Artin A. Shoukas

Load Independence of the Instantaneous Pressure-Volume Ratio of the Canine Left Ventricle and Effects of Epinephrine and Heart Rate on the Ratio

As a means of assessing ventricular performance, we analyzed the time-varying ratio of instantaneous pressure, P(t), to instantaneous volume, V(t), in the canine left ventricle. Intraventricular volume was measured by plethysmography, while the right heart was totally bypassed. The cardiac nerves were sectioned, and an epinephrine infusion was used to alter the contractile state. The instantaneous pressure-volume ratio was defined as E(t) = P(t)/[V(t) – Vd], where Vd is an experimentally determined correction factor. We found that (1) all the E(t) curves thus defined were similar in their basic shape and attained their peak near the end of the ejection phase regardless of the mechanical load, the contractile state, or the heart rate, (2) under a constant heart rate and contractile state extensive changes in preload, afterload, or both did not alter the peak value of E(t), Emax, or the time to Emax from the onset of systole, Tmax, and (3) these parameters of E(t) markedly changed with epinephrine infusion or increases in heart rate. At an epinephrine infusion rate of 2 μg/kg min−1, Emax increased to 12.2 ± 4.5 (SD) mm Hg/ml (N = 9) from its control value of 6.6 ± 1.2 mm Hg/ml before the infusion. Simultaneously, Tmax shortened from 191 ± 29 msec to 157 ± 26 msec. Increases in the paced heart rate proportionally shortened Tmax (45% per 100-beats/min change in heart rate) without any effect on Emax. We concluded that E(t), represented by Emax and Tmax, explicitly reflects the ventricular contractility.

End-systolic pressure/volume ratio: A new index of ventricular contractility

A thesis recently developed from a series of experiments on the isolated canine left ventricle is described. It is claimed that the ventricular presure/volume ratio at end-systole is relatively insensitive to cardiac loading and varies greatly in response to changes in ventricular contractility. The clinical viability of this basic finding rests on the substitution of diameter for volume in this formulation. Diameter can be measured using a noninvasive ultrasonic technique in the clinic. Accordingly, end-systolic pressure/diameter ratio was studied in the isolated preparation and found to be similarly insensitive to loading conditions and sensitive to inotropic interventions. A further analysis of the pressure/diameter ratio in the ventricle of the conscious dog is in progress. In parallel with these studies, use of the pressure/diameter ratio to evaluate contractility in cardiac patients is being tested. The preliminary findings from conscious dogs and clinic patients are briefly discussed.

Arginase Reciprocally Regulates Nitric Oxide Synthase Activity and Contributes to Endothelial Dysfunction in Aging Blood Vessels

Background—Although abnormal l-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. l-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating l-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. Methods and Results—Inhibition of arginase with (S)-(2-boronoethyl)-l-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4±9.4% at 10−5 mol/L, P <0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-l-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P <0.05 and P <0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60±6% versus 39±6%, P <0.05). In addition, BEC restored depressed l-arginine (10−4 mol/L)–dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. Conclusions—These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular l-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Instantaneous pressure-volume relationship of the canine right ventricle.

The instantaneous isovolumic and ejecting pressure-volume relationship of the right ventricle was studied in 11 cross-circulated, isolated canine hearts to characterize the right ventricular contractile state. Accurate measurement of volume was achieved by the use of a water-filled, thin latex balloon in the right ventricle connected to a special volume loading and transducing chamber. Pressure was measured with a miniature pressure transducer mounted within the balloon. Wide variations in loading conditions were achieved by changing the volume of air above the volumetric chamber. The pressure and volume data were collected from multiple beats under a constant contractile state in the same mode of contraction while the left ventricle was vented to air. Linear regression analysis applied to each of the isochronal pressure-volume data sets at 20-msec intervals from the onset of contraction showed a highly linear correlation between the pressure and the volume. Both the slope and the volume intercept of the regression lines changed with time throughout the cardiac cycle. The maximal slope of the regression line (E,max) averaged 2.50 ± 0.49 mm Hg/ml (mean ± SD) for ejecting beats and 2.68 ± 0.55 mm Hg/ml for isovolumic beats. Epinephrine infusions of 12.5 μg/min and 25.0 μg/min increased Emax by 31% and 82%, respectively (P < 0.005). We conclude that: (1) The instantaneous pressure-volume relationships of the right ventricle in the isovolumic and ejecting modes can be regarded as linear, at least within the physiological range; however, these two modes of contraction did not yield an identical relationship. (2) The slope of these pressure-volume relationship curves changes with a change in the contractile state. cire Res 44: 309-315, 1979

Control of total systemic vascular capacity by the carotid sinus baroreceptor reflex

To attain a quantitative understanding of carotid sinus baroreceptor reflex control of cardiac output, we studied the reflex control of total systemic vascular capacity in vagotomized dogs. In experiments measuring blood volume shifts caused by the carotid sinus reflex (series 1), venous return was diverted into a reservoir while cardiac output and central venous pressure were maintained at constant levels. The pressure in the isolated carotid sinuses (ISP) was lowered or raised in 25-mm Hg steps between 75 and 200 mm Hg. This procedure mobilized blood into or out of the reservoir, indicating a decrease or an increase in total vascular capacity, respectively. The mean maximum volume shift, 3.6 ml/kg body weight, occurred in the same ISP region, 135 ± 12.5 mm Hg, where reflex control of total peripheral resistance was strongest. The total volume shift was approximately 7.5 ml/kg for ISP changes from 75 to 200 mm Hg. When mean arterial blood pressure was maintained constant during the ISP step changes, the volume shift almost doubled. In experiments measuring the reflex effect on total systemic vascular compliance (series 2) and in experiments determining the reflex control of arterial compliance (series 3), total systemic vascular and lumped arterial compliances were measured in the same dogs that were used in series 1 experiments. The total systemic vascular and arterial compliances were approximately 2.0 ml/mm Hg kg−1 and 0.0677 ml/mm Hg kg−1, respectively. The reflex did not affect these compliances. We concluded that the reflex controls the total systemic venous capacity to a degree that changes cardiac output potentially by 30–10% per 25-mm Hg change in ISP.

