Maximum Rate Of Shortening Research Articles

Cobalt oxide nanoparticles induce oxidative stress and alter electromechanical function in rat ventricular myocytes

BackgroundNanotoxicology is an increasingly relevant field and sound paradigms on how inhaled nanoparticles (NPs) interact with organs at the cellular level, causing harmful conditions, have yet to be established. This is particularly true in the case of the cardiovascular system, where experimental and clinical evidence shows morphological and functional damage associated with NP exposure. Giving the increasing interest on cobalt oxide (Co3O4) NPs applications in industrial and bio-medical fields, a detailed knowledge of the involved toxicological effects is required, in view of assessing health risk for subjects/workers daily exposed to nanomaterials. Specifically, it is of interest to evaluate whether NPs enter cardiac cells and interact with cell function. We addressed this issue by investigating the effect of acute exposure to Co3O4-NPs on excitation-contraction coupling in freshly isolated rat ventricular myocytes.ResultsPatch clamp analysis showed instability of resting membrane potential, decrease in membrane electrical capacitance, and dose-dependent decrease in action potential duration in cardiomyocytes acutely exposed to Co3O4-NPs. Motion detection and intracellular calcium fluorescence highlighted a parallel impairment of cell contractility in comparison with controls. Specifically, NP-treated cardiomyocytes exhibited a dose-dependent decrease in the fraction of shortening and in the maximal rate of shortening and re-lengthening, as well as a less efficient cytosolic calcium clearing and an increased tendency to develop spontaneous twitches. In addition, treatment with Co3O4-NPs strongly increased ROS accumulation and induced nuclear DNA damage in a dose dependent manner. Finally, transmission electron microscopy analysis demonstrated that acute exposure did lead to cellular internalization of NPs.ConclusionsTaken together, our observations indicate that Co3O4-NPs alter cardiomyocyte electromechanical efficiency and intracellular calcium handling, and induce ROS production resulting in oxidative stress that can be related to DNA damage and adverse effects on cardiomyocyte functionality.

Methods for Local Recovery of Tectonic Stresses Based on Kinematic Data: Physical Inconsistency and False Objectives. Part I

This work criticizes the entrenched views according to which the orientation of the principal tectonic stress axes can be determined from local (in time and space) observations of the kinematic indicators which jointly allow estimating the strain rate of a crustal block under study. The criticized approach ignores or replaces by subjective assumptions the following factors: (1) The block’s interaction with a hosting medium expressed in terms of the equilibrium conditions of the block; (2) The stress rate which (in addition to stresses) can affect the strain rate; (3)The specific macroscopic mechanical properties of the block’s material under the unknown sought stresses, including the ratio of the stress relaxation time to the period of observations. This approach, which is developed in some Solid Earth sciences and mainly in tectonophysics, is referred to in our paper as the method of local kinematic reconstruction (MLKR) of stresses. After briefly surveying the concept of forces and stresses and discussing the importance of studying the tectonic stresses, this paper refutes the MLKR notions based on general arguments and by the example of certain thought experiments. It is shown that the use of the MLKR for the conditions of the Earth’s interior does not guarantee against obtaining the results that fundamentally and drastically differ from the true tectonic stresses. In the studied rock block, depending on the factors ignored in the MLKR, the principal axes of the strain rate tensor, on one hand, and the principal stress axes, on the other hand, can be oriented discordantly in any arbitrary given fashion. In particular, in the processes accompanied by the release of elastic energy, the maximal rate of elongation can be oriented along the axis of maximal compression, whereas the maximal rate of shortening can be aligned with the axis of the maximal tension. In this paper, the deformation processes that are most detrimental to the results of stress reconstruction by the MLKR are revealed. We introduce the notion of the inherited stress-state regime in which the orientation of the axes of principal stresses during the observation period does not depend on the deformation process and, hence, cannot be in principle determined by the MLKR. An attempt to directly locally recover the stress axes from the kinematic data is a false objective because neither the physical meaning of the stress tensor nor the way it is introduced has anything to do with strains. It is concluded that the MLKR is physically inadequate and that the tectonophysical concept of locality should be abandoned in favor of returning to the notions of classical physics, namely, to using the conservation laws. By the example of several guides on tectonophysics, this paper exposes typical errors in understanding the stress reconstruction problem.

