RESUMO
L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVß2b. The CaV1.2 channel's C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels' basal activity in cardiomyocytes.
Assuntos
Canais de Cálcio Tipo L , Miócitos Cardíacos , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/química , Animais , Miócitos Cardíacos/metabolismo , Humanos , Células HEK293 , Ratos , Domínios ProteicosRESUMO
Chagas disease (CD) is caused by the parasitic protozoan T. cruzi. The progression of CD in ~30% of patients results in Chagasic Cardiomyopathy (CCM). Currently, it is known that the inflammatory system plays a significant role in the CCM. Interferon-gamma (IFN-γ) is the major cytokine involved in parasitemia control but has also been linked to CCM. The L-type calcium current (ICa,L) is crucial in the excitation/contraction coupling in cardiomyocytes. Thus, we compared ICa,L and the mechanical properties of cardiomyocytes isolated from infected wild type (WT) and IFN-γ(-/-) mice in the first stage of T. cruzi infection. Using the patch clamp technique, we demonstrated that the infection attenuated ICa,L in isolated cardiomyocytes from the right and left ventricles of WT mice at 15 days post-infection (dpi), which was not observed in the IFN-γ(-/-) cardiomyocytes. However, ICa,L was attenuated between 26 and 30 dpi in both experimental groups. Interestingly, the same profile was observed in the context of the mechanical properties of isolated cardiomyocytes from both experimental groups. Simultaneously, we tracked the mortality and MCP-1, TNF-α, IL-12, IL-6, and IL-10 serum levels in the infected groups. Importantly, the IFN-γ(-/-) and WT mice presented similar parasitemia and serum inflammatory markers at 10 dpi, indicating that the modifications in the cardiomyocyte functions observed at 15 dpi were directly associated with IFN-γ(-/-) deficiency. Thus, we showed that IFN-γ plays a crucial role in the electromechanical remodeling of cardiomyocytes during experimental T. cruzi infection in mice.
RESUMO
Functional and morphological modifications in the brain caused by major mood disorders involve many brain areas, including the hippocampus, leading to cognitive and mood alterations. Cav1.2 channel expression has been found to increase in animals with depressive-like behaviors. Calcium influx through these channels is associated with changes in excitation-transcriptional coupling by several intracellular signal pathways that are regulated by its C-terminus region. However, which of these signaling pathways is activated during the development of depressive-like behaviors is not known. Here, we evaluate the phosphorylation and expression levels of crucial kinases and transcription factors at the hippocampus of rats after 21 days of chronic restraint stress. Our results show that rats subjected to CRS protocol achieve less body weight, have heavier adrenal glands, and exhibit depression-like behaviors such as anhedonia, behavioral despair and decreased social interaction. Cav1.2 mRNA and protein expression levels, plus l-type calcium current amplitude, are also increased in treated rats when compared with control animals. Out of the three main signaling pathways activated by l-type currents, we only observed an increment of CaM-NFAT axis activity with the concomitant increment in Fas ligand expression. Thus, our results suggest that CRS activates specific pathways, and the increased expression of Cav1.2 could lead to neuronal death in the hippocampus.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Depressão/fisiopatologia , Hipocampo/metabolismo , Transdução de Sinais , Glândulas Suprarrenais , Anedonia , Animais , Comportamento Animal , Peso Corporal , Cálcio/metabolismo , Transtorno Depressivo Maior/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ligantes , Masculino , Neurônios/metabolismo , Fosforilação , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , NataçãoRESUMO
In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVß2. To date, five distinct CaVß2 transcriptional start site (TSS) variants (CaVß2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVß2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVß2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVß2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVß2a and smaller in CaVß2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVß2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVß2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Miócitos Cardíacos/metabolismo , Sítio de Iniciação de Transcrição , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/genética , Células Cultivadas , Ventrículos do Coração/citologia , Ventrículos do Coração/crescimento & desenvolvimento , Miócitos Cardíacos/fisiologia , RatosRESUMO
It was previously showed that aqueous leaf extract (AqEx) of Averrhoa carambola depresses the guinea pig atrial inotropism. Therefore, experiments were carried out on guineapig left atrium and on pituitary GH3 cells in order to evaluate the effect of AqEx on the cellular calcium infl ux. The atrium was mounted in an organ chamber (5 mL, Tyrode, 27 ± 0.1 °C, 95% O2, 5 % CO2), stretched to 10 mN, and paced at 2 Hz (0.5 ms, 400 V) and GH3 cells were submitted to a whole cell voltage clamp confi guration. In the atrium, the AqEx (1500 μg/mL) shifted to the right the concentration-effect curve of the positive inotropic effect produced by (±) BAY K 8644, an L-type calcium channel agonist. The AqEx increased EC50 (concentration required to promote 50% of the maximum effect) of the inotropic effect of BAY K 8644 from 7.8 ± 0.38 to 115.1 ± 0.44 nM (N = 3; p < 0.05). In GH3 cells assayed with 500 μg/mL of AqEx, the L-type calcium inward current declined 30 % (from 282 to 190 pA). Nevertheless, the extract did not change the voltage correspondent to the peak current. These data suggest that, at least in part, the negative inotropic effect of AqEx on the guinea pig atrium is due to a reduction of the L-type calcium current.
Em estudo prévio mostrou-se que o extrato aquoso das folhas de Averrhoacarambola (ExAq) reduziu o inotropismo atrial da cobaia. Por isso, este trabalho avaliou se o ExAq interfere com o infl uxo de cálcio através da membrana celular. A investigação foi conduzidaem átrio esquerdo de cobaia, montado em cuba (5 mL, Tyrode, 27 ± 0,1 °C, 95 % O2, 5 % CO2), estirado para uma tensão de repouso de 10 mN e submetido a uma estimulação de 2 Hz (0,5 ms, 400 V). O efeito do ExAq sobre a entrada de cálcio nas células foi avaliado em átrio de cobaia e em células GH3, estas submetidas a patch clamp na confi guração whole cell. No átrio, o ExAq (1500 μg /mL) deslocou para direita a curva concentração-efeito do (±) BAY K 8644 (agonista dos canais de cálcio tipo-L), aumentando a CE50 (concentração capaz de produzir 50 % do efeito máximo) de 7,8 ± 0,38 para 115,1 ± 0,44 nM (N = 3, p < 0,05). Em células GH3, este extrato (500 μg /mL) reduziu de 282 para 190 pA (30 %) a corrente de cálcio, sem contudo alterar a voltagem de pico da curva desta corrente. Estes resultados mostram que, pelo menos em parte, o efeito inotrópico negativo do ExAq em átrio de cobaia se deve a uma diminuição do infl uxo de cálcio pelos canais tipo-L.