Changes In Contractility Research Articles

Hyperpolarized Metabolic and Parametric CMR Imaging of Longitudinal Metabolic-Structural Changes in Experimental Chronic Infarction

BackgroundProlonged ischemia and myocardial infarction are followed by a series of dynamic processes that determine the fate of the affected myocardium toward recovery or necrosis. Metabolic adaptions are considered to play a vital role in the recovery of salvageable myocardium in the context of stunned and hibernating myocardium. ObjectivesThe potential of hyperpolarized pyruvate cardiac magnetic resonance (CMR) alongside functional and parametric CMR as a tool to study the complex metabolic-structural interplay in a longitudinal study of chronic myocardial infarction in an experimental pig model is investigated. MethodsMetabolic imaging using hyperpolarized [1-13C] pyruvate and proton-based CMR including cine, T1/T2 relaxometry, dynamic contrast-enhanced, and late gadolinium enhanced imaging were performed on clinical 3.0-T and 1.5-T MR systems before infarction and at 6 days and 5 and 9 weeks postinfarction in a longitudinal study design. Chronic myocardial infarction in pigs was induced using catheter-based occlusion and compared with healthy controls. ResultsMetabolic image data revealed temporarily elevated lactate-to-bicarbonate ratios at day 6 in the infarcted relative to remote myocardium. The temporal changes of lactate-to-bicarbonate ratios were found to correlate with changes in T2 and impaired local contractility. Assessment of pyruvate dehydrogenase flux via the hyperpolarized [13C] bicarbonate signal revealed recovery of aerobic cellular respiration in the hibernating myocardium, which correlated with recovery of local radial strain. ConclusionsThis study demonstrates the potential of hyperpolarized CMR to longitudinally detect metabolic changes after cardiac infarction over days to weeks. Viable myocardium in the area at risk was identified based on restored pyruvate dehydrogenase flux.

Cell mechanical responses to subcellular perturbations generated by ultrasound and targeted microbubbles

The inherently dynamic and anisotropic microenvironment of cells imposes not only global and slow physical stimulations on cells but also acute and local perturbations. However, cell mechanical responses to transient subcellular physical signals remain unclear. In this study, acoustically activated targeted microbubbles were used to exert mechanical perturbations to single cells. The cellular contractile force was sensed by elastic micropillar arrays, while the pillar deformations were imaged using brightfield high-speed video microscopy at a frame rate of 1k frames per second for the first 10s and then confocal fluorescence microscopy. Cell mechanical responses are accompanied by cell membrane integrity changes. Both processes are determined by the perturbation strength generated by microbubble volumetric oscillations. The instantaneous cellular traction force relaxation exhibits two distinct patterns, correlated with two cell fates (survival or permanent damage). The mathematical modeling unveils that force-induced actomyosin disassembly leads to gradual traction force relaxation in the first few seconds. The perturbation may also influence the far end subcellular regions from the microbubbles and may propagate into connected cells with attenuations and delays. This study carefully characterizes the cell mechanical responses to local perturbations induced by ultrasound and microbubbles, advancing our understanding of the fundamentals of cell mechano-sensing, -responsiveness, and -transduction. Statement of significanceSubcellular physical perturbations commonly exist but haven't been fully explored yet. The subcellular perturbation generated by ultrasound and targeted microbubbles covers a wide range of strength, from mild, intermediate to intense, providing a broad biomedical relevance. With µm2 spatial sensing ability and up to 1ms temporal resolution, we present spatiotemporal details of the instantaneous cellular contractile force changes followed by attenuated and delayed global responses. The correlation between the cell mechanical responses and cell fates highlights the important role of the instantaneous mechanical responses in the entire cellular reactive processes. Supported by mathematical modeling, our work provides new insights into the dynamics and mechanisms of cell mechanics.

