Rate Of Spontaneous Contractions Research Articles

Influence of Remodeled ECM and Co-culture with iPSC-Derived Cardiac Fibroblasts on the Mechanical Function of Micropatterned iPSC-Derived Cardiomyocytes.

In native heart tissue, functions of cardiac fibroblasts (CFs) include synthesis, remodeling, and degradation of the extracellular matrix (ECM) as well as secreting factors that regulate cardiomyocyte (CM) function. The influence of direct co-culture and CF-derived ECM on CM mechanical function are not fully understood. Here we use an engineered culture platform that provides control over ECM geometry and substrate stiffness to evaluate the influence of iPSC-CFs, and the ECM they produce, on the mechanical function of iPSC-CMs. Mechanical analysis was performed using digital image correlation to quantify maximum contractile strain, spontaneous contraction rate, and full-field organization of the contractions. When cultured alone, iPSC-CFs produce and remodel the ECM into fibers following the underlying 15° chevron patterned ECM. The substrates were decellularized and confirmed to have highly aligned fibers that covered a large fraction of the pattern area before reseeding with iPSC-CMs, alone or in co-culture with iPSC-CFs. When seeded on decellularized ECM, larger maximum contractile strains were observed in the co-culture condition compared to the CM Only condition. No significant difference was found in contractile strain between the Matrigel and decellularized ECM conditions; however, the spontaneous contraction rate was lower in the decellularized ECM condition. A methodology for quantifying alignment of cell contraction across the entire field of view was developed based on trajectories approximating the cell displacements during contraction. Trajectory alignment was unaltered by changes in culture or ECM conditions. These combined observations highlight the important role CFs play in vivo and the need for models that enable a quantitative approach to examine interactions between the CFs and CMs, as well as the interactions of these cells with the ECM.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Fluid Osmolarity Modulates the Rate of Spontaneous Contraction of Lymphatic Vessels and Lymph Flow by Means of a Cooperation between TRPV and VRAC Channels.

Lymphatic vessels are capable of sustaining lymph formation and propulsion via an intrinsic mechanism based on the spontaneous contraction of the lymphatic muscle in the wall of lymphatic collectors. Exposure to a hyper- or hypo-osmolar environment can deeply affect the intrinsic contraction rate and therefore alter lymph flow. In this work, we aimed at defining the putative receptors underlying such a response. Functional experiments were conducted in ex vivo rat diaphragmatic specimens containing spontaneously contracting lymphatic vessels that were exposed to either hyper- or hypo-osmolar solutions. Lymphatics were challenged with blockers to TRPV4, TRPV1, and VRAC channels, known to respond to changes in osmolarity and/or cell swelling and expressed by lymphatic vessels. Results show that the normal response to a hyperosmolar environment is a steady decrease in the contraction rate and lymph flow and can be prevented by blocking TRPV1 channels with capsazepine. The response to a hyposmolar environment consists of an early phase of an increase in the contraction rate, followed by a decrease. The early phase is abolished by blocking VRACs with DCPIB, while blocking TRPV4 mainly resulted in a delay of the early response. Overall, our data suggest that the cooperation of the three channels can shape the response of lymphatic vessels in terms of contraction frequency and lymph flow, with a prominent role of TRPV1 and VRACs.

Enhancement of pacing function by HCN4 overexpression in human pluripotent stem cell-derived cardiomyocytes

BackgroundThe number of patients with bradyarrhythmia and the number of patients with cardiac pacemakers are increasing with the aging population and the increase in the number of patients with heart diseases. Some patients in whom a cardiac pacemaker has been implanted experience problems such as pacemaker infection and inconvenience due to electromagnetic interference. We have reported that overexpression of HCN channels producing a pacemaker current in mouse embryonic stem cell-derived cardiomyocytes showed enhanced pacing function in vitro and in vivo. The aim of this study was to determine whether HCN4 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can strengthen the pacing function of the cells.MethodsHuman HCN4 was transduced in the AAVS1 locus of human induced pluripotent stem cells by nucleofection and HCN4-overexpressing iPSC-CMs were generated. Gene expression profiles, frequencies of spontaneous contraction and pacing abilities of HCN4-overexpressing and non-overexpressing iPSC-CMs in vitro were compared.ResultsHCN4-overexpressing iPSC-CMs showed higher spontaneous contraction rates than those of non-overexpressing iPSC-CMs. They responded to an HCN channel blocker and β adrenergic stimulation. The pacing rates against parent iPSC line-derived cardiomyocytes were also higher in HCN4-overexpressing iPSC-CMs than in non-overexpressing iPSC-CMs.ConclusionsOverexpression of HCN4 showed enhancement of If current, spontaneous firing and pacing function in iPSC-CMs. These data suggest this transgenic cell line may be useful as a cardiac pacemaker.

