Reduction in interaction between cGMP and cAMP in dog ventricular myocytes with hypertrophic failure

Baseline function and signal transduction are depressed in hearts with hypertrophic failure. We tested the hypothesis that the effects of cGMP and its interaction with cAMP would be reduced in cardiac myocytes from hypertrophic failing hearts. Ventricular myocytes were isolated from control dogs, dogs with aortic valve stenosis hypertrophy, and dogs with pacing hypertrophic failure. Myocyte function was measured using a video edge detector. Cell contraction data were obtained at baseline, with 8-bromo-cGMP (10(-7), 10(-6), and 10(-5) M), with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA; a cAMP phosphodiesterase (PDE(2)) inhibitor] plus 8-bromo-cGMP, or milrinone (a PDE(3) inhibitor) plus 8-bromo-cGMP. Baseline percent shortening and maximal rates of shortening (R(max)) and relaxation were slightly reduced in hypertrophic myocytes and were significantly lower in failing myocytes (R(max): control dogs, 95.3 +/- 17.3; hypertrophy dogs, 88.2 +/- 5.5; failure dogs, 53.2 +/- 6.4 mum/s). 8-Bromo-cGMP dose dependently reduced myocyte function in all groups. However, EHNA (10(-6) M) and milrinone (10(-6) M) significantly reduced the negative effects of cGMP on cell contractility in control and hypertrophy but not in failing myocytes (R(max) for control dogs: cGMP, -46%; +EHNA, -21%; +milrinone, -19%; for hypertrophy dogs: cGMP, -40%; +EHNA, -13%; +milrinone, -20%; for failure dogs: cGMP, -40%; +EHNA, -29%; +milrinone, -32%). Both combinations of EHNA-cGMP and milrinone-cGMP significantly increased intracellular cAMP in control, hypertrophic, and failing myocytes. These data indicated that the cGMP signaling pathway was preserved in hypertrophic failing cardiac myocytes. However, the interaction of cGMP with the cAMP signaling pathway was impaired in these failing myocytes.     

Cyclic GMP and cyclic AMP induced changes in control and hypertrophic cardiac myocyte function interact through cyclic GMP affected cyclic-AMP phosphodiesterases.

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.     

Functional effects of C-type natriuretic peptide and nitric oxide are attenuated in hypertrophic myocytes from pressure-overloaded mouse hearts

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10(-6) and 10(-5) M) or CNP (10(-8) and 10(-7) M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10(-6) M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.     

Aging induces cardiac diastolic dysfunction, oxidative stress, accumulation of advanced glycation endproducts and protein modification

Evidence suggests that aging, per se, is a major risk factor for cardiac dysfunction. Oxidative modification of cardiac proteins by non-enzymatic glycation, i.e. advanced glycation endproducts (AGEs), has been implicated as a causal factor in the aging process. This study was designed to examine the role of aging on cardiomyocyte contractile function, cardiac protein oxidation and oxidative modification. Mechanical properties were evaluated in ventricular myocytes from young (2-month) and aged (24-26-month) mice using a MyoCam system. The mechanical indices evaluated were peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/- dL/dt). Oxidative stress and protein damage were evaluated by glutathione and glutathione disulfide (GSH/GSSG) ratio and protein carbonyl content, respectively. Activation of NAD(P)H oxidase was determined by immunoblotting. Aged myocytes displayed a larger cell cross-sectional area, prolonged TR90, and normal PS, +/- dL/dt and TPS compared with young myocytes. Aged myocytes were less tolerant of high stimulus frequency (from 0.1 to 5 Hz) compared with young myocytes. Oxidative stress and protein oxidative damage were both elevated in the aging group associated with significantly enhanced p47phox but not gp91phox expression. In addition, level of cardiac AGEs was approximately 2.5-fold higher in aged hearts than young ones determined by AGEs-ELISA. A group of proteins with a molecular range between 50 and 75 kDa with pI of 4-7 was distinctively modified in aged heart using one- or two-dimension SDS gel electrophoresis analysis. These data demonstrate cardiac diastolic dysfunction and reduced stress tolerance in aged cardiac myocytes, which may be associated with enhanced cardiac oxidative damage, level of AGEs and protein modification by AGEs.     

