Qihang Zhang

Cyclic GMP-Induced Reduction in Cardiac Myocyte Function Is Partially Mediated by Activation of the Sarcoplasmic Reticulum Ca2+-ATPase

We tested the hypothesis that the mechanism through which cyclic GMP reduces cardiac function is mediated by activation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Cardiac myocytes were isolated from New Zealand white rabbits (n = 11). Individual ventricular cells were stimulated by electrical field stimulation. The maximal rate of cell shortening and percentage shortening were measured with a video edge detector. Thapsigargin (10–8 mol/l) was used as a specific inhibitor of SERCA. When 8-bromo-cyclic GMP (8-Br-cGMP, 10–7, –6, –5 mol/l) was added to cells, the maximal rate of myocyte shortening (Rmax, µm/s) and percentage shortening were both decreased in a concentration-dependent manner. Rmax decreased 27% from 117 ± 12 at baseline to 85.2 ± 13 when 10–5 mol/l of 8-Br-cGMP was present, and percent shortening was reduced 28% from 6.0 ± 0.5 to 4.3 ± 0.5%. Thapsigargin (10–8 mol/l) increased the maximal rate of myocyte shortening and percent shortening. Addition of thapsigargin prior to 8-Br-cGMP reduced the negative effects of cGMP on myocyte function. The percent shortening decreased only 11% and Rmax decreased 14% with 10–5 mol/l 8-Br-cGMP, which was not significant. Cyclopiazonic acid, another SERCA inhibitor, was also used to test whether 8-Br-cGMP reduced myocyte function through SERCA. The results were similar to those when thapsigargin was used. These results indicated that the cyclic GMP-induced reduction in cardiac myocyte function was partially mediated through the action of the sarcoplasmic reticulum Ca2+-ATPase.

Hypoxia inducible factor-1 improves the actions of nitric oxide and natriuretic peptides after simulated ischemia-reperfusion.

Ischemia-reperfusion reduces the negative functional effects of cyclic GMP in cardiac myocytes. In this study, we tested the hypothesis that upregulation of hypoxic inducible factor-1 (HIF-1) would improve the actions of cyclic GMP signaling following simulated ischemia-reperfusion. HIF-1α was increased with deferoxamine (150 mg/kg for 2 days). Rabbit cardiac myocytes were subjected to simulated ischemia [15 min 95% N2-5% CO2] and reperfusion [reoxygenation] to produce myocyte stunning. Cell function was measured utilizing a video-edge detector. Shortening was examined at baseline and after brain natriuretic peptide (BNP, 10-8, 10-7M) or S-nitroso-N-acetyl-penicillamine (SNAP, 10-6, 10-5M) followed by KT5823 (cyclic GMP protein kinase inhibitor, 10-6M). Kinase activity was measured via a protein phosphorylation assay. Under control conditions, BNP (-30%) and SNAP (-41%) reduced percent shortening, while KT5823 partially restored function (+18%). Deferoxamine treated control myocytes responded similarly. In stunned myocytes, BNP (-21%) and SNAP (-25%) reduced shortening less and KT5823 did not increase function (+2%). Deferoxamine increased the effects of BNP (-38%) and SNAP (-41%) in stunning and restored the effects of KT5823 (+12%). The cyclic GMP protein kinase increased phosphorylation of several proteins in control HIF-1 +/- cells. Phosphorylation was reduced in stunned cells and was restored in deferoxamine treated stunned cells. This study demonstrated that simulated ischemia-reperfusion reduced the negative functional effects of increasing cyclic GMP and this was related to reduced effects of the cyclic GMP protein kinase. Increased HIF-1α protects the functional effects of cyclic GMP thorough maintenance of cyclic GMP protein kinase activity after ischemic-reperfusion.

