Inositol Trisphosphate Receptors and Nuclear Calcium in Atrial Fibrillation.

RATIONALE: The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca2+ regulates gene expression, but the nature and significance of nuclear Ca2+-changes in AF are largely unknown. OBJECTIVE: To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca2+ ([Ca2+]Nuc) and CaMKII (Ca2+/calmodulin-dependent protein kinase-II)-related signaling. METHODS AND RESULTS: Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca2+]Nuc and cytosolic [Ca2+] ([Ca2+]Cyto) were recorded via confocal microscopy. Diastolic [Ca2+]Nuc was greater than [Ca2+]Cyto under control conditions, while resting [Ca2+]Nuc was similar to [Ca2+]Cyto; both diastolic and resting [Ca2+]Nuc increased with AF. IP3R (Inositol-trisphosphate receptor) stimulation produced larger [Ca2+]Nuc increases in AF versus control cardiomyocytes, and IP3R-blockade suppressed the AF-related [Ca2+]Nuc differences. AF upregulated nuclear protein expression of IP3R1 (IP3R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca2+]Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP3R knockdown with short-interfering RNA directed against IP3R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP3R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP3R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP3R1 upregulation and nuclear diastolic Ca2+-loading. CONCLUSIONS: AF increases atrial-cardiomyocyte nucleoplasmic [Ca2+] by IP3R1-upregulation involving miR-26a, leading to enhanced IP3R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Caged ligands to study the role of intracellular GPCRs.

In addition to cell surface membranes, numerous G protein-coupled receptors (GPCRs) are located on intracellular membranes including the nuclear envelope. Although the role of numerous GPCRs at the cell surface has been well characterized, the physiological function of these same receptors located on intracellular membranes remains to be determined. Here, we employ a novel caged Ang-II analog, cAng-II, to compare the effects of the activation of cell surface versus intracellular angiotensin receptors in intact cardiomyocytes. When added extracellularly to HEK 293 cells, Ang-II and photolysed cAng-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R). In contrast unphotolysed cAng-II did not. Cellular uptake of cAng-II was 6-fold greater than that of Ang-II and comparable to the HIV TAT(48-60) peptide. Intracellular photolysis of cAng-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was greater than that induced by extracellular application of Ang-II. We conclude that cell-permeable ligands that can access intracellular GPCRs may evoke responses distinct from those with access restricted to the same receptor located on the cell surface.

Photoreleasable ligands to study intracrine angiotensin II signalling.

KEY POINTS: The renin-angiotensin system plays a key role in cardiovascular physiology and its overactivation has been implicated in the pathogenesis of several major cardiovascular diseases. There is growing evidence that angiotensin II (Ang-II) may function as an intracellular peptide to activate intracellular/nuclear receptors and their downstream signalling effectors independently of cell surface receptors. Current methods used to study intracrine Ang-II signalling are limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we present novel photoreleasable Ang-II analogues used to probe intracellular actions with spatial and temporal precision. The photorelease of intracellular Ang-II causes nuclear and cytosolic calcium mobilization and initiates the de novo synthesis of RNA in cardiac cells, demonstrating the application of the method. ABSTRACT: Several lines of evidence suggest that intracellular angiotensin II (Ang-II) contributes to the regulation of cardiac contractility, renal salt reabsorption, vascular tone and metabolism; however, work on intracrine Ang-II signalling has been limited to indirect approaches because of a lack of selective intracellularly-acting probes. Here, we aimed to synthesize and characterize cell-permeant Ang-II analogues that are inactive without uncaging, but release active Ang-II upon exposure to a flash of UV-light, and act as novel tools for use in the study of intracrine Ang-II physiology. We prepared three novel caged Ang-II analogues, [Tyr(DMNB)(4)]Ang-II, Ang-II-ODMNB and [Tyr(DMNB)(4)]Ang-II-ODMNB, based upon the incorporation of the photolabile moiety 4,5-dimethoxy-2-nitrobenzyl (DMNB). Compared to Ang-II, the caged Ang-II analogues showed 2-3 orders of magnitude reduced affinity toward both angiotensin type-1 (AT1R) and type-2 (AT2R) receptors in competition binding assays, and greatly-reduced potency in contraction assays of rat thoracic aorta. After receiving UV-irradiation, all three caged Ang-II analogues released Ang-II and potently induced the contraction of rat thoracic aorta. [Tyr(DMNB)(4)]Ang-II showed the most rapid photolysis upon UV-irradiation and was the focus of subsequent characterization. Whereas Ang-II and photolysed [Tyr(DMNB)(4)]Ang-II increased ERK1/2 phosphorylation (via AT1R) and cGMP production (AT2R), caged [Tyr(DMNB)(4)]Ang-II did not. Cellular uptake of [Tyr(DMNB)(4)]Ang-II was 4-fold greater than that of Ang-II and significantly greater than uptake driven by the positive-control HIV TAT(48-60) peptide. Intracellular photolysis of [Tyr(DMNB)(4)]Ang-II induced an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n), and initiated 18S rRNA and nuclear factor kappa B mRNA synthesis in adult cardiac cells. We conclude that caged Ang-II analogues represent powerful new tools for use in the selective study of intracrine signalling via Ang-II.

