Increase In Amplitude Of Contractions Research Articles

Abstract Or107: MyBP-HL nonsense mutations fail in sarcomere incorporation leads to modifications in myocyte contraction trough calcium modifications.

Introduction: Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation. AF is the most common cardiac arrhythmia and is associated with ischemic stroke, heart failure, and substantial morbidity and mortality. Recently, our group described a novel sarcomere component, myosin binding protein-H like (MyBP-HL), that is mainly expressed in the cardiac atria. MyBP-HL is composed of two immunoglobulin domains and one fibronectin domain, with homology to last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations prevent his incorporation into the sarcomere. C-MyBP-C truncated peptides are identified by cells as poisoning peptides, and they are rapidly degraded. cMyBP-C nonsense mutations are causative of hypertrophic cardiomyopathy (HCM). The MyBP-HL Arg255stop variant has been described in humans and has been linked to ventricular conduction system abnormalities, atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. The gnomAD database reports 9 nonsense mutations for MYBPHL in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: MYBPHL nonsense mutations fail to incorporate into the sarcomere will lead to contractile disfunction. Results: We demonstrated that MyBP-HL overexpression showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. Nevertheless, nonsense mutations disrupt normal sarcomere localization. MyBP-HL nonsense mutations promote an increase in contraction amplitude and slower relaxation kinetics. Also, we observed an increase in sarcomere length. Similarly, MyBP-HL nonsense mutations lead to increase in amplitude peak of calcium transients, and reduction in velocity to maximal calcium peak. In accordance with calcium modifications, we observed through mass spectrometry an increase in calpain 6 abundance promoted by MyBP-HL nonsense mutations. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament. The failure of MyBP-HL truncating peptides to incorporate into the sarcomere leads to a deficiencies in myocytes contraction patterns, changes in calcium patterns and increase in degradation proteins abundance, which are related to AF prevalence.

Loss of anoctamin 1 reveals a subtle role for BK channels in lymphatic muscle action potentials.

Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCMassay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Effects of ketone body 3-hydroxybutyrate on cardiac and mitochondrial function during donation after circulatory death heart transplantation

Normothermic regional perfusion (NRP) allows assessment of therapeutic interventions prior to donation after circulatory death transplantation. Sodium-3-hydroxybutyrate (3-OHB) increases cardiac output in heart failure patients and diminishes ischemia–reperfusion injury, presumably by improving mitochondrial metabolism. We investigated effects of 3-OHB on cardiac and mitochondrial function in transplanted hearts and in cardiac organoids. Donor pigs (n = 14) underwent circulatory death followed by NRP. Following static cold storage, hearts were transplanted into recipient pigs. 3-OHB or Ringer’s acetate infusions were initiated during NRP and after transplantation. We evaluated hemodynamics and mitochondrial function. 3-OHB mediated effects on contractility, relaxation, calcium, and conduction were tested in cardiac organoids from human pluripotent stem cells. Following NRP, 3-OHB increased cardiac output (P < 0.0001) by increasing stroke volume (P = 0.006), dP/dt (P = 0.02) and reducing arterial elastance (P = 0.02). Following transplantation, infusion of 3-OHB maintained mitochondrial respiration (P = 0.009) but caused inotropy-resistant vasoplegia that prevented weaning. In cardiac organoids, 3-OHB increased contraction amplitude (P = 0.002) and shortened contraction duration (P = 0.013) without affecting calcium handling or conduction velocity. 3-OHB had beneficial cardiac effects and may have a potential to secure cardiac function during heart transplantation. Further studies are needed to optimize administration practice in donors and recipients and to validate the effect on mitochondrial function.

IP3R1 underlies diastolic ANO1 activation and pressure-dependent chronotropy in lymphatic collecting vessels.

