Spontaneous Rhythmic Contractions Research Articles

Real-Time Evaluation of Absolute, Cytosolic, Free Ca2+ and Corresponding Contractility in Isolated, Pressurized Lymph Vessels.

The lymphatic vasculature, now often referred to as "the third circulation," is located in many vital organ systems. A principal mechanical function of the lymphatic vasculature is to return fluid from extracellular spaces back to the central venous ducts. Lymph transport is mediated by spontaneous rhythmic contractions of lymph vessels (LVs). LV contractions are largely regulated by the cyclic rise and fall of cytosolic, free calcium ([Ca2+]i). This paper presents a method to concurrently calculate changes in absolute concentrations of [Ca2+]i and vessel contractility/rhythmicity in real time in isolated, pressurized LVs. Using isolated rat mesenteric LVs, we studied changes in [Ca2+]i and contractility/rhythmicity in response to drug addition. Isolated LVs were loaded with the ratiometric Ca2+-sensing indicator Fura-2AM, and video microscopy coupled with edge-detection software was used to capture [Ca2+]iand diameter measurements continuously in real time. The Fura-2AM signal from each LV was calibrated to the minimum and maximum signal for each vessel and used to calculate absolute [Ca2+]i. Diameter measurements were used to calculate contractile parameters (amplitude, end diastolic diameter, end systolic diameter, calculated flow) and rhythmicity (frequency, contraction time, relaxation time) and correlated with absolute [Ca2+]i measurements.

Anatomical and morphological features of the heart of a domestic dog (Canis Lupus Familiaris L., 1758)

Cardiovascular organopathology in dogs occurs quite often, but is not fully diagnosed. The occurrence of cardiovascular pathology is due to inadequate physical activity, infectious diseases, injuries, blood loss, diseases of the lungs and other systems, hereditary factors, etc. It is known that prevention, diagnosis, surgical intervention and treatment of these pathologies are impossible without knowledge of the morpho-functional parameters of anatomy, histology and physiology. The cardiovascular system in mammals performs vital functions, ensuring the vital activity of the animal and human body. The cardiovascular system is very plastic in relation to the morpho-functional relationship and has not only pronounced hereditary individual traits, but also the ability to quickly adapt to the changing conditions of the organism's existence. It is one of the first to respond to various natural and anthropogenic factors of the environment and physical stress. The study of the cardiovascular system is an urgent issue today. The aim of the study was to study the morphological assessment of macro- and microstructures of the heart of a sexually mature domestic dog (n = 5), class – Mammalia, species – wolf (Canis lupus), subspecies – domestic dog (Canis familiaris). The completed work is a fragment of the scientific topic of the scientific research work of the Department of Normal and Pathological Morphology, Hygiene and Expertise of the Faculty of Veterinary Medicine of the Polish National University on the topic: “Development, morphology and histochemistry of animal organs in normal and pathological conditions”, according to state registration number No. 0113V000900 and scientific topics: “Features of the morphology of the heart of domestic mammals”, state registration No. 0121U108884. For the work, complex morphological research methods were used: anatomical, histological, organo-, histo- and cytometric, statistical, thanks to which new data on the peculiarities of the macro-, histo- and cytomorphometric characteristics of the morphological structures of the heart in the studied animals were presented. The dog's heart has a rounded shape, its absolute weight is 167.58 ± 9.46 g (without epicardial fat – 154.22 ± 8.04 g), relative weight is 0.72 ± 0.005 %. It was established that the microscopic structure of the ventricles and atria of the dog's heart differ by cytometric indicators, depending on their morphofunctional load. Thus, cardiomyocytes of the left ventricle have the largest volume – 2941.76 ± 127.44 μm3, the right one has a smaller volume – 2237.24 ± 103.02 μm3, and the cardiomyocytes of the atria have the smallest volume – 1496.92±98.02 μm3. The volume of the nuclei of cardiomyocytes of the left ventricle is 64.58 ± 5.09 μm3, of the right ventricle – 59.97 ± 5.83 μm3, the volume of cardiomyocytes of the atria is correspondingly smaller – 53.06 ± 6.02 μm3. At the same time, the nuclear-cytoplasmic ratio of cardiomyocytes of the left ventricle is equal to 0.0224±0.0076, cardiomyocytes of the right ventricle are more important – 0.0275 ± 0.0081, and cardiomyocytes of the atria are the most important – 0.0367 ± 0.0105. We associate such ambiguous cytometric parameters of cardiomyocytes with the morphofunctional activity of the ventricles and the functional features of this myocardial tissue, which is capable of spontaneous rhythmic contractions, as a result of which blood moves through a closed vascular system. Information on the morphology of the heart of a domestic dog, including the results of the study of the macro- and microscopic structure of the studied organs, which are presented in the publication, are of great importance for histology and comparative anatomy, and also make a significant contribution to clinical veterinary medicine.