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca²âº in adult cardiac myocytes.

Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca(2+) signaling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with peptide: N-glycosidase F did not alter the electrophoretic mobility of ETB. The absence of N-glycosylation further indicates that these receptors did not originate at the cell surface. Intracellular photolysis of a caged ET-1 analog ([Trp-ODMNB(21)]ET-1) evoked an increase in nucleoplasmic Ca(2+) ([Ca(2+)]n) that was attenuated by inositol 1,4,5-trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate and prevented by pre-treatment with ryanodine. A caged cell-permeable analog of the ETB-selective antagonist IRL-2500 blocked the ability of intracellular cET-1 to increase [Ca(2+)]n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffics directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulates nuclear Ca(2+) signaling.

Regulation of cardiac nitric oxide signaling by nuclear ß-adrenergic and endothelin receptors.

At the cell surface, ßARs and endothelin receptors can regulate nitric oxide (NO) production. ß-adrenergic receptors (ßARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a ß3AR-selective agonist, BRL 37344, increased NO synthesis whereas the ß1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular ß-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear ß3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.

Post-translational modifications, a key process in CD36 function: lessons from the spontaneously hypertensive rat heart.

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of (13)C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to ß-oxidation (-40%) and triglycerides (+2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized ß-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-ß-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

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 [alpha-(32)P]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 NFkappaB 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 NFkappaB mRNA levels at saturating concentrations with approximately 2-fold greater affinity upon nuclear application, suggesting preferential nuclear signaling. AT1R, but not AT2R, stimulation increased [Ca(2+)] in isolated cardiomyocyte nuclei. Inositol 1,4,5-trisphosphate receptor blockade by 2-aminoethoxydiphenyl borate prevented AT1R-mediated Ca(2+) 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 (I to) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac I(to) and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37 degrees C) were paced continuously at 1 or 3 Hz for 24 hours. I to was recorded with whole-cell patch clamp. The 3-Hz pacing reduced I to by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by approximately 30% and approximately 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 I to 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 I to 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 I to 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 I to expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent I to control.

Cellular signaling underlying atrial tachycardia remodeling of L-type calcium current.

Atrial tachycardia (AT) downregulates L-type Ca(2+) current (I(CaL)) and causes atrial fibrillation-promoting electric remodeling. This study assessed potential underlying signal transduction. Cultured adult canine atrial cardiomyocytes were paced at 0, 1, or 3 Hz (P0, P1, P3) for up to 24 hours. Cellular tachypacing (P3) mimicked effects of in vivo AT: decreased I(CaL) and transient outward current (I(to)), unchanged I(CaT), I(Kr), and I(Ks), and reduced action potential duration (APD). I(CaL) was unchanged in P3 at 2 and 8 hours but decreased by 55+/-6% at 24 hours. Tachypacing caused Ca(2+)(i) accumulation in P3 cells at 2 to 8 hours, but, by 24 hours, Ca(2+)i returned to baseline. Ca(v)1.2 mRNA expression was not altered at 2 hours but decreased significantly at 8 and 24 hours (32+/-4% and 48+/-4%, respectively) and protein expression was decreased (47+/-8%) at 24 hours only. Suppressing Ca(2+)(i) increases during tachypacing with the I(CaL) blocker nimodipine or the Ca(2+) chelator BAPTA-AM prevented I(CaL) downregulation. Calcineurin activity increased in P3 at 2 and 8 hours, respectively, returning to baseline at 24 hours. Nuclear factor of activated T cells (NFAT) nuclear translocation was enhanced in P3 cells. Ca(2+)-dependent signaling was probed with inhibitors of Ca(2+)/calmodulin (W-7), calcineurin (FK-506), and NFAT (INCA6): each prevented I(CaL) downregulation. Significant APD reductions ( approximately 30%) at 24 hours in P3 cells were prevented by nimodipine, BAPTA-AM, W-7, or FK-506. Thus, rapid atrial cardiomyocyte activation causes Ca(2+) loading, which activates the Ca(2+)-dependent calmodulin-calcineurin-NFAT system to cause transcriptional downregulation of I(CaL), restoring Ca(2+)i to normal at the cost of APD reduction. These studies elucidate for the first time the molecular feedback mechanisms underlying arrhythmogenic AT remodeling.

