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1.
Am J Physiol Heart Circ Physiol ; 326(3): H599-H611, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38180453

RESUMO

Patient-derived induced pluripotent stem cells (iPSCs) can be differentiated into atrial and ventricular cardiomyocytes to allow for personalized drug screening. A hallmark of differentiation is the manifestation of spontaneous beating in a two-dimensional (2-D) cell culture. However, an outstanding observation is the high variability in this maturation process. We valued that contractile parameters change during differentiation serving as an indicator of maturation. Consequently, we recorded noninvasively spontaneous motion activity during the differentiation of male iPSC toward iPSC cardiomyocytes (iPSC-CMs) to further analyze similar maturated iPSC-CMs. Surprisingly, our results show that identical differentiations into ventricular iPSC-CMs are variable with respect to contractile parameters resulting in two distinct subpopulations of ventricular-like cells. In contrast, differentiation into atrial iPSC-CMs resulted in only one phenotype. We propose that the noninvasive and cost-effective recording of contractile activity during maturation using a smartphone device may help to reduce the variability in results frequently reported in studies on ventricular iPSC-CMs.NEW & NOTEWORTHY Differentiation of induced pluripotent stem cells (iPSCs) into iPSC-derived cardiomyocytes (iPSC-CMs) exhibits a high variability in mature parameters. Here, we monitored noninvasively contractile parameters of iPSC-CM during full-time differentiation using a smartphone device. Our results show that parallel maturations of iPSCs into ventricular iPSC-CMs, but not into atrial iPSC-CMs, resulted in two distinct subpopulations of iPSC-CMs. These findings suggest that our cost-effective method may help to compare iPSC-CMs at the same maturation level.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Masculino , Miócitos Cardíacos , Diferenciação Celular , Fenótipo , Ventrículos do Coração
2.
Life Sci Alliance ; 7(2)2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38012000

RESUMO

In the heart, genetic or acquired mishandling of diastolic [Ca2+] by ryanodine receptor type 2 (RyR2) overactivity correlates with risks of arrhythmia and sudden cardiac death. Strategies to avoid these risks include decrease of Ca2+ release by drugs modulating RyR2 activity or increase in Ca2+ uptake by drugs modulating SR Ca2+ ATPase (SERCA2a) activity. Here, we combine these strategies by developing experimental compounds that act simultaneously on both processes. Our screening efforts identified the new 1,4-benzothiazepine derivative GM1869 as a promising compound. Consequently, we comparatively studied the effects of the known RyR2 modulators Dantrolene and S36 together with GM1869 on RyR2 and SERCA2a activity in cardiomyocytes from wild type and arrhythmia-susceptible RyR2R2474S/+ mice by confocal live-cell imaging. All drugs reduced RyR2-mediated Ca2+ spark frequency but only GM1869 accelerated SERCA2a-mediated decay of Ca2+ transients in murine and human cardiomyocytes. Our data indicate that S36 and GM1869 are more suitable than dantrolene to directly modulate RyR2 activity, especially in RyR2R2474S/+ mice. Remarkably, GM1869 may represent a new dual-acting lead compound for maintenance of diastolic [Ca2+].


Assuntos
Dantroleno , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Humanos , Camundongos , Arritmias Cardíacas/metabolismo , Transporte Biológico , Dantroleno/farmacologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
3.
J Med Chem ; 66(23): 15761-15775, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-37991191

RESUMO

To discover new multifunctional agents for the treatment of cardiovascular diseases, we designed and synthesized a series of compounds with a cyclopropyl alcohol moiety and evaluated them in biochemical assays. Biological screening identified derivatives with dual activity: preventing Ca2+ leak through ryanodine receptor 2 (RyR2) and enhancing cardiac sarco-endoplasmic reticulum (SR) Ca2+ load by activation of Ca2+-dependent ATPase 2a (SERCA2a). The compounds that stabilize RyR2 at micro- and nanomolar concentrations are either structurally related to RyR-stabilizing drugs or Rycals or have structures similar to them. The novel compounds also demonstrate a good ability to increase ATP hydrolysis mediated by SERCA2a activity in cardiac microsomes, e.g., the half-maximal effective concentration (EC50) was as low as 383 nM for compound 12a, which is 1,4-benzothiazepine with two cyclopropanol groups. Our findings indicate that these derivatives can be considered as new lead compounds to improve cardiac function in heart failure.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina , Retículo Sarcoplasmático , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Miócitos Cardíacos , Canal de Liberação de Cálcio do Receptor de Rianodina/farmacologia , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Tiazepinas/química , Tiazepinas/farmacologia
4.
J Mol Cell Cardiol ; 165: 141-157, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35033544

