bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of HSP27 protects canine myocytes from simulated ischemia-reperfusion injury
Authors: Vander Heide Richard S.
Published: March 2015

Previous studies have shown that adult rat myocytes can be protected from simulated ischemia-reperfusion (I/R) injury by small heat shock proteins (sHSPs). However, to date the cardioprotective effect of sHSPs has not been confirmed in adult myocytes from a large animal species. Left ventricular myocytes from adult dogs were cultured and infected with a replication-deficient adenovirus designed to increase expression of the human form of HSP27. The response to simulated I/R injury was compared using morphologic criteria. Virus-infected myocytes expressed two- to threefold more HSP27 and sustained less injury in response to simulated I/R than control cells ( P < 0.001; paired t-test). Canine myocytes can be isolated, cultured, and induced to increase the expression of a foreign protein without significant effects on differentiation and/or viability. Increased expression of HSP27 provides significant protection from simulated I/R injury in adult canine myocytes. Determining the mechanism by which sHSPs protect from lethal cell injury will provide important new insights into the mechanism of irreversible cell injury in adult myocardium.
Electrophysiological consequences of humanI Ks channel expression in adult murine heart
Authors: Chiello Tracy Christine , Cabo Candido , Coromilas James , Kurokawa Junko , Kass Robert S. , Wit Andrew L.
Published: March 2015

We expressed human delayed rectifier K+ cardiac current ( I Ks) channels in the murine heart, which lacks native I Ks, to determine their electrophysiological role. Mice expressing human I Ks channels were anesthetized, and an electrocardiogram and monophasic action potentials (MAP) recorded from the left ventricle. Sinus rate was not different between wild-type mice (WT) and transgenic mice (TG). Infusion of isoproterenol accelerated WT heart rate but not TG. Lack of TG sinus rate responsiveness may have resulted from accumulated outward current in I Kschannels in sinus node. Ventricular MAP duration of TG mice to 50% repolarization (APD50) during ventricular pacing was shorter than WT, likely resulting from outward current through I Ks channels. TG APD50 showed enhanced responsiveness (shortening) to isoproterenol compared with WT. Ventricular tachyarrhythmias were initiated in TG mice by programmed stimulation but not in WT and were accelerated by isoproterenol. I Ks channels impart β-adrenergic sensitivity to the ventricles and may be responsible for ventricular tachyarrhythmias.
Cooperative activation in cardiac muscle: impact of sarcomere length
Authors: Dobesh David P. , Konhilas John P. , de Tombe Pieter P.
Published: March 2015

This study was undertaken to determine the impact of sarcomere length (SL) on the level of cooperative activation of the cardiac myofilament at physiological [Mg2+]. Active force development was measured in skinned rat cardiac trabeculae as a function of free [Ca2+] at five SLs (1.85–2.25 μm; 1 mM free [Mg2+]; 15°C). Only muscle preparations with minimal force rundown during the entire protocol were included in the analysis (average 7.2 ± 1.7%). Median SL was measured by on-line computer video micrometry and controlled within 0.01 μm. Care was taken to ensure a sufficient number of data points in the steep portion of the [Ca2+]-force relationship at every SL to allow for accurate fit of the data to a modified Hill equation. Multiple linear regression analysis of the fit parameters revealed that both maximum, Ca2+-saturated force and Ca2+sensitivity were a significant function of SL ( P < 0.001), whereas the level of cooperativity did not depend on SL ( P = 0.2). Further analysis of the [Ca2+]-force relationships revealed a marked asymmetry that, also, was not affected by SL ( P = 0.2–0.6). Finally, we found that the level of cooperativity in isolated skinned myocardium was comparable to that reported for intact, nonskinned myocardium. Our results suggest that an increase in SL induces an increase in the Ca2+responsiveness of the cardiac sarcomere without affecting the level of cooperativity.
Role of paraventricular nucleus in regulation of sympathetic nerve frequency components
Authors: Kenney Michael J. , Weiss Mark L. , Mendes Tammy , Wang Yan , Fels Richard J.
Published: March 2015

Autospectral and coherence analyses were used to determine the role of and interactions between paraventricular nucleus (PVN) nitric oxide, γ-aminobutyric acid (GABA), and the N-methyl-d-aspartic acid (NMDA)-glutamate receptor in regulation of sympathetic nerve discharge (SND) frequency components in anesthetized rats. Four observations were made. First, PVN microinjection of bicuculline (BIC) (GABAAreceptor antagonist), but not single PVN injections of NMDA (excitatory amino acid) or N G-monomethyl-l-arginine (l-NMMA; a nitric oxide synthase inhibitor), altered SND frequency components. Second, combined PVN microinjections ofl-NMMA and NMDA changed the SND bursting pattern; however, the observed pattern change was different from that produced by PVN BIC and not observed after sinoaortic denervation. Third, PVN microinjection of kynurenic acid prevented and reversed BIC-induced changes in the SND bursting pattern. Finally, vascular resistance (renal and splenic) was significantly increased after PVN BIC microinjection despite the lack of change in the level of renal and splenic SND. These data demonstrate that the PVN contains the neural substrate for altering SND frequency components and suggest complex interactions between specific PVN neurotransmitters and between PVN neurotransmitters and the arterial baroreceptor reflex in SND regulation.
Increases in endothelial Ca2+activate KCachannels and elicit EDHF-type arteriolar dilation via gap junctions
Authors: Ungvari Zoltan , Csiszar Anna , Koller Akos
Published: March 2015