Cardiac Depressant and Vasodialatory Effect of Flaxseed - Basis for the Medicinal use in Hypertension

Linum usitatissimum (Flaxseed) is known to be traditionally used for managing hypertension. In this study, we aim to provide a mechanistic basis for the medicinal use of Flaxseed in hypertension. The high-performance liquid chromatography (HPLC) analysis that we carried out during our study showed the presence of polar compounds (quercetin, nicotinic acid, and nicotinamide) in Flaxseed’s crude extract (Fs.Cr; aqueous methanolic). In anesthetized rats, Fs.Cr reduced arterial blood pressure (BP) dose-dependently (10-100 mg/kg). When tested for its mechanism of action ex vivo, Fs.Cr inhibited both the force and rate of spontaneous contractions in the dose range of 1-10 mg/mL in isolated guinea-pig atria, similar to how verapamil, a standard Ca+2 channel blocker does it. Further, Fs.Cr showed vasodilator effect against the contractions induced by phenylephrine (PE, 1 μM) in rat aortic ring preparations (concentration range: 1-10 mg/mL), whereas no effect was observed against the contractions induced by low K+ (25 mM) as well as high K+ (80 mM). This selective inhibitory effect of Fs.Cr against PE was tested for endothelium-dependent nitric oxide (NO) and/or cholinergic component involvement. The vasodilator effect of Fs.Cr against PE was retested in the absence and presence of atropine in endothelium (E)-intact and E-denuded aorta, but no significant shift was observed in the inhibitory effect of Fs.Cr. Further, Fs.Cr shifted the PE-induced concentration-response curves (CRCs) to the right in a dose-dependent manner (1 and 3 mg/mL). This effect was similar to that of prazosin. All these findings indicate that Flaxseed may mediate its antihypertensive activity by the alpha-1 receptor antagonist and Ca+2 channel blocking-like activity, which may account for its efficacy in hypertension.

3D Co-culture of hiPSC-Derived Cardiomyocytes With Cardiac Fibroblasts Improves Tissue-Like Features of Cardiac Spheroids.

Purpose: Both cardiomyocytes and cardiac fibroblasts (CF) play essential roles in cardiac development, function, and remodeling. Properties of 3D co-cultures are incompletely understood. Hence, 3D co-culture of cardiomyocytes and CF was characterized, and selected features compared with single-type and 2D culture conditions.Methods: Human cardiomyocytes derived from induced-pluripotent stem cells (hiPSC-CMs) were obtained from Cellular Dynamics or Ncardia, and primary human cardiac fibroblasts from ScienCell. Cardiac spheroids were investigated using cryosections and whole-mount confocal microscopy, video motion analysis, scanning-, and transmission-electron microscopy (SEM, TEM), action potential recording, and quantitative PCR (qPCR).Results: Spheroids formed in hanging drops or in non-adhesive wells showed spontaneous contractions for at least 1 month with frequent media changes. SEM of mechanically opened spheroids revealed a dense inner structure and no signs of blebbing. TEM of co-culture spheroids at 1 month showed myofibrils, intercalated disc-like structures and mitochondria. Ultrastructural features were comparable to fetal human myocardium. We then assessed immunostained 2D cultures, cryosections of spheroids, and whole-mount preparations by confocal microscopy. CF in co-culture spheroids assumed a small size and shape similar to the situation in ventricular tissue. Spheroids made only of CF and cultured for 3 weeks showed no stress fibers and strongly reduced amounts of alpha smooth muscle actin compared to early spheroids and 2D cultures as shown by confocal microscopy, western blotting, and qPCR. The addition of CF to cardiac spheroids did not lead to arrhythmogenic effects as measured by sharp-electrode electrophysiology. Video motion analysis showed a faster spontaneous contraction rate in co-culture spheroids compared to pure hiPSC-CMs, but similar contraction amplitudes and kinetics. Spontaneous contraction rates were not dependent on spheroid size. Applying increasing pacing frequencies resulted in decreasing contraction amplitudes without positive staircase effect. Gene expression analysis of selected cytoskeleton and myofibrillar proteins showed more tissue-like expression patterns in co-culture spheroids than with cardiomyocytes alone or in 2D culture.Conclusion: We demonstrate that the use of 3D co-culture of hiPSC-CMs and CF is superior over 2D culture conditions for co-culture models and more closely mimicking the native state of the myocardium with relevance to drug development as well as for personalized medicine.