Trimethylamine-N-Oxide (TMAO)-Induced Impairment of Cardiomyocyte Function and the Protective Role of Urolithin B-Glucuronide.

One of the most recently proposed candidates as a potential trigger for cardiovascular diseases is trimethylamine-N-oxide (TMAO). Possible direct effects of TMAO on myocardial tissue, independent of vascular damage, have been only partially explored so far. In the present study, we assessed the detrimental direct effects of TMAO on cardiomyocyte contractility and intracellular calcium dynamics, and the ability of urolithin B-glucuronide (Uro B-gluc) in counteracting TMAO-induced cell damage. Cell mechanics and calcium transients were measured, and ultrastructural analysis was performed in ventricular cardiomyocytes isolated from the heart of normal adult rats. Cells were either untreated, exposed to TMAO, or to TMAO and Uro B-gluc. TMAO exposure worsened cardiomyocyte mechanics and intracellular calcium handling, as documented by the decrease in the fraction of shortening (FS) and the maximal rate of shortening and re-lengthening, associated with reduced efficiency in the intracellular calcium removal. Ultrastructurally, TMAO-treated cardiomyocytes also exhibited glycogen accumulation, a higher number of mitochondria and lipofuscin-like pigment deposition, suggesting an altered cellular energetic metabolism and a higher rate of protein oxidative damage, respectively. Uro B-gluc led to a complete recovery of cellular contractility and calcium dynamics, and morphologically to a reduced glycogen accumulation. We demonstrated for the first time a direct negative role of TMAO on cardiomyocyte functional properties and the ability of Uro B-gluc in counteracting these detrimental effects.

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

Spinophilin is required for normal morphology, Ca2+ homeostasis and contraction but dispensable for β-adrenergic stimulation of adult cardiomyocytes

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and β-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) β-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for β-adrenergic stimulation of adult cardiomyocytes.

Upregulation of cardiomyocyte ribonucleotide reductase increases intracellular 2 deoxy-ATP, contractility, and relaxation

We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca2+-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca2+ fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca2+], indicating increased contractility is primarily due to increased myofilament activity without altering Ca2+ release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca2+ activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin–myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca2+. This article is part of a Special issue entitled "Possible Editorial".

Short-term in vivo inhibition of nitric oxide synthase with L-NAME influences the contractile function of single left ventricular myocytes in rats

The main purpose of this study was to investigate the effects of short-term L-NAME treatment on the contractile function of left ventricle (LV) myocytes and the expression of proteins related to Ca(2+) homeostasis. Data from Wistar rats treated with L-NAME (L group, n = 20; 0.7g/L in drinking water; 7days) were compared with results from untreated controls (C group, n = 20). Cardiomyocytes from the L group showed increased (p< 0.05) fractional shortening (23%) and maximum rate of shortening (20%) compared with the C group. LV from the L group also showed increased (p< 0.05) expression of the ryanodine receptor 2 and Na(+)/Ca(2+) exchanger proteins (76% and 83%, respectively; p< 0.05). However, the L and C groups showed similar in vivo hemodynamic parameters of cardiac function. In conclusion, short-term NOS inhibition determines an increased expression of Ca(2+) regulatory proteins, which contributes to improving cardiomyocyte contractile function, preserving left ventricular function.

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.

Abstract 1506: Upregulation of Cardiac AT 1 Receptor Signaling Inhibits Myocyte Contractile Performance, [Ca 2+ ] i Transient, and Ca 2+ Current in Alcoholic Dogs: Insights into the Underlying Molecular Mechanism

Background. Chronic alcohol ingestion is accompanied by sustained renin-angiotensin system (RAS) activation followed by progressive left ventricular (LV) myocyte dysfunction. This suggests a role for angiotensin II (AII) in the development of alcoholic cardiomyopathy (ACM). We tested the hypothesis that chronic alcohol intake may cause upregulation of AII AT 1 receptors and altered cardiac inotropic response to AII stimulation, thus contributing to the development of ACM. Methods. We compared LV myocyte AT 1 - and AT 2 -receptor mRNA and protein levels, myocyte contractile function, [Ca 2+ ] i transients ([Ca 2+ ] iT ), and Ca 2+ current (I Ca,L ) in response to AII (10 −6 M) in 16 instrumented dogs over 6 months. Eight animals received alcohol (once per day orally, providing 33% of total daily caloric intake), and eight were controls. Myocytes were isolated from LV myocardial biopsy tissues. Results . Compared with controls, the alcoholic myocytes showed increased AT 1 mRNA (0.69 vs 0.33) and protein levels (0.79 vs 0.37) ( p &lt;0.01). Conversely, AT 2 mRNA (0.18 vs 0.16) and protein concentration (0.16 vs 0.14) were relatively unchanged. Importantly, in alcoholic myocytes, the increased AT 1 receptors were associated with altered myocyte inotropic response to AII. In control myocytes, superfusion of AII (10 −6 M) increased myocyte systolic amplitude (SA, 14.9 vs 12.4%) and the maximum rate of shortening (dL/dt max , 203.1 vs 153.9 μm/s) accompanied by significant increases in [Ca 2+ ] iT (1.52 vs 1.21) and I Ca,L (4.0 vs 3.3 pA/pF). In alcoholic myocytes, this positive response was reversed. AII caused significant decreases in SA (3.4 vs 5.8 %), dL/dt max (50.5 vs 78.9 μm/s), and the maximum rate of relaxation (dR/dt max, 37.5 vs 59.6 μm/s) associated with reduced [Ca 2+ ] iT (0.86 vs 1.12) and I Ca,L (1.5 vs 2.1 pA/pF) ( p &lt;0.05). These AII-induced changes in alcoholic myocytes were abolished by incubation of alcoholic myocytes with an AT 1 receptor blocker, losartan (10 −5 M), or pretreatment with a G i inhibitor, PTX (2 μg/ml, 36°C, 5h). Conclusions. In a canine model, chronic alcohol consumption produces upregulation of LV myocyte AII AT 1 receptors and alters myocyte functional response to AII stimulation, which may promote the development of ACM.

Chronic nitrates blunt the effects of the natriuretic peptides in cardiac myocytes

Chronic exposure to nitrates causes tachyphylaxis to nitric oxide (NO), which reduces the effects of cyclic GMP. We tested the hypothesis that prolonged exposure to NO would not only blunt the effects of NO, but also natriuretic peptides. Cardiac myocytes were isolated from control (N=7) and chronic nitroglycerin (patched, N=7) rabbits. Patched animals received a transdermal nitroglycerin patch (0.3mg/hr for 5 days). Myocyte function was determined at baseline, CNP (C-type natriuretic peptide) or BNP (Brain natriuretic peptide) 10−8M, 10−7M, followed by KT5823 (cyclic GMP protein kinase inhibitor) 10−6M; baseline, SNAP (S-nitroso-N-acetyl-penicilliamine, NO donor) 10−6M, 10−5M, followed by KT5823. Soluble and particulate guanylyl cyclase activities were measured and in vitro phosphoprotein analysis was performed. In control animals, CNP 10−8M, 10−7M significantly reduced percentage shortening from baseline 6.1±1.6% to 5.14±0.5 % and 4.4±0.7%. KT5823 restored percentage shortening to 4.9±0.8%. Similar data were obtained with BNP and SNAP. In patched animals, CNP, BNP, SNAP had no significant effects on percentage shortening. The data on maximal rate of shortening and relaxation were consistent with these results. Guanylyl cyclase activities were not different in the control and patched animals. The myocytes from control and patched animals had similar protein phosphorylation patterns. Our data suggested that in addition to NO, the responses to both natriuretic peptides were downregulated after chronic exposure to nitroglycerin, but these effects were not due to changes in either guanylyl cyclase or cyclic GMP protein kinase, suggesting altered downstream effects.

Chronic nitrates blunt the effects of not only nitric oxide but also natriuretic peptides in cardiac myocytes

Exposure to nitrates causes tachyphylaxis to nitric oxide (NO), which reduces the effects of the second messenger cyclic guanosine-3′,-5′-monophosphate (cyclic GMP). We tested the hypothesis that prolonged exposure to NO would also blunt the effects of natriuretic peptides. Cardiac myocytes were isolated from control ( N = 7) and chronic nitroglycerin (patched, N = 7) rabbits. Patched animals received a transdermal nitroglycerin patch (0.3 mg/h for 5 days). Myocyte function was determined at baseline, after C-type natriuretic peptide (CNP, 10 −8 and 10 −7 M) or brain natriuretic peptide (BNP, 10 −8 and 10 −7 M) or S-nitroso- N-acetyl-penicilliamine (SNAP, a NO donor, 10 −6 and 10 −5 M) followed by KT5823 (a cyclic GMP protein kinase inhibitor, 10 −6 M). Soluble and particulate guanylyl cyclase activities were measured in vitro and phosphoprotein analysis was performed. In control animals, CNP 10 −8 M (5.14 ± 0.5%) and 10 −7 M (4.4 ± 0.7%) significantly reduced percentage shortening from baseline (6.1 ± 1.6%). KT5823 restored percentage shortening to 4.9 ± 0.8%. Similar data were obtained with BNP and SNAP. In patched animals, CNP, BNP, SNAP had no significant effects on percentage shortening. The data on maximal rate of shortening and relaxation were consistent with these results. Guanylyl cyclase activities were not different in the control and patched animals. The myocytes from control and patched animals had similar protein phosphorylation patterns. Our data suggested that in addition to NO, the responses to both natriuretic peptides were downregulated after chronic exposure to nitroglycerin, but these effects were not due to changes in either guanylyl cyclase or cyclic GMP protein kinase, suggesting an altered downstream pathway.

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.

Negative Inotropic Effects of C-Type Natriuretic Peptide are Attenuated in Hypertrophied Ventricular Myocytes Associated with Reduced Cyclic GMP Production

We tested the hypothesis that the negative inotropic effects of C-type natriuretic peptide (CNP) would be diminished in renal hypertensive (one-kidney-one-clip, 1K1C) hypertrophic rabbit hearts and that this attenuated effect would be due either to decreased cyclic GMP production or to reduced signaling. Using isolated control and 1K1C ventricular myocytes, cell shortening data (video edge detection) were collected: (1) at baseline and after CNP 10(-8,-7) M, followed by KT5823 (KT), a cyclic GMP-dependent protein kinase inhibitor; or (2) at baseline, following KT pre-treatment and subsequent CNP 10(-8,-7) M. In addition, cyclic GMP levels were determined by radioimmunoassay at baseline and CNP 10(-7) M. In control myocytes, CNP decreased percent shortening (5.7 +/- 0.4 versus 4.0 +/- 0.4% at 10(-7) M), maximal rate of shortening (58.7 +/- 5.1 versus 45.2 +/- 3.6 microm/sec) and maximal rate of relaxation (57.1 +/- 4.9 versus 44.1 +/- 3.4 microm/sec) in a concentration-dependent manner. These effects were attenuated by subsequent KT administration. CNP failed to produce these negative functional effects in 1K1C myocytes. When pre-treated with KT, CNP had no negative functional effect in either normal and 1K1C myocytes. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes; however, CNP produced a significant rise in cyclic GMP level in control (63.6 +/- 7.8 versus 83.5 +/- 11.3 pmol/10(5) myocytes) but not in 1K1C (49.2 +/- 2.6 versus 52.7 +/- 5.6) myocytes. Thus, CNP acted through the cyclic GMP protein kinase in control myocytes. We conclude that in hypertrophic cardiac myocytes, the decreased effect of CNP was because of decreased production of cyclic GMP.

Modulation of contractility by myocyte-derived arginase in normal and hypertrophied feline myocardium

L-Arginine, the sole substrate for the nitric oxide (NO) synthase (NOS) enzyme in producing NO, is also a substrate for arginase. We examined normal feline hearts and hearts with compensated left ventricular (LV) hypertrophy (LVH) produced by ascending aorta banding. Using Western blot analysis, we examined the abundance of arginase isozymes in crude homogenates and isolated cardiac myocytes obtained from the LVs of normal and LVH hearts. We examined the functional significance of myocyte arginase via measurement of shortening and intracellular calcium in isolated myocytes in the presence and absence of boronoethyl chloride (BEC), a specific pharmacological inhibitor of arginase. Both arginase I and II were detected in crude myocardial homogenates, but only arginase I was present in isolated cardiac myocytes. Arginase I was downregulated in LVH compared with normal. Inhibition of arginase with BEC reduced fractional shortening, maximal rate of shortening (+dL/dt) and relengthening (-dL/dt), and the peak of the free cytosolic calcium transient in normal myocytes but did not affect these parameters in LVH myocytes. These negative inotropic actions of arginase inhibition were associated with increases in cGMP generation. These studies indicate that only arginase I is present in cardiac myocytes where it tends to limit NO and cGMP production with the effect of supporting basal contractility. In experimental LVH induced by pressure overload, our studies demonstrate reduced arginase I expression and reduced functional significance, allowing greater arginine substrate availability for NO/cGMP signaling.

Negative Inotropic Effect of Low-Dose Ethanol in Mouse Ventricular Myocytes is Mediated by Activation of Endothelial Nitric Oxide Synthase

Low doses of ethanol can produce negative functional effects in ventricular myocytes. This may be related to the nitric oxide-cyclic GMP signal transduction system. We tested the hypothesis that ethanol stimulated endothelial nitric oxide synthase and this caused the negative functional effects in cardiac myocytes. This hypothesis was tested in ventricular myocytes isolated from endothelial nitric oxide synthase-knockout (eNOS-/-) and wild-type (WT) control mice. Cell function was determined with a video edge detector at 37 degrees C. Myocytes were administered 5 or 10 mM ethanol alone or after 10(-6) M L-nitro-arginine-methyl ester (L-NAME, nitric oxide synthase inhibitor) or 10(-6) M 1H[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, soluble guanylyl cyclase inhibitor). There were no differences in basal perecentage shortening (2.6+/-0.2% WT versus 2.4+/-0.2% eNOS) or maximal rate of shortening (44+/-6 WT versus 47+/-6 microms eNOS) between groups. In the WT mice, 10 mM ethanol significantly decreased percentage shortening (1.8+/-0.1) and maximal rate of shortening (35+/-4). These effects were blocked by L-NAME and ODQ. In the eNOS-/- mice, these values were not affected by ethanol. Similar data were seen for both maximal rate of shortening and relaxation. These data provide both pharmacological and direct genetic proof that these low doses of ethanol act as a stimulator of endothelial nitric oxide synthase, and this leads to the negative functional effects in ventricular myocytes.

Differential Effects of cGMP Produced by Soluble and Particulate Guanylyl Cyclase on Mouse Ventricular Myocytes

Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) are cGMP-generation systems distributed in different intracellular locations. Our aim was to test the hypothesis that the functional effects of cGMP produced by pGC and sGC on contraction and Ca2+ transients would differ in ventricular myocytes. We measured myocyte shortening from adult mice using a video edge-detector and investigated the functional changes after stimulating pGC with C-type natriuretic peptide (CNP; 10(-8) M and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP; nitric oxide donor; 10(-6) M and 10(-5) M). Significant concentration-dependent decreases in percentage shortening (PCS), maximal rate of shortening (RSmax), and relaxation (RRmax) were produced by CNP. To a similar degree, SNAP concentration-dependently reduced PCS, RSmax, and RRmax. The addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (cGMP-dependent protein kinase inhibitor; 5 x 10(-6) M) or erythro-9-(2-hydroxy-3-nonyl) adenine (cGMP-stimulated cAMP phosphodiesterase inhibitor; 10(-5) M) reduced the responses induced by CNP or SNAP, suggesting that their actions were through cGMP-mediated pathways. While SNAP significantly increased intracellular cGMP concentration by 57%, CNP had little effect on cGMP production. We also found that CNP markedly decreased the amplitude of Ca2+ transients while SNAP had little effect, suggesting the cGMP generated by sGC may decrease myofilament Ca2+ sensitivity. The small amount of cGMP generated by pGC had a major effect in reducing Ca2+ level. This study suggested the existence of compartmentalization for cGMP in ventricular myocytes.

Effects of natriuretic peptides on ventricular myocyte contraction and role of cyclic GMP signaling

Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) act through different receptors and at different potencies to affect cardiac myocyte function. We tested the hypothesis that these three peptides would differentially reduce cardiomyocyte function through their effects on the cyclic GMP signaling pathway. Rabbit ventricular myocytes were isolated and stimulated by electrical field stimulation. Cell function was measured using a video edge detector. ANP BNP or CNP at 10 −9, 10 −8, 10 −7 M were added to the myocytes. Intracellular cyclic GMP was determined using a radioimmunoassay in the absence or presence of ANP, BNP or CNP. All natriuretic peptides decreased myocyte contractility in a similar concentration dependent manner. Myocyte percentage shortening was significantly decreased with all peptides at 10 −7 M compared with baseline (ANP from 5.4±0.4 to 3.9±0.2%; BNP from 5.0±0.2 to 3.5±0.1%; CNP from 5.6±0.3 to 4.0±0.3%). Maximum rate of shortening and relaxation were also decreased similarly and significantly. Intracellular cyclic GMP was significantly increased in myocytes treated with ANP, BNP or CNP (Baseline 1.0±0.2, ANP 2.1±0.2, BNP 2.3±0.3, CNP 2.0±0.2 pmol/10 5 myocytes). Furthermore, inhibition of the cyclic GMP protein kinase with KT5823 caused a reversal in the functional effects of CNP. We concluded that all natriuretic peptides had similar negative effects on ventricular myocyte function and their effects were accompanied by increased cyclic GMP. Blockade the effect of CNP by a cyclic GMP protein kinase inhibitor demonstrated that effects were mediated through the cyclic GMP signaling pathway.

The negative functional effect of C-type natriuretic factor is attenuated in hypertrophic myocytes due to diminished cyclic GMP production

Body. We tested the hypothesis that the negative functional effects of C-type Natriuretic Peptide (CNP) would be diminished in renal hypertensive (One-Kidney-One-Clip, 1K1C) cardiac hypertrophic rabbits and that this attenuated effect is due either to decreased cGMP production or to an altered downstream signaling pathway. Using isolated control and 1K1C ventricular myocytes, cell shortening data (video edge detection) were collected: (1) at baseline and after the addition of CNP 10 −8,−7 M, followed by KT8523, a cGMP-dependent protein kinase inhibitor; or (2) at baseline, following KT pretreatment and following subsequent CNP 10 −8,−7 M. In addition, cGMP levels were determined by radioimmunoassay at baseline and following CNP 10 −7 M. We found that in control myocytes, CNP decreased the percent shortening (5.7 ± 0.4 versus 4.0 ± 0.4%), maximal rate of shortening (58.7 ± 5.1 versus 45.2 μm/s) and maximal rate of relaxation (57.1 ± 4.9 versus 44.1 ± 3.4 μm/s) in a dose-dependent manner ( P < 0.05). All of these effects were reversed with subsequent KT administration. CNP failed to produce these effects in 1K1C myocytes. When pretreated with KT, CNP had no negative effect in both control and 1K1C myocytes. Basal levels of cyclic GMP were similar in control versus 1K1C myocytes (63.6 versus 49.2 pmol/10 5 myocytes, P = NS); however, CNP produced a significant ( P < 0.05) rise in cGMP level in control (63.6 ± 7.8 versus 83.5 ± 11.3) but not in 1K1C (49.2 ± 2.6 versus 52.7 ± 5.6) myocytes. We conclude that in hypertrophic cardiac myocytes, the decreased negative functional effect of CNP is due to a decreased production of cGMP.

Combined renin-angiotensin system blockade and dietary sodium restriction impairs cardiomyocyte contractility.

Blockade of the renin-angiotensin system (RAS) by combined angiotensin-converting enzyme inhibitor and angiotensin type 1 receptor (AT(1)) antagonist treatment with reduced dietary sodium intake produces suppression of cardiac growth and regression of cardiac hypertrophy. The purpose of this study was to investigate whether cardiac growth suppression by combined RAS blockade under conditions of dietary sodium restriction is associated with cardiomyocyte atrophy and contractile dysfunction and whether this intervention modifies cardiomyocyte inotropic responsiveness to angiotensin II (Ang II). Rats were fed a high (4% w/w) or low (0.2% w/w) NaCl diet for six days. Both groups were then given a combined intraperitoneal injection of perindopril (6 mg/kg/day) and losartan (10 mg/kg/day) with maintained dietary treatment for another seven days. At the end of the treatment period, animals were anaesthetised and their hearts were removed and weighed. Left ventricular cardiomyocytes were isolated by enzymatic dissociation and cell dimensions were evaluated. A line scan camera and digital imaging technique were used to assess cardiomyocyte contraction and inotropic responses to exogenous Ang II (10 to 10(-8) M). Dietary treatment alone had no effect on body growth, whereas combined RAS blockade suppressed somatic growth in the low sodium (LS) group, compared with the high sodium (HS) group. This growth suppression in the LS group was also evident in the heart at the organ and cellular level. Studies of cardiomyocyte contraction showed that myocytes from the LS group exhibited contractile instability and depression of contractile performance. Compared with the HS group, myocytes from the LS group showed a significant reduction in maximum cell shortening (6.40+0.17 vs. 7.32+0.16% resting length, p<0.05), and maximum rate of shortening (3.85+0.14 vs. 4.29+0.11 cell length/ms, p<0.05). Myocytes of the HS group exhibited negative inotropic responses to Ang II at all concentrations tested, with a significant reduction in maximum cell shortening by 11 16% after 12 minutes peptide exposure (p<0.05 vs. non-treated control). In comparison, Ang II elicited both positive and negative responses in myocytes from the LS group, with a predominant negative inotropic effect. This study provides evidence that combined RAS blockade treatment under restricted sodium intake conditions can impair cardiomyocyte contractile function in association with cardiomyocyte growth suppression. Chronic RAS blockade qualitatively alters the intrinsic inotropic status and responsiveness of ventricular cardiomyocytes, and this shift is further modulated by dietary sodium intake conditions.

Negative functional effects of cyclic GMP are altered by cyclic AMP phosphodiesterases in rabbit cardiac myocytes

In this study, we tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes would be affected by the actions of cyclic GMP on cyclic AMP phosphodiesterases. Ventricular myocytes from eight rabbits were used to determine the functional and cyclic AMP changes caused by 10 −7, 10 −6, 10 −5 M 8-Bromo-cGMP alone and after the administration of 10 −6 M milrinone (cyclic GMP-inhibited cyclic AMP phosphodiesterase inhibitor) or 10 −6 M erythro-9-(2-Hydroxy-3-3-nonyl)adenine (EHNA, cyclic GMP-stimulated cyclic AMP phosphodiesterase inhibitor). 8-Br-cGMP dose-dependently reduced %shortening by 35±4% of baseline at 10 −5 M. This effect was significantly blunted by EHNA at all doses. The maximum rate of shortening was reduced by 31±3% by 10 −5 M 8-Br-cGMP. This effect of 8-Br-cGMP was significantly enhanced (42±4%) in the milrinone group. A similar pattern was observed in the maximum rate of relaxation data. Cyclic AMP levels were significantly increased from a baseline level of 4.0±0.8 pmol/10 5 myocytes by milrinone (+60%), EHNA (+61%) and 8-Br-cGMP (+47%). The combination of EHNA plus 8-Br-cGMP increased cyclic AMP levels significantly more that the combination of milrinone plus 8-Br-cGMP. Exogenous cyclic GMP reduces myocyte function, while raising cyclic AMP possibly through cyclic GMP-inhibited cyclic AMP phosphodiesterase effects. Blocking cyclic GMP-inhibited cyclic AMP phosphodiesterase enhances the functional effects cyclic GMP, while blocking cyclic GMP-stimulated cyclic AMP phosphodiesterase reduced these effects. The study demonstrated a functional interaction between cyclic GMP and cyclic AMP related to the cyclic GMP affected cyclic AMP phosphodiesterases.