RESUMEN
Although angiotensin II (Ang II) plays an important role in heart disease associated with pump dysfunction, its direct effects on cardiac pump function remain controversial. We found that after Ang II infusion, the developed pressure and +dP/dt(max) in isolated Langendorff-perfused mouse hearts showed a complex temporal response, with a rapid transient decrease followed by an increase above baseline. Similar time-dependent changes in cell shortening and L-type Ca(2+) currents were observed in isolated ventricular myocytes. Previous studies have established that Ang II signaling involves phosphoinositide 3-kinases (PI3K). Dominant-negative inhibition of PI3Kalpha in the myocardium selectively eliminated the rapid negative inotropic action of Ang II (inhibited by approximately 90%), whereas the loss of PI3Kgamma had no effect on the response to Ang II. Consistent with a link between PI3Kalpha and protein kinase C (PKC), PKC inhibition (with GF 109203X) reduced the negative inotropic effects of Ang II by approximately 50%. Although PI3Kalpha and PKC activities are associated with glycogen synthase kinase-3beta and NADPH oxidase, genetic ablation of either glycogen synthase kinase-3beta or p47(phox) (an essential subunit of NOX2-NADPH oxidase) had no effect on the inotropic actions of Ang II. Our results establish that Ang II has complex temporal effects on contractility and L-type Ca(2+) channels in normal mouse myocardium, with the negative inotropic effects requiring PI3Kalpha and PKC activities.
Asunto(s)
Angiotensina II/farmacología , Canales de Calcio Tipo L/metabolismo , Contracción Miocárdica/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Análisis de Varianza , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Ratones , Ratones Noqueados , Miocardio/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteína Quinasa C/genética , Transducción de Señal/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds as a binary complex with phospholamban (PLN). To demonstrate whether overexpression of SLN in the heart might impair cardiac function directly, transgenic (TG) mice with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope) were generated on a phospholamban (PLN) null (PLN KO) background. In NF-SLN TG/PLN KO cardiac microsomes, the apparent affinity of SERCA2a for Ca2+ was decreased compared with non-TG littermate PLN KO hearts. Analyses of isolated NF-SLN/PLN KO cardiomyocytes revealed impaired cardiac contractility, reduced calcium transient peak amplitude, and slower decay kinetics compared to PLN KO animals. In these cardiomyocytes, isoproterenol restored calcium dynamics to the levels seen in PLN KO. Invasive hemodynamic and echocardiographic analyses of NF-SLN/PLN KO mouse cardiac muscle in vivo showed no direct effects of NF-SLN overexpression when compared to PLN KO mice. A possible mechanism for the lack of effects in the whole heart may be a responsiveness to phosphorylation because we determined that NF-SLN can be phosphorylated in cardiomyocytes in response to isoproterenol, and we provide evidence that serine/threonine kinase 16 is a kinase that can phosphorylate NF-SLN. Site-directed mutagenesis showed that SLN Thr-5 is the target site for this kinase. These data show that overexpression of NF-SLN can inhibit SERCA2a in the absence of PLN and that the inhibition of SERCA2a is correlated with impairment of contractility and calcium cycling in cardiomyocytes.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Proteínas Musculares/metabolismo , Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Proteolípidos/metabolismo , Animales , Calcio/análisis , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Cardiotónicos/farmacología , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Isoproterenol/farmacología , Ratones , Ratones Noqueados , Proteínas Musculares/genética , Mutación , Contracción Miocárdica/genética , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación , Fosfotransferasas/análisis , Fosfotransferasas/metabolismo , Proteolípidos/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Activación Transcripcional , Función VentricularRESUMEN
Cardiac and skeletal muscle critically depend on mitochondrial energy metabolism for their normal function. Recently, we showed that apoptosis-inducing factor (AIF), a mitochondrial protein implicated in programmed cell death, plays a role in mitochondrial respiration. However, the in vivo consequences of AIF-regulated mitochondrial respiration resulting from a loss-of-function mutation in Aif are not known. Here, we report tissue-specific deletion of Aif in the mouse. Mice in which Aif has been inactivated specifically in cardiac and skeletal muscle exhibit impaired activity and protein expression of respiratory chain complex I. Mutant animals develop severe dilated cardiomyopathy, heart failure, and skeletal muscle atrophy accompanied by lactic acidemia consistent with defects in the mitochondrial respiratory chain. Isolated hearts from mutant animals exhibit poor contractile performance in response to a respiratory chain-dependent energy substrate, but not in response to glucose, supporting the notion that impaired heart function in mutant animals results from defective mitochondrial energy metabolism. These data provide genetic proof that the previously defined cell death promoter AIF has a second essential function in mitochondrial respiration and aerobic energy metabolism required for normal heart function and skeletal muscle homeostasis.