Basal and ethanol-induced cardiac contractile response in lean and obese zucker rat hearts

Obesity plays a pivotal role in metabolic and cardiovascular diseases. Certain types of obesity may be related to alcohol ingestion, which itself leads to impaired cardiac function. This study analyzed basal and ethanol-induced cardiac contractile response using left-ventricular papillary muscles and myocytes from lean and obese Zucker rats. Contractile properties analyzed include: peak tension development (PTD), peak shortening amplitude (PS), time to PTD/PS (TPT/TPS), time to 90% relaxation/relengthening (RT(90)/TR(90)) and maximal velocities of contraction/shortening and relaxation/relengthening (+/-VT and +/-dL/dt). Intracellular Ca(2+) transients were measured as fura-2 fluorescence intensity (DeltaFFI) changes and fluorescence decay time (FDT). In papillary muscles from obese rats, the baseline TPT and RT(90) were significantly prolonged accompanied with low to normal PTD and +/-VT compared to those in lean rats. Muscles from obese hearts also exhibited reduced responsiveness to postrest potentiation, increase in extracellular Ca(2+) concentration, and norepinephrine. By contrast, in isolated myocytes, obesity reduced PS associated with a significant prolonged TR(90), normal TPS and +/-dL/dt. Intracellular Ca(2+) recording revealed decreased resting Ca(2+) levels and prolonged FDT. Acute ethanol exposure (80-640 mg/dl) caused comparable concentration-dependent inhibitions of PTD/PS and DeltaFFI, associated with reduced +/-VT in both groups. Collectively, these results suggest altered cardiac contractile function and unchanged ethanol-induced depression in obesity.     

C-peptide exerts cardioprotective effects in myocardial ischemia-reperfusion

Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in cardiac dysfunction. C-peptide, a cleavage product of proinsulin to insulin processing, induces nitric oxide (NO)-mediated vasodilation. NO is reported to attenuate cardiac dysfunction caused by PMNs after ischemia-reperfusion (I/R). Therefore, we hypothesized that C-peptide could attenuate PMN-induced cardiac dysfunction. We examined the effects of C-peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. C-peptide (70 nmol/kg iv) given 4 or 24 h before I/R significantly improved coronary flow (P < 0.05), left ventricular developed pressure (LVDP) (P < 0.01), and the maximal rate of development of LVDP (+dP/dt(max)) compared with I/R hearts obtained from rats given 0.9% NaCl (P < 0.01). N(G)-nitro-L-arginine methyl ester (L-NAME) (50 micromol/l) blocked these cardioprotective effects. In addition, C-peptide significantly reduced cardiac PMN infiltration from 183 +/- 24 PMNs/mm(2) in untreated hearts to 44 +/- 10 and 58 +/- 25 PMNs/mm(2) in hearts from 4- and 24-h C-peptide-treated rats, respectively. Rat PMN adherence to rat superior mesenteric artery exposed to 2 U/ml thrombin was significantly reduced in rats given C-peptide compared with rats given 0.9% NaCl (P < 0.001). Moreover, C-peptide enhanced basal NO release from rat aortic segments. These results provide evidence that C-peptide can significantly attenuate PMN-induced cardiac contractile dysfunction in the isolated perfused rat heart subjected to I/R at least in part via enhanced NO release.     

Effects of cyclic GMP and its protein kinase on the contraction of ventricular myocytes from hearts after cardiopulmonary arrest

Hearts undergoing cardiopulmonary arrest and resuscitation have depressed function and may have changes in signal transduction. We hypothesized that the cyclic GMP (cGMP) signaling pathway would be altered in the post-resuscitation heart. This was studied in ventricular myocytes from 7 anesthetized open-chest rabbits. Cardiopulmonary arrest was achieved for 10 min through ventricular fibrillation and respirator shutdown. After cardiopulmonary arrest, respiration was resumed, the heart was defibrillated, and the heart recovered for 15 min. Seven additional rabbits served as controls. Myocyte function was measured via a video edge detector. Myocytes were treated with 8-bromo-cGMP (10(-5)-10(-6) mol/L) followed by KT5823 (10(-6) mol/L, cGMP protein kinase inhibitor). The baseline percent shortening was significantly depressed in the cardiac arrest myocytes compared with control (3.3 +/- 0.1 vs. 5.5 +/- 0.3%). Treatment with 8-Br-cGMP similarly and dose-dependently reduced cell contraction in both cardiac arrest (-24%) and control (-25%) myocytes. The negative effect of 8-Br-cGMP was partially reversed by KT5823 in control myocytes, but not in the arrest group, indicating reduced involvement of cGMP protein kinase. Multiple proteins were specifically phosphorylated when cGMP was present, but the degree of phosphorylation was significantly less in myocytes after cardiac arrest. The data suggested that the basal contraction was reduced, but the functional response to 8-Br-cGMP was preserved in myocytes from cardiopulmonary arrested hearts. The results also indicated that the action of cGMP appeared to be mainly through non-cGMP protein kinase pathways in the post-resuscitation heart.     