RESUMO

Axial tubule junctions with the sarcoplasmic reticulum control the rapid intracellular Ca2+-induced Ca2+ release that initiates atrial contraction. In atrial myocytes we previously identified a constitutively increased ryanodine receptor (RyR2) phosphorylation at junctional Ca2+ release sites, whereas non-junctional RyR2 clusters were phosphorylated acutely following ß-adrenergic stimulation. Here, we hypothesized that the baseline synthesis of 3',5'-cyclic adenosine monophosphate (cAMP) is constitutively augmented in the axial tubule junctional compartments of atrial myocytes. Confocal immunofluorescence imaging of atrial myocytes revealed that junctin, binding to RyR2 in the sarcoplasmic reticulum, was densely clustered at axial tubule junctions. Interestingly, a new transgenic junctin-targeted FRET cAMP biosensor was exclusively co-clustered in the junctional compartment, and hence allowed to monitor cAMP selectively in the vicinity of junctional RyR2 channels. To dissect local cAMP levels at axial tubule junctions versus subsurface Ca2+ release sites, we developed a confocal FRET imaging technique for living atrial myocytes. A constitutively high adenylyl cyclase activity sustained increased local cAMP levels at axial tubule junctions, whereas ß-adrenergic stimulation overcame this cAMP compartmentation resulting in additional phosphorylation of non-junctional RyR2 clusters. Adenylyl cyclase inhibition, however, abolished the junctional RyR2 phosphorylation and decreased L-type Ca2+ channel currents, while FRET imaging showed a rapid cAMP decrease. In conclusion, FRET biosensor imaging identified compartmentalized, constitutively augmented cAMP levels in junctional dyads, driving both the locally increased phosphorylation of RyR2 clusters and larger L-type Ca2+ current density in atrial myocytes. This cell-specific cAMP nanodomain is maintained by a constitutively increased adenylyl cyclase activity, contributing to the rapid junctional Ca2+-induced Ca2+ release, whereas ß-adrenergic stimulation overcomes the junctional cAMP compartmentation through cell-wide activation of non-junctional RyR2 clusters.


Assuntos
Adenilil Ciclases , Canal de Liberação de Cálcio do Receptor de Rianodina , Adenilil Ciclases/metabolismo , Adrenérgicos , Cálcio/metabolismo , Sinalização do Cálcio , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo
5.
Front Physiol ; 12: 777770, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34955889

RESUMO

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E GSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E GSH increase. Importantly, ß-adrenergic stimulation resulted in excessive E GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically ß-adrenergic stimulation significantly increased mitochondrial E GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial E GSH oxidization in RyR2-R2474S cardiomyocytes.

7.
Pflugers Arch ; 467(10): 2229-34, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25547873

RESUMO

Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.


Assuntos
Cardiomegalia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Cardiomegalia/patologia , Fibrose , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Canais de Cátion TRPC/genética
8.
Phytomedicine ; 21(6): 787-92, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24680617