In skeletal muscle arterioles, the pathway leading to non-nitric oxide (NO), non-prostaglandin-mediated endothelium-derived hyperpolarizing factor (EDHF)-type dilations is not well characterized. To elucidate some of the steps in this process, simultaneous changes in endothelial intracellular Ca2+concentration ([Ca2+]i) and the diameter of rat gracilis muscle arterioles (∼60 μm) to acetylcholine (ACh) were measured by fura 2 microfluorimetry (in the absence of NO and prostaglandins). ACh elicited rapid increases in endothelial [Ca2+]i(101 ± 7%), followed by substantial dilations (73 ± 2%, coupling time: 1.3 ± 0.2 s) that were prevented by endothelial loading of an intracellular Ca2+chelator [1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid]. Arteriolar dilations to ACh were also inhibited by intraluminal administration of the Ca2+-activated K+(KCa) channel blockers charybdotoxin plus apamin or by palmitoleic acid, an uncoupler of myoendothelial gap junctions without affecting changes in endothelial [Ca2+]i. The presence of large conductance KCachannels on arteriolar endothelial cells was demonstrated with immunohistochemisty. We propose that in skeletal muscle arterioles, EDHF-type mediation is evoked by an increase in endothelial [Ca2+]i, which by activating endothelial KCachannels elicits hyperpolarization that is conducted via myoendothelial gap junctions to the smooth muscle resulting in decreases in [Ca2+]iand consequently dilation.
Ventricular expression of natriuretic peptides inNpr1 −/− mice with cardiac hypertrophy and fibrosis
Authors: Ellmers L. J. , Knowles J. W. , Kim H.-S. , Smithies O. , Maeda N. , Cameron V. A.
Published: March 2015

Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that regulate blood pressure and volume, and exert their biological actions via the natriuretic peptide receptor-A gene ( Npr1). Mice lacking Npr1 ( Npr −/−) have marked cardiac hypertrophy and fibrosis disproportionate to their increased blood pressure. This study examined the relationships between ANP and BNP gene expression, immunoreactivity and fibrosis in cardiac tissue, circulating ANP levels, and ANP and BNP mRNA during embryogenesis in Npr1 −/− mice. Disruption of the Npr1 signaling pathway resulted in augmented ANP and BNP gene and ANP protein expression in the cardiac ventricles, most pronounced for ANP mRNA in females [414 ± 57 in Npr1 −/− ng/mg and 124 ± 25 ng/mg in wild-type (WT) by Taqman assay, P < 0.001]. This increased expression was highly correlated to the degree of cardiac hypertrophy and was localized to the left ventricle (LV) inner free wall and to areas of ventricular fibrosis. In contrast, plasma ANP was significantly greater than WT in male but not female Npr1 −/− mice. Increased ANP and BNP gene expression was observed in Npr1 −/− embryos from 16 days of gestation. Our study suggests that cardiac ventricular expression of ANP and BNP is more closely associated with local hypertrophy and fibrosis than either systemic blood pressure or circulating ANP levels.
Integration of cornea and cardiorespiratory afferents in the nucleus of the solitary tract of the rat
Authors: Boscan Pedro , Paton Julian F. R.
Published: March 2015

We determined the activity of neurons within the nucleus of the solitary tract (NTS) after stimulation of the cornea and assessed whether this input affected the processing of baroreceptor and peripheral chemoreceptor inputs. In an in situ, unanesthetized decerebrate working heart-brain stem preparation of the rat, noxious mechanical or electrical stimulation was applied to the cornea, and extracellular single unit recordings were made from NTS neurons. Cornea nociceptor stimulation evoked bradycardia and an increase in the cycle length of the phrenic nerve discharge. Of 90 NTS neurons with ongoing activity, corneal stimulation excited 51 and depressed 39. There was a high degree of convergence to these NTS neurons from either baroreceptors or chemoreceptors. The excitatory synaptic response in 12 of 19 baroreceptive and 10 of 15 chemoreceptive neurons was attenuated significantly during concomitant electrical stimulation of the cornea. This inhibition was GABAA receptor mediated, being blocked by pressure ejection of bicuculline. Thus the NTS integrates information from corneal receptors, some of which converges onto neurons mediating reflexes from baroreceptors and chemoreceptors to inhibit these inputs.
Effects of homocysteine on intracellular nitric oxide and superoxide levels in the renal arterial endothelium
Authors: Li Ningjun , Yi Fu-Xian , Rute Elizabeth , Zhang David X. , Slocum Glenn R. , Zou Ai-Ping
Published: March 2015