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.     

Altered cardiac excitation-contraction coupling in ventricular myocytes from spontaneously diabetic BB rats

Cardiac excitation-contraction (E-C) coupling abnormalities in chemically induced diabetes have been well defined. Heart dysfunction has also been reported in diabetes of genetic origin. The purpose of this study was to determine whether heart dysfunction in genetically predisposed diabetes is attributable to impaired E-C coupling at the cellular level. Myocytes were isolated from 1-yr-old BioBreed (BB) spontaneously diabetic-prone (BB/DP) rats and their diabetic-resistant littermates (BB/DR). Mechanical properties were evaluated by use of a video edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening and relengthening (+/-dL/dt). Intracellular Ca(2+) handling was evaluated with fura 2 fluorescent dye. Myocytes from spontaneously diabetic hearts exhibited a depressed PS, prolonged TPS and TR(90), and reduced +/-dL/dt. Consistent with the mechanical response, myocytes from the BB/DP group displayed reduced resting and peak intracellular Ca(2+) concentration associated with a slowed Ca(2+)-transient decay. Furthermore, myocytes from BB/DP hearts were less responsive to increases in extracellular Ca(2+) and norepinephrine and equally responsive to increases in stimulation frequency and KCl compared with those from the BB/DR group. These results suggest that the genetic diabetic state produces altered cardiac E-C coupling, in part, because of abnormalities of the myocyte, similar to that demonstrable after chemically induced diabetes or during human diabetes.     

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 Ca(2+)-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(-5)M 8-bromo-cyclic GMP (cGMP), 10(-8), 10(-7)M C-type natriuretic peptide (CNP), or 10(-6), 10(-5)M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca(2+) 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(-5)M 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 Ca(2+) transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca(2+) 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.     

Effect of SIN-1 in rat ventricular myocytes: interference with beta-adrenergic stimulation

We have examined the effects of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on Ca(2+) transients, L-type Ca(2+) current (I(Ca,L)), and cGMP/cAMP content in electrically-stimulated rat ventricular myocytes in the absence and presence of the beta-adrenergic stimulation with isoproterenol. SIN-1 had no effect at low concentrations, but decreased the amplitude of electrically-induced Ca(2+) transients at higher concentrations. SIN-1 attenuated the increase in Ca(2+) transients induced by isoproterenol in a concentration-dependent manner. SIN-1 Also reduced the amplitude of caffeine-induced Ca(2+) transients, and the increase in I(Ca,L) induced by isoproterenol. These effects of SIN-1 were associated with an increased cGMP and a decreased cAMP content in ventricular myocytes in either the absence or presence of isoproterenol. These data suggest that the inhibitory effect of SIN-1 on basal and beta-adrenergic stimulated Ca2+ signal in ventricular myocytes could be due to the depression in the SR function and I(Ca,L), possibly mediated by a cGMP/cAMP-dependent mechanism. Taken together, the present study supports the idea that NO acts as an inhibitory modulator of the cardiac function during pathological conditions associated with an abnormal production of NO such as septic shock.     

Impaired cardiac function and IGF-I response in myocytes from calmodulin-diabetic mice: role of Akt and RhoA

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)) and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) was evaluated as Ca(2+)-induced Ca(2+) release [difference in fura 2 fluorescent intensity (Delta FFI)] and fluorescence decay rate (tau). Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, phospholamban (PLB), Na(+)-Ca(2+) exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, +/-dL/dt, and Delta FFI associated with prolonged TPS, TR(90), and tau. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and Delta FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA.     