Cyclic GMP protein kinase activity is reduced in thyroxine‐induced hypertrophic cardiac myocytes

1. We tested the hypothesis that the cGMP‐dependent protein kinase has major negative functional effects in cardiac myocytes and that the importance of this pathway is reduced in thyroxine (T4; 0.5 mg/kg per day for 16 days) hypertrophic myocytes.

Role of phospholamban in cyclic GMP mediated signaling in cardiac myocytes.

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca2+-ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10-6, 10-5M 8-bromo-cyclic GMP (cGMP), 10-8, 10-7M C-type natriuretic peptide (CNP), or 10-6, 10-5M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca2+ concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10-5M caused a significant decrease in %shortening (3.6±0.2% to 2.3±0.1%) in WT, but little change in PLB-KO myocytes (3.4±0.1% to 3.2±0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca2+ transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca2+ transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban.

Increased cerebral oxygen consumption in Eker rats and effects of N-methyl-D-aspartate blockade: Implications for autism

Because there is a strong correlation between tuberous sclerosis and autism, we used a tuberous sclerosis model (Eker rat) to test the hypothesis that these animals would have an altered regional cerebral O2 consumption that might be associated with autism. We also examined whether the altered cerebral O2 consumption was related to changes in the importance of N‐methyl‐D‐aspartate (NMDA) receptors. Young (4 weeks) male control Long Evans (N = 14) and Eker (N = 14) rats (70–100 g) were divided into control and CGS‐19755 (10 mg/kg, competitive NMDA antagonist)‐treated animals. Cerebral regional blood flow (14C‐iodoantipyrine) and O2 consumption (cryomicrospectrophotometry) were determined in isoflurane‐anesthetized rats. NMDA receptor protein levels were determined by Western immunoblotting. We found significantly increased basal O2 consumption in the cortex (6.2 ± 0.6 ml O2/min/100 g Eker vs. 4.7 ± 0.4 Long Evans), hippocampus, cerebellum, and pons. Regional cerebral blood flow was also elevated in Eker rats at baseline, but cerebral O2 extraction was similar. CGS‐19755 significantly lowered O2 consumption in the cortex (2.8 ± 0.3), hippocampus, and pons of the Long Evans rats but had no effect on cortex (5.8 ± 0.8) or other regions of the Eker rats. Cerebral blood flow followed a similar pattern. NMDA receptor protein levels (NR1 subunit) were similar between groups. In conclusion, Eker rats had significantly elevated cerebral O2 consumption and blood flow, but this was not related to NMDA receptor activation. In fact, the importance of NMDA receptors in the control of basal cerebral O2 consumption was reduced. This might have important implications in the treatment of autism.

T4-induced cardiac hypertrophy disrupts cyclic GMP mediated responses to brain natriuretic peptide in rabbit myocardium

Brain natriuretic peptide (BNP) affects the regulation of myocardial metabolism through the production of cGMP and these effects may be altered by cardiac hypertrophy. We tested the hypothesis that BNP would cause decreased metabolism and function in the heart and cardiac myocytes by increasing cGMP and that these effects would be disrupted after thyroxine-induced cardiac hypertrophy (T4). Open-chest control and T4 rabbits were instrumented to determine local effects of epicardial BNP (10(-3) M). Function of isolated cardiac myocytes was examined with BNP (10(-8)-10(-7) M) with or without KT5823 (10(-6) M, cGMP protein kinase inhibitor). Cyclic GMP levels were measured in myocytes. In open-chest controls, O2 consumption was reduced in the BNP area of the subepicardium (6.6+/-1.3 ml O2/min/100 g versus 8.9+/-1.4 ml O2/min/100 g) and subendocardium (9.4+/-1.3 versus 11.3+/-0.99). In T4 animals, functional and metabolic rates were higher than controls, but there was no difference between BNP-treated and untreated areas. In isolated control myocytes, BNP (10(-7) M) reduced percent shortening (PSH) from 6.5+/-0.6 to 4.3+/-0.4%. With KT5823 there was no effect of BNP on PSH. In T4 myocytes, BNP had no effect on PSH. In control myocytes, BNP caused cGMP levels to rise from 279+/-8 to 584+/-14 fmol/10(5) cells. In T4 myocytes, baseline cGMP levels were lower (117+/-2 l) and were not significantly increased by BNP. Thus, BNP caused decreased metabolism and function while increasing cGMP in control. These effects were lost after T4 due to lack of cGMP production. These data indicated that the effects of BNP on heart function operated through a cGMP-dependent mechanism, and that this mechanism was disrupted in T4-induced cardiac hypertrophy.