English

Many patients in Africa often utilize herbal medicine for the management of hyperactive gut disorders such as diarrhea and abdominal colic. There is therefore a need to scientifically evaluate efficacy of this herbal remedies. This study investigated the myorelaxant effect of Carissa edulis (Forssk.) Vahl and its possible mechanism of action using isolated rabbit jejunum preparations. Pieces of jejunum were isolated from adult New Zealand white rabbits. They were then mounted in an organ bath containing Tyrode’s solution. The rate and force of jejunal contraction were recorded and analyzed using a Powerlab that was coupled to Chart5 software for windows. The effects of the extract (0.1-10.0 mg/ml) on spontaneous contraction were investigated. Its effect (3.0 mg/ml) was also studied in the presence of LG-NAME (nitric oxide synthase inhibitor), methylene blue (soluble guanylyl cyclase inhibitor) and propranolol (non-selective β adrenergic receptors antagonist). The extract dose-dependent significantly decreased the force but not the rate of spontaneous rabbit jejunal contraction. Among the blockers used, only LG -NAME significantly blocked the effect of the extract. Aqueous root bark extracts of C. edulis possess significant myorelaxant effect on isolated rabbit jejunum. This appears to be mediated through stimulation of nitric oxide release by nitric oxide synthase. Key words: Carissa edulis, diarrhea, jejunum, motility, myorelaxant.

Bioengineering Cardiac Tissue Constructs With Adult Rat Cardiomyocytes.

Bioengineering cardiac tissue constructs with adult cardiomyocytes may help treat adult heart defects and injury. In this study, we fabricated cardiac tissue constructs by seeding adult rat cardiomyocytes on a fibrin gel matrix and analyzed the electromechanical properties of the formed cardiac tissue constructs. Adult rat cardiomyocytes were isolated with a collagenase type II buffer using an optimized Langendorff perfusion system. Cardiac tissue constructs were fabricated using either indirect plating with cardiomyocytes that were cultured for 1 week and dedifferentiated or with freshly isolated cardiomyocytes. The current protocol generated (3.1 ± 0.5) × 10 (n = 5 hearts) fresh cardiomyocytes from a single heart. Tissue constructs obtained by both types of plating contracted up to 30 days, and electrogram (ECG) signals and contractile twitch forces were detected. The constructs bioengineered by indirect plating of dedifferentiated cardiomyocytes produced an ECG R wave amplitude of 15.1 ± 5.2 µV (n = 7 constructs), a twitch force of 70-110 µN, and a spontaneous contraction rate of about 390 bpm. The constructs bioengineered by direct plating of fresh cardiomyocytes generated an ECG R wave amplitude of 6.3 ± 2.5 µV (n = 8 constructs), a twitch force of 40-60 µN, and a spontaneous contraction rate of about 230 bpm. This study successfully bioengineered three-dimensional cardiac tissue constructs using primary adult cardiomyocytes.

Channelopathy as a SUDEP Biomarker in Dravet Syndrome Patient-Derived Cardiac Myocytes.

SummaryDravet syndrome (DS) is a severe developmental and epileptic encephalopathy with a high incidence of sudden unexpected death in epilepsy (SUDEP). Most DS patients carry de novo variants in SCN1A, resulting in Nav1.1 haploinsufficiency. Because SCN1A is expressed in heart and in brain, we proposed that cardiac arrhythmia contributes to SUDEP in DS. We generated DS patient and control induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). We observed increased sodium current (INa) and spontaneous contraction rates in DS patient iPSC-CMs versus controls. For the subject with the largest increase in INa, cardiac abnormalities were revealed upon clinical evaluation. Generation of a CRISPR gene-edited heterozygous SCN1A deletion in control iPSCs increased INa density in iPSC-CMs similar to that seen in patient cells. Thus, the high risk of SUDEP in DS may result from a predisposition to cardiac arrhythmias in addition to seizures, reflecting expression of SCN1A in heart and brain.