RESUMO

Tetra-acetylajugasterone C (TAAC) was found to be one of the naturally occurring compounds of the Cameroonian medicinal plant Vitex cienkowskii which is responsible for a vasorelaxant activity of an extract of this plant. The evaluation of the underlying mechanisms for the relaxing effect of TAAC was determined using aortic rings of rats and mice. TAAC produced a concentration-dependent relaxation in rat artery rings pre-contracted with 1µM noradrenaline (IC50: 8.40µM) or 60mM KCl (IC50: 36.30µM). The nitric oxide synthase inhibitor l-NAME (100µM) and the soluble guanylate cyclase inhibitor ODQ (10µM) significantly attenuated the vasodilatory effect of TAAC. TAAC also exerted a relaxing effect in aorta of wild-type mice (cGKI(+/+); IC50=13.04µM) but a weaker effect in aorta of mice lacking cGMP-dependent protein kinase I (cGKI(-/-); IC50=36.12µM). The involvement of calcium channels was studied in rings pre-incubated in calcium-free buffer and primed with 1µM noradrenaline prior to addition of calcium to elicit contraction. TAAC (100µM) completely inhibited the resulting calcium-induced vasoconstriction. The same concentration of TAAC showed a stronger effect on the tonic than on the phasic component of noradrenaline-induced contraction. This study shows that TAAC, a newly detected constituent of Vitex cienkowskii contributes to the relaxing effect of an extract of the plant. The effect is partially mediated by the involvement of the NO/cGMP pathway of the smooth muscle but additionally inhibition of calcium influx into the cell may play a role.


Assuntos
Ecdisterona/análogos & derivados , Endotélio Vascular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Vitex/química , Animais , Aorta/efeitos dos fármacos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ecdisterona/isolamento & purificação , Ecdisterona/farmacologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Norepinefrina/farmacologia , Casca de Planta , Extratos Vegetais/química , Caules de Planta , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Guanilil Ciclase Solúvel , Vasodilatadores/isolamento & purificação
9.
Physiol Rev ; 94(1): 303-26, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24382889

RESUMO

The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that ß-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVß2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Humanos , Cinética , Fosforilação/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos
10.
FASEB J ; 28(3): 1044-8, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24253251

RESUMO

α1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the α1-adrenergic-mediated signaling in vascular smooth muscle. α1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the α1-adrenergic-induced contraction and the α1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1(-/-) mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2(-/-) mice) but still present in the heterozygous control mice. α1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the α1-adrenergic-induced CaV1.2 channel activity.


Assuntos
Músculo Liso Vascular/fisiologia , Fosfolipase D/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Animais , Cálcio/fisiologia , Camundongos , Camundongos Knockout , Contração Muscular , Fosfolipase D/genética
11.
Cardiovasc Res ; 100(2): 280-7, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23832809

RESUMO

AIMS: Signalling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (Trpc) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signalling. We tested this concept by investigating the role of cGMP/cGKI signalling on vascular tone and peripheral resistance using Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), Trpc1(-/-)/3(-/-)/6(-/-), and SM-specific cGKI(-/-) (sm-cGKI(-/-)) mice. METHODS AND RESULTS: α-Adrenergic stimulation induced similar contractions in L-NG-nitroarginine methyl ester (l-NAME)-treated aorta and comparably increased peripheral pressure in hind limbs from all mouse lines investigated. After α-adrenergic stimulation, 8-Br-cGMP diminished similarly aortic tone and peripheral pressure in control, Trpc6(-/-), Trpc3(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice but not in sm-cGKI(-/-) mice. In untreated aorta, α-adrenergic stimulation induced similar contractions in the aorta from control and Trpc3(-/-) mice but larger contractions in sm-cGKI(-/-), Trpc6(-/-), Trpc3(-/-)/6(-/-), and Trpc1(-/-)/3(-/-)/6(-/-) mice, indicating a functional link between cGKI and Trpc6 channels. Trpc3 channels were detected by immunocytochemistry in both isolated aortic smooth muscle cells (SMCs) and aortic endothelial cells (ECs), whereas Trpc6 channels were detected only in ECs. Phenylephrine-stimulated Ca(2+) levels were similar in SMCs from control (Ctr) and Trpc6(-/-) mice. Carbachol-stimulated Ca(2+) levels were reduced in ECs from Trpc6(-/-) mice. Stimulated Ca(2+) levels were lowered by 8-Br-cGMP in Ctr but not in Trpc6(-/-) ECs. CONCLUSIONS: The results suggest that cGKI and Trpc1,3,6 channels are not functionally coupled in vascular SM. Deletion of Trpc6 channels impaired endothelial cGKI signalling and vasodilator tone in the aorta.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/fisiologia , GMP Cíclico/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPC/fisiologia , Vasoconstrição , Animais , Cálcio/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Fenilefrina/farmacologia , Vasoconstrição/efeitos dos fármacos
12.
Methods Mol Biol ; 1020: 17-50, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23709024

