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1.
J Mol Cell Cardiol ; 182: 44-53, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37433391

RESUMO

Cardiac excitation-contraction coupling (ECC) depends on Ca2+ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca2+ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca2+ sparks, which summate to form a cell-wide Ca2+ transient that switches on contraction. Voltage (Vm) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca2+ spark timing, but Ca2+ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the Vm-dependence of evoked Ca2+ spark probability (Pspark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca2+ spark latency showed a U-shaped Vm-dependence, while repolarising steps from 50 mV produced Ca2+ spark latencies that increased monotonically with Vm. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of âˆ¼ 5:1 for the Ca2+ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (Pcpl âˆ¼ 0.5) between each LCC opening and IC activation. The presence of âˆ¼ 4 ICs per couplon reduced Ca2+ spark latency and increased Pspark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease Pspark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the Vm- and time-dependence of Pspark, and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca2+ release.


Assuntos
Sinalização do Cálcio , Miócitos Cardíacos , Ratos , Animais , Miócitos Cardíacos/metabolismo , Ventrículos do Coração/metabolismo , Acoplamento Excitação-Contração , Canais Iônicos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Retículo Sarcoplasmático/metabolismo
2.
J Mol Cell Cardiol ; 172: 52-62, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35908686

RESUMO

Loss of ventricular action potential (AP) early phase 1 repolarization may contribute to the impaired Ca2+ release and increased risk of sudden cardiac death in heart failure. Therefore, restoring AP phase 1 by augmenting the fast transient outward K+ current (Itof) might be beneficial, but direct experimental evidence to support this proposition in failing cardiomyocytes is limited. Dynamic clamp was used to selectively modulate the contribution of Itof to the AP and Ca2+ transient in both normal (guinea pig and rabbit) and in failing rabbit cardiac myocytes. Opposing native Itof in non-failing rabbit myocytes increased Ca2+ release heterogeneity, late Ca2+ sparks (LCS) frequency and AP duration. (APD). In contrast, increasing Itof in failing myocytes and guinea pig myocytes (the latter normally lacking Itof) increased Ca2+ transient amplitude, Ca2+ release synchrony, and shortened APD. Computer simulations also showed faster Ca2+ transient decay (mainly due to fewer LCS), decreased inward Na+/Ca2+ exchange current and APD. When the Itof conductance was increased to ~0.2 nS/pF in failing cells (a value slightly greater than seen in typical human epicardial myocytes), Ca2+ release synchrony improved and AP duration decreased slightly. Further increases in Itof can cause Ca2+ release to decrease as the peak of the bell-shaped ICa-voltage relationship is passed and premature AP repolarization develops. These results suggest that there is an optimal range for Itof enhancement that may support Ca2+ release synchrony and improve electrical stability in heart failure with the caveat that uncontrolled Itof enhancement should be avoided.


Assuntos
Insuficiência Cardíaca , Miócitos Cardíacos , Humanos , Coelhos , Cobaias , Animais , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Ventrículos do Coração , Sódio , Cálcio
3.
J Mol Cell Cardiol ; 164: 29-41, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34823101

RESUMO

The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 µmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.


Assuntos
Insuficiência Cardíaca , Canais de Potássio Shal , Potenciais de Ação/fisiologia , Animais , Células HEK293 , Humanos , Miócitos Cardíacos/metabolismo , Coelhos , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Transgenes
4.
Proc Natl Acad Sci U S A ; 117(5): 2687-2692, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31969455

RESUMO

Sudden death in heart failure patients is a major clinical problem worldwide, but it is unclear how arrhythmogenic early afterdepolarizations (EADs) are triggered in failing heart cells. To examine EAD initiation, high-sensitivity intracellular Ca2+ measurements were combined with action potential voltage clamp techniques in a physiologically relevant heart failure model. In failing cells, the loss of Ca2+ release synchrony at the start of the action potential leads to an increase in number of microscopic intracellular Ca2+ release events ("late" Ca2+ sparks) during phase 2-3 of the action potential. These late Ca2+ sparks prolong the Ca2+ transient that activates contraction and can trigger propagating microscopic Ca2+ ripples, larger macroscopic Ca2+ waves, and EADs. Modification of the action potential to include steps to different potentials revealed the amount of current generated by these late Ca2+ sparks and their (subsequent) spatiotemporal summation into Ca2+ ripples/waves. Comparison of this current to the net current that causes action potential repolarization shows that late Ca2+ sparks provide a mechanism for EAD initiation. Computer simulations confirmed that this forms the basis of a strong oscillatory positive feedback system that can act in parallel with other purely voltage-dependent ionic mechanisms for EAD initiation. In failing heart cells, restoration of the action potential to a nonfailing phase 1 configuration improved the synchrony of excitation-contraction coupling, increased Ca2+ transient amplitude, and suppressed late Ca2+ sparks. Therapeutic control of late Ca2+ spark activity may provide an additional approach for treating heart failure and reduce the risk for sudden cardiac death.