Pressure-dependent chronotropy of murine lymphatic collecting vessels relies on the activation of the Ca2+-activated chloride channel encoded by Anoctamin 1 (Ano1) in lymphatic muscle cells. Genetic ablation or pharmacological inhibition of ANO1 results in a significant reduction in basal contraction frequency and essentially complete loss of pressure-dependent frequency modulation by decreasing the rate of the diastolic depolarization phase of the ionic pacemaker in lymphatic muscle cells (LMCs). Oscillating Ca2+ release from sarcoendoplasmic reticulum Ca2+ channels has been hypothesized to drive ANO1 activity during diastole, but the source of Ca2+ for ANO1 activation in smooth muscle remains unclear. Here, we investigated the role of the inositol triphosphate receptor 1 (Itpr1; Ip3r1) in this process using pressure myography, Ca2+ imaging, and membrane potential recordings in LMCs of ex vivo pressurized inguinal-axillary lymphatic vessels from control or Myh11CreERT2;Ip3r1fl/fl (Ip3r1ismKO) mice. Ip3r1ismKO vessels had significant reductions in contraction frequency and tone but an increased contraction amplitude. Membrane potential recordings from LMCs of Ip3r1ismKO vessels revealed a depressed diastolic depolarization rate and an elongation of the plateau phase of the action potential (AP). Ca2+ imaging of LMCs using the genetically encoded Ca2+ sensor GCaMP6f demonstrated an elongation of the Ca2+ flash associated with an AP-driven contraction. Critically, diastolic subcellular Ca2+ transients were absent in LMCs of Ip3r1ismKO mice, demonstrating the necessity of IP3R1 activity in controlling ANO1-mediated diastolic depolarization. These findings indicate a critical role for IP3R1 in lymphatic vessel pressure-dependent chronotropy and contractile regulation.

PD23-01 EFFECT OF OPIOID RECEPTOR ANTAGONIST ON BLADDER UNDERACTIVITY INDUCED BY PROLONGED PUDENDAL AFFERENT STIMULATION

You have accessJournal of UrologyCME1 Apr 2023PD23-01 EFFECT OF OPIOID RECEPTOR ANTAGONIST ON BLADDER UNDERACTIVITY INDUCED BY PROLONGED PUDENDAL AFFERENT STIMULATION Michael Pintauro, Jianan Jian, Jicheng Wang, Bing Shen, Zhijun Shen, Khari Goosby, Joseph Scolieri, Avanish Madhavaram, Jonathan Beckel, William De Groat, Christopher Chermansky, and Changfeng Tai Michael PintauroMichael Pintauro More articles by this author , Jianan JianJianan Jian More articles by this author , Jicheng WangJicheng Wang More articles by this author , Bing ShenBing Shen More articles by this author , Zhijun ShenZhijun Shen More articles by this author , Khari GoosbyKhari Goosby More articles by this author , Joseph ScolieriJoseph Scolieri More articles by this author , Avanish MadhavaramAvanish Madhavaram More articles by this author , Jonathan BeckelJonathan Beckel More articles by this author , William De GroatWilliam De Groat More articles by this author , Christopher ChermanskyChristopher Chermansky More articles by this author , and Changfeng TaiChangfeng Tai More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003296.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Fowler Syndrome, or non-obstructive urinary retention, is seen in young female patients. Although the cause is unknown, it is theorized that aberrant sensory nerves from the urethral sphincter cause inhibition of the central nervous system, thereby leading to urinary retention. Prior studies have suggested that the opioid receptor may be implicated in bladder overactivity and underactivity. We aimed to evaluate opioid receptor inhibition on bladder function in a feline model of Fowler’s Syndrome. METHODS: A total of 10 cats were anesthetized, ureters externalized via laparotomy, and urethral catheter placed. A tripolar cuff electrode was placed on the pudendal nerve. Multiple cystometrograms (CMGs) were performed to determine baseline bladder function (Control). Multiple pudendal nerve stimulations (PNS) were performed at 30-minute cycles (5 Hz, 0.2 ms) were applied at 4-6 times the intensity required to elicit anal contraction for 2-5 hours. The result was a stable, underactive bladder state (Post-PNS 1). Next, naloxone (1mg/kg IV) was administered, and CMGs obtained (Post-NX). A single 30-minute PNS was then performed to evaluate the combined effect of naloxone and PNS (Post-PNS 2). Lastly, a CMG was performed to examine any recovery (Post-Control). Primary outcomes were bladder capacity and contraction amplitude (contractility). RESULTS: Application of PNS produced a significant increase in bladder capacity and decrease in bladder amplitude, indicative of a state of bladder underactivity (Figure 1, * denotes significance p<0.05). Administration of naloxone significantly reduced the bladder capacity and increased contraction amplitude. Re-application of PNS led fully overcame the naloxone effect on capacity; however, the amplitude is a combined effect. This effect of re-stimulation lasted only for a short period (15-20 minutes) and Post-Control demonstrate a reduction in capacity indicating effects of the still present naloxone overcoming the PNS. CONCLUSIONS: Pharmacologic opioid receptor antagonism increased bladder contractility and reduced bladder capacity to baseline in our feline model of non-obstructive urinary retention. This is a new target for possible pharmacotherapy in the treatment of patients with Fowler's Syndrome. Source of Funding: 5R01DK121698-02 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e670 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Pintauro More articles by this author Jianan Jian More articles by this author Jicheng Wang More articles by this author Bing Shen More articles by this author Zhijun Shen More articles by this author Khari Goosby More articles by this author Joseph Scolieri More articles by this author Avanish Madhavaram More articles by this author Jonathan Beckel More articles by this author William De Groat More articles by this author Christopher Chermansky More articles by this author Changfeng Tai More articles by this author Expand All Advertisement PDF downloadLoading ...