GABA-B(1) receptors are coupled to the ERK1/2 MAP kinase pathway in the absence of GABA-B(2) subunits.

In the current model of gamma-aminobutyric acid (GABA) B receptor function, there is a requirement for GABA-B(1/2) heterodimerisation for targetting to the cell surface. However, different lines of evidence suggest that the GABA-B(1) subunit can form a functional receptor in the absence of GABA-B(2). We observed coupling of endogenous GABA-B(1) receptors in the DI-TNC1 glial cell line to the ERK pathway in response to baclofen even though these cells do not express GABA-B(2). GABA-B(1A) receptors were also able to mediate a rapid, transient, and dose-dependent activation of the ERK1/2 MAP kinase pathway when transfected alone into HEK 293 cells. The response was abolished by G(i/o) and MEK inhibition, potentiated by inhibitors of phospholipase C and protein kinase C and did not involve PI-3-kinase activity. Finally, using bioluminescence resonance energy transfer and co-immunoprecipitation, we show the existence of homodimeric GABA-B(1A) receptors in transfected HEK293 cells. Altogether, our observations show that GABA-B(1A) receptors are able to activate the ERK1/2 pathway despite the absence of surface targetting partner GABA-B(2) in both HEK 293 cells and the DI-TNC1 cell line.

Heterodimers of adenylyl cyclases 2 and 5 show enhanced functional responses in the presence of Galpha s.

Recent studies have demonstrated that adenylyl cyclase isoforms can form both homo- and heterodimers and that this may be the basic functional unit of these enzymes (see Cooper, D.M.F. and Crossthwaite, A.J. (2006) Trends. Pharmacol. Sci. 8:426-431). Here, we show that adenylyl cyclases 2 and 5 can form a functional heterodimeric complex in HEK293 cells using a combination of BRET, confocal imaging, co-immunoprecipitation and assays of adenylyl cyclase activity. The AC2/5 complex is formed constitutively and is stable in the presence of receptor or forskolin stimulation. The complex formed by AC2/5 is also much more sensitive to the presence of Galpha(s) and forskolin than either of the parent AC isoforms themselves. Finally, we also show that this complex can be detected in native tissues as AC2 and AC5 were localized to the same structures in adult mouse ventricular myocytes and neonatal mouse cardiac fibroblasts and could be co-immunoprecipitated from lysates of mouse heart.

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms.

Recently identified, angiopoietin-1 (Ang1) and -2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities. As inflammation exists in a mutually dependent association with angiogenesis, we sought to determine if Ang1 and/or Ang2 could modulate proinflammatory activities, namely P-selectin translocation, in bovine aortic endothelial cells (EC) and dissect the mechanisms implicated. P-selectin, an adhesion molecule found in the Weibel-Palade bodies of EC, is translocated rapidly to the cell surface upon EC activation during inflammatory processes. Herein, we report that Ang1 and Ang2 (1 nM) are capable of mediating a rapid Tie2 phosphorylation as well as a rapid and transient endothelial P-selectin translocation maximal within 7.5 min (125% and 100% increase, respectively, over control values). In addition, we demonstrate for the first time that angiopoietin-mediated endothelial P-selectin translocation is calcium-dependent and regulated through phospholipase C-gamma activation.

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels.

gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability. These channels are functionally regulated by GABA-B receptors. Possible physical interactions between GABA-B receptor and Kir 3 channels expressed in HEK cells were evaluated using Bioluminescence Resonance Energy Transfer (BRET) experiments, co-immunoprecipitation and confocal microscopy. Our data indicate that Kir 3 channels and Gbetagamma subunits can interact with the GABA-B(1) subunits independently of the GABA-B(2) subunit or Kir 3.4 which are ultimately responsible for their targetting to the cell surface. Thus signalling complexes containing GABA-B receptors, G proteins and Kir channels are formed shortly after biosynthesis most likely in the endoplasmic reticulum.

Using caged ligands to study intracrine endothelin signaling in intact cardiac myocytes.