Negative Functional Effects of Natriuretic Peptides Are Attenuated in Hypertrophic Cardiac Myocytes by Reduced Particulate Guanylyl Cyclase Activity

We tested the hypothesis that the negative functional effects of natriuretic peptides would be blunted in thyroxine (T4)-induced hypertrophic cardiac myocytes. We also studied the causes of these changes. Ventricular myocytes were obtained from control (n = 8) and T4 (0.5 mg/kg/16 days) treated rabbit hearts (n = 7). Cell shortening parameters were studied with a video edge detector. We also determined particulate (pGC) and soluble (sGC) guanylyl cyclase activity and cyclic GMP levels. Myocyte function was examined at baseline and after brain natriuretic peptide (BNP 10−7,−6 M) or C-type natriuretic peptide (CNP 10−7,−6 M) or zaprinast (cyclic GMP phosphodiesterase inhibitor 10−6M) followed by BNP or CNP. Baseline function was similar in control and T4 myocytes. BNP (5.7 ± 0.2 to 4.3 ± 0.1%) and CNP (5.7 ± 0.4 to 4.2 ± 0.2%) significantly reduced percent shortening in control myocytes. These reductions were not observed with T4 (BNP, 5.7 ± 0.6 to 5.6 ± 0.6; CNP, 5.6 ± 0.4 to 5.5 ± 0.5). BNP and CNP responded similarly after zaprinast. Baseline cyclic GMP was similar in control and T4, but BNP only increased cyclic GMP in controls. The activity of pGC was similar at baseline in control and T4, but the stimulated activity was significantly lower in T4 myocytes. Both basal and stimulated sGC activity were similar in control and hypertrophic myocytes. These results demonstrated that the ability of natriuretic peptides to reduce ventricular myocyte function was blunted in T4 hypertrophic myocytes. This blunted response was related to the reduced ability of natriuretic peptides to increase cyclic GMP levels due to a reduced stimulated particulate guanylyl cyclase activity.

Cyclic GMP reduces myocardial stunning through non-cyclic GMP protein kinase mechanisms

We tested the hypothesis that myocardial stunning would be reduced by increased cyclic GMP and cGMP protein kinase activity. Hearts were instrumented in eight open-chest anesthetized dogs. The left anterior descending coronary artery (LAD) was occluded for 15 minutes followed by a 30-minute recovery and infusion of 8-Bromo-cGMP (0.1 and 1 μg/kg/min) during functional and metabolic data collection. Myocytes from circumflex and LAD regions were then used to obtain data at baseline, with 8-Br-cGMP (10−7,−6,−5 M) and KT5823 10−6 M, cGMP protein kinase inhibitor. The in vivo time delay of regional shortening increased significantly from 55 ± 12 to 99 ± 3 msec following stunning, but was reduced to 81 ± 2 by 1 μg/kg/min 8-Br-cGMP. The % regional work during systole decreased during stunning (93 ± 2 to 76 ± 8%), but was restored by 8-Br-cGMP (91 ± 7). Stunning lengthened the time of myocyte contraction and relaxation and reduced baseline shortening. 8-Br-cGMP reduced myocyte shortening in both regions. However, KT5823 only restored myocyte shortening in controls. These data indicated that regional myocardial stunning could be reduced by cyclic GMP but this appeared to be through non-cGMP protein kinase mechanisms.