Role of adenosine triphosphate and protein kinase A in the force-frequency relationship in isolated rat cardiomyocytes

The physiological heart rate of rodents is around 4-6 Hz, although a stimulus frequency of 1 Hz is generally used in isolated cardiomyocytes to study changes in the contraction-relaxation cycle in cardiac muscle physiology and pathophysiology. Our study investigated the contraction parameters in isolated cardiomyocytes at 1, 2 and 4 Hz stimulation, and the roles of ATP and protein kinase A (PKA) in the force-frequency relationship in isolated cardiomyocytes. The contraction of the cell and intracellular Ca2+ changes were recorded simultaneously during cell stimulation by applying pulses of 6-8 V amplitude with frequencies of 1, 2 and 4 Hz. The increase in stimulus frequency caused a significant decrease in the percentage of shortening, relaxation times, slowing of the relaxation rate, and a significant increase in diastolic Ca2+ levels, but had no effect on the contraction rate and Ca2+ transients. Administration of ATP and N6-benzoyladenosine-3'-5'-cyclic monophosphate (6-BNZ-cAMP) caused an increase in contraction amplitude and speed which were proportional to the stimulus frequency but had no effect on the relaxation times. The experimental results show that the force-stimulus frequency has a negative correlation in isolated myocytes and that energy metabolism and the ?-adrenergic system may be responsible for this relationship.

Involvement of gap junctions in the increased contraction of bovine oviducts at pre-ovulation observed in vitro.

Spontaneous contraction of oviductal smooth muscle is essential for gamete transport to the fertilization site in mammals. This study sheds light on the mechanism of elevated contraction amplitude in the bovine oviductal isthmus just before ovulation. Rhythmic contraction of the oviducts is essential for transporting gametes and embryos at peri-ovulation; however, its regulatory mechanism during the estrous cycle is unclear. Meanwhile, it is reported that ion currents regulate muscle contraction. Our study aimed to clarify the involvement of ion channels and gap junctions in regulating oviductal motility during the estrous cycle in cattle. The isthmic sections of bovine oviducts collected just after ovulation (0-4 days after ovulation), at the mid-late luteal stage (10-17 days), and at the follicular stage (1-3 days before ovulation) were used in the experiments. The frequency and amplitude of contraction of the oviductal strips in the longitudinal direction were examined using the Magnus system. The frequency was not different among the estrous stages. Conversely, the amplitude was significantly higher at the follicular stage. The blockers of voltage-dependent calcium channels, both IP3 receptor and ryanodine receptors, chloride channel, and gap junction reduced the amplitude. Additionally, mRNA and protein expression of GJA1, a component of the gap junction, in the smooth muscle tissues of the oviductal isthmus were significantly higher in the follicular stage. In addition, estradiol-17β (E2; 1.0 ng/mL) significantly increased GJA1 mRNA expression in cultured smooth muscle tissues after 24 h and GJA1 protein expression in cultured smooth muscle cells after 48 h. These results suggest that local levels of E2 in the oviductal isthmus ipsilateral to an ovary with a dominant follicle support the increased contraction amplitude of bovine ipsilateral oviducts by elevating the gap junction expression.

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models.

Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical 'doses' using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

P-333 The influence of hormonal stimulation on uterine peristalsis measured by ultrasound speckle tracking in women with IVF/ICSI treatment compared to normal ovulating women

Abstract Study question To investigate possible effects of ovarian stimulation during IVF/ICSI on uterine peristalsis features using ultrasound speckle tracking compared to normal ovulating women. Summary answer Patients with a normal uterus treated with exogenous hormones during IVF/ICSI treatment show significantly increased contraction amplitude and power parameters compared to normal ovulating women. What is known already Uterine peristalsis plays a role in procreation. Subjective visual inspection of uterine peristalsis is extensively researched throughout the menstrual cycle. Studies on uterine peristalsis in IVF/ICSI patients show higher frequency of uterine contractions during the periovulatory phase. Recently, a new automated quantitative method was validated to objectively analyse uterine strain using speckle tracking. Besides objective insight in frequency, also information on amplitude, power and coordination of motion is available. The effect of exogenous hormones on uterine contraction features has not objectively been studied using this new method. Study design, size, duration This multicentre prospective observational cohort study was performed between 2020 and 2022 in the Catharina Hospital in Eindhoven, the Netherlands, Embryolab Fertility Centre in Thessaloniki, Greece and University of Naples, Federico II in Naples, Italy. 30 normal ovulating patients served as controls. The primary outcomes were the contraction features frequency, amplitude, direction, velocity, power and coordination in women undergoing IVF/ICSI on the day of follicle aspiration compared to controls on cycle day 9 – 14. Participants/materials, setting, methods 19 women undergoing IVF/ICSI were included regardless of the treatment cycle number and stimulation protocol. Indication for IVF/ICSI treatment was male subfertility (n = 8), idiopathic subfertility (n = 5), tubal factor (n = 2), primary ovarian insufficiency (n = 2) or combined (n = 2), all women had normal uteri on transvaginal ultrasound. The control group consisted of 30 healthy pre-menopausal women with normal uteri on ultrasound and spontaneous regular cycles. Main results and the role of chance Each patient underwent a 2.5 – 4 minute transvaginal ultrasound of the uterus in mid-sagittal plane. The various contraction features were extracted using a quantitative dedicated speckle tracking algorithm analysis studying motion of the subendometrial junctional zone. Mean age of IVF/ICSI patients was 32.7 years and mean BMI was 25.6. Mean age of healthy controls was 33.8 years, BMI was 23.4, and cycle length was 27.44 days. Characteristics of IVF/ICSI patients and healthy volunteers showed no statistically significant differences. Both groups predominantly consisted of nulliparous women. In IVF/ICSI patients the contraction amplitude was significantly higher compared to controls (0.062 vs 0.040; p &amp;lt; 0.001). The contraction power (combination of amplitude and frequency) appeared to be significantly higher in IVF/ICSI patients compared to controls (1528.18 vs 683.44; p &amp;lt; 0.001). No significant differences were found in contraction frequency and coordination. Even though the mean coordination did not show statistically significant differences, the values were less scattered in IVF/ICSI patients, with a much smaller range of coordination values (1.04 – 3.06) compared to healthy volunteers (1.02 – 50.33). Limitations, reasons for caution The reported difference between IVF/ICSI patients and controls in contraction amplitude and power may not solely be attributed to the administration of exogenous hormones. The difference may also be due to an underlying cause for infertility in these patients. Another limitation of this study is the small sample size. Wider implications of the findings Future research should explore which uterine contraction profile is associated with viable pregnancy: during the natural menstrual cycle, or when treated with hormones. The question arises whether it is preferred to transfer the embryo in the stimulated cycle or implement a freeze-all policy and transfer during the natural menstrual cycle. Trial registration number NCT02310802

Acute effects of cardiac contractility modulation on human induced pluripotent stem cell-derived cardiomyocytes.

