Left Atrial Myocardium in Arterial Hypertension.

Arterial hypertension affects ≈ 1 billion people worldwide. It is associated with increased morbidity and mortality and responsible for millions of deaths each year. Hypertension mediates damage of target organs including the heart. In addition to eliciting left ventricular hypertrophy, dysfunction and heart failure, hypertension also causes left atrial remodeling that may culminate in atrial contractile dysfunction and atrial fibrillation. Here, we will summarize data on the various aspects of left atrial remodeling in (essential) hypertension gathered from studies on patients with hypertension and from spontaneously hypertensive rats, an animal model that closely mimics cardiac remodeling in human hypertension. Analyzing the timeline of remodeling processes, i.e., distinguishing between alterations occurring in prehypertension, in early hypertension and during advanced hypertensive heart disease, we will derive the potential mechanisms underlying left atrial remodeling in (essential) hypertension. Finally, we will discuss the consequences of these remodeling processes for atrial and ventricular function. The data imply that left atrial remodeling is multifactorial, starts early in hypertension and is an important contributor to the progression of hypertensive heart disease, including the development of atrial fibrillation and heart failure.

Activation of PKC results in improved contractile effects and Ca2+ cycling by inhibition of PP2A-B56α.

Protein phosphatase 2A (PP2A) represents a heterotrimer that is responsible for the dephosphorylation of important regulatory myocardial proteins. This study was aimed to test whether the phosphorylation of PP2A-B56α at Ser41 by PKC is involved in the regulation of myocyte Ca2+ cycling and contraction. For this purpose, heart preparations of wild-type (WT) and transgenic mice overexpressing the nonphosphorylatable S41A mutant form (TG) were stimulated by administration of the direct PKC activator phorbol 12-myristate 13-acetate (PMA), and functional effects were studied. PKC activation was accompanied by the inhibition of PP2A activity in WT cardiomyocytes, whereas this effect was absent in TG. Consistently, the increase in the sarcomere length shortening and the peak amplitude of Ca2+ transients after PMA administration in WT cardiomyocytes was attenuated in TG. However, the costimulation with 1 µM isoprenaline was able to offset these functional deficits. Moreover, TG hearts did not show an increase in the phosphorylation of the myosin-binding protein C after administration of PMA but was detected in corresponding WT. PMA modulated voltage-dependent activation of the L-type Ca2+ channel (LTCC) differently in the two genotypes, shifting V1/2a by +1.5 mV in TG and by -2.4 mV in WT. In the presence of PMA, ICaL inactivation remained unchanged in TG, whereas it was slower in corresponding WT. Our data suggest that PKC-activated enhancement of myocyte contraction and intracellular Ca2+ signaling is mediated by phosphorylation of B56α at Ser41, leading to a decrease in PP2A activity.NEW & NOTEWORTHY The importance of the serine-41 phosphorylation site on B56α in reducing PP2A activity was demonstrated for the first time using a transgenic mutation model. Direct activation of PKC inhibits PP2A, leading to increased phosphorylation of MyBP-C in cardiomyocytes. The increased phosphorylation of contractile proteins is influenced by the PKC-phosphoB56α-PP2A signaling cascade resulting in improved intracellular Ca2+ handling and enhanced contractility and relaxation. PKC-mediated inhibition of PP2A also leads to modulation of the LTCC activation and inactivation kinetics.

Inward Rectifier K+ Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM-IbΔC-X.

BACKGROUND Transgenic mice (TG) with heart-directed overexpresion of the isoform of the transcription factor cyclic adenosine monophosphate response element modulator (CREM), CREM-IbΔC-X, display spontaneous atrial fibrillation (AF) and action potential prolongation. The remodeling of the underlying ionic currents remains unknown. Here, we investigated the regulatory role of CREM-IbΔC-X on the expression of K+ channel subunits and the corresponding K+ currents in relation to AF onset in TG atrial myocytes. METHODS AND RESULTS ECG recordings documented the absence or presence of AF in 6-week-old (before AF onset) and 12-week-old TG (after AF onset) and wild-type littermate mice before atria removal to perform patch clamp, contractility, and biochemical experiments. In TG atrial myocytes, we found reduced repolarization reserve K+ currents attributed to a decrease of transiently outward current and inward rectifier K+ current with phenotype progression, and of acetylcholine-activated K+ current, age independent. The molecular determinants of these changes were lower mRNA levels of Kcnd2/3, Kcnip2, Kcnj2/4, and Kcnj3/5 and decreased protein levels of K+ channel interacting protein 2 (KChIP2 ), Kir2.1/3, and Kir3.1/4, respectively. After AF onset, inward rectifier K+ current contributed less to action potential repolarization, in line with the absence of outward current component, whereas the acetylcholine-induced action potential shortening before AF onset (6-week-old TG mice) was smaller than in wild-type and 12-week-old TG mice. Atrial force of contraction measured under combined vagal-sympathetic stimulation revealed increased sensitivity to isoprenaline irrespective of AF onset in TG. Moreover, we identified Kcnd2, Kcnd3, Kcnj3, and Kcnh2 as novel CREM-target genes. CONCLUSIONS Our study links the activation of cyclic adenosine monophosphate response element-mediated transcription to the proarrhythmogenic electrical remodeling of atrial inward rectifier K+ currents with a role in action potential duration, resting membrane stability, and vagal control of the electrical activity.

Induction of ICER is superseded by smICER, challenging the impact of ICER under chronic beta-adrenergic stimulation.

ICER (inducible cAMP early repressor) isoforms are transcriptional repressors encoded by the Crem (cAMP responsive element modulator) gene. They were linked to the regulation of a multitude of cellular processes and pathophysiological mechanisms. Here, we show for the first time that two independent induction patterns for CREM repressor isoforms exist in the heart, namely for ICER and smICER (small ICER), which are induced in response to ß-adrenergic stimulation in a transient- and saturation-like manner, respectively. This time-shifted induction pattern, driven by two internal promoters in the Crem gene, leads to the predominant transcription of smIcer after prolonged ß-adrenergic stimulation. Using an ICER knockout mouse model with preserved smICER induction, we show that the transient-like induction of Icer itself has minor effects on gene regulation, cardiac hypertrophy or contractile function in the heart. We conclude that the functions previously linked to ICER may be rather attributed to smICER, also beyond the cardiac background.

Annexin A4 N-terminal peptide inhibits adenylyl cyclase 5 and limits ß-adrenoceptor-mediated prolongation of cardiac action potential.

Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over ß-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, ß-adrenoceptor (ßAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.

HDAC (Histone Deacetylase) Inhibitor Valproic Acid Attenuates Atrial Remodeling and Delays the Onset of Atrial Fibrillation in Mice.

BACKGROUND: A structural, electrical and metabolic atrial remodeling is central in the development of atrial fibrillation (AF) contributing to its initiation and perpetuation. In the heart, HDACs (histone deacetylases) control remodeling associated processes like hypertrophy, fibrosis, and energy metabolism. Here, we analyzed, whether the HDAC class I/IIa inhibitor valproic acid (VPA) is able to attenuate atrial remodeling in CREM-IbΔC-X (cAMP responsive element modulator isoform IbΔC-X) transgenic mice, a mouse model of extensive atrial remodeling with age-dependent progression from spontaneous atrial ectopy to paroxysmal and finally long-lasting AF. METHODS: VPA was administered for 7 or 25 weeks to transgenic and control mice. Atria were analyzed macroscopically and using widefield and electron microscopy. Action potentials were recorded from atrial cardiomyocytes using patch-clamp technique. ECG recordings documented the onset of AF. A proteome analysis with consecutive pathway mapping identified VPA-mediated proteomic changes and related pathways. RESULTS: VPA attenuated many components of atrial remodeling that are present in transgenic mice, animal AF models, and human AF. VPA significantly ( P<0.05) reduced atrial dilatation, cardiomyocyte enlargement, atrial fibrosis, and the disorganization of myocyte's ultrastructure. It significantly reduced the occurrence of atrial thrombi, reversed action potential alterations, and finally delayed the onset of AF by 4 to 8 weeks. Increased histone H4-acetylation in atria from VPA-treated transgenic mice verified effective in vivo HDAC inhibition. Cardiomyocyte-specific genetic inactivation of HDAC2 in transgenic mice attenuated the ultrastructural disorganization of myocytes comparable to VPA. Finally, VPA restrained dysregulation of proteins in transgenic mice that are involved in a multitude of AF relevant pathways like oxidative phosphorylation or RhoA (Ras homolog gene family, member A) signaling and disease functions like cardiac fibrosis and apoptosis of muscle cells. CONCLUSIONS: Our results suggest that VPA, clinically available, well-tolerated, and prescribed to many patients for years, has the therapeutic potential to delay the development of atrial remodeling and the onset of AF in patients at risk.

Progressive impairment of atrial myocyte function during left ventricular hypertrophy and heart failure.

Hypertensive heart disease (HHD) can cause left ventricular (LV) hypertrophy and heart failure (HF). It is unclear, though, which factors may contribute to the transition from compensated LV hypertrophy to HF in HHD. We hypothesized that maladaptive atrial remodeling with impaired atrial myocyte function would occur in advanced HHD and may be associated with the emergence of HF. Experiments were performed on atrial myocytes and tissue from old (15-25months) normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) with advanced HHD. Based on the absence or presence of elevated lung weight, a sign of lung congestion and heart failure, SHR were divided into a non-failing (SHR-NF) and failing (SHR-HF) group. Compared with WKY, SHR exhibited elevated blood pressure, LV hypertrophy and left atrial (LA) hypertrophy with increased LA expression of markers of hypertrophy and fibrosis. SHR-HF were distinguished from SHR-NF by aggravated hypertrophy and fibrosis. SHR-HF atrial myocytes exhibited reduced contractility and impaired SR Ca2+ handling. Moreover, in SHR the expression and phosphorylation of SR Ca2+-regulating proteins (SERCA2a, calsequestrin, RyR2 and phospholamban) showed negative correlation with increasing lung weight. Increasing stimulation frequency (1-2-4Hz) of atrial myocytes caused a progressive increase in arrhythmogenic Ca2+ release (including alternans), which was observed most frequently in SHR-HF. Thus, in old SHR with advanced HHD there is profound structural and functional atrial remodeling. The occurrence of HF in SHR is associated with LA and RA hypertrophy, increased atrial fibrosis, impaired atrial myocyte contractility and SR Ca2+ handling and increased propensity for arrhythmogenic Ca2+ release. Therefore, functional remodeling intrinsic to atrial myocytes may contribute to the transition from compensated LV hypertrophy to HF in advanced HHD and an increased propensity of atrial arrhythmias in HF.

Characterization of the Genetic Program Linked to the Development of Atrial Fibrillation in CREM-IbΔC-X Mice.

BACKGROUND: Reduced expression of genes regulated by the transcription factors CREB/CREM (cAMP response element-binding protein/modulator) is linked to atrial fibrillation (AF) susceptibility in patients. Cardiomyocyte-directed expression of the inhibitory CREM isoform CREM-IbΔC-X in transgenic mice (TG) leads to spontaneous-onset AF preceded by atrial dilatation and conduction abnormalities. Here, we characterized the altered gene program linked to atrial remodeling and development of AF in CREM-TG mice. METHODS AND RESULTS: Atria of young (TGy, before AF onset) and old (TGo, after AF onset) TG mice were investigated by mRNA microarray profiling in comparison with age-matched wild-type controls (WTy/WTo). Proteomic alterations were profiled in young mice (8 TGy versus 8 WTy). Annotation of differentially expressed genes revealed distinct differences in biological functions and pathways before and after onset of AF. Alterations in metabolic pathways, some linked to altered peroxisome proliferator-activated receptor signaling, muscle contraction, and ion transport were already present in TGy. Electron microscopy revealed significant loss of sarcomeres and mitochondria and increased collagen and glycogen deposition in TG mice. Alterations in electrophysiological pathways became prominent in TGo, concomitant with altered gene expression of K+-channel subunits and ion channel modulators, relevant in human AF. CONCLUSIONS: The most prominent alterations of the gene program linked to CREM-induced atrial remodeling were identified in the expression of genes related to structure, metabolism, contractility, and electric activity regulation, suggesting that CREM transgenic mice are a valuable experimental model for human AF pathophysiology.

Enhanced nucleoplasmic Ca2+ signaling in ventricular myocytes from young hypertensive rats.

Arterial hypertension causes left ventricular (LV) myocyte hypertrophy. Alterations in nuclear Ca2+ may be involved in regulation of histone acetylation, transcription and hypertrophy. Regulation of nuclear Ca2+ in hypertension, however, is unknown. Therefore, we elucidated cellular mechanisms underlying nuclear Ca2+ regulation in LV myocytes from hypertensive versus normotensive rats and evaluated possible consequences for Ca2+-dependent regulation of histone acetylation. LV myocytes and myocyte nuclei were isolated from young spontaneously hypertensive rats (SHR) shortly after development of hypertension. Normotensive Wistar-Kyoto rats (WKY) served as controls. Cytoplasmic and nucleoplasmic Ca2+ transients (CaTs) were imaged simultaneously using linescan confocal microscopy and Fluo-4. LV myocytes and nuclei from SHR exhibited hypertrophy. Cytoplasmic and nucleoplasmic CaTs were increased in SHR. The increase in nucleoplasmic Ca2+, however, exceeded the increase in cytoplasmic Ca2+, indicating enhanced nuclear Ca2+ signaling in SHR. Ca2+ load of sarcoplasmic reticulum and perinuclear Ca2+ stores was also increased in SHR, while fractional release from both stores remained unchanged. Intranuclear Ca2+ propagation was accelerated in SHR, associated with preserved density of nuclear envelope invaginations and elevated nuclear expression of nucleoporins and SR-Ca2+-ATPase, SERCA2a. Nuclear Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) expression was elevated and histone deacetylases exhibited redistribution from nucleus to cytosol associated with increased histone acetylation in SHR. Thus, in early hypertension, there is remodeling of nuclear Ca2+ handling resulting in enhanced nuclear Ca2+ signaling. Enhanced nuclear Ca2+ signaling, in turn, increases nuclear localization and activity of CaMKIIδ driving nuclear export of histone deacetylases and increased histone acetylation.

The angiotensin receptor-associated protein Atrap is a stimulator of the cardiac Ca2+-ATPase SERCA2a.

AIMS: The angiotensin II type 1 receptor-associated protein (Atrap) is highly expressed in the heart, but its function in the heart is unknown. We hypothesized that cardiac Atrap may interact with proteins other than the AT1 receptor. METHODS AND RESULTS: To identify potential novel interacting partners of Atrap, pull-down assays were performed. Sequencing by MALDI-MS of the isolated complexes showed that Atrap interacts with the cardiac Ca(2+)-ATPase SERCA2a. The interaction between Atrap and SERCA2a was confirmed by co-immunoprecipitation and by surface plasmon resonance (SPR) spectroscopy. Atrap enhanced the SERCA-dependent Ca(2+) uptake in isolated SR membrane vesicles. Furthermore, sarcomere shortenings and [Ca(2+)]i transients (CaTs) were determined in ventricular myocytes isolated from Atrap-/- and wild-type (WT) mice. The amplitudes of CaTs and sarcomere shortenings were similar in Atrap-/- and WT myocytes. However, the CaT decay and sarcomere re-lengthening were prolonged in Atrap-/- myocytes. To further evaluate the functional relevance of the Atrap-SERCA2a interaction in vivo, left-ventricular function was assessed in WT and Atrap-/- mice. The heart rates (564 ± 10 b.p.m. vs. 560 ± 11 b.p.m.; P = 0.80) and ejection fractions (71.3 ± 1.3 vs. 72 ± 1.8%; P = 0.79) were similar in WT and Atrap-/- mice, respectively (n = 15 for each genotype). However, the maximum filling rate (dV/dtmax) was markedly decreased in Atrap-/- (725 ± 48 µL/s) compared with WT mice (1065 ± 122 µL/s; P = 0.01; n = 15). CONCLUSION: We identified Atrap as a novel regulatory protein of the cardiac Ca(2+)-ATPase SERCA2a. We suggest that Atrap enhances the activity of SERCA2a and, consequently, facilitates ventricular relaxation.

Early subcellular Ca2+ remodelling and increased propensity for Ca2+ alternans in left atrial myocytes from hypertensive rats.

AIMS: Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias. METHODS AND RESULTS: Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls. Early after the onset of hypertension, at 3 months of age, there were no differences in Ca2+ transients (CaTs) or expression and phosphorylation of Ca2+ handling proteins between SHR and WKY. At 7 months of age, when left ventricular (LV) hypertrophy had progressed and markers of fibrosis were increased in left atrium, CaTs (at 1 Hz stimulation) were still unchanged. Subcellular alterations in Ca2+ handling were observed, however, in SHR atrial myocytes including (i) reduced expression of the α1C subunit of and reduced Ca2+ influx through L-type Ca2+ channels, (ii) reduced expression of ryanodine receptors with increased phosphorylation at Ser2808, (iii) decreased activity of the Na+ / Ca2+ exchanger (at unaltered intracellular Na+ concentration), and (iv) increased SR Ca2+ load with reduced fractional release. These changes were associated with an increased propensity of SHR atrial myocytes to develop frequency-dependent, arrhythmogenic Ca2+ alternans. CONCLUSIONS: In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become more prone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

Urocortin 2 stimulates nitric oxide production in ventricular myocytes via Akt- and PKA-mediated phosphorylation of eNOS at serine 1177.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca(2+)-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]i and cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]i and cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

Regulation and function of Cav3.1 T-type calcium channels in IGF-I-stimulated pulmonary artery smooth muscle cells.

Arterial smooth muscle cells enter the cell cycle and proliferate in conditions of disease and injury, leading to adverse vessel remodeling. In the pulmonary vasculature, diverse stimuli cause proliferation of pulmonary artery smooth muscle cells (PASMCs), pulmonary artery remodeling, and the clinical condition of pulmonary hypertension associated with significant health consequences. PASMC proliferation requires extracellular Ca(2+) influx that is intimately linked with intracellular Ca(2+) homeostasis. Among the primary sources of Ca(2+) influx in PASMCs is the low-voltage-activated family of T-type Ca(2+) channels; however, up to now, mechanisms for the action of T-type channels in vascular smooth muscle cell proliferation have not been addressed. The Ca(v)3.1 T-type Ca(2+) channel mRNA is upregulated in cultured PASMCs stimulated to proliferate with insulin-like growth factor-I (IGF-I), and this upregulation depends on phosphatidylinositol 3-kinase/Akt signaling. Multiple stimuli that trigger an acute rise in intracellular Ca(2+) in PASMCs, including IGF-I, also require the expression of Ca(v)3.1 Ca(2+) channels for their action. IGF-I also led to cell cycle initiation and proliferation of PASMCs, and, when expression of the Ca(v)3.1 Ca(2+) channel was knocked down by RNA interference, so were the expression and activation of cyclin D, which are necessary steps for cell cycle progression. These results confirm the importance of T-type Ca(2+) channels in proper progression of the cell cycle in PASMCs stimulated to proliferate by IGF-I and suggest that Ca(2+) entry through Ca(v)3.1 T-type channels in particular interacts with Ca(2+)-dependent steps of the mitogenic signaling cascade as a central component of vascular remodeling in disease.

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels.

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.

T-type calcium channels are regulated by hypoxia/reoxygenation in ventricular myocytes.

Low-voltage-activated calcium channels are reexpressed in ventricular myocytes in pathological conditions associated with hypoxic episodes, but a direct relation between oxidative stress and T-type channel function and regulation in cardiomyocytes has not been established. We aimed to investigate low-voltage-activated channel regulation under oxidative stress in neonatal rat ventricular myocytes. RT-PCR measurements of voltage-gated Ca(2+) (Ca(v))3.1 and Ca(v)3.2 mRNA levels in oxidative stress were compared with whole cell patch-clamp recordings of T-type calcium current. The results indicate that hypoxia reduces T-type current density at -30 mV (the hallmark of this channel) based on the shift of the voltage dependence of activation to more depolarized values and downregulation of Ca(v)3.1 at the mRNA level. Upon reoxygenation, both Ca(v)3.1 mRNA levels and the voltage dependence of total T-type current are restored, although differently for activation and inactivation. Using Ni(2+), we distinguished different effects of hypoxia/reoxygenation on the two current components. Long-term incubation in the presence of 100 microM CoCl(2) reproduced the effects of hypoxia on T-type current activation and inactivation, indicating that the chemically induced oxidative state is sufficient to alter T-type calcium current activity, and that hypoxia-inducible factor-1alpha is involved in Ca(v)3.1 downregulation. Our results demonstrate that Ca(v)3.1 and Ca(v)3.2 T-type calcium channels are differentially regulated by hypoxia/reoxygenation injury, and, therefore, they may serve different functions in the myocyte in response to hypoxic injury.

Uncompetitive antagonism of AMPA receptors: Mechanistic insights from studies of polyamine toxin derivatives.

Philanthotoxins are uncompetitive antagonists of Ca2+-permeable AMPA receptors presumed to bind to the pore-forming region, but a detailed molecular mechanism for this interaction is missing. Here a small library of novel philanthotoxins was designed and synthesized using a solid-phase strategy. The biological activities were investigated at cloned and "native" AMPA receptors using electrophysiological techniques. A distinct relationship between length of the polyamine moiety and the location of a secondary amino group was observed. Fitting the data to the Woodhull equation allowed the first experimental demonstration of the relative location and orientation of the philanthotoxin molecule in the receptor. These results were corroborated by in silico studies using a homology model of the AMPA receptor ion channel. Together these studies provide strong evidence for a molecular mechanism by which polyamine toxins antagonize the AMPA receptor ion channel and provide the basis for rational development of uncompetitive antagonists of AMPA receptors.

The cellular mechanisms involved in the vasodilator effect of nebivolol on the renal artery.

Nebivolol is known as a highly selective beta1-adrenoceptor antagonist. Based on the reported vasodilator effect of nebivolol, we examined the cellular mechanisms by which the drug induces renal artery vasodilation, an issue of potential relevance for condition associated with high blood pressure. To this purpose, myograph and patch-clamp techniques were used. Small mouse renal arteries were placed in the myograph chamber, and after the optimal concentration for the vasodilator effect of nebivolol was established (50 microM), the arteries were further investigated to assess the potential contribution of nitric oxide (NO) and of Ca2+ ions to the nebivolol-induced effect, by exposing the arteries to the specific inhibitors such as N(G)-nitro-L-arginine methylester (L-NAME, 100 microM), ethylenglycol-bis-(beta-amino-ethylen ester) N,N'-tetraacetic acid (EGTA, 4 microM) and thapsigargin (1 microM). The expression of NO synthase was evaluated by the Western-blot technique. Using myograph and patch-clamp techniques applied on intact renal artery, we investigated the role of beta2-adrenoceptors, of myoendothelial junctions and of Ca(2+)-activated K+ channels in the vasodilatory effects of nebivolol, using 100 microM butoxamine, 40 microM 18 beta-glycyrrhetinic acid, 1 mM tetraethylammonium, and 100 nM iberiotoxin, respectively. The results showed that the cellular mechanisms of the vasodilator effect of nebivolol on the renal artery entail (i) activation of the endothelial beta2-adrenoceptor, (ii) participation of [Ca2+]i, (iii) increase in NO and eNOS, and (iv) activation of Ca(2+)-activated K+ channels. The cellular mechanisms underlying vasodilator effect of nebivolol on the artery explain the favorable effect of this drug in hypertension.

Methoctramine analogues inhibit responses to capsaicin and protons in rat dorsal root ganglion neurons.

We have investigated the possibility that vanilloid receptors have a binding site for polyamines and determined the consequences of binding to such a site. Whole-cell and single-channel patch-clamp recordings were used to investigate the effect of the tetraamine, methoctramine, and 16 of its analogues on capsaicin and proton induced responses of foetal rat dorsal root ganglion neurons. All but two methoctramine analogues inhibited responses to 10 microM capsaicin with IC50 values in the range of 2-70 microM at a holding potential of -100 mV. Inhibition was generally non-competitive and voltage-dependent. Methoctramine at 10 microM reduced the single channel mean open time (>3-fold), but also increased the mean closed time (1.7-fold). Sustained responses to pH 5.4 were antagonized by methoctramine with similar potency to capsaicin responses. Similar data were obtained with adult rat dorsal root ganglion neurons. These data indicate that methoctramine analogues bind to vanilloid receptors to inhibit their function.

A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction.

Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold-sensitive neurones were pre-selected by screening for an increase in [Ca(2+)](i) on cooling; 49 % of them were also excited by 0.5 microM capsaicin. Action potentials and voltage-gated currents of cold-sensitive neurones were clearly distinct from those of cold-insensitive neurones. All cold-sensitive neurones expressed an inward current activated by cold and sensitised by (-)-menthol, which was absent from cold-insensitive neurones. This current was carried mainly by Na(+) ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage-gated Ca(2+) entry; a minor fraction of Ca(2+) entry was voltage-independent. Application of (-)-menthol shifted the threshold temperatures of the cold-induced depolarisation and the inward current to the same extent, indicating that the cold- and menthol-activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)-isomer being a less effective agonist than the (-)-isomer. Extracellular Ca(2+) modulated the cold- and menthol-activated current in a similar way to its action on intact cold receptors: lowered [Ca(2+)](o) sensitised the current, while raised [Ca(2+)](o) antagonised the menthol-induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca(2+) and was mediated by a rise in [Ca(2+)](i). This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin-sensitive neurones, capsaicin application caused a profound depression of the cold-activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold-activated current towards warmer temperatures. The current was blocked by 50 microM capsazepine and 100 microM SKF 96365. We conclude that the cold- and menthol-activated current is the major mechanism responsible for cold-induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.