Intermittent exposure to chlorpyrifos results in cardiac hypertrophy and oxidative stress in rats

Forbidden in some countries due to its proven toxicity to humans, chlorpyrifos (CPF) still stands as an organophosphate pesticide (OP) highly used worldwide. Cardiotoxicity assessment is an unmet need in pesticide regulation and should be deeply studied through different approaches to better inform and generate an appropriate regulatory response to OP use. In the present study, we used our 4-week intermittent OP exposure model in rats to address the CPF effects on cardiac morphology allied with cardiovascular functional and biomolecular evaluation. Rats were intermittently treated with CPF at doses of 7 mg/kg and 10 mg/kg or saline (i.p.) and assessed for cardiac morphology (cardiomyocyte diameter and collagen content), cardiopulmonary Bezold-Jarisch reflex (BJR) function, cardiac autonomic tone, left ventricle (LV) contractility, cardiac expression of NADPH oxidase (Nox2), catalase (CAT), superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2) and cardiac levels of advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). Plasma butyrylcholinesterase (BuChE) and brainstem acetylcholinesterase (AChE) were also measured. Intermittent exposure to CPF induced cardiac hypertrophy, increasing cardiomyocyte diameter and collagen content. An impairment of cardioinhibitory BJR responses and an increase in cardiac vagal tone were also observed in CPF-treated animals without changes in LV contractility. CPF exposure increased cardiac Nox-2, CAT, SOD1, and TBARS levels and inhibited plasma BuChE and brainstem AChE activities. Our data showed that intermittent exposure to CPF induces cardiac hypertrophy together with cardiovascular reflex impairment, imbalance of autonomic tone and oxidative stress, which may bring significant cardiovascular risk to individuals exposed to OP compounds seasonally.

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform. The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling. The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

Helicobacter pylori-infected human neutrophils exhibit impaired chemotaxis and a uropod retraction defect.

Helicobacter pylori is a major human pathogen that colonizes the gastric mucosa and plays a causative role in development of peptic ulcers and gastric cancer. Neutrophils are heavily infected with this organism in vivo and play a prominent role in tissue destruction and disease. Recently, we demonstrated that H. pylori exploits neutrophil plasticity as part of its virulence strategy eliciting N1-like subtype differentiation that is notable for profound nuclear hypersegmentation. We undertook this study to test the hypothesis that hypersegmentation may enhance neutrophil migratory capacity. However, EZ-TAXIScan™ video imaging revealed a previously unappreciated and progressive chemotaxis defect that was apparent prior to hypersegmentation onset. Cell speed and directionality were significantly impaired to fMLF as well as C5a and IL-8. Infected cells oriented normally in chemotactic gradients, but speed and direction were impaired because of a uropod retraction defect that led to cell elongation, nuclear lobe trapping in the contracted rear and progressive narrowing of the leading edge. In contrast, chemotactic receptor abundance, adhesion, phagocytosis and other aspects of cell function were unchanged. At the molecular level, H. pylori phenocopied the effects of Blebbistatin as indicated by aberrant accumulation of F-actin and actin spikes at the uropod together with enhanced ROCKII-mediated phosphorylation of myosin IIA regulatory light chains at S19. At the same time, RhoA and ROCKII disappeared from the cell rear and accumulated at the leading edge whereas myosin IIA was enriched at both cell poles. These data suggest that H. pylori inhibits the dynamic changes in myosin IIA contractility and front-to-back polarity that are essential for chemotaxis. Taken together, our data advance understanding of PMN plasticity and H. pylori pathogenesis.

Contractility assessment of human iPSC-derived cardiomyocytes by using a motion vector system and measuring cell impedance

Predicting drug-induced cardiotoxicity during the non-clinical stage is important to avoid severe consequences in the clinical trials of new drugs. Human iPSC-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac safety assessments in drug development. To date, multi-electrode array system (MEA) has been a widely used as a tool for the assessment of proarrhythmic risk with hiPSC-CMs. Recently, new methodologies have been proposed to assess in vitro contractility, such as the force and velocity of cell contraction, using hiPSC-CMs. Herein, we focused on an imaging-based motion vector system (MV) and an electric cell-substrate impedance sensing system (IMP). We compared the output signals of hiPSC-CMs from MV and IMP in detail and observed a clear correlation between the parameters. In addition, we assessed the effects of isoproterenol and verapamil on hiPSC-CM contraction and identified a correlation in the contractile change of parameters obtained with MV and IMP. These results suggest that both assay systems could be used to monitor hiPSC-CM contraction dynamics.