Peculiarities of macro- and cytometric assessment of morphological structures of the domestic pig heart

The cardiovascular system, which includes the heart, is one of the essential systems of the human and animal body. With its participation, the blood supply of the organs takes place; it promotes the outflow of lymph from the organs and its transport into the veins, and it helps to implement the functions of the organs of immune protection, endocrine, and nervous systems. The work aims to conduct a macro-, histo-, and cytomorphometric assessment of the morphological structures of the heart of the domestic pig using complex research methods (macroscopic, histological, morphometric). Functionally mature, clinically healthy animals (n = 7) of Sus scrofa f. domestica were used for the work. According to organometallic studies, the absolute weight of a pig's heart is 487.4 ± 8.12 g; the relative weight is 0.290 ± 0.004%, and the net weight of the heart is 461.4 ± 8.0 g. According to its linear dimensions, a domestic pig's heart is elongated-narrowed (conical) type, as evidenced by the heart development index of 155.1 ± 6.3%. According to the functional load of the muscle tissues of the myocardium of the heart and its separate morphological structures (ventricles and atria) when performing particular work during spontaneous rhythmic contractions, the absolute mass of the ventricles and atria is different. The enormous mass is characteristic of the left ventricle, then the right, and the smallest for the left and right atria. Therefore, the ventricles of the heart are more functionally developed, as evidenced by the ratio coefficient (1:0.79) of the mass of the ventricles to the net mass of the heart, respectively, the ratio coefficient (1:0.21) of the mass of the atria to the net mass of the heart and the ratio coefficient (1:0.27) atrial mass to ventricular mass. Cardiomyocytes of the left ventricle have the most significant volume, cardiomyocytes of the right ventricle have a smaller volume, and cardiomyocytes of the atria have the smallest volume. Their nuclear volumes have similar values, respectively, in the left ventricle – 77.16 ± 2.01 μm3, the right ventricle – 76.02 ± 2.43 μm3, and the atrium – 75.97 ± 3.24 μm3, and the nuclear-cytoplasmic ratio is different from them: the smallest nuclear-cytoplasmic ratio, respectively characteristic of cardiomyocytes of the left ventricle, significantly more for cardiomyocytes of the right ventricle and significantly the most for cardiomyocytes of the atria. The scientific results of morphoarchitectonics, organo-, histo-, and cytometry of the heart of the domestic pig presented in the publication supplement the information on the morphological structure of the heart of domestic animals in the relevant sections of histology, comparative anatomy and are a significant contribution to clinical cardiology.

Spontaneous body wall contractions stabilize the fluid microenvironment that shapes host-microbe associations.

The freshwater polyp Hydra is a popular biological model system; however, we still do not understand one of its most salient behaviors, the generation of spontaneous body wall contractions. Here, by applying experimental fluid dynamics analysis and mathematical modeling, we provide functional evidence that spontaneous contractions of body walls enhance the transport of chemical compounds from and to the tissue surface where symbiotic bacteria reside. Experimentally, a reduction in the frequency of spontaneous body wall contractions is associated with a changed composition of the colonizing microbiota. Together, our findings suggest that spontaneous body wall contractions create an important fluid transport mechanism that (1) may shape and stabilize specific host-microbe associations and (2) create fluid microhabitats that may modulate the spatial distribution of the colonizing microbes. This mechanism may be more broadly applicable to animal-microbe interactions since research has shown that rhythmic spontaneous contractions in the gastrointestinal tracts are essential for maintaining normal microbiota.

Intermediate conductance Ca2+-activated potassium channels are activated by functional coupling with stretch-activated nonselective cation channels in cricket myocytes.

Cooperative gating of localized ion channels ranges from fine-tuning excitation-contraction coupling in muscle cells to controlling pace-making activity in the heart. Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels. The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction. To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity, we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data. In this study, we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+. The single-channel conductance of the IK channel was 62 pS, and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent. These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes. Second, the Ca2+ influx pathway that activates the IK channel was investigated. The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels. Finally, we investigated the proximity between SA and IK channels. The removal of extracellular Ca2+, administration of Ca2+ to the microscopic region in a pipette, and application of membrane stretching stimulation increased SA channel activity, followed by IK channel activity. Membrane stretch-induced SA and IK channel activity were positively correlated. However, the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette. These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity. In conclusion, functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions.