Assuntos
Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Acoplamento Excitação-Contração , Insuficiência Cardíaca/fisiopatologia , Humanos , Miócitos Cardíacos/metabolismo
5.
Exp Physiol ; 104(5): 654-666, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30786093

RESUMO

NEW FINDINGS: What is the central question of this study? What is the cellular basis of the protection conferred on the heart by overexpression of caveolin-3 (Cav-3 OE) against many of the features of heart failure normally observed in vivo? What is the main finding and its importance? Cav-3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t-tubular ICa , but not local t-tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav-3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release. ABSTRACT: Caveolin-3 (Cav-3) is an 18 kDa protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted and excitation-contraction coupling (ECC) is impaired. Previous work has suggested that Cav-3 overexpression (OE) is cardio-protective, but the effect of Cav-3 OE on these cellular changes is unknown. We therefore investigated whether Cav-3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks' transverse aortic constriction (TAC). Cav-3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t-tubule regularity and density, and impaired local Ca2+ release at the t-tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav-3 OE compared to WT mice, and t-tubular Ca2+ current (ICa ) density was maintained. These data suggest that Cav-3 OE helps prevent hypertrophy and loss of t-tubular ICa following TAC, but that other factors disrupt local Ca2+ release.


Assuntos
Canais de Cálcio/metabolismo , Caveolina 3/metabolismo , Insuficiência Cardíaca/fisiopatologia , Animais , Sinalização do Cálcio , Cardiomegalia , Caveolina 3/genética , Constrição Patológica/fisiopatologia , Ecocardiografia , Insuficiência Cardíaca/genética , Ventrículos do Coração , Masculino , Camundongos , Miócitos Cardíacos/metabolismo , Circulação Pulmonar , Retículo Sarcoplasmático/metabolismo , Volume Sistólico , Vasodilatação
6.
Am J Physiol Heart Circ Physiol ; 315(5): H1101-H1111, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30028203

RESUMO

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current ( ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Caveolina 3/deficiência , Ventrículos do Coração/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Miócitos Cardíacos/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Potenciais de Ação , Animais , Caveolina 3/genética , Regulação para Baixo , Predisposição Genética para Doença , Ventrículos do Coração/patologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/patologia , Fenótipo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia , Função Ventricular Esquerda
7.
Proc Natl Acad Sci U S A ; 115(30): E7073-E7080, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29991602

RESUMO

Cardiac transverse (t-) tubules carry both electrical excitation and solutes toward the cell center but their ability to transport small molecules is unclear. While fluorescence recovery after photobleaching (FRAP) can provide an approach to measure local solute movement, extraction of diffusion coefficients is confounded by cell and illumination beam geometries. In this study, we use measured cellular geometry and detailed computer modeling to derive the apparent diffusion coefficient of a 1-kDa solute inside the t-tubular system of rabbit and mouse ventricular cardiomyocytes. This approach shows that diffusion within individual t-tubules is more rapid than previously reported. T-tubule tortuosity, varicosities, and the presence of longitudinal elements combine to substantially reduce the apparent rate of solute movement. In steady state, large (>4 kDa) solutes did not freely fill the t-tubule lumen of both species and <50% of the t-tubule volume was available to solutes >70 kDa. Detailed model fitting of FRAP data suggests that solute diffusion is additionally restricted at the t-tubular entrance and this effect was larger in mouse than in rabbit. The possible structural basis of this effect was investigated using electron microscopy and tomography. Near the cell surface, mouse t-tubules are more tortuous and filled with an electron-dense ground substance, previously identified as glycocalyx and a polyanionic mesh. Solute movement in the t-tubule network of rabbit and mouse appears to be explained by their different geometric properties, which impacts the use of these species for understanding t-tubule function and the consequences of changes associated with t-tubule disease.