The Cardiac Power Index during Abdominal Open Aortic Surgery: Intraoperative Insights into the Cardiac Performance-A Retrospective Observational Analysis.

Background: The Cardiac Power Index (CPI) measures the rate of energy output generated by the heart and correlates this with in-hospital mortality due to cardiogenic shock. In open aortic surgery, both aortic clamping and unclamping expose the heart to abrupt variations of the left ventricle afterload, preload, and contractility, with possible hemodynamic impairment. We investigated how aortic-cross clamping (Ao-XC) and unclamping (Ao-UC) procedures affect the CPI during open aortic surgery. Methods: We retrospectively analyzed our surgical database of 67 patients submitted to open surgical aortic repair at Humanitas Research Hospital, Milan. Patients were monitored by an EV1000-FloTrac SystemTM (Edwards Lifescience, Irvine, CA, USA) beyond the standard intra-operative hemodynamic monitoring. The primary outcome was the variation of basal CPI after aortic clamping and unclamping. Secondary outcomes were variations of the cardiac index (CI), mean arterial pressure (MAP), heart rate, and lactate during aortic clamping and after unclamping. The CPI was computed as: (CI × MAP)/451. Results: The CPI changed significantly after aortic unclamping. CPI: basal = 0.39 ± 0.1 W/m2, after Ao-XC = 0.39 ± 0.1 W/m2, and after Ao-UC = 0.44 ± 0.2 W/m2, p < 0.05. The CI changed during both cross-clamping and unclamping (p < 0.0001), whilst the MAP and heart rate did not during any phase of the surgery. Five subjects (8.3%) needed inotropic support after cross-clamping. Their basal CPI was lower than the general population: 0.31 ± 0.11 W/m2 vs. 0.39 ± 0.1 W/m2. Conclusions: The CPI describes the adaptation of the cardiac function to the changes in preload, contractility, and afterload occurring during aortic cross-clamping and unclamping. It may be used to explore the cardiac performance in real-time and predict cardiac impairment in the intraoperative period in a minimally invasive way, similar to ventriculo-arterial coupling parameters.

Prostate cancer cells of increasing metastatic potential exhibit diverse contractile forces, cell stiffness, and motility in a microenvironment stiffness-dependent manner.

During metastasis, all cancer types must migrate through crowded multicellular environments. Simultaneously, cancers appear to change their biophysical properties. Indeed, cell softening and increased contractility are emerging as seemingly ubiquitous biomarkers of metastatic progression which may facilitate metastasis. Cell stiffness and contractility are also influenced by the microenvironment. Stiffer matrices resembling the tumor microenvironment cause metastatic cells to contract more strongly, further promoting contractile tumorigenic phenotypes. Prostate cancer (PCa), however, appears to deviate from these common cancer biophysics trends; aggressive metastatic PCa cells appear stiffer, rather than softer, to their lowly metastatic PCa counterparts. Although metastatic PCa cells have been reported to be more contractile than healthy cells, how cell contractility changes with increasing PCa metastatic potential has remained unknown. Here, we characterize the biophysical changes of PCa cells of various metastatic potential as a function of microenvironment stiffness. Using a panel of progressively increasing metastatic potential cell lines (22RV1, LNCaP, DU145, and PC3), we quantified their contractility using traction force microscopy (TFM), and measured their cortical stiffness using optical magnetic twisting cytometry (OMTC) and their motility using time-lapse microscopy. We found that PCa contractility, cell stiffness, and motility do not universally scale with metastatic potential. Rather, PCa cells of various metastatic efficiencies exhibit unique biophysical responses that are differentially influenced by substrate stiffness. Despite this biophysical diversity, this work concludes that mechanical microenvironment is a key determinant in the biophysical response of PCa with variable metastatic potentials. The mechanics-oriented focus and methodology of the study is unique and complementary to conventional biochemical and genetic strategies typically used to understand this disease, and thus may usher in new perspectives and approaches.

MEA-integrated cantilever platform for comparison of real-time change in electrophysiology and contractility of cardiomyocytes to drugs

Drug-induced cardiotoxicity is a potentially severe side effect that can alter the contractility and electrophysiology of the cardiomyocytes. Cardiotoxicity is generally assessed through animal models using conventional drug screening platforms. Despite significant developments in drug screening platforms, the difficulty in measuring electrophysiology and contractile profile together affects the investigation of cardiotoxicity in potential drugs. Some drugs can prove to be more toxic to contractility than electrophysiology, which demands the need for a reliable, dual, and simultaneous drug screening platform. Herein, we propose the microelectrode array integrated SU-8 cantilever for dual and simultaneous measurement of electrophysiology and contractility of cardiomyocytes. The SU-8 cantilever is integrated with microelectrode array (C-MEA) using conventional photolithographic techniques. Drug tests are conducted to verify the feasibility of the C-MEA platform using three cardiovascular drugs. Clinically recognized drugs, quinidine and verapamil, are used to activate both the hERG channel and the contractile characteristics of cardiomyocytes. The effect of ion channel blockers on the field potential duration (FPD) of the cardiomyocytes is compared with several contractility-based parameters. The contraction-relaxation duration (CRD) profile is relatively close to that of FPD in tested drugs (half-maximal (IC50) toxicities are 1.093 μM (FPD) and 1.924 μM (CRD) for quinidine and 166.2 nM (FPD) and 459.4 nM (CRD) for verapamil). Blebbistatin, a known myosin II inhibitor, primarily affects the contractile profile of cardiomyocytes but not their field potential, with no evident correlation between contractility and field potential profiles. The proposed cantilever-based mechano-electrophysiology measurements platform provides a promising and accurate means to assess cardiotoxicity.

Changes in diaphragm contractility in cigarette smoking-exposed and smoking cessation rats are associated with alterations in mitochondrial morphology and homeostasis.

The effects of cigarette smoking (CS) cessation on the diaphragm are unknown, as are the CS-induced diaphragmatic mitochondrial changes. We examined the changes in diaphragm contractility, as well as alterations in mitochondrial morphology, function and homoeostasis during CS exposure and after cessation. Rats were randomly divided into CS exposure and CS cessation groups: 3-month CS (S3), 6-month CS (S6), 6-month CS followed by 3-month cessation (S6N3). The changes in the diaphragm were investigated, including contractile properties, the ultrastructure, mitochondrial function and the expression of markers of mitochondrial homoeostasis. CS caused irreversible histological disruption and functional depression in the lungs, along with significantly declines in diaphragmatic contractility and more severely in extensor digitorum longus muscular contractility. Such declines were recovered after 3-month CS cessation. CS exposure disrupted the diaphragmatic mitochondrial morphology and function (S6), which was significantly alleviated in the S6N3 group. The mitochondrial homoeostasis was depressed (S6), as indicated by the downregulation of Pink1 and Mfn1. Interestingly, the Mfn1 level was recovered after smoking cessation (S6N3). In conclusion, smoking cessation eased CS-induced diaphragmatic dysfunction and mitochondrial deregulation, which are likely associated with deregulated mitochondrial homoeostasis.

Echocardiographic and Cardiac MRI Comparison of Longitudinal Strain and Strain Rate in Cancer Patients Treated with Immune Checkpoint Inhibitors.

Background: Immune checkpoint inhibitor (ICI)-induced cardiac side effects in cancer patients are increasingly being recognized and can be fatal. There is no standardized cardiac imaging test to examine the effects of ICIs in myocardial morphology and function. Objective: To study the utility of echocardiography and cardiac MRI in examining regional and global changes arising from ICI-induced myocarditis and cardiomyopathy in high-risk subjects suspected to have developed ICI cardiomyopathy. Methods: We studied eight consecutive patients referred for cardiac MRI (CMR) from a comprehensive cancer center for suspected ICI-induced myocarditis and compared the data with sixteen age-matched controls. Using newly developed strain analysis algorithms, we measured myocardial strain and strain rates using echocardiography and CMR. Then, we compared the mean longitudinal strain and strain rates derived from echocardiography and CMR in the same ICI-treated cohort of patients (n = 8). They underwent both of these imaging studies with images taken 24–48 h apart and followed up prospectively within the same hospital course. Results: All our cases had preserved ejection fraction (EF) > 50%. Echocardiogram showed reduced mean systolic longitudinal strain (LS, %) (ICI: −12.381 ± 4.161; control: −19.761 ± 1.925; p < 0.001), peak systolic strain rate (SRS, s−1) (ICI: −0.597 ± 0.218; control: −0.947 ± 0.135; p = 0.002) and early diastolic strain rate (SRE, s−1) (ICI: 0.562 ± 0.295; control: 1.073 ± 0.228; p = 0.002) in ICI-treated cases. Direct comparison between the echocardiogram vs. CMR obtained within the same hospital course demonstrated strong a correlation of LS scores (r = 0.83, p = 0.012) and SRS scores (r = 0.71, p = 0.048). The Bland–Altman plots showed that 95% of the data points fitted within the ±1.96 SD of the mean difference, suggesting an agreement among these two imaging modalities. Conclusion: In this feasibility cohort study, both echocardiography- and CMR-based strain indices illustrate changes in myocardial contractility and relaxation suggestive of ICI-induced cardiomyopathy. Our data, after validation in a larger cohort, can form the basis of myocardial imaging in cancer patients treated with ICIs.

Heart rate changes in swimming exercised 12-13-week-old white experimental rats

The aim of the study was to determine the heart rate in 12-13-week-old rats under conditions of a 6-week dosed swimming load. In general, rat swimming is considered one of the best methods for studying cardiological parameters and the adaptive capabilities of the heart. As part of our study, we ensured 12-13-weeks-old male rats to undergo dosed swimming exercises for 4 and 6 weeks, at the end of each week we studied the electrocardiographic data of rats in order to assess the dynamics of changes in the frequency of contractions in experimental groups. The rats were divided into 3 groups: 1) a control group that did not receive any load (n=6); 2) The frst study group was subjected to a daily 30-minute swimming load for 4 weeks with a weight in the amount of 3% of the body weight of the rat (n=6) attached to the tail, 3) The second study group, which was subjected to a daily 60-minute swimming exercise for 6 weeks, the weight of the load attached to the tail amounted to 4% of the weight of the rat (n=6).&nbsp; Significant changes in cardiac contractility were observed only in experimental rats (30-minute exercise with a load of 3% of body weight or a 60-minute load with a load of 4% of body weight). In most rats, initial tachycardia was followed by prolonged bradycardia. The mentioned study can be considered as another step forward in the study of the pathogenetic mechanisms of pathological changes in rhythm and frequency that occur during exercise.

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes.

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

BACKGROUNDThe mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC.AIMTo reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODSMale rats (n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTSThe proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions.CONCLUSIONIn UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Abstract P1015: Construction Of A Pro-cardiogenic Extracellular Matrix: How Stem-cell Derived Epicardium Enhances Cardiac Regeneration Through Fibronectin

Background: Human embryonic stem cell-derived epicardium (hESC-Epis) has been shown to improve the function of hESC-derived cardiomyocytes (hESC-CM) in vitro and post-transplantation into animal myocardial infarction models. However, the underlying mechanisms mediating these effects remain poorly characterised. Bulk RNA sequencing of hESC-Epis suggests a role for genes related to extracellular matrix remodeling in mediating these effects, with fibronectin (FN1) being highly implicated. Study Objective: To examine the role of FN1 in hESC-CM/Epi crosstalk through gain and loss of function studies. Methods: A tetracycline inducible FN1 knockdown system was used on hESC-CMs and Epis within the setting of 3D-engineered heart tissue. Changes in contractility and calcium handling were measured. Recombinant human plasma FN1 (rhFN) was then used to determine the effect of FN1 supplementation on hESC-CM function. Results: FN1 knockdown in hESC-Epis resulted in significant abrogation of the beneficial effects on hESC-CM force generation and calcium handling. Knockdown of FN1 in hESC-CMs however significantly attenuated contractility of the hESC-CMs but had no effects on calcium handling. When rhFN was added to hESC-CMs in increasing doses, hESC-CM contractility was improved in a dose dependent fashion to the same extent as seen with hESC-Epis co-cultures. However, rhFN supplementation did not result in significant improvements in calcium handling. Conclusions: hESC-CM and Epi crosstalk improving hESC-CM function may rely heavily on the presence of FN1. FN1 secreted by both cell types are shown to be of importance in this crosstalk with artificial supplementation of rhFN being able to recapitulate some beneficial effects of hESC-Epis on hESC-CMs. Further studies are required to determine the role of specific isoforms of secreted FN1 on hESC-CM maturity and what mechanism underlies these effects. This opens up the possibility of improving cell-based therapy for heart failure post-myocardial infarction using recombinant FN1 supplementation.

Abstract P1024: 3d Cardiac Tissue Rings With Exogenous Extracellular Matrix As A Model For Cardiac Fibrosis

Introduction: Cardiac fibrosis, or excess deposition of extracellular matrix (ECM) within heart muscle, acts as a protective mechanism following cardiac injury, but causes long-term heart stiffening and impaired contraction. In vitro models aid in understanding cardiac disease mechanisms, but current fibrosis models lack (i) native-like ECM and (ii) interactions between multiple cardiac cell types. Objective: Our 3D model containing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) and cardiac fibroblasts (CF) with exogenous ECM will reproducibly assess contractile changes under fibrotic conditions. Methods: Human GCaMP-encoded iPSC-CM (day 19) and CF (3:1) were combined in a custom agarose ring mold with collagen I (Col) (0.02 or 0.5 mg/mL), laminin (Lam) (0.01 mg/mL), both Col+Lam (0.03 mg/mL), Matrigel (0.03 mg/mL), or no ECM, n=5 for each condition. Concentrations were based on in vivo ratios of Col and Lam in literature. After 24 hours, cells self-assembled into 3D rings (Fig. 1a). Results: All conditions formed robust rings; average thickness = 460 μm (Fig.1b, c). Spontaneous beating resumed 48 hours after seeding. After 3 days in culture, Ca 2+ flux videos were captured; rings without ECM displayed shorter contraction durations, as did Col+Lam rings (Fig. 1d, e). This suggests that exogenous ECM in ratios found in healthy hearts may aid in contraction. Conclusion: This ring platform provides a reproducible cardiac fibrosis model with native-like ECM conditions and interactions between multiple cardiac cell types. Further assessments of ring stiffness and additional ECM conditions will enable this model to be used in therapeutic development.

The Role of the Left Atrium: From Multimodality Imaging to Clinical Practice: A Review.

In recent years, new interest is growing in the left atrium (LA). LA functional analysis and measurement have an essential role in cardiac function evaluation. Left atrial size and function are key elements during the noninvasive analysis of diastolic function in several heart diseases. The LA represents a “neuroendocrine organ” with high sensitivity to the nervous, endocrine, and immune systems. New insights highlight the importance of left atrial structural, contractile, and/or electrophysiological changes, introducing the concept of “atrial cardiomyopathy”, which is closely linked to underlying heart disease, arrhythmias, and conditions such as aging. The diagnostic algorithm for atrial cardiomyopathy should follow a stepwise approach, combining risk factors, clinical characteristics, and imaging. Constant advances in imaging techniques offer superb opportunities for a comprehensive evaluation of LA function, underlying specific mechanisms, and patterns of progression. In this literature review, we aim to suggest a practical, stepwise algorithm with integrative multimodality imaging and a clinical approach for LA geometry and functional analysis. This integrates diastolic flow analysis with LA remodelling by the application of traditional and new diagnostic imaging techniques in several clinical settings such as heart failure (HF), atrial fibrillation (AF), coronary artery disease (CAD), and mitral regurgitation (MR).