Cardiac striatin interacts with caveolin-3 and calmodulin in a calcium sensitive manner and regulates cardiomyocyte spontaneous contraction rate

Impaired cardiomyocyte contraction rate is detrimental to cardiac function and often lethal. Despite advancements in the field, there is a paucity of information regarding the coordination of molecules implicated in regulating the heart rate. Striatin (STRN) is a dynamic protein with binding domains to calmodulin (CaM) and caveolin (Cav), both of which are regulators of myocardial function. However, its role in cardiomyocyte contraction is not yet determined. Herein, we show that STRN is expressed in cardiomyocytes and is more abundant in atrial myocardium than in ventricles. Cardiac expression of STRN (protein and mRNA) was developmentally regulated with the highest expression being at neonatal stage (day one) and the lowest in adult rats (13 weeks). CaM pulldown assay indicated that the interaction of cardiac STRN with CaM and caveolin-3 (Cav-3) was calcium sensitive. Interestingly, the overexpression of STRN induced an increase (∼2-fold) in the rate of the spontaneous contraction of cultured cardiomyocytes, while the knockdown of STRN reduced their contraction rate (∼40%). The expression level of STRN was inversely proportional to the interaction of Cav-3 with the CaM/STRN complex. Collectively, our data delineate a novel role for STRN in regulating cardiomyocyte spontaneous contraction rate and the dynamics of the STRN/Cav-3/CaM complex.

P3495Striatin regulates the dynamics of the Caveolin-3/Calmodulin complex and modulates cardiomyocyte spontaneous contraction rate

P3495Striatin regulates the dynamics of the Caveolin-3/Calmodulin complex and modulates cardiomyocyte spontaneous contraction rate

Electrical Stimulation of Artificial Heart Muscle: A Look Into the Electrophysiologic and Genetic Implications.

Development of tissue-engineered hearts for treatment of myocardial infarction or biologic pacemakers has been hindered by the production of mostly arrhythmic or in-synergistic constructs. Electrical stimulation (ES) of these constructs has been shown to produce tissues with greater twitch force and better adrenergic response. To further our understanding of the mechanisms underlying the effect of ES, we fabricated a bioreactor capable of delivering continuous or intermittent waveforms of various types to multiple constructs simultaneously. In this study, we examined the effect of an intermittent biphasic square wave on our artificial heart muscle (AHM) composed of neonatal rat cardiac cells and fibrin gel. Twitch forces, spontaneous contraction rates, biopotentials, gene expression profiles, and histologic observations were examined for the ES protocol over a 12 day culture period. We demonstrate improved consistency between samples for twitch force and contraction rate, and higher normalized twitch force amplitudes for electrically stimulated AHMs. Improvements in electrophysiology within the AHM were noted by higher conduction velocities and lower latency in electrical response for electrically stimulated AHMs. Genes expressing key electrophysiologic and structural markers peaked at days 6 and 8 of culture, only a few days after the initiation of ES. These results may be used for optimization strategies to establish protocols for producing AHMs capable of replacing damaged heart tissue in either a contractile or electrophysiologic capacity. Optimized AHMs can lead to alternative treatments to heart failure and alleviate the limited donor supply crisis.

Application based on the Canny edge detection algorithm for recording contractions of isolated cardiac myocytes

Background and objectiveThe isolated cardiomyocyte preparation is amenable to several experimental approaches not suitable to the myocardial tissue, which has allowed the gain of important information on the pathophysiology of the cardiac muscle. Thus, the development of techniques for functional studies in this preparation is important. The goal of the present study was to develop a computer program to extract contraction traces generated by cyclic cell shortening from cardiomyocyte video image files. MethodsThe Canny algorithm, widely used for computer vision, was implemented for cell edge recognition and continuous tracking, so that changes in cardiomyocyte length could be monitored. The program was applied to demonstrate the effect of classical inotropic maneuvers on contraction parameters, as well as to assess the development of spontaneous activity in response to defibrillator-like electrical shocks in rat isolated cardiomyocytes. ResultsThe method resulted in successful monitoring of variations in cell length during both electrically-triggered and post-shock spontaneous contractions, of which the rate was significantly related to shock strength. ConclusionsThe proposed approach might be useful for analysis of contractile activity of isolated muscle cells, and allows detection of even the typically low-amplitude noisy spontaneous contractile events. Additionally, the experimental data suggest that the rate of spontaneous contraction could be used as an index of shock-induced electrical membrane damage.

Lymphatic pumping: mechanics, mechanisms and malfunction.