Hypertension Induces Contractile Dysfunction in Rat Mesenteric Lymph Vessels

Hypertension predisposes patients to a cluster of deadly cardiovascular and metabolic diseases involving inflammation, dyslipidemia, and fluid imbalance. Current medications fail to normalize blood pressure in 25% of hypertensive patients; therefore, we considered the possibility that the lymphatic circulation provides a new therapeutic target to alleviate hypertension and its associated co‐morbidities. The intrinsic driving force for the propulsion of lymph fluid is the robust, spontaneous rhythmic contractions of lymphatic muscle cells (LMCs) that facilitate the lymph flow which helps maintain fluid balance and the transport of lipids and immune cells. Thus, evaluation of lymphatic contractions and estimation of lymph flow during hypertension is important to understand the possibility of targeting lymphatic circulation in hypertension. Angiotensin II (Ang II) is a key regulator of blood pressure, and increased levels of Ang II are known to induce hypertension. However, the role of Ang II mediated hypertension on lymphatic contractile dysfunction is still a matter of debate. Here, we evaluated the rhythmic contractions of LVs isolated from Ang II infused hypertensive rats (AHT LVs) compared to normotensive controls (NT LVs). Ang II containing osmotic mini pumps (500 ng/kg/min) were implanted subcutaneously in adult (9‐13 weeks old) male Sprague Dawley (SD) rats for 14 days. Rat mesenteric LVs were dissected free from fat and then cannulated and pressurized (4‐5 mmHg) in a perfusion bath filled with physiological salt solution (PSS). Changes in diameter were monitored by real‐time edge detection software. Baseline rhythmic contractions were recorded for ~45 minutes and then a L‐type calcium channel antagonist, nifedipine (NIF; 1 µmol/L) was added to the bath for 10 mins followed by washout with calcium free PSS. AHT LVs had significantly increased calcium dependent tone to 14.3% ± 5.1% (n=8) compared to NT LVs, which remained at 5.2% ± 1.1% (n=8). AHT LVs also exhibited a larger response to nifedipine with 14.0% ± 6.5% (n=6) increase in dilation compared to NT LVs 3.9% ± 0.9% (n=8). Contractile traces also showed visible dysrhythmia, defined as an increased variation in contraction interval, in LVs isolated from AHT rats at 32.4% ± 14.5% (n=8) when compared with NT LVs at 2.3% ± 1.1% (n=8). An increased variance for the time to relaxation also was observed in AHT LVs (7.4% ± 4.6%; n=8) compared to NT LVs (0.6% ± 0.2%; n=8). These differences ultimately resulted in a significant decrease in calculated flow in AHT LVs (62.0 nL/s ± 8.4 nL/s; n=5) compared to NT LVs (92.4 nL/s ± 4.3 nL/s; n=5). Our data suggest that Ang II mediated hypertension can cause lymphatic contractile dysfunction leading to loss of lymph flow and may be a potential mechanism by which hypertension increases the risk of associated co‐morbidities. Studies are ongoing to assess the changes in lymph flow in vivo during Ang II mediated hypertensive versus normotensive rats.

Morphology and specifics of morphometry of lungs and myocardium of heart ventricles of cattle, sheep and horses

Respiratory organs and cardiovascular system are interconnected and perform extremely vital functions of the organism, the main goals being performing gas exchange with the environment and emitting carbon dioxide produced in the organism into the environment. Therefore, we carried out comparative histo- and cytomorphometric evaluation of morphological structures of the heart and lungs of cattle, sheep and horses. Using complex methods of research, we determined specifics of microscopic structure and histometric parameters of parenchyma of the lungs and myocardium of the ventricles of the hearts of clinically healthy animals in species aspect – cattle, sheep and horses. The studies revealed that the histoarchitectonics of the studied animals were similar in structure, characteristic of other species of agricultural mammals, and distinct morphometric peculiarities of their histostructures. The study indicated that the respiratory zone of the lungs is most developed in horses. This parameter was lower in ruminants – cattle and sheep. Connective tissue septum of parenchyma of lungs was better expressed in cattle and sheep, and less in horses. At the same time, mean volume of lung alveoli in clinically healthy animals varied: being highest in horses – 699 ± 106 thou µm3, then cattle – 337 ± 43 thou µm3 and sheep – 158 ± 37 thou µm3. Such variation in histometric parameters of parenchyma of the lungs in experimental animals indicates adaptive specifics of the organism of animals in terms of living conditions. Therefore, the respiratory zone was most developed in the lungs of horses, animals that experience significant physical and physiological load on corresponding organs and systems. As a result of histometric studies of myocardium, we determined patterns of sizes of thickness of its cardiomyocytes and volume of their nuclei. We determined that in myocardium of the heart ventricles, in the same microscope field of view, cardiomyocytes of varying thicknesses occur. At the same time, thickness of cardiomyocytes, their mean volume of nuclei in the ventricle myocardium were expressed the most in cattle, then in horses and sheep, and their histometric parameters in myocardium of the left ventricle of the heart in the experimental animals were higher than in the right one. Therefore, thickness of cardiomyocytes of the left ventricle in cattle equaled 14.06 ± 0.41 µm, and volume of nuclei of cardiomyocytes respectively 124.55 ± 7.99 µm3. Similar changes in such parameters of histometry were found in sheep and horses. We attribute such varying histometric parameters of the thickness of cardiomyocytes and volume of their nuclei in myocardium of the left ventricle of the heart in experimental animals, compared with such parameters in the right ventricle, to the activities of the ventricles (the left one generally functions as a pump, right one – as a volumetric) and functional specifics of this myocardium tissue, which is capable of spontaneous rhythmic contractions, resulting in blood flow in the vessels: cardiomyocytes of the left ventricle carry greater load, promoting blood flow in vessels of greater (somatic) blood circulation, respectively cardiomyocytes of the right ventricle – less load, promoting blood flow in vessels of lesser (pulmonary) blood circulation.