Intracrine signaling refers to the activation of receptors located within the cell and many intracrine receptors have been localized to the nuclear membrane. The presence and function of nuclear receptors have been studied in isolated nuclei. Much less information is available concerning the function of these receptors within the context of intact cells due, in part, to difficulties in accessing the intracellular receptor without activating those at the cell surface. Here, we describe the use of caged agonists to study intracrine signaling in intact, living cells. The caging moiety permits cells to be loaded with a functionally "inert" ligand. After washing the cells free of extracellular caged ligand, a brief exposure to UV releases the native ligand within the cell. The actual duration of UV irradiation required is a function of the type of caging moiety employed and where it is incorporated into the ligand. Cells may then be assessed for changes in morphology, second messenger production, cellular signaling, or gene expression by confocal fluorescence microscopy, immunoblotting, or transcriptomic techniques.

Hypoxia in early life is associated with lasting changes in left ventricular structure and function at maturity in the rat.

BACKGROUND: There is a growing population of adults with repaired cyanotic congenital heart disease. These patients have increased risk of impaired cardiac health and premature death. We hypothesized that hypoxia in early life before surgical intervention causes lasting changes in left ventricular structure and function with physiological implications in later life. METHODS: Sprague-Dawley rats reared initially hypoxic conditions (FiO(2)=0.12) for days 1-10 of life were compared to rats reared only in ambient air. Cellular morphology and viability were compared among LV cardiomyocytes and histological analyses were performed on LV myocardium and arterioles. Intracellular calcium transients and cell shortening were measured in freshly-isolated cardiomyocytes, and mitochondrial hexokinase 2 (HK2) expression and activity were determined. Transthoracic echocardiography was used to assess LV function in anesthetized animals. RESULTS: Cardiomyocytes from adult animals following hypoxia in early life had greater cellular volumes but significantly reduced viability. Echocardiographic analyses revealed LV hypertrophy and diastolic dysfunction, and alterations in cardiomyocyte calcium transients and cell shortening suggested impaired diastolic calcium reuptake. Histological analyses revealed significantly greater intima-media thickness and decreased lumen area in LV arterioles from hypoxic animals. Alterations in mitochondrial HK2 protein distribution and activity were also observed which may contribute to cardiomyocyte fragility. CONCLUSIONS: Hypoxia in early life causes lasting changes in left ventricular structure and function that may negatively influence myocardial and vascular responses to physiological stress in later life. These data have implications for the growing population of adults with repaired or palliated cyanotic congenital heart disease.

Characterization of a novel MK3 splice variant from murine ventricular myocardium.

p38 MAP kinase (MAPK) isoforms alpha, beta, and gamma, are expressed in the heart. p38alpha appears pro-apoptotic whereas p38beta is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2(-/-) mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38alpha/beta inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38alpha and p38beta, but a poor substrate for p38delta and p38gamma. GST-MK3.2 was poorly phosphorylated by p38alpha and p38beta and not phosphorylated by p38delta and p38gamma. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.

Effect of pressure overload-induced hypertrophy on the expression and localization of p38 MAP kinase isoforms in the mouse heart.

p38 mitogen-activated protein kinases (MAPKs) are serine/threonine specific protein kinases that respond to cellular stress and regulate a broad range of cellular activities. There are four major isoforms of p38 MAPK: alpha, beta, gamma, and delta. To date, the prominent isoform in heart has been thought to be p38alpha. We examined the expression of each p38 isoform at both the mRNA and protein level in murine heart. mRNA for all four p38 isoforms was detected. p38gamma and p38delta were expressed at protein levels comparable to p38alpha and 38beta, respectively. In the early phase of pressure-overload hypertrophy (1-7 days after constriction of the transverse aorta), the abundance of p38beta, p38gamma and p38delta mRNA increased; however, no corresponding changes were detected at the protein level. Confocal immunofluorescence studies revealed p38alpha and p38gamma in both the cytoplasm and nucleus. In the established phase of hypertrophy induced by chronic pressure overload (7-28 days after constriction of the transverse aorta), p38gamma immunoreactivity accumulated in the nucleus whereas the distribution of p38alpha remained unaffected. Hence, both p38alpha and p38gamma are prominent p38 isoforms in heart and p38gamma may play a role in mediating the changes in gene expression associated with cardiac remodeling during pressure-overload hypertrophy.

Characterization of the expression and regulation of MK5 in the murine ventricular myocardium.