RESUMO

cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes-prkg1 and prkg2-code for cGKs, namely, cGKI and cGKII. In mammals, two isozymes, cGKIα and cGKIß, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets, and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxtaglomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major downstream effectors of many, but not all, signalling events of the NO/cGMP and the ANP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night, and endochondral bone growth. This chapter focuses on the involvement of cGKs in cardiovascular and non-cardiovascular processes including cell growth and metabolism.


Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Transdução de Sinais , Adipócitos/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Osso e Ossos/metabolismo , Sistema Cardiovascular/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Sistema Nervoso/metabolismo
13.
Pflugers Arch ; 465(7): 955-64, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23338940

RESUMO

Cardiac CaV1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the CaV1.2 channel is the target of Ca(2+)-dependent and Ca(2+)-independent regulation of the channel. Recent studies on C-terminal truncated forms of the CaV1.2 channel reported neonatal death, reduced CaV1.2 current, and failure of ß-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the CaV1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca(2+) currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca(2+) channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the CaV1.2 channel but a twofold increase in the level of mRNA for the CaV1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of CaV1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated CaV1.2 channel with an upregulation of the CaV1.3 channel.


Assuntos
Potenciais de Ação , Canais de Cálcio Tipo L/metabolismo , Deleção de Genes , Miócitos Cardíacos/fisiologia , Transcrição Gênica , Animais , Ácido Aspártico/genética , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Colforsina/farmacologia , Coração Fetal/citologia , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Estrutura Terciária de Proteína
14.
J Biol Chem ; 287(27): 22616-25, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22589547

RESUMO

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.


Assuntos
Canais de Cálcio Tipo L/genética , Calmodulina/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/mortalidade , Motivos de Aminoácidos/genética , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Antiarrítmicos/farmacologia , Sítios de Ligação/genética , Cálcio/metabolismo , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Captopril/farmacologia , Cardiomiopatia Dilatada/tratamento farmacológico , Células Cultivadas , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/mortalidade , Metoprolol/farmacologia , Camundongos , Camundongos Mutantes , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Estrutura Terciária de Proteína/genética , Taxa de Sobrevida
15.
J Biol Chem ; 287(27): 22584-92, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22589548

RESUMO

Phosphorylation of the cardiac ß subunit (Ca(v)ß(2)) of the Ca(v)1.2 L-type Ca(2+) channel complex has been proposed as a mechanism for regulation of L-type Ca(2+) channels by various protein kinases including PKA, CaMKII, Akt/PKB, and PKG. To test this hypothesis directly in vivo, we generated a knock-in mouse line with targeted mutation of the Ca(v)ß(2) gene by insertion of a stop codon after proline 501 in exon 14 (mouse sequence Cacnb2; ßStop mouse). This mutation prevented translation of the Ca(v)ß(2) C terminus that contains the relevant phosphorylation sites for the above protein kinases. Homozygous cardiac ßStop mice were born at Mendelian ratio, had a normal life expectancy, and normal basal L-type I(Ca). The regulation of the L-type current by stimulation of the ß-adrenergic receptor was unaffected in vivo and in cardiomyocytes (CMs). ßStop mice were cross-bred with mice expressing the Ca(v)1.2 gene containing the mutation S1928A (SAßStop) or S1512A and S1570A (SFßStop) in the C terminus of the α(1C) subunit. The ß-adrenergic regulation of the cardiac I(Ca) was unaltered in these mouse lines. In contrast, truncation of the Ca(v)1.2 at Asp(1904) abolished ß-adrenergic up-regulation of I(Ca) in murine embryonic CMs. We conclude that phosphorylation of the C-terminal sites in Ca(v)ß(2), Ser(1928), Ser(1512), and Ser(1570) of the Ca(v)1.2 protein is functionally not involved in the adrenergic regulation of the murine cardiac Ca(v)1.2 channel.


Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Receptores Adrenérgicos beta/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Sequência de Bases , Canais de Cálcio Tipo L/genética , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletrocardiografia , Feminino , Deleção de Genes , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fosforilação/fisiologia , Estrutura Terciária de Proteína/fisiologia
16.
FASEB J ; 26(4): 1745-54, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22253479

RESUMO

Brief contact of the duodenal mucosa with luminal acid elicits a long-lasting bicarbonate (HCO(3)(-)) secretory response, which is believed to be the primary protective mechanism against mucosal damage. Here, we show that cGMP-dependent protein kinase type I-knockout (cGKI(-/-)) mice are unable to respond to a physiological H(+) stimulus with a HCO(3)(-) secretory response and spontaneously develop duodenal ulcerations. Smooth muscle-selective cGKI knock-in rescued the motility disturbance but not the defective HCO(3)(-) secretion. Proton-induced HCO(3)(-) secretion was not attenuated by selective inactivation of the cGKI gene in interstitial cells of Cajal or in enterocytes, but was abolished by inactivation of cGKI in neurons (ncGKI(-/-)). cGKI was expressed in the brainstem nucleus tractus solitarius that connects the afferent with the efferent N. vagus. Accordingly, truncation of the subdiaphragmal N. vagus significantly diminished proton-induced HCO(3)(-) secretion in wild-type mice, whereas stimulation of the subdiaphragmal N. vagus elicited a similar HCO(3)(-) secretory response in cGKI(-/-), ncGKI(-/-) and wild-type mice. These findings show that protection of the duodenum from acid injury requires neuronal cGKI.


Assuntos
Ácidos/metabolismo , Bicarbonatos/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Duodeno/metabolismo , Neurônios/enzimologia , Animais , Colforsina/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/genética , Úlcera Duodenal/metabolismo , Úlcera Duodenal/patologia , Duodeno/anatomia & histologia , Células Intersticiais de Cajal/citologia , Células Intersticiais de Cajal/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Núcleo Solitário/citologia , Núcleo Solitário/metabolismo , Vagotomia , Nervo Vago/metabolismo
17.
Eur J Pharmacol ; 670(1): 266-71, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21914444

RESUMO

cGMP-dependent kinase I (cGKI) is a major mediator of smooth muscle relaxation and exists in two isoforms, α and ß. Both isoforms are supposed to mediate their effects via different intracellular signaling pathways. To verify this concept, the kinetics of relaxation mediated by either isoform was analyzed in gastric fundus smooth muscle from mice. Muscles from mice that express selectively the Iα or Iß isoform of cGKI in smooth muscle (sm-cGKIα or sm-cGKIß mice) were compared to muscles from conventional cGKI(-/-) mice. Fundus muscles were contracted by carbachol and then relaxed by 8-Br-cGMP or by electrical field stimulation (EFS). The time course of relaxation by 8-Br-cGMP was not different between muscles from sm-cGKIα and sm-cGKIß mice. EFS induced a fast transient relaxation in muscles from sm-cGKIα and sm-cGKIß mice that was blocked by the NO synthase inhibitor L-NAME. Recovery from this relaxation was about 4-times slower in muscles from sm-cGKIα mice than in muscles from sm-cGKIß mice. The different kinetic of recovery from relaxation after EFS in sm-cGKIα and sm-cGKIß mice suggests that different signaling pathways exist for each cGKI isoform in vivo in fundus muscles.


Assuntos
GMP Cíclico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Relaxamento Muscular , Músculo Liso/citologia , Músculo Liso/fisiologia , Transdução de Sinais , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Estimulação Elétrica , Feminino , Fundo Gástrico , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Cinética , Masculino , Camundongos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos
18.
J Biol Chem ; 286(39): 33863-71, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21832054