Enhancement of engineered cardiac tissues by promotion of hiPSC-cardiomyocyte proliferation

Abstract Background/Introduction Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modelling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Purpose Determining the effects of the induction of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocyte proliferation post-fabrication. Methods hiPSCs were differentiated into cardiomyocytes, embedded with or without CHIR990121 at three concentrations in a collagen pre-gel, and cast. The engineered cardiac tissues were then cultured in the absence or presence of CHIR99021 for up to 35 days. Hydrogels and engineered cardiac tissues were analysed utilizing rheology and assays to determine viability, proliferation, calcium flow, and contractility. Results Here, we show that the integration of CHIR99021 in collagen I hydrogels promotes proliferation of hiPSC-cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered cardiac tissues. Presence of CHIR99021 has no effect on the gelation kinetic or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ∼2-fold within 7 days post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 days in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increases speed of contraction and contraction amplitude, and reduces peak-to-peak time. The CHIR99021-stimulated engineered cardiac tissues exhibited spontaneous rhythmic contractions for at least 35 days. Conclusion Collectively, our data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac tissues by increasing cardiomyocyte density and reducing arrhythmia. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

CHIR99021 Promotes hiPSC-Derived Cardiomyocyte Proliferation in Engineered 3D Microtissues.

Cardiac tissue engineering is a promising strategy to generate human cardiac tissues for modeling cardiac diseases, screening for therapeutic drugs, and repairing the injured heart. Yet, several issues remain to be resolved including the generation of tissues with high cardiomyocyte density. Here, it is shown that the integration of the glycogen synthase kinase-3β inhibitor CHIR99021 in collagen I hydrogels promotes proliferation of human-induced pluripotent stem cell-derived (hiPSC) cardiomyocytes post-fabrication improving contractility of and calcium flow in engineered 3D cardiac microtissues. CHIR99021 has no effect on the gelation kinetics or the mechanical properties of collagen I hydrogels. Analysis of cell density and proliferation based on Ki-67 staining indicates that integration of CHIR99021 together with external CHIR99021 stimulation increases hiPSC-cardiomyocyte number by ≈2-fold within 7 d post-fabrication. Analysis of the contractility of engineered cardiac tissues after another 3 d in the absence of external CHIR99021 shows that CHIR99021-induced hiPSC-cardiomyocyte proliferation results in synchronized calcium flow, rhythmic beating, increased speed of contraction and contraction amplitude, and reduced peak-to-peak time. The CHIR99021-stimulated engineered cardiac microtissues exhibit spontaneous rhythmic contractions for at least 35 d. Collectively, the data demonstrate the potential of induced cardiomyocyte proliferation to enhance engineered cardiac microtissues by increasing cardiomyocyte density.

Differential effects of β-methylphenylethylamine and octopamine on contractile parameters of the rat gastrointestinal tract

This study tested the effects of β-methylphenylethylamine (β-MPEA) and octopamine on contractile parameters of the gastrointestinal tract in rats. We hypothesized that some of their effects result from interactions with trace amine (TA)-associated receptors or serotoninergic 5-hydroxytryptamine (5-HT) receptors. β-MPEA-induced contractions in rat gastric fundus strips under resting tonus conditions, but induced relaxation in preparations that were previously contracted with carbachol. Octopamine relaxed gastric fundus strips maintained at resting tonus or contracted with carbachol. The contractile effect of β-MPEA was reduced by cyproheptadine and methiothepin, antagonists of excitatory 5-HT receptors. The relaxing effect of β-MPEA on gastric fundus was insensitive to pretreatment with N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl)benzamide (EPPTB) and tropisetron, antagonists of TA1 and 5-HT4 receptors, respectively. Both EPPTB and tropisetron inhibited the relaxant effects of octopamine on carbachol-contracted preparations. Contrarily, EPPTB did not reduce the relaxant effects of RO5263397 (TA1 agonist) or zacopride (5-HT4 agonist). Octopamine, but not β-MPEA, delayed the gastrointestinal transit of a liquid test meal in awaken rats. In isolated preparations of the small intestine under resting conditions, β-MPEA did not alter the basal tonus, but octopamine relaxed it. Intestinal preparations previously contracted with carbachol relaxed after the addition of octopamine and decreased the magnitude of their spontaneous rhythmic contractions in a tropisetron-dependent manner. Thus, β-MPEA and octopamine exerted pharmacological actions on the rat gastrointestinal tract. The excitatory effects of β-MPEA involved 5-HT receptors. Octopamine inhibited the rat gut contractility through the likely involvement of 5-HT4 and TA receptors. Overall, octopamine effectively inhibited rat gastrointestinal transit.