A combination of extrinsic (passive) and intrinsic (active) forces move lymph against a hydrostatic pressure gradient in most regions of the body. The effectiveness of the lymph pump system impacts not only interstitial fluid balance but other aspects of overall homeostasis. This review focuses on the mechanisms that regulate the intrinsic, active contractions of collecting lymphatic vessels in relation to their ability to actively transport lymph. Lymph propulsion requires not only robust contractions of lymphatic muscle cells, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neural influences. Lymphatic contractile dysfunction, barrier dysfunction and valve defects are common themes among pathologies that directly involve the lymphatic system, such as inherited and acquired forms of lymphoedema, and pathologies that indirectly involve the lymphatic system, such as inflammation, obesity and metabolic syndrome, and inflammatory bowel disease.

The effect of methanol rhizome extract of Nymphaea lotus Linn. (Nymphaeaceae) in animal models of diarrhoea

Ethnopharmacological relevanceNymphaea lotus, which is widely distributed throughout tropical Africa, enjoys a number of ethnomedical uses in Nigeria. Traditionally, the rhizomes of N. lotus are used to cure diarrhoea. Aim of studyThis study aims to evaluate the antidiarrhoeal activity of the methanol rhizome extract of N. lotus plant in laboratory animals. Materials and methodsThe extract was screened for activity against castor oil-induced diarrhoea and magnesium sulphate-induced diarrhoea as well as effect on gastric transit time in mice. The effect of methanol rhizome extract of Nymphaea lotus on the perfused isolated tissue preparation was also determined. ResultsFor castor oil-induced diarrhoea, the extract at doses of 200, 400 and 800mg/kg produced significant reduction in the frequency of diarrhoea (at p<0.001, p<0.001 and p<0.01 respectively). The extract at 800mg/kg produced a significant delay in onset of diarrhoea (p<0.05) comparable to loperamide (3mg/kg). The frequency of magnesium sulphate-induced diarrhoea was also significantly reduced in the groups treated with 200, 400 and 800mg/kg of the extract at p<0.001, p<0.001 and p<0.01 respectively. At doses of 200mg/kg and 400mg/kg, the protection produced was comparable to loperamide, 3mg/kg.All treated groups produced significant reduction in the transit of charcoal meal along the intestinal tract at p<0.001. The extract at low concentration (4×10−4–6.4×10−2mg/ml) had contractile effect on the tone of contraction of the rabbit jejunum while at higher concentrations (8×10−2–512×10−2mg/ml) produced significant reduction in the tone and rate of spontaneous contraction of rabbit jejunum. The extract at lower concentrations (4×10−4–2×10−2mg/ml) has no effect on contraction of the guinea pig ileum while higher concentrations (4×10−2–512×10−2mg/ml) produced significant relaxant activity on guinea pig ileum. ConclusionThis study has shown that the methanol rhizome extract of N. lotus has antidiarrhoeal properties thus justifying its use by the local population for this purpose.

Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.

Pharmacological Basis for the Medicinal Use of Wrightia tinctoria in Hypertension and Dyslipidemia

This study was aimed to offer a possible pharmacological basis regarding the remedial utilization of Wrightia tinctoria in hypertension and dyslipidemia in certain South Asian traditional systems of medicine, using in vivo and in vitro assays. The aqueous methanolic extract of W. tinctoria seeds (Wt.Cr) caused a dose-dependent (1-10 mg/kg) decrease in arterial pressure in anesthetized rats. In the right atria of isolated guinea pigs, Wt.Cr equally inhibited force and rate of spontaneous atrial contractions. When tested on phenylephrine-, high K(+)-, and low K(+)-induced vasoconstrictions in isolated rat aorta, Wt.Cr produced a concentration-dependent vasorelaxation, the most potent being against low K(+)-induced contraction. It also created a rightward shift in the Ca(++) concentration-response curves and suppressed phenylephrine control peaks in a Ca(++)-free environment. In the rat model of tyloxapol-induced dyslipidemia, Wt.Cr produced a decline in the serum levels of total cholesterol and triglycerides. In high fat diet-induced dyslipidemia, it decreased serum total cholesterol and low-density lipoprotein cholesterol, improved high-density lipoprotein cholesterol, and prevented the increase in average body weights by causing a decrease in diet consumption. These data suggest that Wt.Cr(++) lowers blood pressure through a combination of K(+)-channel opening and Ca(++)-channel blocking effects along with antidyslipidemic and weight-reducing properties.