The present study was designed to test the hypothesis that homocysteine (Hcys) reduces intracellular nitric oxide (NO) concentrations ([NO]i) and stimulates superoxide (O[Formula: see text]·) production in the renal arterial endothelium, thereby resulting in endothelial dysfunction. With the use of fluorescence microscopic imaging analysis, a calcium ionophore, A-23187 (2 μM), and bradykinin (2 μM) were found to increase endothelial [NO]iin freshly dissected lumen-opened small renal arteries loaded with 4,5-diaminofluorescein diacetate (DAF-2DA; 10 μM). Preincubation of the arteries with l-Hcys (20–40 μM) significantly attenuated the increase in endothelial [NO]i. However, l-Hcys had no effect on NO synthase activity in the renal arteries, as measured by the conversion rate of [3H]arginine to [3H]citrulline, but it concentration dependently decreased DAF-2DA-sensitive fluorescence induced by PAPA-NONOate in the solution, suggesting thatl-Hcys reduces endothelial [NO]iby its scavenging action. Because other thiol compounds such asl-cysteine and glutathione were also found to reduce [NO]i, it seems that decreased NO is not the only mechanism resulting in endothelial dysfunction or arteriosclerosis in hyperhomocysteinemia (hHcys). By analysis of intracellular O[Formula: see text]· levels using dihydroethidium trapping, we found that only l-Hcys among the thiol compounds studied markedly increased O[Formula: see text]· levels in the renal endothelium. These results indicate that l-Hcys inhibits the agonist-induced NO increase but stimulates O[Formula: see text]· production within endothelial cells. These effects ofl-Hcys on [NO]iand [O[Formula: see text]·] may contribute to endothelial injury associated with hHcys.
Left ventricular resynchronization therapy in a canine model of left bundle branch block
Authors: Liu Lili , Tockman Bruce , Girouard Steven , Pastore Joseph , Walcott Greg , KenKnight Bruce , Spinelli Julio
Published: March 2015

Positive responses to left (LV) and biventricular (BV) stimulation observed in heart failure patients with left bundle branch block (LBBB) suggest a possible mechanism of LV resynchronization. An anesthetized canine LBBB model was developed using radio frequency ablation. Before and after ablation, LV pressure derivative over time (dP/d t) and aortic pulse pressure (PP) were assessed during normal sinus rhythm with right ventricle (RV), LV, or BV stimulation combined with four atrioventricular delays in six dogs. In three more dogs, M-mode echocardiograms of septal and LV posterior wall motion were obtained before and after LBBB and during LV stimulation. LBBB caused QRS widening and hemodynamics deterioration. Before ablation, stimulation alone worsened LV dP/d t and PP. After ablation, LV and BV stimulation maximally increased LV dP/d t by 16% and PP by 7% ( P < 0.001), whereas little improvement was observed during RV stimulation. M-mode echocardiogram showed that LBBB resulted in a paradoxical septal wall motion that was corrected by LV stimulation. In conclusion, LV and BV stimulation improved cardiac function in a canine LBBB model via resynchronization of LV excitation and contraction.
Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells
Authors: Ray John , Noll Frank , Daut Jürgen , Hanley Peter J.
Published: March 2015

The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential (ΔΨ) of intact guinea pig cardiac muscle were investigated at 37°C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and ΔΨ was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl-β-cyclodextrin was used as fatty acid carrier. Application of 400 μM oleate or linoleate increased resting heat rate by ∼30% and ∼25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40–80 μM oleate nor 40 μM linoleate affected ΔΨ. At a higher concentration (400 μM) both oleate and linoleate increased TMRE fluorescence by ∼20% of maximum, obtained using 2,4-dinitrophenol (100 μM), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease ΔΨ in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium.
Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries
Authors: Zhang David X. , Zou Ai-Ping , Li Pin-Lan
Published: March 2015

We tested the hypothesis that ceramide induces endothelial dysfunction in small coronary arteries via NADPH oxidase-mediated superoxide and resulting peroxynitrite formation. With the use of dihydroethidium as a superoxide indicator, C2-ceramide was found to increase superoxide production in the endothelial cells of small coronary arteries, which was inhibited by the NADPH oxidase inhibitors N-vanillylnonanamide, apocynin, and diphenylene iodonium. NADPH oxidase expression was confirmed in endothelial cells, as indicated by the immunoblotting of its subunits gp91phoxand p47phox. C2-ceramide increased NADPH oxidase activity by 52%, which was blocked by NADPH oxidase inhibitors but not by inhibitors of NO synthase, xanthine oxidase, and mitochondrial electron transport chain enzymes. By Western blot analysis, ceramide-induced NADPH oxidase activation was found to be associated with the translocation of p47phoxto the membrane. In isolated and pressurized small coronary arteries, N-vanillylnonanamide, apocynin, or uric acid, a peroxynitrite scavenger, largely restored the inhibitory effects of ceramide on bradykinin- and A-23187-induced vasorelaxation. With the use of nitrotyrosine as a marker, C2-ceramide was found to increase peroxynitrite in small coronary arteries, which could be blocked by uric acid. We conclude that NADPH oxidase-mediated superoxide production and subsequent peroxynitrite formation mediate ceramide-induced endothelial dysfunction in small coronary arteries.
ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel
Authors: Lebuffe Gilles , Schumacker Paul T. , Shao Zuo-Hui , Anderson Travis , Iwase Hirotoro , Vanden Hoek Terry L.
Published: March 2015