The relationship between external and internal load parameters in 3 × 3 basketball tournaments

Purpose3 × 3 basketball games are characterized by high-intensity accelerations and decelerations, and a high number of changes of direction and jumps. It is played in tournament form with multiple games per day. Therefore, optimal regeneration is crucial for maintaining a high performance level over the course of the tournament. To elucidate how load of a match affects the athletes' bodies (i.e., internal load), muscular responses to the load of 3 × 3 games were analyzed. We aimed to investigate changes in contractility of the m. rectus femoris (RF) and m. gastrocnemius medialis (GC) in response to the load of single 3 × 3 games and a 3 × 3 tournament.MethodsInertial movement analysis was conducted to capture game load in 3 × 3. Changes in contractility were measured using tensiomyography (TMG). During a two-day tournament, TMG measurements were conducted in the morning and after each game. Additionally, off-game performance analysis consisting of jump and change-of-direction (COD) tests was conducted the day before the tournament.ResultsSignificant changes of the muscle contractility were found for GC with TMG values being higher in the baseline than in the post-game measurements. In contrast to athletes of the GC group, athletes of the RF group responded with either decreased or increased muscle contractility after a single 3 × 3 game. A significant correlation between external and internal load parameters could not be shown. Concerning off-game performance, significant correlations can be reported for COD test duration, CMJ height and ∆Vc as well as COD test duration and ∆Dm. No systematic changes in muscle contractility were found over the course of the tournament in RF and GC.ConclusionThe athletes' external 3 × 3 game load and their performance level did not seem to affect muscular contractility after a single 3 × 3 game or a complete 3 × 3 tournament within this investigation. This might indicate that elite athletes can resist external load without relevant local muscular fatigue. With respect to the course of the tournament, it can therefore be concluded that the breaks between games seem to be sufficient to return to the initial level of muscle contractility.

The Acute Effects of Leptin on the Contractility of Isolated Rat Atrial and Ventricular Cardiomyocytes.

Leptin is a pleiotropic peptide playing an important role in the regulation of cardiac functions. It is not clear whether leptin directly modulates the mechanical function of atrial cardiomyocytes. We compared the acute effects of leptin on the characteristics of mechanically non-loaded sarcomere shortening and cytosolic Ca2+ concentration ([Ca2+]i) transients in single rat atrial and ventricular cardiomyocytes. We also studied the functional properties of myosin obtained from cardiomyocytes using an in vitro motility assay and assessed the sarcomeric protein phosphorylation. Single cardiomyocytes were exposed to 5, 20, and 60 nM leptin for 60 min. In ventricular cardiomyocytes, 60 nM leptin depressed sarcomere shortening amplitude and decreased the rates of shortening and relaxation. These effects were accompanied by a decrease in the phosphorylation of cMyBP-C, an increase in Tpm phosphorylation, and a slowdown of the sliding velocity of thin filaments over myosin in the in vitro motility assay. In contrast, in atrial cardiomyocytes, the phosphorylation of cMyBP-C and TnI increased, and the characteristics of sarcomere shortening did not change. Leptin had no effect on the characteristics of [Ca2+]i transients in ventricular cardiomyocytes, while 5 nM leptin prolonged [Ca2+]i transients in atrial cardiomyocytes. Thus, leptin-induced changes in contractility of ventricular cardiomyocytes may be attributed to the direct effects of leptin on cross-bridge kinetics and sarcomeric protein properties rather than changes in [Ca2+]i. We also suggest that the observed differences between atrial and ventricular cardiomyocytes may be associated with the peculiarities of the expression of leptin receptors, as well as signaling pathways in the atrial and ventricular myocardium.