BK Channels Are Activated by Functional Coupling With L-Type Ca2+ Channels in Cricket Myocytes.

Large-conductance calcium (Ca2+)-activated potassium (K+) (BK) channel activation is important for feedback control of Ca2+ influx and cell excitability during spontaneous muscle contraction. To characterize endogenously expressed BK channels and evaluate the functional relevance of Ca2+ sources leading to BK activity, patch-clamp electrophysiology was performed on cricket oviduct myocytes to obtain single-channel recordings. The single-channel conductance of BK channels was 120 pS, with increased activity resulting from membrane depolarization or increased intracellular Ca2+ concentration. Extracellular application of tetraethylammonium (TEA) and iberiotoxin (IbTX) suppressed single-channel current amplitude. These results indicate that BK channels are endogenously expressed in cricket oviduct myocytes. Ca2+ release from internal Ca2+ stores and Ca2+ influx via the plasma membrane, which affect BK activity, were investigated. Extracellular Ca2+ removal nullified BK activity. Administration of ryanodine and caffeine reduced BK activity. Administration of L-type Ca2+ channel activity regulators (Bay K 8644 and nifedipine) increased and decreased BK activity, respectively. Finally, the proximity between the L-type Ca2+ channel and BK was investigated. Administration of Bay K 8644 to the microscopic area within the pipette increased BK activity. However, this increase was not observed at a sustained depolarizing potential. These results show that BK channels are endogenously expressed in cricket oviduct myocytes and that BK activity is regulated by L-type Ca2+ channel activity and Ca2+ release from Ca2+ stores. Together, these results show that functional coupling between L-type Ca2+ and BK channels may underlie the molecular basis of spontaneous rhythmic contraction.

515 Repurposing of FDA-approved lead drugs as potentiators of uterine contractility in postpartum hemorrhage

Current medical options for management of postpartum hemorrhage preclude universal application due to their potential for adverse side effects. The objective of this study was to test the ex vivo efficacy of uterine-selective, FDA-approved drugs with no known cardiopulmonary toxicities repurposed for use at the human myometrial level. Lead drugs that selectively regulate human myometrial contractility were identified in prior studies using in vitro high throughput drug screening for calcium mobilization in myometrial cells. In this study, functional ex vivo isometric contractility was assessed via organ bath assays to determine uterotonic activity of mefloquine and pimavanserin on the human myometrial tissue. Human myometrial tissue was obtained at the time of cesarean delivery from non-laboring women meeting inclusion and exclusion criteria. Myometrial tissue strips (∼6-8 per biopsy) were submerged in organ baths (37°C, 95%O2, 5%CO2) containing Kreb’s Bicarbonate Solution and suspended under 3-gram isometric conditions. Tissues that displayed rhythmic spontaneous contractions were treated with cumulative concentrations (10pM – 0.1mM) of either mefloquine, pimavanserin or vehicle control (DMSO) at 20min intervals. Contractile activity was recorded and analyzed for frequency, amplitude, and area under the curve. Drug response relative to spontaneous contractile activity was analyzed, and non-linear regression models were used to generate concentration-response curves. Comparisons of fit were performed to determine if the non-linear curves of mefloquine and pimavanserin were significantly different (p<0.05) from DMSO. We performed 57 experiments in myometrial samples obtained from 12 women. There was no significant difference in the amplitude, frequency, or area under the curve between the vehicle control, mefloquine, and pimavanserin. Neither mefloquine nor pimavanserin induces ex vivo uterotonic activity in human myometrial tissue.

Carfilzomib Induces Cardiotoxicity Via ß5/ß2-Specific Proteasome Subunit Inhibition Pattern