Cardiac contractility modulation (CCM) is an intracardiac therapy whereby nonexcitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. We previously evaluated the effects of CCM in isolated adult rabbit ventricular cardiomyocytes and found a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) to develop a robust model to evaluate CCM in vitro. HiPSC‐CMs (iCell Cardiomyocytes2, Fujifilm Cellular Dynamic, Inc.) were studied in monolayer format plated on flexible substrate. Contractility, calcium handling, and electrophysiology were evaluated by fluorescence‐ and video‐based analysis (CellOPTIQ, Clyde Biosciences). CCM pulses were applied using an A‐M Systems 4100 pulse generator. Robust hiPSC‐CMs response was observed at 14 V/cm (64 mA) for pacing and 28 V/cm (128 mA, phase amplitude) for CCM. Under these conditions, hiPSC‐CMs displayed enhanced contractile properties including increased contraction amplitude and faster contraction kinetics. Likewise, calcium transient amplitude increased, and calcium kinetics were faster. Furthermore, electrophysiological properties were altered resulting in shortened action potential duration (APD). The observed effects subsided when the CCM stimulation was stopped. CCM‐induced increase in hiPSC‐CMs contractility was significantly more pronounced when extracellular calcium concentration was lowered from 2 mM to 0.5 mM. This study provides a comprehensive characterization of CCM effects on hiPSC‐CMs. These data represent the first study of CCM in hiPSC‐CMs and provide an in vitro model to assess physiologically relevant mechanisms and evaluate safety and effectiveness of future cardiac electrophysiology medical devices.

Ouabain worsens diastolic sarcomere length in myocytes from a cardiomyopathy mouse model

Diastolic dysfunction is a major feature of hypertrophic cardiomyopathy (HCM). Data from patient tissue and animal models associate increased Ca2+ sensitivity of myofilaments with altered Na+ and Ca2+ ion homeostasis in cardiomyocytes with diastolic dysfunction. In this study, we tested the acute effects of ouabain on ventricular myocytes of an HCM mouse model. The effects of ouabain on contractility and Ca2+ transients were tested in intact adult mouse ventricular myocytes (AMVMs) of Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Concentration-response assessment of contractile function revealed low sensitivity of AMVMs to ouabain (10 μM) compared to literature data on human cardiomyocytes (100 nM). Three hundred μM ouabain increased contraction amplitude (WT ~1.8-fold; KI ~1.5-fold) and diastolic intracellular Ca2+ in both WT and KI (+12–18%), but further decreased diastolic sarcomere length in KI cardiomyocytes (−5%). Western Blot analysis of whole heart protein extracts revealed 50% lower amounts of Na+/K+ ATPase (NKA) in KI than in WT. Ouabain worsened the diastolic phenotype of KI cardiomyocytes at concentrations which did not impair WT diastolic function. Ouabain led to an elevation of intracellular Ca2+, which was poorly tolerated in KI showing already high cytosolic Ca2+ at baseline due to increased myofilament Ca2+ sensitivity. Lower amounts of NKA in KI could amplify the need to exchange excessive intracellular Na+ for Ca2+ and thereby explain the general tendency to higher diastolic Ca2+ in KI.