Assuntos
Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Miócitos Cardíacos/citologia , Coelhos
8.
Exp Physiol ; 103(5): 652-665, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29473235

RESUMO

NEW FINDINGS: What is the central question of this study? Heart failure is associated with redistribution of L-type Ca2+ current (ICa ) away from the t-tubule membrane to the surface membrane of cardiac ventricular myocytes. However, the underlying mechanism and its dependence on severity of pathology (hypertrophy versus failure) are unclear. What is the main finding and its importance? Increasing severity of response to transverse aortic constriction, from hypertrophy to failure, was accompanied by graded loss of t-tubular ICa and loss of regulation of ICa by caveolin 3. Thus, the pathological loss of t-tubular ICa , which contributes to impaired excitation-contraction coupling and thereby cardiac function in vivo, appears to be attributable to loss of caveolin 3-dependent stimulation of t-tubular ICa . ABSTRACT: Previous work has shown redistribution of L-type Ca2+ current (ICa ) from the t-tubules to the surface membrane of rat ventricular myocytes after myocardial infarction. However, whether this occurs in all species and in response to other insults, the relationship of this redistribution to the severity of the pathology, and the underlying mechanism, are unknown. We have therefore investigated the response of mouse hearts and myocytes to pressure overload induced by transverse aortic constriction (TAC). Male C57BL/6 mice underwent TAC or equivalent sham operation 8 weeks before use. ICa and Ca2+ transients were measured in isolated myocytes, and expression of caveolin 3 (Cav3), junctophilin 2 (Jph2) and bridging integrator 1 (Bin1) was determined. C3SD peptide was used to disrupt Cav3 binding to its protein partners. Some animals showed cardiac hypertrophy in response to TAC with little evidence of heart failure, whereas others showed greater hypertrophy and pulmonary congestion. These graded changes were accompanied by graded cellular hypertrophy, t-tubule disruption, decreased expression of Jph2 and Cav3, and decreased t-tubular ICa density, with no change at the cell surface, and graded impairment of Ca2+ release at t-tubules. C3SD decreased ICa density in control but not in TAC myocytes. These data suggest that the graded changes in cardiac function and size that occur in response to TAC are paralleled by graded changes in cell structure and function, which will contribute to the impaired function observed in vivo. They also suggest that loss of t-tubular ICa is attributable to loss of Cav3-dependent stimulation of ICa .


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cardiomegalia/metabolismo , Caveolina 3/metabolismo , Insuficiência Cardíaca/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Cardiomegalia/fisiopatologia , Acoplamento Excitação-Contração/fisiologia , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos
9.
Biochem Pharmacol ; 150: 120-130, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29378180

RESUMO

Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ±â€¯1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound's effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Canal de Potássio ERG1/metabolismo , Proteínas Interatuantes com Canais de Kv/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Compostos de Fenilureia/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio Shal/metabolismo , Tetrazóis/farmacologia , Canal de Potássio ERG1/agonistas , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/agonistas , Proteínas do Tecido Nervoso/agonistas , Canais de Potássio/agonistas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/metabolismo , Canais de Potássio Shal/agonistas
10.
Circ Res ; 122(3): 473-478, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29282211

RESUMO

RATIONALE: The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. OBJECTIVE: To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. METHODS AND RESULTS: We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. CONCLUSIONS: These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Acoplamento Excitação-Contração/fisiologia , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo L/fisiologia , Microscopia Confocal , Coelhos , Trocador de Sódio e Cálcio/fisiologia , Sístole
11.
Am J Physiol Heart Circ Physiol ; 314(3): H521-H529, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29101175

RESUMO

ß2-Adrenoceptors and L-type Ca2+ current ( ICa) redistribute from the t-tubules to the surface membrane of ventricular myocytes from failing hearts. The present study investigated the role of changes in caveolin-3 and PKA signaling, both of which have previously been implicated in this redistribution. ICa was recorded using the whole cell patch-clamp technique from ventricular myocytes isolated from the hearts of rats that had undergone either coronary artery ligation (CAL) or equivalent sham operation 18 wk earlier. ICa distribution between the surface and t-tubule membranes was determined using formamide-induced detubulation (DT). In sham myocytes, ß2-adrenoceptor stimulation increased ICa in intact but not DT myocytes; however, forskolin (to increase cAMP directly) and H-89 (to inhibit PKA) increased and decreased, respectively, ICa at both the surface and t-tubule membranes. C3SD peptide (which decreases binding to caveolin-3) inhibited ICa in intact but not DT myocytes but had no effect in the presence of H-89. In contrast, in CAL myocytes, ß2-adrenoceptor stimulation increased ICa in both intact and DT myocytes, but C3SD had no effect on ICa; forskolin and H-89 had similar effects as in sham myocytes. These data show the redistribution of ß2 -adrenoceptor activity and ICa in CAL myocytes and suggest constitutive stimulation of ICa by PKA in sham myocytes via concurrent caveolin-3-dependent (at the t-tubules) and caveolin-3-independent mechanisms, with the former being lost in CAL myocytes. NEW & NOTEWORTHY In ventricular myocytes from normal hearts, regulation of the L-type Ca2+ current by ß2-adrenoceptors and the constitutive regulation by caveolin-3 is localized to the t-tubules. In heart failure, the regulation of L-type Ca2+ current by ß2-adrenoceptors is redistributed to the surface membrane, and the constitutive regulation by caveolin-3 is lost.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Caveolina 3/metabolismo , Insuficiência Cardíaca/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Insuficiência Cardíaca/fisiopatologia , Masculino , Infarto do Miocárdio/fisiopatologia , Transporte Proteico , Ratos Wistar , Receptores Adrenérgicos beta 2/metabolismo
12.
J Gerontol A Biol Sci Med Sci ; 73(6): 711-719, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29236992

RESUMO

Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.


Assuntos
Envelhecimento/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Caveolina 3/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Masculino , Camundongos , Camundongos Transgênicos
14.
J Mol Cell Cardiol ; 108: 1-7, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28483597

RESUMO

Transverse-axial tubules (TTs) are key structures involved in cardiac excitation-contraction coupling and can become deranged in disease. Although optical measurement of TTs is frequently employed to assess TT abundance and regularity, TT dimensions are generally below the diffraction limit of optical microscopy so determination of tubule size is problematic. TT diameter was measured by labeling both local surface membrane area and volume with fluorescent probes (FM4-64 and calcein, respectively), correcting image asymmetry by image processing and using the relationship between surface area and volume for a geometric primitive. This method shows that TTs have a mean (±SEM) diameter of 356±18nm in rabbit and 169±15nm in mouse (p<0.001). Rabbit TT diameters were more variable than those of mouse (p<0.01) and the smallest TT detected was 41nm in mouse and the largest 695nm in rabbit. These estimates are consistent with TT diameters derived from the more limited sampling of high-pressure frozen samples by electron tomography (which examines only a small fraction of the cell volume). Other measures of TT abundance and geometry (such as volume, membrane fractions and direction) were also derived. On the physiological time scale of E-C coupling (milliseconds), the average TT electrical space constant is ~175µm in rabbit and ~120µm in mouse and is ~50% of the steady-state space constant. This is sufficient to ensure reasonable electrical uniformity across normal cells. The image processing strategy and shape-based 3D approach to feature quantification is also generally applicable to other problems in quantification of sub-cellular anatomy.


Assuntos
Ventrículos do Coração/citologia , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Animais , Camundongos , Miócitos Cardíacos/ultraestrutura , Coelhos , Retículo Sarcoplasmático/ultraestrutura
15.
Physiol Rep ; 5(5)2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28270595

RESUMO

Transient outward potassium current (Ito) in the heart underlies phase 1 repolarization of cardiac action potentials and thereby affects excitation-contraction coupling. Small molecule activators of Ito may therefore offer novel treatments for cardiac dysfunction, including heart failure and atrial fibrillation. NS5806 has been identified as a prototypic activator of canine Ito This study investigated, for the first time, actions of NS5806 on rabbit atrial and ventricular Ito Whole cell patch-clamp recordings of Ito and action potentials were made at physiological temperature from rabbit ventricular and atrial myocytes. 10 µmol/L NS5806 increased ventricular Ito with a leftward shift in Ito activation and accelerated restitution. At higher concentrations, stimulation of Ito was followed by inhibition. The EC50 for stimulation was 1.6 µmol/L and inhibition had an IC50 of 40.7 µmol/L. NS5806 only inhibited atrial Ito (IC50 of 18 µmol/L) and produced a modest leftward shifts in Ito activation and inactivation, without an effect on restitution. 10 µmol/L NS5806 shortened ventricular action potential duration (APD) at APD20-APD90 but prolonged atrial APD NS5806 also reduced atrial AP upstroke and amplitude, consistent with an additional atrio-selective effect on Na+ channels. In contrast to NS5806, flecainide, which discriminates between Kv1.4 and 4.x channels, produced similar levels of inhibition of ventricular and atrial Ito NS5806 discriminates between rabbit ventricular and atrial Ito, with mixed activator and inhibitor actions on the former and inhibitor actions against the later. NS5806 may be of significant value for pharmacological interrogation of regional differences in native cardiac Ito.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Coração/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Canais de Potássio/agonistas , Tetrazóis/farmacologia , Potenciais de Ação/fisiologia , Animais , Flecainida/farmacologia , Coração/fisiologia , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Coelhos , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
16.
PLoS One ; 11(6): e0156862, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27281038

RESUMO

BACKGROUND: T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue. METHODS: Cardiac tissue was obtained from young female Landrace White pigs (45-75 kg, 5-6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT). RESULTS & CONCLUSION: The spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 µm, n = 17) than in either ventricular cells (1.33±0.05 µm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 µm, n = 5, P<0.05). These data demonstrate considerable heterogeneity between pig cardiomyocytes in the extent of t-tubule network, which correlated with cell size.


Assuntos
Sinalização do Cálcio , Átrios do Coração/ultraestrutura , Ventrículos do Coração/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Sarcolema/ultraestrutura , Animais , Tamanho Celular , Acoplamento Excitação-Contração , Feminino , Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Microscopia Confocal , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Suínos
17.
Clin Exp Pharmacol Physiol ; 43(1): 88-94, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26466753

RESUMO

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3',5'-monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation-contraction coupling in ventricular preparations from rat hearts. The study used 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca(2+) spark frequency from about 7 to 32/100 µm(3)/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca(2+) and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca(2+)]o) revealed no effect of Epac activation on either the amplitude of Ca(2+) transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura-2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm(2) vs cpTOME 23 ± 5 mN/mm(2), P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca(2+)]o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca(2+) sensitivity, an effect that was blocked by addition of KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.


Assuntos
Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Depressão Sináptica de Longo Prazo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Animais , Acoplamento Excitação-Contração , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/fisiologia , Ratos
18.
PLoS One ; 10(12): e0144309, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26713852

RESUMO

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca2+-handling. We investigated the effects of ascending aortic banding (AoB) on Ca2+-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca2+-release at the cell edge whereas Ca2+ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na+/Ca2+ exchanger, SR Ca2+ ATPase or phospholamban. Mathematical modeling indicated that [Ca2+]i transients at the cell center were accounted for by simple centripetal diffusion of Ca2+ released at the cell edge. In contrast, caffeine (10 mM) induced Ca2+ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca2+-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca2+ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca2+-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca2+-release.


Assuntos
Fibrilação Atrial/fisiopatologia , Átrios do Coração/patologia , Miócitos Cardíacos/fisiologia , Animais , Sinalização do Cálcio , Crescimento Celular , Células Cultivadas , Átrios do Coração/fisiopatologia , Hipertrofia , Masculino , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
19.
J Mol Cell Cardiol ; 86: 23-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26103619

RESUMO

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~18weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation-contraction coupling observed in heart failure.


Assuntos
Canais de Cálcio Tipo L/biossíntese , Cálcio/metabolismo , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Humanos , Isoquinolinas/administração & dosagem , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Técnicas de Patch-Clamp , Ratos , Sulfonamidas/administração & dosagem
20.
J Mol Cell Cardiol ; 84: 170-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25953258

RESUMO

Evidence from animal models suggest that t-tubule changes may play an important role in the contractile deficit associated with heart failure. However samples are usually taken at random with no regard as to regional variability present in failing hearts which leads to uncertainty in the relationship between contractile performance and possible t-tubule derangement. Regional contraction in human hearts was measured by tagged cine MRI and model fitting. At transplant, failing hearts were biopsy sampled in identified regions and immunocytochemistry was used to label t-tubules and sarcomeric z-lines. Computer image analysis was used to assess 5 different unbiased measures of t-tubule structure/organization. In regions of failing hearts that showed good contractile performance, t-tubule organization was similar to that seen in normal hearts, with worsening structure correlating with the loss of regional contractile performance. Statistical analysis showed that t-tubule direction was most highly correlated with local contractile performance, followed by the amplitude of the sarcomeric peak in the Fourier transform of the t-tubule image. Other area based measures were less well correlated. We conclude that regional contractile performance in failing human hearts is strongly correlated with the local t-tubule organization. Cluster tree analysis with a functional definition of failing contraction strength allowed a pathological definition of 't-tubule disease'. The regional variability in contractile performance and cellular structure is a confounding issue for analysis of samples taken from failing human hearts, although this may be overcome with regional analysis by using tagged cMRI and biopsy mapping.


Assuntos
Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Contração Miocárdica , Miócitos Cardíacos/patologia , Adulto , Cardiomiopatia Dilatada/complicações , Feminino , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Processamento de Imagem Assistida por Computador , Imagem Cinética por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Aglutininas do Germe de Trigo/metabolismo , Adulto Jovem
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