BACKGROUND All proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment, Bortezomib (BTZ), Ixazomib (IXA) and Carfilzomib (CFZ), by design target the rate-limiting ß5 proteolytic proteasome subunit, but differ in their co-inhibition of the ß1 or ß2 proteolytic subunits at higher doses. CFZ is unique in inducing ß5/ß2 co-inhibition in a dose-dependent manner, which results in increased proteotoxic stress and higher cytotoxicity compared to the ß5/ß1 co-inhibition pattern mediated by BTZ or IXA. CFZ treatment, in particular at higher doses, is not only associated with increased clinical activity, but also with significantly higher cardiac toxicity compared to BTZ. The molecular basis for this difference is poorly understood, and mechanism-based pharmacological alleviation strategies are lacking. We hypothesized that the acute cardiotoxicity of CFZ is related to its unique ß5/ß2 inhibition pattern. METHODS Isolated primary murine cardiomyocytes showing spontaneous rhythmical contractions were used to assess the functional cardiotoxicity of PI in vitro. Cardiomyocytes were treated with the PI BTZ, CFZ or with highly-selective, subunit (ß1, ß2, ß5)-specific PI for 1h. Proteasome inhibition was directly verified using subunit-selective activity-based chemical probes. Contractility was assessed with motion vector image analysis, and calcium transients were examined by confocal microscopy after transduction of cardiomyocytes with a GCaMP vector. Differentially accumulated proteins after 1h PI treatment were quantitatively compared by liquid chromatography/mass spectrometry (LC/MS). In vivo, electrical changes and alterations in heart rate were monitored by electrocardiography (ECG). Calcium transients, ECG signals and LC/MS data were analyzed with R software version 3.5.1 (2018-07-02). RESULTS Treatment of cardiomyocytes with CFZ or with the combination of ß5/ß2-targeting subunit-specific PI impaired contractility in vitro in contrast to BTZ, or to co-inhibition of ß5/ß1 proteasome subunits. In vivo, the CFZ-type proteasome inhibition triggered acute bradycardia. Treatment of cardiomyocytes with CFZ induced a shift of intracellular calcium pools from the endoplasmic reticulum (ER) to the cytosol, consistent with ER-to-cytosol translocation of calcium described upon ER stress induction. Co-treatment with CFZ and cycloheximide, an inhibitor of protein synthesis, rescued cardiomyocytes from CFZ-induced functional impairment in vitro, suggesting that the accumulation of specific proteins is involved in CFZ-induced cardiomyocyte dysfunction. Quantitative proteomic comparison of primary cardiomyocytes treated by either CFZ-type or BTZ-type proteasome inhibition for 1h identified a selective accumulation of proteins of the retinoic acid pathway in CFZ-treated cardiomyocytes. Interestingly, co-treatment of cardiomyocytes with all-trans-retinoic acid (ATRA) prevented CFZ-induced acute cardiotoxicity in vitro. CONCLUSION Our data suggests that CFZ specifically impairs cardiac contractility through its unique ß5/ß2 proteasome subunit inhibition pattern, which results in more effective functional proteasome inhibition, protein accumulation and proteotoxic stress. The shift of intracellular calcium pools in cardiomyocytes upon CFZ treatment mirrors contractility impairment, and the specific changes identified in the retinoic acid pathway suggest ATRA as a potential drug candidate to alleviate CFZ-induced cardiac toxicity. Disclosures No relevant conflicts of interest to declare.

Pharmacological Profile of KUL-7211, a Selective -Adrenoceptor Agonist, in Isolated Ureteral Smooth Muscle

Since, in the human ureter, both β2- and β3-adrenoceptors mediate adrenergic-stimulation-induced relaxation, selective β2-/β3-adrenoceptor agonists might prove clinically useful for relieving ureteral colic and promoting stone passage. We evaluated the β-adrenoceptor subtype selectivity and ureteral-relaxing efficacy of (-)-2-[4-(2-{[(1S,2R)-2-hydroxy-2-(4-hydroxyphenyl)-1-methylethyl]amino}ethyl)phenyloxy]acetic acid (KUL-7211), a new β-adrenoceptor agonist, in vitro. In rat isolated organs, its selectivities, for inhibition of spontaneous uterine contraction (mediated via β2-adrenergic stimulation) and inhibition of colonic contraction (via β3-adrenergic stimulation) versus increase in atrial rate (via β1-adrenergic stimulation), were 56.3 and 242.2, respectively. KUL-7211 relaxed 80-mM-KCl-induced tonic contractions in both rabbit (pD2 value: 5.86 ± 0.13, whose ureteral relaxation is mediated via β2-adrenergic stimulation) and canine (pD2 value: 6.52 ± 0.16, via β3-adrenergic stimulation) isolated ureters in a concentration-dependent manner. These KUL-7211-induced relaxing effects were antagonized by ICI-118,551 (selective β2-adrenoceptor antagonist, pKB value: 8.91 ± 0.24) in the rabbit ureter and by bupranolol (non-selective β-adernoceptor antagonist, pKB value: 6.85 ± 0.12) in the canine ureter. KUL-7211 also reduced the spontaneous rhythmic contraction in a canine ureteral spiral preparation in a concentration-dependent manner, the pD2 value being 6.83 ± 0.20. These data clearly demonstrate that KUL-7211 selectively stimulates both ureteral β2- and β3-adrenoceptors and potently relaxes ureteral smooth muscle. KUL-7211 may be a novel and useful medication for relieving ureteral colic and promoting stone passage in urolithiasis patients.