Brain Natriuretic Peptide Reverses the Effects of Myocardial Stunning in Rabbit Myocardium

We tested the hypothesis that brain natriuretic peptide (BNP) would decrease the effects of myocardial stunning in rabbit hearts. We also examined the mechanisms responsible for these effects. In two groups of anesthetized open-chest rabbits, myocardial stunning was produced by 2 15-min occlusions of the left anterior descending artery separated by 15 min of reperfusion. The treatment group had BNP (10–3 mol/l) topically applied to the stunned area. Hemodynamic and functional parameters were measured. Coronary flow and O2 extraction were used to determine myocardial O2 consumption. In separate animals, we measured the function of isolated control and simulated ischemia (95% N2/5% CO2, 15 min)-reperfusion ventricular myocytes with BNP or C-type natriuretic peptide (10–8–10–7 mol/l) followed by KT5823 (10–6 mol/l, cyclic GMP protein kinase inhibitor). In the in vivo control group, baseline delay to contraction was 47 ± 4 ms and after stunning it increased to 71 ± 10 ms. In the treatment group, baseline delay to contraction was 40 ± 7 ms, and after stunning and BNP it did not significantly increase (43 ± 6 ms). Neither stunning nor BNP administration affected regional O2 consumption. In control myocytes, BNP (10–7 mol/l) decreased the percent shortening from 6.7 ± 0.4 to 4.5 ± 0.2%; after KT5823 administration, the percent shortening increased to 5.4 ± 0.5%. In ischemia-reperfusion myocytes, BNP (10–7 mol/l) decreased the percent shortening less from 5.0 ± 0.5 to 3.8 ± 0.2%; KT5823 administration did not increase the percent shortening (3.8 ± 0.2%). BNP similarly and significantly increased cyclic GMP levels in control and stunned myocytes. The data illustrated that BNP administration reversed the effects of stunning and its mechanism may be independent of the cyclic GMP protein kinase.

SERCA Inhibition Limits the Functional Effects of Cyclic GMP in Both Control and Hypertrophic Cardiac Myocytes

The negative functional effects of cyclic GMP are controlled by the sarcoplasmic reticulum calcium-ATPase (SERCA). The effects of cyclic GMP are blunted in cardiac hypertrophy. We tested the hypothesis that the interaction between cyclic GMP and SERCA would be reduced in hypertrophic cardiac myocytes. Myocytes were isolated from 7 control and 7 renal-hypertensive hypertrophic rabbits. Control and hypertrophic myocytes received 8-bromo-cGMP (8-Br-cGMP; 10–7, 10–6, 10–5 mol/l), the SERCA blocker thapsigargin (10–8 mol/l) followed by 8-Br-cGMP, or the SERCA blocker, cyclopiazonic acid (CPA; 10–7 mol/l) followed by 8-Br-cGMP. Percent shortening and maximal rate of shortening and relaxation were recorded using a video edge detector. Changes in cytosolic Ca2+ were assessed in fura 2-loaded myocytes. In controls, 8-Br-cGMP caused a significant 36% decrease in percent shortening from 5.8 ± 0.4 to 3.7 ± 0.3%. Thapsigargin and CPA did not affect basal control or hypertrophic myocyte function. When 8-Br-cGMP was given following thapsigargin or CPA, the negative effects of 8-Br-cGMP on control myocyte function were reduced. In hypertrophic myocytes, 8-Br-cGMP caused a smaller but significant 17% decrease in percent shortening from 4.7 ± 0.2 to 3.9 ± 0.1%. When 8-Br-cGMP was given following thapsigargin or CPA, no significant changes occurred in hypertrophic cell function. Intracellular Ca2+ transients responded in a similar manner to changes in cell function in control and hypertrophic myocytes. These results show that the effects of cyclic GMP were reduced in hypertrophic myocytes, but this was not related to SERCA. In presence of SERCA inhibitors, the responses to cyclic GMP were blunted in hypertrophic as well as control myocytes.