ON THE MILK EJECTION MECHANISM IN COWS UPON AN INCREASE IN ONE-TIME MILK YIELD

Sympathetic nervous system regulates milk flow through the udder duct system. However, the mechanisms of contraction of the alveoli upon accumulation of different amounts of milk in the udder have not been studied. In the paper, it is found for the first time that the excretion of an increased amount of milk is accompanied by a change in the parameters of the udder blood supply caused by an increase in the contractile response of the alveolar complex. Thus, the goal of the paper is to investigate the contractile reaction of the alveolar complex and lactation in cows with an increased single-milking yield. Nine black-and-white dairy cows of the 2-5th calving were tested, 7 times each, in the first half of lactation (All-Russian Scientific Research Institute of Physiology, Biochemistry and Nutrition of Farm Animals, Kaluga Province). A serial ADU-1 milking machine and a counter sensor (Latvia) were used to record milking parameters. The single-milking yield ranged from 3.4 to 6.7 kg in the control period and was 24.5 % higher (p < 0.001) in the test period. The volumetric blood flow velocity (VBFV) in the udder was assessed by the electromagnetic flowmetry method. A blood flow sensor (Nihon Kohden, Japan) was fixed on the external pudendal artery of the udder. The average VBFV values were recorded for 3 minutes prior to milking (initial period) and in milking. The points characterizing udder blood supply were marked on the VBFV curve, i.e., the beginning of udder irritation, a sharp increase in the blood supply, its maximum and the baseline restoration. The time intervals until a sharp increase in VBFV and an increased blood supply period were calculated. The average and maximum VBFV values and an increase in volumetric blood flow velocity during milking in relation to the initial indicators were determined. The parameters of the udder blood supply were used to assess the contractile response of the alveolar complex. The latent period until milk secretion was determined based on the time from the beginning of udder irritation to a sharp increase in udder blood supply. It was established that the single-milking yield affects the parameters of milking and the udder blood supply. The increased single-milking yield led to a shorter period for removal of the first portion of milk (p < 0.001), an increase in the average intensity (p < 0.001) and maximum intensity (p < 0.001) of milk removal as well as an increase in the machine-milking period (p <0.001). The increased milk secretion was also accompanied by an increase in the udder blood supply. I.e., the period before the udder VBFV increased sharply shortened (p < 0.05), while the average (p < 0.05) and maximum (p < 0.05) VBFV values during milking, as well as period of intensive blood supply to the udder (p < 0.001) increased. The increased blood flow during the test period resulted from an increased contractile response of the myoepithelium and alveoli. The intensive contraction of the alveoli, with the increased contraction amplitude and duration, leads to a shorter latent period. The milk let-down and secretion are under regulation of the sympathetic nervous system. Probably, the excitation of the udder sympathetic nervous system depends on the amount of accumulated milk. The udder tone during milk ejection determines both the alveoli contraction and the rate of milk movement through the milk ducts and its removal through the nipple. The increased udder tone when low filling with milk resulted in a delayed let-down, delayed first portion of milk, low contractile response of the alveoli, and low milk removal. With an increase in the amount of milk in the udder, its tone decreases, resulting in a shorter latent period and faster removal of the first portion of milk, strengthening the contractile response of the alveoli and increasing the intensity of milk removal. Therefore, the latent period of secretion of the first portion of milk can be proposed to characterize the tone of the udder sympathetic nervous system.

О МЕХАНИЗМЕ МОЛОКООТДАЧИ У КОРОВ ПРИ ПОВЫШЕНИИ РАЗОВОГО УДОЯ

Sympathetic nervous system regulates milk flow through the udder duct system. However, the mechanisms of contraction of the alveoli upon accumulation of different amounts of milk in the udder have not been studied. In the paper, it is found for the first time that the excretion of an increased amount of milk is accompanied by a change in the parameters of the udder blood supply caused by an increase in the contractile response of the alveolar complex. Thus, the goal of the paper is to investigate the contractile reaction of the alveolar complex and lactation in cows with an increased single-milking yield. Nine black-and-white dairy cows of the 2-5th calving were tested, 7 times each, in the first half of lactation (All-Russian Scientific Research Institute of Physiology, Biochemistry and Nutrition of Farm Animals, Kaluga Province). A serial ADU-1 milking machine and a counter sensor (Latvia) were used to record milking parameters. The single-milking yield ranged from 3.4 to 6.7 kg in the control period and was 24.5 % higher (p < 0.001) in the test period. The volumetric blood flow velocity (VBFV) in the udder was assessed by the electromagnetic flowmetry method. A blood flow sensor (Nihon Kohden, Japan) was fixed on the external pudendal artery of the udder. The average VBFV values were recorded for 3 minutes prior to milking (initial period) and in milking. The points characterizing udder blood supply were marked on the VBFV curve, i.e., the beginning of udder irritation, a sharp increase in the blood supply, its maximum and the baseline restoration. The time intervals until a sharp increase in VBFV and an increased blood supply period were calculated. The average and maximum VBFV values and an increase in volumetric blood flow velocity during milking in relation to the initial indicators were determined. The parameters of the udder blood supply were used to assess the contractile response of the alveolar complex. The latent period until milk secretion was determined based on the time from the beginning of udder irritation to a sharp increase in udder blood supply. It was established that the single-milking yield affects the parameters of milking and the udder blood supply. The increased single-milking yield led to a shorter period for removal of the first portion of milk (p < 0.001), an increase in the average intensity (p < 0.001) and maximum intensity (p < 0.001) of milk removal as well as an increase in the machine-milking period (p <0.001). The increased milk secretion was also accompanied by an increase in the udder blood supply. I.e., the period before the udder VBFV increased sharply shortened (p < 0.05), while the average (p < 0.05) and maximum (p < 0.05) VBFV values during milking, as well as period of intensive blood supply to the udder (p < 0.001) increased. The increased blood flow during the test period resulted from an increased contractile response of the myoepithelium and alveoli. The intensive contraction of the alveoli, with the increased contraction amplitude and duration, leads to a shorter latent period. The milk let-down and secretion are under regulation of the sympathetic nervous system. Probably, the excitation of the udder sympathetic nervous system depends on the amount of accumulated milk. The udder tone during milk ejection determines both the alveoli contraction and the rate of milk movement through the milk ducts and its removal through the nipple. The increased udder tone when low filling with milk resulted in a delayed let-down, delayed first portion of milk, low contractile response of the alveoli, and low milk removal. With an increase in the amount of milk in the udder, its tone decreases, resulting in a shorter latent period and faster removal of the first portion of milk, strengthening the contractile response of the alveoli and increasing the intensity of milk removal. Therefore, the latent period of secretion of the first portion of milk can be proposed to characterize the tone of the udder sympathetic nervous system.