RESUMO

The carboxyl-terminal intracellular tail of the L-type Ca(2+) channel CaV1.2 modulates various aspects of channel activity.For example, deletion of the carboxyl-terminal sequence at Ser-1905 increased CaV1.2 currents in an expression model. To verify this finding in an animal model, we inserted three stop codons at the corresponding Asp-1904 in the murine CaV1.2 gene. Mice homozygous for the Stop mutation (Stop/Stop mice)were born at a Mendelian ratio but died after birth. Stop/Stop hearts showed reduced beating frequencies and contractions.Surprisingly, Stop/Stop cardiomyocytes displayed reduced IBa and a minor expression of the CaV1.2Stop protein. In contrast,expression of the CaV1.2Stop protein was normal in pooled smooth muscle samples from Stop/Stop embryos. As the CaV1.2 channel exists in a cardiac and smooth muscle splice variant, HK1 and LK1, respectively, we analyzed the consequences of the deletion of the carboxyl terminus in the respective splice variant using the rabbit CaV1.2 clone expressed in HEK293 cells.HEK293 cells transfected with the HK1Stop channel showed a reduced IBa and CaV1.2 expression. Treatment with proteasome inhibitors increased the expression of HK1Stop protein and IBa in HEK293 cells and in Stop/Stop cardiomyocytes indicating that truncation of CaV1.2 containing the cardiac exon 1a amino terminus results in proteasomal degradation of the translated protein. In contrast, HEK293 cells transfected with the LK1Stop channel had normal IBa and CaV1.2 expression. These findings indicate that absence of the carboxyl-terminal tail differentially determines the fate of the cardiac and smooth muscle splice variant of the CaV1.2 channel in the mouse.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Códon de Terminação , Insuficiência Cardíaca/metabolismo , Doenças do Recém-Nascido/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Processamento Alternativo/genética , Animais , Canais de Cálcio Tipo L/genética , Modelos Animais de Doenças , Embrião de Mamíferos/metabolismo , Células HEK293 , Insuficiência Cardíaca/genética , Humanos , Recém-Nascido , Doenças do Recém-Nascido/genética , Camundongos , Contração Miocárdica/genética , Especificidade de Órgãos/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Coelhos
19.
J Biol Chem ; 286(30): 26702-7, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21665954

RESUMO

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Antineoplásicos Hormonais/farmacologia , Canais de Cálcio Tipo L/genética , Células Cultivadas , Ventrículos do Coração/patologia , Camundongos , Camundongos Mutantes , Miocárdio/patologia , Miócitos Cardíacos/patologia , Tamoxifeno/farmacologia
20.
FASEB J ; 24(8): 2651-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20371628

RESUMO

Here we present functional and biochemical evidence for a Ca(2+) channel (Ca(V)1.2)/protein kinase C (PKC) signaling complex being a key player in muscarinic regulation of urinary bladder smooth muscle. Muscarinic stimulation induced Ca(2+) signals and concomitant contractions in detrusor muscle from mice that were dependent on functional Ca(2+) channels. These signals were still present in muscles being depolarized by 85 mM extracellular K(+). Muscarinic-induced contractions were reduced by a PKC inhibitor [bisindolylmaleimide I (BIM-I)] and a phospholipase D (PLD) inhibitor (1-butanol). A phorbol ester (PDBu) enlarged muscarinic-induced Ca(2+) signals and contractions. The effects of BIM-I and PDBu were inhibited by isradipine and/or absent in muscles from Ca(V)1.2-deficient mice. Both carbachol and PDBu increased Ca(V)1.2 channel currents in isolated bladder myocytes. Blue native-PAGE electrophoresis revealed that Ca(V)1.2, PKC, and PLD are closely associated in muscles being previously stimulated by carbachol. Immunoprecipitation using anti-Ca(V)1.2 followed by Western blotting demonstrated that Ca(V)1.2 and PKC are coupled in stimulated muscles from wild-type mice. Autoradiography on immunoprecipitates showed that Ca(V)1.2 is a substrate for PKC-mediated phosphorylation. These findings suggest that a signaling complex consisting of Ca(V)1.2, PKC, and, probably, PLD controls muscarinic-mediated phasic contraction of urinary bladder smooth muscle.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Músculo Liso/metabolismo , Proteína Quinase C/metabolismo , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Animais , Potenciais da Membrana , Camundongos , Contração Muscular , Músculo Liso/fisiologia , Fosforilação , Potássio , Suínos , Bexiga Urinária/citologia
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