, as opposed to the severe deterioration observed in age-matched mdx hearts, which also displayed two to three times more lactate dehydrogenase release than mdx/GC+/0. At the metabolic level, mdx/GC+/0 displayed a pattern of substrate selection for energy production that was similar to that of their mdx counterparts, but levels of citric acid cycle intermediates were significantly higher (36 ± 8%), suggesting improved mitochondrial function. Finally, the ability of dystrophin-deficient hearts to resist sarcolemmal damage induced in vivo by increasing the cardiac workload acutely with isoproterenol was enhanced by the presence of the transgene and even more so by inhibiting cGMP breakdown using the phosphodiesterase inhibitor sildenafil (44.4 ± 1.0% reduction in cardiomyocyte damage). Overall, these findings demonstrate that enhancing cGMP signaling, specifically downstream and independent of NO formation, in the dystrophin-deficient heart improves contractile performance, myocardial metabolic status, and sarcolemmal integrity and thus constitutes a potential clinical avenue for the treatment of the dystrophin-related cardiomyopathies.

G protein‐coupled receptor signalling in the cardiac nuclear membrane: evidence and possible roles in physiological and pathophysiological function

Abstract  G protein‐coupled receptors (GPCRs) play key physiological roles in numerous tissues, including the heart, and their dysfunction influences a wide range of cardiovascular diseases. Recently, the notion of nuclear localization and action of GPCRs has become more widely accepted. Nuclear‐localized receptors may regulate distinct signalling pathways, suggesting that the biological responses mediated by GPCRs are not solely initiated at the cell surface but may result from the integration of extracellular and intracellular signalling pathways. Many of the observed nuclear effects are not prevented by classical inhibitors that exclusively target cell surface receptors, presumably because of their structures, lipophilic properties, or affinity for nuclear receptors. In this topical review, we discuss specifically how angiotensin‐II, endothelin, β‐adrenergic and opioid receptors located on the nuclear envelope activate signalling pathways, which convert intracrine stimuli into acute responses such as generation of second messengers and direct genomic effects, and thereby participate in the development of cardiovascular disorders.

G protein-coupled receptors in and on the cell nucleus: a new signaling paradigm?

Signaling from internalizing and endosomal receptors has almost become a classic GPCR paradigm in the last several years. However, it has become clear in recent years that GPCRs also elicit signals when resident at other subcellular sites including the endoplasmic reticulum, Golgi apparatus, and the nucleus. In this review we discuss the nature, function, and trafficking of nuclear GPCR signaling complexes, as well as potential sources of endogenous and exogenous ligands. Finally, we pose a series of questions that will need to be answered in the coming years to confirm and extend this as a new paradigm for GPCR signaling.

Nuclear-delimited Angiotensin Receptor-mediated Signaling Regulates Cardiomyocyte Gene Expression

Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene expression. Here, we assessed the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors on the nuclear envelope mediate effects on cardiomyocyte gene expression. Immunoblots of nucleus-enriched fractions from isolated cardiomyocytes indicated the presence of AT1R and AT2R proteins that copurified with the nuclear membrane marker nucleoporin-62 and histone-3, but not markers of plasma (calpactin-I), Golgi (GRP-78), or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [α-32P]UTP (e.g. 36.0 ± 6.0 cpm/ng of DNA control versus 246.4 ± 15.4 cpm/ng of DNA Ang-II, 390.1 ± 15.5 cpm/ng of DNA L-162313 (AT1), 180.9 ± 7.2 cpm/ng of DNA CGP42112A (AT2), p < 0.001). Ang-II application to cardiomyocyte nuclei enhanced NFκB mRNA expression, a response that was suppressed by co-administration of AT1R (valsartan) and/or AT2R (PD123177) blockers. Dose-response experiments with Ang-II applied to purified cardiomyocyte nuclei versus intact cardiomyocytes showed greater increases in NFκB mRNA levels at saturating concentrations with ∼2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca2+] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca2+ release and attenuated AT1R-mediated transcription initiation responses. We conclude that cardiomyocyte nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene expression, with potentially important mechanistic and therapeutic implications.

Mechanisms Underlying Rate-Dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes

Transient outward K+ current (Ito) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac Ito and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. Ito was recorded with whole-cell patch clamp. The 3-Hz pacing reduced Ito by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by ≈30% and ≈40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced Ito downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented Ito downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca2+i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na+/Ca2+ exchange and sarcoplasmic reticulum Ca2+-ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented Ito reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates Ito expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent Ito control.

Expression, distribution and regulation of sex steroid hormone receptors in mouse heart

The effects of sex hormones on the heart are dependent on the presence and distribution of sex steroid hormone receptors (SSHR) in cardiac tissue. This study used subcellular fractionation, Western blot analysis and densitometry to characterize the subcellular distribution and abundance of estrogen receptor (ER) α, ERβ and androgen receptor (AR) in atrial and ventricular tissue from male and female mice. The results showed that in both atrial and ventricular tissue ERα was primarily found in the sarcolemma, whereas ERβ and AR were predominantly located in the nucleus and cytosol. Interestingly, ERα expression was greater in the ventricles compared to the atria, whereas ERβ and AR expression were similar in both heart chambers. Furthermore, the distribution and abundance of SSHR in the atria and ventricles did not differ between sexes. This study also showed that a reduction in hormone levels (as a result of ovariectomy) resulted in a significant increase in the abundance of ERα in the ventricular sarcolemmal fraction. Overall, the results suggest ERα, ERβ and AR distribution and expression are not sex dependent in the mouse heart. However, it appears that ERα expression is chamber specific and that, in certain cases, hormone levels can modulate the subcellular location of SSHRs.

Essential Role of Protein Kinase Cδ in Platelet Signaling, αIIbβ3 Activation, and Thromboxane A2 Release

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCδ, but not PKCα or PKCβ, is required for collagen-induced phospholipase C-dependent signaling, activation of αIIbβ3, and platelet aggregation. Analysis of PKCδ phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by αIIbβ3 outside-in signaling. Moreover, PKCδ triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A2, which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCδ signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A2.

Nuclear β-adrenergic receptors modulate gene expression in adult rat heart.

Both β(1)- and β(3)-adrenergic receptors (β(1)ARs and β(3)ARs) are present on nuclear membranes in adult ventricular myocytes. These nuclear-localized receptors are functional with respect to ligand binding and effector activation. In isolated cardiac nuclei, the non-selective βAR agonist isoproterenol stimulated de novo RNA synthesis measured using assays of transcription initiation (Boivin et al., 2006 Cardiovasc Res. 71:69-78). In contrast, stimulation of endothelin receptors, another G protein-coupled receptor (GPCR) that localizes to the nuclear membrane, resulted in decreased RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of different mitogen-activated protein kinase (MAPK) and PI3K/PKB pathways. Components of p38, JNK, and ERK1/2 MAP kinase cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine, in the presence of isoproterenol, converted the activation of the βAR from stimulatory to inhibitory with regards to RNA synthesis, while ERK1/2, JNK and p38 inhibition reduced both basal and isoproterenol-stimulated activity. Analysis by qPCR indicated an increase in the expression of 18S rRNA following isoproterenol treatment and a decrease in NFκB mRNA. Further qPCR experiments revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB, while ERK1/2 and PKB inhibition substantially reversed this effect. Our results suggest that GPCRs on the nuclear membrane regulate nuclear functions such as gene expression and this process is modulated by activation/inhibition of downstream protein kinases within the nucleus.