MK5, a member of the MAPK-activated protein kinase family, is highly expressed in the heart. Whereas MK2 and MK3 are activated by p38 MAPK, MK5 has also been shown to be activated by ERK3 and ERK4. We studied the regulation of MK5 in mouse heart. mRNA for 5 splice variants (MK5.1-5.5), including the original form (MK5.1), was detected. MK5 comprises 14 exons: exon 12 splicing was modified in MK5.2, MK5.3, and MK5.5. MK5.2 and MK5.5 lacked 6 bases at the 3'-end of exon 12, whereas MK5.3 lacked exon 12, resulting in a frame shift and premature termination of translation at codon 3 of exon 13. MK5.4 and MK5.5 lacked exons 2-6, encoding kinase subdomains I-VI, and were kinase-dead. All 5 MK5 variants were detected at the mRNA level in all mouse tissues examined; however, their relative abundance was tissue-specific. Furthermore, the relative abundance of variant mRNA was altered both during hypertrophy and postnatal cardiac development, suggesting that the generation or the stability of MK5 variant mRNAs is subject to regulation. When expressed in HEK293 cells, MK5.1, MK5.2 and MK5.3 were nuclear whereas MK5.4 and MK5.5 were cytoplasmic. A p38 MAPK activator, anisomycin, induced the redistribution of each variant. In contrast, MK5 co-immunoprecipitated ERK3, but not ERK4 or p38 alpha, in control and hypertrophying hearts. GST pull-down assays revealed unbound ERK4 and p38 alpha but no free MK5 or ERK3 in heart lysates. Hence, 1) in heart MK5 complexes with ERK3 and 2) MK5 splice variants may mediate distinct effects thus increasing the functional diversity of ERK3-MK5 signaling.

Gbetagamma is a negative regulator of AP-1 mediated transcription.

Following stimulation of G protein-coupled receptors (GPCRs) at the cell surface, heterotrimeric G proteins are activated. Both Galpha and Gbetagamma subunits regulate specific effectors to transmit signals received by the receptor. Recent data suggest potential nuclear localization or translocation of the Gbetagamma subunit. Here, we show that co-expression of the Gbetagamma dimer decreased phorbol 12-myristate 13-acetate (PMA)-stimulated AP-1 gene reporter activity in HEK293 cells as well as the AP-1 dependent gonadotropin-releasing hormone-stimulated human follicle-stimulating hormone beta reporter activity in LbetaT2 gonadotrope cells. Further, we identify Fos transcription factors as novel interactors of the Gbeta1 subunit, using protein fragment complementation assays, as well as co-immunoprecipitation in vivo and in vitro. Fos proteins dimerize with Jun proteins to form activator protein-1 (AP-1) transcription factor complexes, which regulate target gene expression. Gbetagamma did not interfere with the dimerization of Fos and Jun or their ability to bind DNA. Rather, Gbetagamma co-localized with the AP-1 complex in the nucleus and recruited histone deacetylases (HDACs) to inhibit AP-1 transcriptional activity. Our data indicate a novel role for Gbetagamma subunits as transcriptional regulators.

Association of neonatal hypoxia with lasting changes in left ventricular gene expression: an animal model.

OBJECTIVE: Innovations in pediatric cardiovascular surgery have resulted in significant improvements in survival for children with congenital heart disease. In adults with such disease, however, surgical morbidity and mortality remain significant. We hypothesized that hypoxemia in early life causes lasting changes in gene expression in the developing heart and that such changes may persist into later life, affecting the physiology of the adult myocardium. METHODS: Microarray expression analyses were performed with left ventricular tissue from 10- and 90-day-old rats exposed to hypoxia (inspired oxygen fraction 0.12) for the first 10 days after birth then subsequently reared in ambient air and with tissue from age-matched rats reared entirely in ambient air. Changes in expression of selected genes were confirmed with real-time reverse transcriptase polymerase chain reaction. Left ventricular cardiomyocytes were isolated from adult animals in both groups, and cellular morphology and viability were compared. RESULTS: Microarray analyses revealed significant changes in 1945 and 422 genes in neonates and adults, respectively. Changes in genes associated with adaptive vascular remodeling and energy homeostasis, as well as regulation of apoptosis, were confirmed by real-time reverse transcriptase polymerase chain reaction. The viability of cardiomyocytes isolated from hypoxic animals was significantly lower than in those from control animals (36.7% +/- 13.3% vs 85.0% +/- 2.9%, P = .024). CONCLUSIONS: Neonatal hypoxia is associated with significant changes in left ventricular gene expression in both neonatal and adult rats. This may have physiologic implications for the adult myocardium.