Florentina Pluteanu

Caveolin-3 Regulates Protein Kinase A Modulation of the CaV3.2 (α1H) T-type Ca2+ Channels

Voltage-gated T-type Ca2+ channel Cav3.2 (α1H) subunit, responsible for T-type Ca2+ current, is expressed in different tissues and participates in Ca2+ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Cav3.2 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 Ca2+ in different cells. We examined the localization and regulation of the Cav3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Cav3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Cav3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Cav3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Cav3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (ICa,T) and adenovirus (AdCav3.2)-mediated increase in peak Cav3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of ICa,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Cav3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca2+ signaling during normal and pathological processes.

Caveolin-3 regulates the protein kinase A modulation of CaV3.2 (α1H) T-type Ca2+ channels

Voltage-gated T-type Ca2+ channel Cav3.2 (α1H) subunit, responsible for T-type Ca2+ current, is expressed in different tissues and participates in Ca2+ entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Cav3.2 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 Ca2+ in different cells. We examined the localization and regulation of the Cav3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Cav3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Cav3.1 and Cav3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Cav3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Cav3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Cav3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (ICa,T) and adenovirus (AdCav3.2)-mediated increase in peak Cav3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of ICa,T by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Cav3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca2+ signaling during normal and pathological processes.

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.

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.

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.

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.

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.

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.