Venous Vasomotion.

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.

Smooth muscle relaxant activity of Crocus sativus (saffron) and its constituents: possible mechanisms.

We praise the article by Mokhtari-Zaer et al. (2015) published in the Avicenna Journal of Phytomedicine on the effects of saffron on smooth muscle activity (Mokhtari-Zaer et al., 2015). It was a well-designed and interesting study on the therapeutic properties of Crocus sativus which is a precious plant cultivated in our country. We do believe that along with all other medicinal effects, saffron or its active constituents, primarily crocin and crocetin, could also have some clinical implications in the gynecology field. Historically and particularly in traditional Persian medicine, saffron has been regarded as an abortifacient agent (Hosseinzadeh and Nassiri-Asl, 2013; Schmidt et al., 2007). Farmer women exposed to saffron had increased rates of miscarriage (Ajam et al., 2014). This could be due to the fact that saffron stimulates uterine contractions. However, in Table 1 of the article by Mokhtari-Zaer et al. (2015), a relaxant effect of saffron on uterine contraction was mentioned (Mokhtari-Zaer et al., 2015). Concerning various components of saffron including crocin, picrocrocin, crocetin, and safranal, each could have different actions on muscular tissue. As an antispasmodic, saffron is used for stomach pain by helping digestion and improving appetite. It also reduces tension and alleviates symptoms of premenstrual syndrome and renal colic (Agha-Hosseini et al., 2008; Moshiri et al., 2006; Kashani et al., 2018). In one study, the impact of saffron on cervical ripening in term pregnant women was evaluated for the first time in the form of a clinical trial (Sadi et al., 2016). They concluded that saffron could induce cervical priming which is a perquisite for vaginal delivery by improving the bishop score. Cervical ripening caused by prostaglandins such as misoprostol or prostaglandin E1 is associated with simultaneous increase in uterine muscle contractility which is apparently in contrast with the effects of saffron as a relaxing agent (Vahdat et al., 2015). In a study by Sadraei et al, the effects of Crocus sativus extracts on uterus contractions were assessed in vitro. Following removal of the uteri of rats, spontaneous rhythmic contractions were induced using potassium chloride (KCL). According to their results, Crocus sativus increased these contractions and showed spasmodic action on muscle fibers (Sadraei et al., 2003). Likewise, in other studies, saffron had stimulatory effects on uterus as a result of myogenic and neurogenic actions resulting in prolonged bleeding, premature birth and abortion (Tafazoli et al., 2004; Modaghegh et al., 2008). This stimulatory effect of saffron on uterine musculature, however, is sometimes desirable in obstetrics to enhance the process of labor and also in gynecology for facilitating the surgical procedures on the uterus (Sadi et al., 2016; Vahdat et al., 2015). Thus, the exact mechanism of action of saffron derivatives on uterus and cervix has not been fully elucidated yet. In all the studies mentioned, the effects of the plant extract were assessed without purifying or differentiating its different elements which could have different or even opposing actions. With the availability of saffron tablets in Iran which mainly consists of crocin, with the brand name of Krocina, we believe that it would be much easier to assess the impact of this particular component of saffron on uterine or cervical tissue to clarify the exact mechanisms underlying abortion or induction of labor. In the end, we congratulate Mokhtari-Zaer et al on their article and we appreciate the Avicenna Journal of Phytomedicine editorial board for their judicious concern on this topic. We hope to read well-controlled randomized clinical trials regarding the beneficiary effects of saffron in future

Bioengineering functional smooth muscle with spontaneous rhythmic contraction in vitro

Oriented smooth muscle layers in the intestine contract rhythmically due to the action of interstitial cells of Cajal (ICC) that serve as pacemakers of the intestine. Disruption of ICC networks has been reported in various intestinal motility disorders, which limit the quality and expectancy of life. A significant challenge in intestinal smooth muscle engineering is the rapid loss of function in cultured ICC and smooth muscle cells (SMC). Here we demonstrate a novel approach to maintain the function of both ICC and SMC in vitro. Primary intestinal SMC mixtures cultured on feeder cells seeded electrospun poly(3-caprolactone) scaffolds exhibited rhythmic contractions with directionality for over 10 weeks in vitro. The simplicity of this system should allow for wide usage in research on intestinal motility disorders and tissue engineering, and may prove to be a versatile platform for generating other types of functional SMC in vitro.