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (KATP) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H2O2, or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by KATP channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the KATP channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to KATP channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N ω-nitro-l-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by KATP channel activation. Coadministration of H2O2 and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the KATP channel. We conclude that ROS can trigger preconditioning by causing activation of the KATP channel, which then induces generation of ROS and NO that are both required for preconditioning protection.
Viscoelastic properties of pressure overload hypertrophied myocardium: effect of serine protease treatment
Authors: Stroud Jason D. , Baicu Catalin F. , Barnes Mary A. , Spinale Francis G. , Zile Michael R.
Published: March 2015

To determine whether and to what extent one component of the extracellular matrix, fibrillar collagen, contributes causally to abnormalities in viscoelasticity, collagen was acutely degraded by activation of endogenous matrix metalloproteinases (MMPs) with the serine protease plasmin. Papillary muscles were isolated from normal cats and cats with right ventricular pressure overload hypertrophy (POH) induced by pulmonary artery banding. Plasmin treatment caused MMP activation, collagen degradation, decreased the elastic stiffness constant, and decreased the viscosity constant in both normal and POH muscles. Thus, whereas many mechanisms may contribute to the abnormalities in myocardial viscoelasticity in the POH myocardium, changes in fibrillar collagen appear to play a predominant role.
A novel approach to blood plasma viscosity measurement using fluorescent molecular rotors
Authors: Haidekker Mark A. , Tsai Amy G. , Brady Thomas , Stevens Hazel Y. , Frangos John A. , Theodorakis Emmanuel , Intaglietta Marcos
Published: March 2015

Molecular rotors, a group of fluorescent molecules with viscosity-dependent quantum yield, were tested for their suitability to act as fluorescence-based plasma viscometers. The viscosity of samples of human plasma was modified by the addition of pentastarch (molecular mass 260 kDa, 10% solution in saline) and measured with a Brookfield viscometer. Plasma viscosity was 1.6 mPa · s, and the mixtures ranged up to 4.5 mPa · s (21°C). The stimulated light emission of the molecular rotors mixed in the plasma samples yielded light intensity that was nonoverlapping and of significantly different intensity for viscosity steps down to 0.3 mPa · s ( n = 5, P < 0.0001). The mathematical relationship between intensity ( I) and viscosity (η) was found to be η = (κ I)ν. After calibration and scaling the fluorescence based measurement had an average deviation versus the conventional viscometric measurements that was <1.8%. These results show the suitability of molecular rotors for fast, low-volume biofluid viscosity measurements achieving accuracy and precision comparable to mechanical viscometers.
Skeletal muscle blood flow and flow heterogeneity during dynamic and isometric exercise in humans
Authors: Laaksonen Marko S. , Kalliokoski Kari K. , Kyröläinen Heikki , Kemppainen Jukka , Teräs Mika , Sipilä Hannu , Nuutila Pirjo , Knuuti Juhani
Published: March 2015

The effects of dynamic and intermittent isometric knee extension exercises on skeletal muscle blood flow and flow heterogeneity were studied in seven healthy endurance-trained men. Regional muscle blood flow was measured using positron emission tomography (PET) and an [15O]H2O tracer, and electromyographic (EMG) activity was recorded in the quadriceps femoris (QF) muscle during submaximal intermittent isometric and dynamic exercises. QF blood flow was 61% ( P = 0.002) higher during dynamic exercise. Interestingly, flow heterogeneity was 13% ( P = 0.024) lower during dynamic compared with intermittent isometric exercise. EMG activity was significantly higher ( P < 0.001) during dynamic exercise, and the change in EMG activity from isometric to dynamic exercise was tightly related to the change in blood flow in the vastus lateralis muscle ( r = 0.98, P < 0.001) but not in the rectus femoris muscle ( r = −0.09, P = 0.942). In conclusion, dynamic exercise causes higher and less heterogeneous blood flow than intermittent isometric exercise at the same exercise intensity. These responses are, at least partly, related to the increased EMG activity.
A new cardioprotective agent, JTV519, improves defective channel gating of ryanodine receptor in heart failure
Authors: Kohno Masateru , Yano Masafumi , Kobayashi Shigeki , Doi Masahiro , Oda Tetsuro , Tokuhisa Takahiro , Okuda Shinichi , Ohkusa Tomoko , Kohno Michihiro , Matsuzaki Masunori
Published: March 2015

Defective interaction between FKBP12.6 and ryanodine receptors (RyR) is a possible cause of cardiac dysfunction in heart failure (HF). Here, we assess whether the new cardioprotective agent JTV519 can correct it in tachycardia-induced HF. HF was induced in dogs by 4-wk rapid ventricular pacing, and sarcoplasmic reticulum (SR) was isolated from left ventricular muscles. In failing SR, JTV519 increased the rate of Ca2+release and [3H]ryanodine binding. RyR were then labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetamide. In failing SR, the polylysine induced a rapid change in methylcoumarin acetamide fluorescence, presumably because the channel opening preceding the Ca2+release was smaller than in normal SR (consistent with a decreased rate of Ca2+release in failing SR), and JTV519 increased it. In conclusion, JTV519, a new 1,4-benzothiazepine derivative, corrected the defective channel gating in RyR (increase in both the rapid conformational change and the subsequent Ca2+release rate) in HF.
Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release
Authors: Harris M. Brennan , Blackstone Michele A. , Ju Hong , Venema Virginia J. , Venema Richard C.
Published: March 2015

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37°C, 42°C, or 45°C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 ± 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42°C. We also observed a 7.7 ± 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45°C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42°C. Heat shock in rats (core temperature: 42°C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.
Induction of apoptosis in vascular smooth muscle cells by mechanical stretch
Authors: Sotoudeh Mohammad , Li Yi-Shuan , Yajima Noriyuki , Chang Chih-Chieh , Tsou Tsui-Chun , Wang Yibin , Usami Shunichi , Ratcliffe Anthony , Chien Shu , Shyy John Y.-J.
Published: March 2015

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH2-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-α (TNF-α) receptor-1 and its association with TNF-α receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretch-induced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis.
Role of PKC in autocrine regulation of rat ventricular K+currents by angiotensin and endothelin
Authors: Shimoni Yakhin , Liu Xiu-Fang
Published: March 2015

Transient and sustained K+currents were measured in isolated rat ventricular myocytes obtained from control, steptozotocin-induced (Type 1) diabetic, and hypothyroid rats. Both currents, attenuated by the endocrine abnormalities, were significantly augmented by in vitro incubation (>6 h) with the angiotensin-converting enzyme inhibitor quinapril or the angiotensin II (ANG II) receptor blocker saralasin. Western blots indicated a parallel increase in Kv4.2 and Kv1.2, channel proteins that underlie the transient and (part of the) sustained currents. Under diabetic and hypothyroid conditions, both currents were also augmented by an endothelin receptor blocker (PD142893) or by an endothelin-converting enzyme inhibitor. Kv4.2 density was also enhanced by PD142893. Incubation (>5 h) with the PKC inhibitor bis-indolylmaleimide augmented both currents, whereas the PKC activator dioctanoyl-rac-glycerol (DiC8) prevented the augmentation of currents by quinapril. DiC8 also prevented the augmentation of Kv4.2 density by quinapril. Specific peptides that activate PKC translocation indicated that PKC-ε and not PKC-δ is involved in ANG II action on these currents. In control myocytes, quinapril and PD142893 augmented the sustained late current but had no effect on peak current. It is concluded that an autocrine release of angiotensin and endothelin in diabetic and hypothyroid conditions attenuates K+currents by suppressing the synthesis of some K+channel proteins, with the effects mediated at least partially by PKC-ε.
Impairment of nitric oxide synthase-dependent dilatation of cerebral arterioles during infusion of nicotine
Authors: Fang Qin , Sun Hong , Mayhan William G.
Published: March 2015

The effects of nicotine on nitric oxide synthase (NOS)-dependent reactivity of cerebral arterioles remain uncertain. Our first goal was to examine whether infusion of nicotine alters NOS-dependent reactivity of cerebral arterioles. Our second goal was to examine the mechanisms that may account for the effects of nicotine on cerebral arterioles. We measured the diameter of pial arterioles to NOS-dependent (ADP and acetylcholine) and NOS-independent (nitroglycerin) agonists before and after the infusion of nicotine (2 μg · kg−1· min−1iv for 30 min, followed by a maintenance dose of 0.35 μg · kg−1· min−1). ADP- and acetylcholine-induced vasodilatation was impaired after the infusion of nicotine. In contrast, nicotine did not alter vasodilatation to nitroglycerin. Next, we examined whether the impaired responses of pial arterioles during infusion of nicotine may be related to oxygen radicals. We found that application of superoxide dismutase or tetrahydrobiopterin during infusion of nicotine could prevent impaired NOS-dependent vasodilatation. Thus acute exposure of cerebral vessels to nicotine specifically impairs NOS-dependent dilatation via the production of oxygen radicals possibly related to an alteration in the utilization of tetrahydrobiopterin.
Early effects of metabolic inhibition on intracellular Ca2+in toad pacemaker cells: involvement of Ca2+stores
Authors: Ju Yue-Kun , Allen David G.
Published: March 2015

The early effects of metabolic inhibition on intracellular Ca2+concentration ([Ca2+]i), Ca2+current, and sarcoplasmic reticulum (SR) Ca2+content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca2+]i(Ca2+transients) was reduced after 5-min exposure to 2 mM NaCN from 338 ± 30 to 189 ± 37 nM ( P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 ± 2 to 12 ± 4 beats/min ( P < 0.002, n = 9). It has been proposed that CN−acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca2+current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca2+transients and firing rate. CN−caused an insignificant fall of Ca2+current (23 ± 11%) but a substantial reduction of SR Ca2+content (by 65 ± 5%), whereas clotrimazole produced a larger reduction of Ca2+current and did not affect the SR Ca2+content. Thus the main effect of CN−does not seem to be through inhibition of cytochrome P-450. In conclusion, CN−appears to reduce Ca2+release from the SR mainly by reducing SR Ca2+content. A likely cause of the decreased SR content is reduced Ca2+uptake by the SR pump.
Structural response of microcirculatory networks to changes in demand: information transfer by shear stress
Authors: Pries A. R. , Reglin B. , Secomb T. W.
Published: March 2015

