Efficient Ex Vivo Screening of Agents Targeting Thrombospondin1-Induced Vascular Dysfunction Using a Digital Multiwire Myograph System

Homeostasis of vascular tone is intricately and delicately maintained systemically and locally, by autonomic nerves and hormones in the blood and by intimal vasoactive substances, respectively. The balance can be acutely or chronically interrupted secondary to many alterations, especially under pathological conditions. Excessive matricellular glycoprotein thrombospondin 1 (TSP1) levels in circulation have been found to play an important role in ischemia-reperfusion injuries of different organs, by acutely suppressing vasorelaxation and chronically remodeling vascular bed. Our laboratory has been interested in identifying new drug moieties, which can selectively and effectively counteract TSP1-induced vascular dysfunction, in order to address associated clinical complications. Preliminary studies using computational docking and molecular models revealed potential drug candidates for further evaluation via vascular functional bioassay to prove the antagonism using an ex vivo vascular model. Herein, we described an efficient screening method for the identification of active drug candidates, by adapting a multiwire myograph system to perform a protocol with different treatments, in the presence of pathological levels of TSP1. We discussed the promising pharmacological evaluation results and suggested suitable modification for versatile applications. We also described the necessity of pre-determination of optimal resting tension to obtain the maximal response, if the experimental test model is different from those with determined optimal resting tension.

Vasorelaxation endothelium-independent of the ethyl acetate phase from aerial parts of Solanum paludosum Moric. envolves channels-calcium L-type blockade

Previous results showed the ethyl acetate phase (SP-AcOEt), obtained from aerial parts of Solanum paludosum, relaxed the aorta isolated in the endothelium-dependent and -independent manner. The vasorelaxant effects of SP-AcOEt was not characterized on aorta rings endothelium-denuded, thus this work aimed to elucidate the mechanisms endothelium-independent vasorelaxation on rat isolated aorta. The aorta was isolated from Wistar rats and mounted in glass baths containing 6 mL of normal Krebs physiological solution with pH at 7.4. The preparation was maintained at 37ºC and bubbled continuously with a mixture of 95% O2 and 5% CO2. Aortic rings were maintained for 1 hour by a resting tension of 1g and next were contracted with phenylephrine after the sustained contraction ACh was added to access the integrity of the endothelium. SP-AcOEt relaxed pre-contracted aorta by KCl-30mM or -80mM in a similar manner, suggesting blockade CaV, but not channel-K+ participating. SP-AcOEt also inhibited the contraction induced by CaCl2 and relaxed pre-contracted aorta by (±)-BayK8644 (EC50 = 16.9±1.3 μg/mL), which confirms the involvement of L-type CaV blockade. SP-AcOEt presented vasorelaxation endothelium-independent that involves L-type CaV blockade.

Effect of intraoperative neuromuscular blockade on postoperative sore throat and hoarseness in patients undergoing spinal surgery: a prospective observational study

Intraoperative neuromuscular blockade affects the resting tension between the vocal cords. We assessed the effect of neuromuscular blockade on postoperative sore throat and hoarseness following tracheal intubation in patients undergoing lumbar spinal surgery in the prone position. Altogether, 99 patients were included; 50 patients did not receive neuromuscular blockade, and 49 patients received moderate neuromuscular blockade during the maintenance of anesthesia. Neuromuscular blockade was performed depending on the use of intraoperative neurophysiological monitoring. The number of intubation attempts, time to achieve tracheal intubation, and duration of intubation were recorded accordingly. The incidence and severity of postoperative sore throat and hoarseness was assessed at 1, 6, and 24 h after surgery. The overall cumulative incidence of postoperative sore throat (60% vs. 59%, respectively; P = 1.000) and postoperative hoarseness (68% vs. 61%, respectively; P = 0.532) did not differ between the no neuromuscular blockade and moderate neuromuscular blockade. The incidence and severity of postoperative sore throat and hoarseness was also not different between the moderate and no neuromuscular blockade at each time point after surgery. Nevertheless, the incidences of postoperative sore throat and hoarseness were quite high. Further studies investigating strategies to alleviate them are warranted accordingly.

Non-axisymmetric shapes of biological membranes from locally induced curvature

In various biological processes such as endocytosis and caveolae formation, the cell membrane is locally deformed into curved configurations. Previous theoretical and computational studies to understand membrane morphologies resulting from locally induced curvature are often limited to axisymmetric shapes, which severely restricts the physically admissible morphologies. Under the restriction of axisymmetry, past efforts predict that the cell membrane buds at low resting tensions and stalls at a flat pit at high resting tensions. In this work, we lift the restriction of axisymmetry by employing recent theoretical and numerical advances to understand arbitrarily curved and deforming lipid bilayers. Our non-axisymmetric morphologies reveal membrane morphologies which agree well with axisymmetric studies—however only if the resting tension of the membrane is low. When the resting tension is moderate to high, we show that (i) axisymmetric invaginations are unstable; and (ii) non-axisymmetric ridge-shaped structures are energetically favorable. We further study the dynamical effects resulting from the interplay between intramembrane viscous flow and induced curvature, and find the rate at which the locally induced curvature increases is a key determinant in the formation of ridges. In particular, we show that axisymmetric buds are favored when the induced curvature is rapidly increased, while non-axisymmetric ridges are favored when the curvature is slowly increased: The rate of change of induced curvature affects the intramembrane viscous flow of lipids, which can impede the membrane’s ability to transition into ridges. We conclude that the appearance of non-axisymmetric ridges indicates that axisymmetry cannot be generally assumed when understanding processes involving locally induced curvature. Our results hold potentially relevant implications for biological processes such as endocytosis, and physical phenomena like phase separation in lipid bilayers.

Hypothermia elongates the contraction-relaxation cycle in explanted human failing heart decreasing the time for ventricular filling during diastole

Targeted temperature management is part of the standardized treatment for patients in cardiac arrest. Hypothermia decreases cerebral oxygen consumption and induces bradycardia; thus, increasing the heart rate may be considered to maintain cardiac output. We hypothesized that increasing heart rate during hypothermia would impair diastolic function. Human left ventricular trabeculae obtained from explanted hearts of patients with terminal heart failure were stimulated at 0.5 Hz, and contraction-relaxation cycles were recorded. Maximal developed force (Fmax), maximal rate of development of force [(dF/d t)max], time to peak force (TPF), time to 80% relaxation (TR80), and relaxation time (RT = TR80 − TPF) were measured at 37, 33, 31, and 29°C. At these temperatures, stimulation frequency was increased from 0.5 to 1.0 and to 1.5 Hz. At 1.5 Hz, concentration-response curves for the β-adrenergic receptor (β-AR) agonist isoproterenol were performed. Fmax, TPF, and RT increased when temperature was lowered, whereas (dF/d t)max decreased. At all temperatures, increasing stimulation frequency increased Fmax and (dF/d t)max, whereas TPF and RT decreased. At 31 and 29°C, resting tension increased at 1.5 Hz, which was ameliorated by β-AR stimulation. At all temperatures, maximal β-AR stimulation increased Fmax, (dF/d t)max, and maximal systolic force, whereas resting tension decreased progressively with lowering temperature. β-AR stimulation reduced TPF and RT to the same extent at all temperatures, despite the more elongated contraction-relaxation cycle at lower temperatures. Diastolic dysfunction during hypothermia results from an elongation of the contraction-relaxation cycle, which decreases the time for ventricular filling. Hypothermic bradycardia protects the heart from diastolic dysfunction and increasing the heart rate during hypothermia should be avoided. NEW & NOTEWORTHY Decreasing temperature increases the duration of the contraction-relaxation cycle in the human ventricular myocardium, significantly reducing the time for ventricular filling during diastole. During hypothermia, increasing heart rate further reduces the time for ventricular filling and in some situations increases resting tension further impairing diastolic function. Modest β-adrenergic receptor stimulation can ameliorate these potentially detrimental changes during diastole while improving contractile force generation during targeted temperature management.

V-Y Advancement Flap for Defects of the Lid–Cheek Junction

We report a series of 10 patients who underwent inferolaterally based V-Y advancement flaps for reconstruction of defects involving the lower eyelid and infraorbital cheek junction. Defects ranged from 1.7 to 2.9 cm in largest diameter, and patients ranged from 59 to 84 years of age. All patients had excellent functional and cosmetic outcomes without subsequent surgical or laser revision. There were no instances of flap necrosis, hematoma, or ectropion. Vertically oriented V-Y flaps are often underused in this setting largely due to the perceived increase risk of ectropion. We describe a modification of the flap with lateral orientation that both diminishes the downward tension vector, which threatens ectropion, and conceals incision scars within resting tension lines, providing superior functional and aesthetic outcomes. Our series demonstrates that a properly designed and well-executed inferiorly based V-Y advancement flap can be used as a safe reconstructive modality for defects involving the lid–cheek junction.

Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension

Piezo1 ion channels mediate the conversion of mechanical forces into electrical signals and are critical for responsiveness to touch in metazoans. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents evoked by membrane stretch in three patch configurations, while simultaneously visualizing and measuring membrane geometry. Building on this approach, we developed protocols to minimize resting membrane curvature and tension prior to probing Piezo1 activity. We find that Piezo1 responds to lateral membrane tension with exquisite sensitivity as compared to other mechanically activated channels and that resting tension can drive channel inactivation, thereby tuning overall mechanical sensitivity of Piezo1. Our results explain how Piezo1 can function efficiently and with adaptable sensitivity as a sensor of mechanical stimulation in diverse cellular contexts.

Heart failure with preserved ejection fraction: Defining the function of ROS and NO

The understanding of complex molecular mechanisms underlying heart failure (HF) is constantly under revision. Recent research has paid much attention to understanding the growing number of patients that exhibit HF symptoms yet have an ejection fraction similar to a normal phenotype. Termed heart failure with preserved ejection fraction (HFpEF), this novel hypothesis traces its roots to a proinflammatory state initiated in part by the existence of comorbidities that create a favorable environment for the production of reactive oxygen species (ROS). Triggering a cascade that involves reduced nitric oxide (NO) availability, elevated ROS levels in the coronary endothelium eventually contribute to hypertrophy and increased resting tension in cardiomyocytes. Improved understanding of the molecular pathways associated with HFpEF has led to studies that concentrate on reducing ROS production in the heart, boosting NO availability, and increasing exercise capacity for HFpEF patients. This review will explore the latest research into the role of ROS and NO in the progression of HFpEF, as well as discuss the encouraging results of numerous therapeutic studies.