Superficial peroneal neuromodulation of persistent bladder underactivity induced by prolonged pudendal afferent nerve stimulation in cats.

The purpose of this study is to determine whether superficial peroneal nerve stimulation (SPNS) can reverse persistent bladder underactivity induced by prolonged pudendal nerve stimulation (PNS). In 16 α-chloralose-anesthetized cats, PNS and SPNS were applied by nerve cuff electrodes. Skin surface electrodes were also used for SPNS. Bladder underactivity consisting of a significant increase in bladder capacity to 157.8 ± 10.9% of control and a significant reduction in bladder contraction amplitude to 56.0 ± 5.0% of control was induced by repetitive (4-16 times) application of 30-min PNS. SPNS (1 Hz, 0.2 ms) at 1.5-2 times threshold intensity (T) for inducing posterior thigh muscle contractions was applied either continuously (SPNSc) or intermittently (SPNSi) during a cystometrogram (CMG) to determine whether the stimulation can reverse the PNS-induced bladder underactivity. SPNSc or SPNSi applied by nerve cuff electrodes during the prolonged PNS inhibition significantly reduced bladder capacity to 124.4 ± 10.7% and 132.4 ± 14.2% of control, respectively, and increased contraction amplitude to 85.3 ± 6.2% and 75.8 ± 4.7%, respectively. Transcutaneous SPNSc and SPNSi also significantly reduced bladder capacity and increased contraction amplitude. Additional PNS applied during the bladder underactivity further increased bladder capacity, whereas SPNSc applied simultaneously with the PNS reversed the increase in bladder capacity. This study indicates that a noninvasive superficial peroneal neuromodulation therapy might be developed to treat bladder underactivity caused by abnormal pudendal nerve somatic afferent activation that is hypothesized to occur in patients with Fowler's syndrome.

Experimental faecal peritonitis alters response to 5-hydroxytryptamine in mesenteric lymphatic vessels

Aim. To compare the reactions to 5-hydroxytryptamine (5-HT, serotonin) in the mesenteric lymphangions upon endothelial denudation and during experimental faecal peritonitis as compared with the control lymphangions. Materials and Methods. Experiments were performed on the intestinal lymph trunk lymphangions of rats using a pressure myograph system. We examined the changes in contraction frequency and amplitude as well as tonic reactions upon the addition of 5-HT (10-8-10-4 M). Experimental peritonitis was caused by an intraperitoneal injection of feces. Results. Faecal peritonitis reduced contraction frequency and amplitude in mesenteric lymphangions. 5-HT increased contraction amplitude only at low concentration (10-8 M) and did not alter the tonic reactions. Upon endothelial denudation, serotonin inhibited contraction frequency and amplitude in the lymphangions. As 5-HT stimulates motility through 5-НТ2 receptors and α2-adrenoceptors on endothelial cells, faecal peritonitis abates the sensitivity of such receptors to 5-HT. Conclusion. In experimental faecal peritonitis, alterations in lymphatic vessels are reminiscent of those in blood vessels. Endothelial dysfunction disturbs the response of lymphatic vessels to 5-HT.

