Modulation of the ?-adrenergic (?-AR) response is crucial in heart failure progression. NO from neuronal NOS (nNOS) is necessary to sustain the ?-AR response, but the underlying mechanisms are unknown. We have shown that plasma membrane calcium ATPase (PMCA4) interacts with nNOS, which is calcium/calmodulin dependent, and regulates nNOS-dependent NO production. Furthermore, we found that PMCA4 overexpression ablated the ?-AR inotropic response in the heart (Oceandy et al., 2007). Here, we aimed to investigate the mechanisms behind this observation. In transgenic mice overexpressing PMCA4 in the heart and in neonatal rat cardiomyocytes (NRCM) with adenoviral overexpression of PMCA4, a significant 3-fold increase in phosphorylation of phospholamban (PLB) at serine 16, as well as of troponin-I at serine 22 & 23, was observed prior to ?-AR stimulation (n=5, p<0.05). As a result of increased baseline phosphorylation, the relative increase in PLB and troponin-I phosphorylation following isoproterenol treatment was ablated. PMCA4 overexpression reduced nNOS activity by 65±6.2%, NO by 21.4±5.1% and cGMP by 24±5% (n=8, p<0.05). In addition, cAMP levels were increased by 32±5.5% (n=8, p<0.05) in response to PMCA4 overexpression leading to stimulation of PKA activity and subsequent PLB phosphorylation. Fluorescence resonance emission transfer technology (FRET) in the presence of isoform-specific phospho-diesterase (PDE) inhibitors for PDE2, 3 & 4 was used to determine the crosstalk between cAMP/cGMP. Only PDE2 activity was reduced by 68% at the membrane (assessed using the membrane EPAC1-mp cAMP sensor) but not in the cytoplasm (using cytoplasmic EPAC1 cAMP sensor) following PMCA4 overexpression (n=5, p<0.05). These findings show that PMCA4 determines a highly ordered and spatially confined sequence of events: PMCA4 inhibits nNOS mediated NO and cGMP production; the reduction in local cGMP leads to inhibition of PDE2, which is known to be activated by cGMP; reduced PDE2 activity elevates local cAMP, which in turn activates PKA. The latter phosphorylates PLB as well troponin-I making the cell refractory to further ?-AR stimulation. These results identify PMCA4 as a novel key player in the complex membranous network which determines the ?-AR response.
The Cardiac Sarcolemmal Calcium Pump Isoform 4 (PMCA4) Modulates the beta-adrenergic Response Through Alterations in Spatially Confined Cyclic Nucleotides
Di Benedetto Giulietta;
2009
Abstract
Modulation of the ?-adrenergic (?-AR) response is crucial in heart failure progression. NO from neuronal NOS (nNOS) is necessary to sustain the ?-AR response, but the underlying mechanisms are unknown. We have shown that plasma membrane calcium ATPase (PMCA4) interacts with nNOS, which is calcium/calmodulin dependent, and regulates nNOS-dependent NO production. Furthermore, we found that PMCA4 overexpression ablated the ?-AR inotropic response in the heart (Oceandy et al., 2007). Here, we aimed to investigate the mechanisms behind this observation. In transgenic mice overexpressing PMCA4 in the heart and in neonatal rat cardiomyocytes (NRCM) with adenoviral overexpression of PMCA4, a significant 3-fold increase in phosphorylation of phospholamban (PLB) at serine 16, as well as of troponin-I at serine 22 & 23, was observed prior to ?-AR stimulation (n=5, p<0.05). As a result of increased baseline phosphorylation, the relative increase in PLB and troponin-I phosphorylation following isoproterenol treatment was ablated. PMCA4 overexpression reduced nNOS activity by 65±6.2%, NO by 21.4±5.1% and cGMP by 24±5% (n=8, p<0.05). In addition, cAMP levels were increased by 32±5.5% (n=8, p<0.05) in response to PMCA4 overexpression leading to stimulation of PKA activity and subsequent PLB phosphorylation. Fluorescence resonance emission transfer technology (FRET) in the presence of isoform-specific phospho-diesterase (PDE) inhibitors for PDE2, 3 & 4 was used to determine the crosstalk between cAMP/cGMP. Only PDE2 activity was reduced by 68% at the membrane (assessed using the membrane EPAC1-mp cAMP sensor) but not in the cytoplasm (using cytoplasmic EPAC1 cAMP sensor) following PMCA4 overexpression (n=5, p<0.05). These findings show that PMCA4 determines a highly ordered and spatially confined sequence of events: PMCA4 inhibits nNOS mediated NO and cGMP production; the reduction in local cGMP leads to inhibition of PDE2, which is known to be activated by cGMP; reduced PDE2 activity elevates local cAMP, which in turn activates PKA. The latter phosphorylates PLB as well troponin-I making the cell refractory to further ?-AR stimulation. These results identify PMCA4 as a novel key player in the complex membranous network which determines the ?-AR response.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