Endothelial Arginase II A Novel Target for the Treatment of Atherosclerosis

Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E–null (ApoE−/−) and wild-type mice fed a high cholesterol diet. In ApoE−/− mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II−/− mice) prevents high-cholesterol diet–dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein–dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.

Nitric Oxide Regulation of Myocardial Contractility and Calcium Cycling: Independent Impact of Neuronal and Endothelial Nitric Oxide Synthases

Abstract— The mechanisms by which nitric oxide (NO) influences myocardial Ca2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dtmax normalized to end-diastolic volume; dP/dt-EDV) by 51±5% (P <0.001) and lusitropy (tau; &tgr;) by 20.3±2.0% (P <0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1−/−) (15±2% increase in dP/dt-EDV; P <0.001 versus WT; and no change in &tgr;; P <0.01 versus WT). Isolated myocytes from NOS1−/− (≈2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca2+ transients ([Ca2+]i) compared with WT. SR Ca2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca2+]i at 4 Hz increased 107±23% above 1 Hz response) but not in NOS1−/− (13±26%; P <0.01 versus WT). In contrast, mice lacking NOS3 (NOS3−/−) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca2+]i, and SR Ca2+ stores. NOS1−/− had increased SR Ca2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.

Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2(-)) production than young. Acute inhibition of both NOS, with N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-amino- 6-boronohexanoic acid (ABH), significantly reduced O2(-) production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692-702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2(-) production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.

Knockdown of arginase I restores NO signaling in the vasculature of old rats

Arginase, expressed in endothelial cells and upregulated in aging blood vessels, competes with NO synthase (NOS) for l-arginine, thus modulating vasoreactivity and attenuating NO signaling. Moreover, arginase inhibition restores endothelial NOS signaling and l-arginine responsiveness in old rat aorta. The arginase isoform responsible for modulating NOS, however, remains unknown. Because isoform-specific arginase inhibitors are unavailable, we used an antisense (AS) oligonucleotide approach to knockdown arginase I (Arg I). Western blot and quantitative PCR confirmed that Arg I is the predominant isoform expressed in endothelialized aortic rings and is upregulated in old rats compared with young. Aortic rings from 22-month-old rats were incubated for 24 hours with sense (S), AS oligonucleotides, or medium alone (C). Immunohistochemistry, immunoblotting, and enzyme assay confirmed a significant knockdown of Arg I protein and arginase activity in AS but not S or C rings. Conversely, calcium-dependent NOS activity and vascular metabolites of NO was increased in AS versus S or C rings. Acetylcholine (endothelial-dependent) vasorelaxant responses were enhanced in AS versus S or C treated rings. In addition, 1H-oxadiazolo quinoxalin-1-one (10 &mgr;mol/L), a soluble guanylyl cyclase inhibitor, increased the phenylephrine response in AS compared with S and C rings suggesting increased NO bioavailability. Finally, l-arginine (0.1 mmol/L)-induced relaxation was increased in AS versus C rings. These data support our hypothesis that Arg I plays a critical role in the pathobiology of age-related endothelial dysfunction. AS oligonucleotides may, therefore, represent a novel therapeutic strategy against age-related vascular endothelial dysfunction.

Inducible NO Synthase–Dependent S-Nitrosylation and Activation of Arginase1 Contribute to Age-Related Endothelial Dysfunction

Endothelial function is impaired in aging because of a decrease in NO bioavailability. This may be, in part, attributable to increased arginase activity, which reciprocally regulates NO synthase (NOS) by competing for the common substrate, l-arginine. However, the high Km of arginase (>1 mmol/L) compared with NOS (2 to 20 &mgr;mol/L) seemingly makes direct competition for substrate unlikely. One of the mechanisms by which NO exerts its effects is by posttranslational modification through S-nitrosylation of protein cysteines. We tested the hypothesis that arginase1 activity is modulated by this mechanism, which serves to alter its substrate affinity, allowing competition with NOS for l-arginine. We demonstrate that arginase1 activity is altered by S-nitrosylation, both in vitro and ex vivo. Furthermore, using site-directed mutagenesis we demonstrate that 2 cysteine residues (C168 and C303) are able to undergo nitrosylation. S-Nitrosylation of C303 stabilizes the arginase1 trimer and reduces its Km value 6-fold. Finally, arginase1 nitrosylation is increased (and thus its Km decreased) in blood vessels from aging rats, likely contributing to impaired NO bioavailability and endothelial dysfunction. This is mediated by inducible NOS, which is expressed in the aging endothelium. These findings suggest that S-nitrosylated arginase1 can compete with NOS for l-arginine and contribute to endothelial dysfunction in the aging cardiovascular system.