Abstract 334: Acute Effects of Nonexcitatory Electrical Stimulation During Systole in Human-induced Pluripotent Stem Cell Derived Cardiomyocytes

Cardiac contractility modulation (CCM) is a cardiac therapy whereby non-excitatory electrical simulations are delivered during the absolute refractory period of the cardiac cycle. CCM is indicated for patients with heart failure (Class III/IV) and reduced ejection fraction. We previously evaluated the effects of CCM and found isolated adult rabbit cardiomyocytes (rabbit-CMs) display a transient increase in calcium and contractility. In the present study, we sought to extend these results to human cardiomyocytes using human stem-cell cardiomyocytes (hiPSC-CMs). hiPSC-CMs were studied in both monolayer and isolated cells and results compared to that of rabbit-CMs. Contractility, calcium handling and electrophysiology was evaluated by image and fluorescence analysis. CCM was applied with a custom device covering a range of pulse parameters including standard clinical parameters (Figure 1). Pacing threshold was determined and stimulation performed at twice the diastolic capture threshold. Robust contractile response was observed at 16 V/cm, 70 mA for pacing and 28 V/cm, 120 mA for CCM. Under these conditions hiPSC-CMs and rabbit-CMs displayed increased contraction amplitude of 30 % (1.79 a.u., CCM vs 1.38 a.u., Control) and 22 % (3.67 a.u., CCM vs 3.00 a.u., Control) respectively and faster kinetics that subsided when the CCM signal was eliminated. This study provides a comprehensive characterization of the effects of CCM on hiPSC-CMs and rabbit-CMs in parallel under identical experimental conditions. These data provide the first study of CCM in hiPSC-CMs and demonstrates the potential utility of hiPSC-CMs to evaluate safety and effectiveness of cardiac medical devices.

Early Inhibition of Retinoic Acid Signaling Rapidly Generates Cardiomyocytes Expressing Ventricular Markers from Human Induced Pluripotent Stem Cells

Current protocols for the differentiation of cardiomyocytes from human induced pluripotent stem cells (iPSCs) generally require prolonged time in culture and result in heterogeneous cellular populations. We present a method for the generation of beating cardiomyocytes expressing specific ventricular markers after just 14 days. Addition of the pan-retinoic acid receptor inverse agonist BMS 493 to human iPSCs for the first 8 days of differentiation resulted in increased protein expression of the ventricular isoform of myosin regulatory light chain (MLC2V) from 18.7% ± 1.72% to 55.8% ± 11.4% (p <0.0001) in cells co-expressing the cardiac muscle protein troponin T (TNNT2). Increased MLC2V expression was also accompanied by a slower beating rate (49.4 ± 1.53 vs. 93.0 ± 2.81 beats per minute, p <0.0001) and increased contraction amplitude (201% ± 8.33% vs. 100% ± 10.85%, p <0.0001) compared to untreated cells. Improved directed differentiation will improve in vitro cardiac modeling.

Easy Applied Gelatin-Based Hydrogel System for Long-Term Functional Cardiomyocyte Culture and Myocardium Formation.

Harnessing biomaterials for in vitro tissue construction has long been a research focus because of its powerful potentials in tissue engineering and pharmaceutical industry. Myocardium is a critical cardiac tissue with complex multiple muscular layers. Considering the specific characters of native cardiac tissues, it is necessary to design a biocompatible and biomimetic platform for cardiomyocyte culture and myocardium formation with sustained physiological function. In this study, we developed gelatin-based hydrogels chemically cross-linked by genipin, a biocompatible cross-linker, as cell culture scaffolds. Moreover, to achieve and maintain the functionality of myocardium, for instance, well-organized cardiomyocytes and synchronized contractile behavior, we fabricated gelatin-based hydrogels with patterned microstructure using a microcontact printing technique. Furthermore, graphene oxide (GO), with unprecedented physical and chemical properties, has also been incorporated into gelatin for culturing cardiomyocytes. Our results show that micropatterned genipin-cross-linked gelatin hydrogels are very helpful to promote alignment and maturation of neonatal rat ventricular cardiomyocytes. More interestingly, the presence of GO significantly enhances the functional performance of cardiomyocytes, including an increase in contraction amplitude and cardiac gene expression. The cultured cardiomyocytes reach a well-synchronized contraction within 48 h of cell seeding and keep beating for up to 3 months. Our study provides a new and easy-to-use gelatin-based scaffold for improving physiological function of engineered cardiac tissues, exhibiting promising applications in cardiac tissue engineering and drug screening.

Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐derived cardiomyocytes in serum‐free medium

Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemically defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibroblasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tissue displayed the typical fiber morphology with massive striation. An increase in contraction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemically defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro.