RESUMEN
Sorcin is a Ca2+ binding protein implicated in the regulation of intracellular Ca2+ cycling and cardiac excitation-contraction coupling. Structural and human genetic studies suggest that a naturally occurring sequence variant encoding L112-sorcin disrupts an E-F hand Ca2+ binding domain and may be responsible for a heritable form of hypertension and hypertrophic heart disease. We generated transgenic mice overexpressing L112-sorcin in the heart and characterized the effects on Ca2+ regulation and cardiac function both in vivo and in dissociated cardiomyocytes. Hearts of sorcin(F112L) transgenic mice were mildly dilated but ventricular function was preserved and systemic blood pressure was normal. Sorcin(F112L) myocytes were smaller than control cells and displayed complex alterations in Ca2+ regulation and contractility, including a slowed inactivation of L-type Ca2+ current, enhanced Ca2+ spark width, duration, and frequency, and increased Na+-Ca2+ exchange activity. In contrast, mice with cardiac-specific overexpression of wild-type sorcin displayed directionally opposite effects on L-type Ca2+ channel function and Ca2+ spark behavior. These data further define the role of sorcin in cardiac excitation-contraction coupling and highlight its negative regulation of SR calcium release. Our results also suggest that additional factors may be responsible for the development of cardiac hypertrophy and hypertension in humans expressing the L112-sorcin sequence variant.
Asunto(s)
Proteínas de Unión al Calcio/genética , Mutación Missense , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/fisiología , Simulación por Computador , Humanos , Immunoblotting , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Miocardio/citología , Retículo Sarcoplasmático/metabolismoRESUMEN
To evaluate the effect of sorcin on cardiac excitation-contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad-sorcin). A beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees C) was significantly reduced in Ad-sorcin-transfected myocytes compared with control myocytes (2.10+/-0.05% [n=311] versus 2.42+/-0.06% [n=312], respectively; P<0.001). Action potential duration (at 20 degrees C) was significantly less in the Ad-sorcin group (458+/-22 ms, n=11) compared with the control group (520+/-19 ms, n=10; P<0.05). In voltage-clamped, fura 2-loaded myocytes (20 degrees C), a reduced peak-systolic and end-diastolic [Ca2+]i was observed after Ad-sorcin transfection. L-type Ca2+ current amplitude and time course were unaffected. Caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+-Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+-extrusion kinetics in Ad-sorcin-transfected cells. Higher NCX activity after Ad-sorcin transfection was confirmed by measuring the NCX current-voltage relationship. beta-Escin-permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine-mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin-overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+-transient amplitude predominantly by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation-contraction coupling.
Asunto(s)
Adenoviridae/genética , Proteínas de Unión al Calcio/biosíntesis , Contracción Miocárdica/fisiología , Miocitos Cardíacos/metabolismo , Animales , Cafeína/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Proteínas de Unión al Calcio/genética , Células Cultivadas , Escina/farmacología , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Líquido Intracelular/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Níquel/farmacología , Técnicas de Placa-Clamp , Permeabilidad/efectos de los fármacos , ARN Mensajero/metabolismo , Conejos , Sarcolema/efectos de los fármacos , Sarcolema/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Contracción Miocárdica/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/genética , Cardiomiopatías/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Cinética , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/química , Miocardio/metabolismo , Oocitos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/análisis , Transfección , Xenopus laevisRESUMEN
Adherens junctions and desmosomes are responsible for mechanically coupling myocytes in the heart and are found closely apposed to gap junction plaques at the intercalated discs of cardiomyocytes. It is not known whether loss of cardiac gap junctions, such as described in cardiac disease states, may influence the expression patterns of other intercalated disc-associated proteins. We investigated whether the major cardiac gap junction protein connexin43 (Cx43) may be responsible for regulating adherens junctions, desmosomes and their associated catenins, in terms of abundance and localization at the intercalated discs of cardiomyocytes. In order to study the effect of loss of cardiac gap junctions on the intercalated disc-associated proteins, we used a combination of immunoblotting, immunofluorescence with confocal microscopy and electron microscopy to evaluate heart tissue from mice with cardiac-specific conditional knockout of Cx43. We found that the cardiac adherens junctions, desmosomes and their associated catenins, as well as vinculin and ZO-1, maintain their normal abundance, structural appearance and localization in the absence of Cx43. We conclude from these data that Cx43 is not required for the organization of the cell adhesion junctions and their associated catenins at the intercalated disc in the adult cardiac myocyte.