PIEZORESISTIVE STRAIN SENSOR APPLICATION IN EVALUATION OF MOUSE AORTIC MEDIA CUSHIONS EFFECTIVENESS AND SPONTANEOUS MYOGENIC CONTRACTION

The aortic media realized Windkessel vessel functions and maintain sustained ventricle ejection balance during cardiac circle. Wheatstone bridge circuit piezoresistive strain sensor had desirable sensing properties to investigate aortic cushion features. In this study, Wheatstone bridge sensor was used to evaluate quick stretching-induced aortic efficient cushions and spontaneous myogenic contractions. Mice aortic specimens were loosely hooked and stabilized to [Formula: see text][Formula: see text]mm stainless steel pin and strain sensor, whereas the other side was hooked and shows increasing specimen length. Specimen isometric tension and rhythmic spontaneous myogenic contraction were recorded. Isometric tension and spontaneous myogenic response at initial length ([Formula: see text] and ultimate length ([Formula: see text] were evaluated. Aortic specimen significantly eliminated mechanical rigid oscillations. The recovery to baseline time was significantly shortened at [Formula: see text] ([Formula: see text][Formula: see text]ms and [Formula: see text] ms at [Formula: see text] and [Formula: see text], respectively, but [Formula: see text][Formula: see text]ms and [Formula: see text][Formula: see text]ms in no-load test). High Ca[Formula: see text] incubation prolonged the recovery time to baseline at [Formula: see text] and [Formula: see text] ([Formula: see text][Formula: see text]ms and [Formula: see text][Formula: see text]ms, respectively) and suggested Ca[Formula: see text] decreased efficient cushion. Moreover, strain sensor successfully recorded the enhanced rhythmic spontaneous myogenic contractions in isometric specimen. Wheatstone bridge circuit sensor reflected the significance of efficient cushions under mechanical preload, which absolutely captured rhythmic myogenic contractions of mice aortic specimen.

Wine polyphenol resveratrol inhibits contractions of isolated rat uterus by activation of smooth muscle inwardly rectifying potassium channels

Resveratrol is a phytoalexin produced in a number of plant species including grapes. The benefit of resveratrol to health is widely reported. Resveratrol has been found to promote relaxation of non-pregnant and pregnant uterus, but its mechanism of action is unclear. The aims of our study were to investigate the involvement of inwardly rectifying potassium channels (Kir) in inhibitory effects of resveratrol on three models of contractions of non-pregnant rat uterus: the spontaneous rhythmic contractions (SRC), oxytocin-elicited phasic contractions and tonic oxytocin-elicited contractions.Uterine strips were obtained from virgin female Wistar rats in oestrus. Strips were mounted into organ bath for recording isometric tension in Krebs-Ringer solution. Experiments followed a multiple curve design. In order to test the involvement of Kirchannels in a mechanism of action of resveratrol(1-100 ?M),BaCl2 (1 mM),a antagonist of inwardly rectifying pota?ssium channels was used. Resveratrol induced a concentration-dependent relaxation of all models of contractions. BaCl2 antagonized the response to resveratrolon SRC and oxytocin-elicited phasic contractions. Relaxation achieved by resveratrolon tonic oxytocin-elicited concentrations was insensitive to BaCl2.The antagonism of resveratrol effects by inwardly rectifying potassium channels antagonist suggests that Kir channels are involved in resveratrol action on phasic contractions of rat uterus. Inhibitory effect of resveratrol on tonic contractions did not include Kir channels.

Early Adaptation of Small Intestine After Massive Small Bowel Resection in Rats.

Background:It is important that the residual bowel adapts after massive resection. The necessary intestinal adaptation is a progressive recovery from intestinal failure through increase in absorptive surface area and functional capacity and includes both morphological and functional adaptations.Objectives:The aim of this study was to investigate intestinal morphological and functional adaptations of small bowel syndrome (SBS) model rats (SBS1W) 7 days after bowel resection.Materials and Methods:Male sprague–dawley rats (n = 20/group) underwent either a 75% proximal small bowel resection (SBS1W group) or a control operation (control group). Markers of morphological adaptation were revealed by TEM analysis of H&E-stained tissue samples. The intestinal barrier condition was assessed by BT, and sIgA concentration in intestinal mucus was measured by ELISA. Contractility and the slow wave rhythm of the entire intestinal remnant were measured and recorded.Results:The SBS1W group experienced more weight loss than control group and had a clearly different intestinal morphology as revealed in TEM images. Compared with control rats, the SBS1W group had a lower sIgA concentration in intestinal mucus and higher BT to lymph nodes (70% vs 40%; level I), portal blood (40% vs 10%; level II), and peripheral blood (60% vs 30%; level III). Disorder of spontaneous rhythmic contraction, irregular amplitude, and slow frequency were detected in the SBS1W group by a muscle strips test. Similarly, the slow wave of the entire intestinal remnant in the SBS1W group was irregular and uncoordinated.Conclusions:The finding of intestinal adaptation following massive SBR in SBS1W rats provides more understanding of the mechanisms of progressive recovery from the intestinal failure that underlies SBS. The mechanical, chemical, immunological, and biological barriers were all impaired at 7 days following bowel resection, indicating that the SBS model rats were still in the intestinal adaptation phase.