The Role And Mechanism Of Neuronal Nitric Oxide Synthase In Angiotensin ? Induced Cardiomyocyte Relaxation

Objective:To investigate the effect and mechanism of angiotensin ?(Ang ?)in promoting cardiomyocyte relaxation by upregulating neuronal nitric oxide synthase(n NOS).Method:Left ventricular cardiomyocytes were isolated in the Langendorff perfusion system using collagenase ?.The Ion Optix system was used to measure changes in sarcomere length(degree of contraction and relaxation)of cardiomyocytes induced by electrical stimulation.Fluorescence ratio of Fura-2 was used to detect[Ca²⁺]i.Myofilament calcium sensitivity was assessed by a phase plan of Fura-2 ratio to sarcomere length.Western blot was used to detect the myocardial cell lysates in rats.Nitric oxide is detected by measuring nitrite levels in cardiomyocytes.Results:1.Cardiomyocytes were treated with angiotensin ? for 30 min,1 h,2 h,3 h and 6 h,and contractile responses were measured.At 30 minutes,relaxation is slightly delayed(P<0.001).TR50 recovered to its base level after 1 or 2 hours(P=0.6).After treatment with Ang ? for 3 h,the cardiomyocyte diastolic rate increased significantly(P<0.001)and maintained this level for 6 hours(P=0.001).2.Similar rapid relaxation was observed at lower doses of angiotensin ?(3 h)(10 n M:P=0.03,100n M:P<0.001).Ang ? significantly promoted diastolic response at all tested concentrations(10 n M?100 n M?1?M).3.Losartan inhibited Ang ?-induced rapid relaxation during cardiomyocyte preconditioning(3h)(P=0.53),AT2R antagonist PD 123319 blocked the effect of Ang ? on cardiomyocyte(P=0.59),and Mas R antagonist A779 did not block the effect of Ang ? on myocardial relaxation(P<0.001).4.The expression of n NOS protein was significantly increased in cardiomyocytes treated with angiotensin ?(3 h,P=0.01),and the production of NO(nitrite content)was also higher(3 h,P<0.0001).Pretreatment of cardiomyocytes with selective n NOS inhibitor SMTC inhibited the enhancement of nitric oxide by angiotensin ?(P=0.4).5.SMTC completely prevented angiotensin ? from promoting relaxation of cardiomyocytes.L-NAME also prevented angiotensin ? from promoting relaxation of cardiomyocytes.6.PKA inhibitor did not block angiotensin ? in promoting cardiomyocyte dilation.calmodulin kinase ?(Ca MK?)inhibitor KN-93 did not block Ang ?-induced rapid cardiomyocyte dilation(P<0.001).7.Soluble guanylate cyclase inhibitor(ODQ)or PKG inhibitor(KT 5823)did not affect basal muscle cell diastole,but inhibited Ang ?-induced rapid diastole(P=0.9).Ang ?(3 h)significantly increased PLN-Ser¹⁶without affecting total PLN(P=0.003).SMTC preconditioning blocked the angiotensin ?-induced phosphorylation of PLN-Ser¹⁶(P=0.7).KT 5823 also inhibited phosphorylation of PLN-Ser¹⁶(P=0.9).Conclusions:Angiotensin ? upregulated the expression and activity of n NOS protein,and the upregulated n NOS promoted cardiomyocyte diastole through c GMP/PKG-dependent phosphorylation of PLN-Ser¹⁶.