Role of phosphoinositide 3-kinase {alpha}, protein kinase C, and L-type Ca2+ channels in mediating the complex actions of angiotensin II on mouse cardiac contractility

Abstract

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.

Figure 1

A. Representative left ventricle (LV) pressure traces (left) and +dP/dt_max (right, n=4) of mouse hearts during infusion of AngII (3 nmol/L). Hearts were perfused using the Langendorff method at a constant perfusion pressure. B. Top panels: +dP/dt_max time curse during AngII infusion at 3 nmol/L (left, n=5) and 30 nmol/L (right, n=4). Hearts were perfused at a constant coronary flow rate in the presence of a vasodilator (P1075, 100 nmol/L). Lower panels: Summary of peak early-phase decreases (left) and peak late-phase increases (right) in +dP/dt_max of hearts treated with 3, 30, or 300 (n=4) nmol/L AngII.

Figure 2

A. Representative cell shortening traces of ventricular myocytes without (0 min) and with (5 and 15 min) AngII treatment (30 nmol/L), in the absence (top panels) and presence (bottom panels) of an AT₁ receptor inhibitor (irbersartan, 10 μmol/L). The downward deflections from baselines indicate cell length decrease (shortening). Cell shortening was expressed as a percentage of the baseline diastolic length. Recordings were done at 36 °C and myocytes were stimulated at 1Hz. B. Summary of AngII on cell shortening (n=13–14) in the absence (left) and presence (right) of irbersartan. *p<0.01, #p<0.05 vs. con (0 min).

Figure 3

A. Time course (left) and representative traces (right) of L-type Ca²⁺ currents recorded at 0 mV from ventricular myocytes with AngII (30 nmol/L) perfusion (open circles) or without AngII (filled circles). Capacitance currents in right panel were removed for clarity. Currents were recorded at 22 °C with patch-clamp technique using amphotericin B-perforated configuration. B. Summary of AngII-induced changes in amplitude of L-type Ca²⁺ currents recorded at 0 mV (n=7). *p<0.05, #p<0.01 vs. baseline currents.

Figure 4

A. Time courses of +dP/dt_max during AngII (30 nmol/L) infusion in wild-type hearts (left, n=5) and DN-PI3Kα hearts (right, n=7). Mouse hearts were perfused at a constant flow rate in the presence of a vasodilator (P1075, 100 nmol/L). B. Summary of peak decreases (left) and peak increases (right) of +dP/dt_max during AngII infusion in wild-type hearts (blank bars) and DN-PI3Kα hearts (filled bars). * p<0.01 vs. wild-type.

Figure 5

A. Time courses of +dP/dt_max of wild-type C57BL/6 mouse hearts during AngII (30 nmol/L) infusion in the absence (left, n=10) and presence (right, n=7) of a PKC inhibitor (GF 109203X, 1 μmol/L). Hearts were perfused at a constant flow rate in the presence of a vasodilator (P1075, 100 nmol/L). B. Summary of peak decreases (left) and peak increases (right) of +dP/dt_max during AngII infusion in the absence (blank bars) or presence (filled bars) of GF 109203X. *p<0.05, vs. control.