Uric acid decreases NO production and increases arginase activity in cultured pulmonary artery endothelial cells

Elevated levels of serum uric acid (UA) are commonly associated with primary pulmonary hypertension but have generally not been thought to have any causal role. Recent experimental studies, however, have suggested that UA may affect various vasoactive mediators. We therefore tested the hypothesis that UA might alter nitric oxide (NO) levels in pulmonary arterial endothelial cells (PAEC). In isolated porcine pulmonary artery segments (PAS), UA (7.5 mg/dl) inhibits acetylcholine-induced vasodilation. The incubation of PAEC with UA caused a dose-dependent decrease in NO and cGMP production stimulated by bradykinin or Ca(2+)-ionophore A23187. We explored cellular mechanisms by which UA might cause reduced NO production focusing on the effects of UA on the l-arginine-endothelial NO synthase (eNOS) and l-arginine-arginase pathways. Incubation of PAEC with different concentrations of UA (2.5-15 mg/dl) for 24 h did not affect l-[(3)H]arginine uptake or activity/expression of eNOS. However, PAEC incubated with UA (7.5 mg/dl; 24 h) released more urea in culture media than control PAEC, suggesting that arginase activation might be involved in the UA effect. Kinetic analysis of arginase activity in PAEC lysates and rat liver and kidney homogenates demonstrated that UA activated arginase by increasing its affinity for l-arginine. An inhibitor of arginase (S)-(2-boronoethyl)-l-cysteine prevented UA-induced reduction of A23187-stimulated cGMP production by PAEC and abolished UA-induced inhibition of acetylcholine-stimulated vasodilation in PAS. We conclude that UA-induced arginase activation is a potential mechanism for reduction of NO production in PAEC.     

Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia-reperfusion via nitric oxide mechanism

Caveolin-1 is a protein constituent of cell membranes. The caveolin-1 scaffolding region (residues 82-101) is a known inhibitor of protein kinase C. Inhibition of protein kinase C results in maintained nitric oxide (NO) release from the endothelium, which attenuates cardiac dysfunction after ischemia-reperfusion (I/R). Therefore, we hypothesized that the caveolin-1 scaffolding region of the molecule, termed caveolin-1 peptide, might attenuate postischemia polymorphonuclear neutrophil (PMN)-induced cardiac dysfunction. We examined the effects of caveolin-1 peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts reperfused with PMNs. Caveolin-1 peptide (165 or 330 microg) given intravenously 1 h before I/R significantly attenuated postischemic PMN-induced cardiac dysfunction, as exemplified by left ventricular developed pressure (LVDP) (P < 0.01) and the maximal rate of developed pressure (+dP/dt(max)) (P < 0.01), compared with I/R hearts obtained from rats given 0.9% NaCl. In addition, caveolin-1 peptide significantly reduced cardiac PMN infiltration from 195 +/- 5 PMNs/mm2 in untreated hearts to 103 +/- 5 and 60 +/- 5 PMNs/mm2 in hearts from 165 and 330 microg caveolin-1 peptide-treated rats, respectively (P < 0.01). PMN adherence to the rat coronary vasculature was also significantly reduced in rats given either 165 or 330 microg caveolin-1 peptide compared with rats given 0.9% NaCl (P < 0.01). Moreover, caveolin-1 peptide-treated rat aortas exhibited a 2.2-fold greater basal release of NO than vehicle-treated aortas (P < 0.01), and this was inhibited by NG-nitro-L-arginine methyl ester. These results provide evidence that caveolin-1 peptide significantly attenuated PMN-induced post-I/R cardiac contractile dysfunction in the isolated perfused rat heart, probably via enhanced release of endothelium-derived NO.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

sGC{alpha}1 mediates the negative inotropic effects of NO in cardiac myocytes independent of changes in calcium handling

In the heart, nitric oxide (NO) modulates contractile function; however, the mechanisms responsible for this effect are incompletely understood. NO can elicit effects via a variety of mechanisms including S-nitrosylation and stimulation of cGMP synthesis by soluble guanylate cyclase (sGC). sGC is a heterodimer comprised of a β(1)- and an α(1)- or α(2)-subunit. sGCα(1)β(1) is the predominant isoform in the heart. To characterize the role of sGC in the regulation of cardiac contractile function by NO, we compared left ventricular cardiac myocytes (CM) isolated from adult mice deficient in the sGC α(1)-subunit (sGCα(1)(-/-)) and from wild-type (WT) mice. Sarcomere shortening under basal conditions was less in sGCα(1)(-/-) CM than in WT CM. To activate endogenous NO synthesis from NO synthase 3, CM were incubated with the β(3)-adrenergic receptor (β(3)-AR) agonist BRL 37344. BRL 37344 decreased cardiac contractility in WT CM but not in sGCα(1)(-/-) myocytes. Administration of spermine NONOate, an NO donor compound, did not affect sarcomeric shortening in CM of either genotype; however, in the presence of isoproterenol, addition of spermine NONOate reduced sarcomere shortening in WT but not in sGCα(1)(-/-) CM. Neither BRL 37344 nor spermine NONOate altered calcium handling in CM of either genotype. These findings suggest that sGCα(1) exerts a positive inotropic effect under basal conditions, as well as mediates the negative inotropic effect of β(3)-AR signaling. Additionally, our work demonstrates that sGCα(1)β(1) is required for NO to depress β(1)/β(2)-AR-stimulated cardiac contractility and that this modulation is independent of changes in calcium handling.     

Overexpression of arginase in the aged mouse penis impairs erectile function and decreases eNOS activity: influence of in vivo gene therapy of anti-arginase

Since both increased nitric oxide (NO) synthase (NOS) abundance and diminished NO signaling have been reported in the aging penis, the role of NO in the adaptations of aging remains controversial. Here we tested the hypothesis that arginase, an enzyme that competes with NOS for the substrate l-arginine, contributes to erectile dysfunction with advanced age in the B6/129 mouse strain. Arginase protein abundance, mRNA expression, and enzyme activity were elevated in aged compared with young penile endothelial cells. In addition, endothelial NOS (NOS3) protein abundance was greater in aged versus young penile endothelial cells, whereas NOS activity and cGMP levels were reduced. Calcium-dependent l-arginine-to-l-citrulline conversion and cGMP formation increased significantly in aged mouse penes in the presence of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). However, there was no effect on l-arginine-to-l-citrulline conversion or cGMP accumulation in the endothelium from young mouse penes. To assess the functional role of arginase in the inhibition of NOS pathway responsiveness in the penis, we evaluated the effects of ABH and an adeno-associated virus encoding an antisense sequence to arginase I (AAVanti-arginase) on erectile function in vivo. ABH and AAVanti-arginase enhanced endothelium-dependent erectile responses in the aged mice without altering endothelium-independent responses. Paralleling our in vitro observations, ABH or AAVanti-arginase did not affect vascular responses in the young mice. Inhibition of the arginase pathway improves endothelial function in the aging penile circulation, suggesting that the arginase pathway may be exploited to improve erectile dysfunction associated with aging.     

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to β-adrenergic agonists is eliminated when the heart is perfused with N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the β-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 μM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with l-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed β-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the β-stimulated calcium current.     

cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation

Nitric oxide (NO) has concentration-dependent biphasic myocardial contractile effects. We tested the hypothesis, in isolated rat hearts, that NO cardiostimulation is primarily non-cGMP dependent. Infusion of 3-morpholinosydnonimine (SIN-1, 10(-5) M), which may participate in S-nitrosylation (S-NO) via peroxynitrite formation, increased the rate of left ventricular pressure rise (+dP/dt; 19 +/- 4%, P < 0.001, n = 11) without increasing effluent cGMP or cAMP. Superoxide dismutase (SOD; 150 U/ml) blocked SIN-1 cardiostimulation and led to cGMP elaboration. Sodium nitroprusside (10(-10)-10(-7) M), an iron nitrosyl compound, did not augment +dP/dt but increased cGMP approximately eightfold (P < 0.001), whereas diethylamine/NO (DEA/NO; 10(-7) M), a spontaneous NO. donor, increased +dP/dt (5 +/- 2%, P < 0.05, n = 6) without augmenting cGMP. SIN-1 and DEA/NO +dP/dt increase persisted despite guanylyl cyclase inhibition with 1H-(1,2,4)oxadiazolo-(4,3,-a)quinoxalin-1-one (10(-5) M, P < 0.05 for both donors), suggesting a cGMP-independent mechanism. Glutathione (5 x 10(-4) M, n = 15) prevented SIN-1 cardiostimulation, suggesting S-NO formation. SIN-1 also produced SOD-inhibitable cardiostimulation in vivo in mice. Thus peroxynitrite and NO donors can stimulate myocardial contractility independently of guanylyl cyclase activation, suggesting a role for S-NO reactions in NO/peroxynitrite-positive inotropic effects in intact hearts.