Cardiovascular inhibitory properties of &lt;i&gt;Lens culinaris&lt;/i&gt;

In present study, we report the blood pressure lowering, vasodilatory and cardio-depressant activities of Lens culinaris. The crude extract of L. culinaris induced dose-dependent (3-30 mg/kg) fall in the arterial pressure of rats under anesthesia. When tested in rat aortic ring preparations, L. culinaris at concentration range of 0.03-5.0 mg/mL relaxed high K+ (80 mM) and phenylephrine (1 ?M)-induced contractions, like that caused by verapamil. In isolated guinea-pig atria, L. culinaris caused inhibition of atrial force and rate of spontaneous contractions, similar to that exhibited by verapamil. These data indicate that L. culinaris exhibits blood pressure lowering potential, mediated possibly through Ca++ channel blockade mechanism.

P381The right atrioventricular pacemaker ring in the avian heart, myth or reality?

Background: In the avian heart right atrioventricular (AV) valve there is a large, unicuspid, muscular flap, in remarkable contrast to the fibrous structure characteristic of the mammalian tricuspid valve. Histological studies showed that in addition to a sinus node, AV node, His bundle, left and right bundle branches, the birds also have the right AV Purkinje ring and a special middle bundle branch. The atrioventricular Purkinje ring is located in the atrial sheet of the AV muscular valve along all its base length. Purpose: To perform electrophysiological identification of cells on the atrial side of the chicken right muscular AV valve. Methods: Experiments were conducted on spontaneously contracting right muscular AV valve and contiguous atrial muscle preparations isolated from nine adult chicken hearts using the standard microelectrode technique. Results: The rate of spontaneous contractions of isolated preparations of muscular valve was 121 ± 7 beats/min. Three types of action potentials (APs) were registered from the atrial surface of the valve: APs of working myocardium cells, APs of pacemaker cells and APs of conducting cells. A working myocardium AP with a constant level of membrane potential during phase 4 was the most frequently recorded voltage waveform in the atrial surface of the muscular valve. Pacemaker APs have much lesser values of a maximum upstroke rate (4.3 ± 0.6 vs 193.9 ± 4.9 V/s), an amplitude (73.4 ± 1.7 vs 108.7 ± 1.0 mV ) but higher an amplitude of diastolic depolarization (7.7 ± 0.8 vs 1.4 ± 0.1 mV) than conducting APs (P<0.05). Conducting APs are usually registered from the part of the free edge of the valve, attached to the right ventricle free wall and also from the areas adjacent to dorsal and ventral sides of the interventricular septum. Detailed microelectrode mapping of APs in an isolated muscular valve preparation revealed the presence of the pacemaker zone on the valve atrial side which is permanently localized along the whole length of its base from ventral to dorsal attachment of the valve to the interventricular septum where the fibrous tissue is present. Conclusion: Our study provides evidence of the regularity of the course and the electrophysiological organization of the pacemaker zone in isolated chicken muscular valve. Location of found out pacemaker zone in the muscular valve coincide with histological description of topography of valve component of the right AV Purkinje ring. We believe that we have offered the proof that the avian right AV Purkinje ring is in reality the right AV pacemaker ring. This study was supported by the RFBR.

Optimizing a spontaneously contracting heart tissue patch with rat neonatal cardiac cells on fibrin gel.

Engineered cardiac tissues have been constructed with primary or stem cell-derived cardiac cells on natural or synthetic scaffolds. They represent a tremendous potential for the treatment of injured areas through the addition of tensional support and delivery of sufficient cells. In this study, 1-6 million (M) neonatal cardiac cells were seeded on fibrin gels to fabricate cardiac tissue patches, and the effects of culture time and cell density on spontaneous contraction rates, twitch forces and paced response frequencies were measured. Electrocardiograms and signal volume index of connexin 43 were also analysed. Patches of 1-6 M cell densities exhibited maximal contraction rates in the range 305-410 beats/min (bpm) within the first 4 days after plating; low cell density (1-3 M) patches sustained rhythmic contraction longer than high cell density patches (4-6 M). Patches with 1-6 M cell densities generated contractile forces in the range 2.245-14.065 mN/mm3 on days 4-6. Upon patch formation, a paced response frequency of approximately 6 Hz was obtained, and decreased to approximately 3 Hz after 6 days of culture. High cell density patches contained a thicker real cardiac tissue layer, which generated higher R-wave amplitudes; however, low-density patches had a greater signal volume index of connexin 43. In addition, all patches manifested endothelial cell growth and robust nuclear division. The present study demonstrates that the proper time for in vivo implantation of this cardiac construct is just at patch formation, and patches with 3-4 M cell densities are the best candidates. Copyright © 2014 John Wiley & Sons, Ltd.