Matching blood flow to metabolic demand in terminal vascular beds involves coordinated changes in diameters of vessels along flow pathways, requiring upstream and downstream transfer of information on local conditions. Here, the role of information transfer mechanisms in structural adaptation of microvascular networks after a small change in capillary oxygen demand was studied using a theoretical model. The model includes diameter adaptation and information transfer via vascular reactions to wall shear stress, transmural pressure, and oxygen levels. Information transfer is additionally effected by conduction along vessel walls and by convection of metabolites. The model permits selective blocking of information transfer mechanisms. Six networks, based on in vivo data, were considered. With information transfer, increases in network conductance and capillary oxygen supply were amplified by factors of 4.9 ± 0.2 and 9.4 ± 1.1 (means ± SE), relative to increases when information transfer was blocked. Information transfer by flow coupling alone, in which increased shear stress triggers vascular enlargement, gave amplifications of 4.0 ± 0.3 and 4.9 ± 0.5. Other information transfer mechanisms acting alone gave amplifications below 1.6. Thus shear-stress-mediated flow coupling is the main mechanism for the structural adjustment of feeding and draining vessel diameters to small changes in capillary oxygen demand.
Heart size-independent analysis of myocardial function in murine pressure overload hypertrophy
Authors: Takaoka Hideyuki , Esposito Giovanni , Mao Lan , Suga Hiroyuki , Rockman Howard A.
Published: March 2015

Pressure overload cardiac hypertrophy may be a compensatory mechanism to normalize systolic wall stress and preserve left ventricular (LV) function. To test this concept, we developed a novel in vivo method to measure myocardial stress (ς)-strain (ɛ) relations in normal and hypertrophied mice. LV volume was measured using two pairs of miniature omnidirectional piezoelectric crystals implanted orthogonally in the endocardium and one crystal placed on the anterior free wall to measure instantaneous wall thickness. Highly linear ς-ε relations were obtained in control ( n = 7) and hypertrophied mice produced by 7 days of transverse aortic constriction (TAC; n = 13). Administration of dobutamine in control mice significantly increased the load-independent measure of LV contractility, systolic myocardial stiffness. In TAC mice, systolic myocardial stiffness was significantly greater than in control mice (3,156 ± 1,433 vs. 1,435 ± 467 g/cm2, P < 0.01), indicating enhanced myocardial contractility with pressure overload. However, despite the increased systolic performance, both active (time constant of LV pressure decay) and passive (diastolic myocardial stiffness constant) diastolic properties were markedly abnormal in TAC mice compared with control mice. These data suggest that the development of cardiac hypertrophy is associated with a heightened contractile state, perhaps as an early compensatory response to pressure overload.
Rho-Rho kinase pathway is involved in the regulation of myogenic tone and pump activity in isolated lymph vessels
Authors: Hosaka Kayoko , Mizuno Risuke , Ohhashi Toshio
Published: March 2015

To evaluate whether or not Rho-Rho kinase pathway is involved in the regulation of mechanical activity of lymph vessels, effects of Y-27632 and okadaic acid on lymph pump activity and myogenic, pressure- and agonist-induced tone were examined in isolated rat lymph vessels. Y-27632 caused a significant dilation with a cessation of the lymph pump activity. Y-27632 also produced a dose-related dilation of the lymph vessels precontracted by norepinephrine (NE)-, U-46619- or 80 mM KCl. Okadaic acid significantly constricted the lymph vessels and reduced the frequency of the lymph pump activity. Okadaic acid also produced a dose-related constriction of the lymph vessels precontracted by NE or U-46619. The Y-27632-induced decrease of the frequency of lymph pump activity was significantly reversed by the pretreatment with okadaic acid. In the presence of Y-27632, the pressure-mediated tone of the lymph vessel was significantly decreased. On the other hand, okadaic acid significantly increased the pressure-mediated tone. These findings suggest that Rho kinase and myosin phosphatase activity in lymphatic smooth muscles may contribute to the regulation of lymph pump activity and may be also involved in the control of myogenic pressure- and agonist-induced tone.
Enhanced IPC by activation of pertussis toxin-sensitive and -insensitive G protein-coupled purinoceptors
Authors: Ninomiya Hideki , Otani Hajime , Lu Kejie , Uchiyama Takamichi , Kido Masakuni , Imamura Hiroji
Published: March 2015

Extracellular ATP plays an important role in ischemic preconditioning (IPC) through the activation of P2ypurinoceptors. This study examined whether ATP-stimulated P2ypurinoceptors are coupled to pertussis toxin (PTX)-insensitive G protein and whether activation of this pathway enhances myocardial protection afforded by IPC. The rat was treated with PTX for 48 h, and the heart was then isolated and buffer perfused. The heart underwent IPC by three cycles of 5-min ischemia and 5-min reperfusion before 25 min of global ischemia. Isovolumic left ventricular function was measured, and functional recovery at 30 min after reperfusion was taken as an end point of myocardial protection. PTX pretreatment partially inhibited functional protection by IPC. Treatment with 100 μM 8-( p-sulfophenyl) theophylline (SPT) during IPC had no further effect on PTX-induced inhibition of functional protection by IPC, whereas suramin (300 μM) or reactive blue (RB) (10 μM) completely abolished myocardial protection in the preconditioned heart pretreated with PTX. Supplementation with adenosine (30 μM), ATP (30 μM), or UTP (50 μM) significantly enhanced IPC-induced functional protection, although preconditioning with these nucleotides without IPC had no protective effect. Adenosine-enhanced IPC was inhibited by pretreatment with PTX and SPT but not by suramin or RB, whereas ATP-enhanced IPC was inhibited by suramin or RB in combination with PTX pretreatment. On the other hand, UTP-enhanced IPC was not affected by PTX pretreatment but was inhibited by suramin or RB. Adenosine supplemented IPC without PTX pretreatment and ATP supplemented IPC with PTX pretreatment were not affected by nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (100 μM). Although the protein kinase C inhibitor Ro318425 (0.3 μM) or tyrosine kinase inhibitor genistein (50 μM) had no significant effect on the functional protection afforded by adenosine-supplemented IPC without PTX pretreatment and ATP-supplemented IPC with PTX pretreatment, the combination of Ro318425 and genistein attenuated functional protection afforded by both the purinoceptor agonist-supplemented IPC. These results suggest the crucial involvement of PTX-sensitive and -insensitive G protein coupled purinoceptors in enhanced IPC by supplementation with adenosine, ATP, and UTP.
Muscle LIM protein deficiency leads to alterations in passive ventricular mechanics
Authors: Omens Jeffrey H. , Usyk Taras P. , Li Zuangjie , McCulloch Andrew D.
Published: March 2015

Accumulating evidence indicates that cytoskeletal defects may be an important pathway for dilated cardiomyopathy and eventual heart failure. Targeted disruption of muscle LIM protein (MLP) has previously been shown to result in dilated cardiomyopathy with many of the clinical signs of heart failure, although the effects of MLP disruption on passive ventricular mechanics and myocyte architecture are not known. We used the MLP knockout model to examine changes in passive ventricular mechanics and laminar myofiber sheet architecture. Pressure-volume and pressure-strain relations were altered in MLP knockout mice, in general suggesting a less compliant tissue in the dilated hearts. Transmural laminar myocyte structure was also altered in this mouse model, especially near the epicardium. A mathematical model of the heart showed a likely increase in passive tissue stiffness in the MLP-deficient (−/−) heart. These results suggest that the disruption of the cytoskeletal protein MLP results in less compliant passive tissue and concomitant structural alterations in the three-dimensional myocyte architecture that may in part explain the ventricular dysfunction in the dilated heart.
Oxytocinergic regulation of cardiovascular function: studies in oxytocin-deficient mice
Authors: Michelini Lisete C. , Marcelo Marialuisa C. , Amico Janet , Morris Mariana
Published: March 2015

Oxytocin (OT) has been implicated in the cardiovascular responses to exercise, stress, and baroreflex adjustments. Studies were conducted to determine the effect of genetic manipulation of the OT gene on blood pressure (BP), heart rate (HR), and autonomic/baroreflex function. OT knockout (OTKO −/−) and control +/+ mice were prepared with chronic arterial catheters. OTKO −/− mice exhibited a mild hypotension (102 ± 3 vs. 110 ± 3 mmHg). Sympathetic and vagal tone were tested using β1-adrenergic and cholinergic blockade (atenolol and atropine). Magnitude of sympathetic and vagal tone to the heart and periphery was not significantly different between groups. However, there was an upward shift of sympathetic tone to higher HR values in OTKO −/− mice. This displacement combined with unchanged basal HR led to larger responses to cholinergic blockade (+77 ± 25 vs. +5 ± 15 beats/min, OTKO −/− vs. control +/+ group). There was also an increase in baroreflex gain (−13.1 ± 2.5 vs. −4.1 ± 1.2 beats · min−1 · mmHg−1, OTKO −/− vs. control +/+ group) over a smaller BP range. Results show that OTKO −/− mice are characterized by 1) hypotension, suggesting that OT is involved in tonic BP maintenance; 2) enhanced baroreflex gain over a small BP range, suggesting that OT extends the functional range of arterial baroreceptor reflex; and 3) shift in autonomic balance, indicating that OT reduces the sympathetic reserve.
Fas pathway is a critical mediator of cardiac myocyte death and MI during ischemia-reperfusion in vivo
Authors: Lee Peiyee , Sata Masataka , Lefer David J. , Factor Stephen M. , Walsh Kenneth , Kitsis Richard N.
Published: March 2015

Fas is a widely expressed cell surface receptor that can initiate apoptosis when activated by its ligand (FasL). Whereas Fas abundance on cardiac myocytes increases in response to multiple pathological stimuli, direct evidence supporting its role in the pathogenesis of heart disease is lacking. Moreover, controversy exists even as to whether Fas activation induces apoptosis in cardiac myocytes. In this study, we show that adenoviral overexpression of FasL, but not β-galactosidase, results in marked apoptosis both in cultures of primary neonatal cardiac myocytes and in the myocardium of intact adult rats. Myocyte killing by FasL is a specific event, because it does not occur in lpr (lymphoproliferative) mice that lack functional Fas. To assess the contribution of the Fas pathway to myocardial infarction (MI) in vivo, lpr mice were subjected to 30 min of ischemia followed by 24 h of reperfusion. Compared with wild-type mice, lpr mice exhibited infarcts that were 62.3% smaller with 63.8% less myocyte apoptosis. These data provide direct evidence that activation of Fas can induce apoptosis in cardiac myocytes and that Fas is a critical mediator of MI due to ischemia-reperfusion in vivo.
Epitope-dependent localization of estrogen receptorα, but not -β, in en face arterial endothelium
Authors: Dan Pauline , Cheung Joyce C. Y. , Scriven David R. L. , Moore Edwin D. W.
Published: March 2015

Rapid, nongenomic effects of 17β-estradiol (E2) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ERα or ERβ antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55-kDa ERα isoform. Three-dimensional images of cells labeled with these antibodies and markers for the nucleus and caveolin-1 were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ERα in the nucleus and cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the epitope of the antibody. Human ovarian surface epithelium produced similar results; but in rat myometrium, the distribution was epitope independent and nuclear. ERβ distribution was predominately intranuclear and epitope independent. A small amount of ERα colocalized with ERβ within the nucleus. The results were identical in both arterial preparations and insensitive to E2. We postulate that the different ERα conformations at the membrane, in the nucleus, and between different cell types allow E2to trigger cell- and location-specific signaling cascades.
Indexes of diastolic RV function: load dependence and changes after chronic RV pressure overload in lambs
Authors: Leeuwenburgh Boudewijn P. J. , Steendijk Paul , Helbing Willem A. , Baan Jan
Published: March 2015

Diastolic function is a major determinant of ventricular performance, especially when loading conditions are altered. We evaluated biventricular diastolic function in lambs and studied possible load dependence of diastolic parameters [minimum first derivative of pressure vs. time (dP/d t min) and time constant of isovolumic relaxation (τ)] in normal ( n = 5) and chronic right ventricular (RV) pressure-overloaded ( n = 5) hearts by using an adjustable band on the pulmonary artery (PAB). Pressure-volume relations were measured during preload reduction to obtain the end-diastolic pressure-volume relationship (EDPVR). In normal lambs, absolute dP/d t min and τ were lower in the RV than in the left ventricle whereas the chamber stiffness constant ( b) was roughly the same. After PAB, RV τ and dP/d t min were significantly higher compared with control. The RV EDPVR indicated impaired diastolic function. During acute pressure reduction, both dP/d t min and τ showed a relationship with end-systolic pressure. These relationships could explain the increased dP/d t min but not the increased τ-value after banding. Therefore, the increased τ after banding reflects intrinsic myocardial changes. We conclude that after chronic RV pressure overload, RV early relaxation is prolonged and diastolic stiffness is increased, both indicative of impaired diastolic function.
Reduction of myocardial infarct size by fluvastatin
Authors: Tiefenbacher C. P. , Kapitza J. , Dietz V. , Lee C.-H. , Niroomand F.
Published: March 2015

Statins have a variety of cardioprotective properties following chronic treatment. In contrast, little is known about the acute effects. Reperfusion acutely injures the heart by activation of neutrophils as well as endothelial cells. Because statins are known to influence the processes pathogenetically involved, we hypothesized that acute application of statins attenuates the sequelae of cardiac reperfusion. In rats, myocardial infarction (MI) was induced by ligature of the left coronary artery followed by reperfusion. Myocardial blood flow (MBF) was determined by H2 clearance and regional myocardial function (fractional thickening, FT) by pulsed Doppler. MI size was measured by triphenyltetrazolium chloride (TTC) staining, neutrophil extravasation by determination of myeloperoxidase (MPO) activity, and nitric oxide generation via measurement of cGMP. Treatment with fluvastatin, administered intravenously 20 min before the onset of ischemia, significantly attenuated the decline of FT and MBF at the end of the reperfusion period and significantly reduced MI size. Furthermore, fluvastatin induced a significant reduction of MPO activity and an increase of cGMP level compared with the control group. The effect of fluvastatin was completely abolished following pretreatment of NG-nitro-l-arginine methyl ester (l-NAME). These findings suggest that acute application of fluvastatin reduces MI size and attenuates reperfusion injury. We propose that the underlying mechanism is at least partially an inhibition of inflammation and endothelial dysfunction by preventing the activation and extravasation of neutrophils.