Passive Tension Research Articles

Submaximal eccentric resistance training increases serial sarcomere number and improves dynamic muscle performance in old rats.

The age-related loss of muscle mass is partly accounted for by the loss of sarcomeres in series, contributing to declines in muscle mechanical performance. Resistance training biased to eccentric contractions increases serial sarcomere number (SSN) in young muscle, however, maximal eccentric training in old rats previously did not alter SSN and worsened performance. A submaximal eccentric training stimulus may be more conducive to adaptation for aged muscle. The purpose of this study was to assess whether submaximal eccentric training can increase SSN and improve mechanical function in old rats. Twelve 32-month-old male F344/BN rats completed 4 weeks of submaximal (60% maximum) eccentric plantar-flexion training 3 days/week. Pre- and post-training, we assessed in-vivo maximum isometric torque at a stretched and neutral ankle angle, the passive torque-angle relationship, and the isotonic torque-velocity-power relationship. The soleus and medial gastrocnemius (MG) were harvested for SSN measurements via laser diffraction, with the untrained leg as a control. SSN increased 11% and 8% in the soleus and MG, respectively. Training also shifted optimal torque production towards longer muscle lengths, reduced passive torque 42%, and increased peak isotonic power 23%. Submaximal eccentric training was beneficial for aged muscle adaptations, increasing SSN, reducing muscle passive tension, and improving dynamic contractile performance.

Abstract P453: Dissecting tunica responsibility in arterial stress relaxation: Smooth muscle need not apply

Perivascular adipose tissue (PVAT) is being recognized as an important arterial tunica. Stress relaxation, a gradual reduction in stress over time while under a constant strain, is considered a property of smooth muscle (ex. intestine, uterus, bladder). While aortic PVAT (aPVAT) contains a microvasculature, it does not contain organized smooth muscle and isolated PVAT stress relaxes to a greater extent than the vessel itself. This raised the idea that other tunicas lacking smooth muscle may also stress relax. Here we hypothesize that appropriately oriented and functional smooth muscle was not necessary for stress relaxation to occur. To test this, we used histochemical staining and isometric contractility of thoracic aorta from the male Dahl salt-sensitive (SS) rat. Some rings of aorta were separated into adventitia (no muscle), media (muscle), and PVAT (no muscle), and responses of these isolates compared to the intact aortic ring (muscle + integration of tunicas). Masson Trichrome (MT) and Verhoeff Van Gieson (VVG) staining validated the dissection of the different tunicas with minimal contamination of cells from other tunicas. Stress relaxation was measured after increasing, cumulative amounts of passive tension (6 grams total) was applied in the presence or absence of Ca 2+ , essential for smooth muscle contraction. Each step was followed by challenge with a maximum concentration of the a 1 adrenergic agonist phenylephrine (PE, 10 -5 M). Stress relaxation occurred in all tunicas at each passive tension, whether or not Ca 2+ was present (figures). In fact, loss of Ca 2+ improved stress relaxation in adventitia and PVAT. Importantly, in the presence of Ca 2+ , PE contraction was observed at each passive tension in tissues containing smooth muscle: media (min, 250 mg PT: 26.67 +/- 8.51 mg; max, 6000 mg PT: 589.59 +/- 114.53 mg) and aorta + PVAT (min, 250 mg PT: 562.61 +/- 238.45 mg; max, 6000 mg PT: 1322.91 +/- 352.73mg). Little to no measurable contraction was recorded in the adventitia and aPVAT. There was little to no measurable contraction in tunicas in the absence of Ca 2+ . Taken together, these data suggest that all tunicas of the rat thoracic aorta stress relax and can do so without the presence of organized smooth muscle. Of all tunicas, PVAT demonstrated the greatest ability to stress relax. Collectively, these findings force reconsideration of individual responsibilities of arterial tunicas, all of which participate in the (dys)function of an artery.

Abstract P455: Integrins play a role in stress relaxation of perivascular adipose tissue

Perivascular adipose tissue (PVAT) is well known for being anti-contractile (reducing potency, maximum) in healthy tissues. We discovered a new function of PVAT, the ability to stress relax in response to a stretch. This is of note because stress relaxation has been attributed largely to smooth muscle, of which PVAT has none that is organized in a functional layer. Here we test the hypothesis that stress relaxation depends on the interactions of the cell membrane delimited heterodimer integrins with collagen. Our model is the thoracic aorta of the male Dahl SS rat on a normal diet. The PVAT and aorta were physically separated for most assays. Results from snRNA seq in PVAT, histochemistry and isometric contractility were used. Collagen was evident in PVAT as stained by Masson Trichrome. snRNA seq of whole PVAT determined expression of many collagen isoforms and integrins, with mRNA for both the α1 and β1 integrin being most highly expressed. Isolated aortic rings and separated PVAT were challenged with 2 or 4 grams of passive tension. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1β1 distintegrin obtustatin (5 uM) or general inhibitor RGD peptide (1 mM). RGD peptide but not obtustatin reduced the ability of the isolated PVAT ring to maintain applied tone at both the 2 and 4 gram challenge (~15% gain in relaxation/loss of tone, figure ). By contrast, stress relaxation in the aorta itself was not reduced by either inhibitor. In addition to a1b1 and collagen interaction, cell-cell communication inference identified integrins av and a5, two major RGD motif containing isoforms, as potential partners of collagens with fibroblast-like cells most likely involved. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so at least in part through integrin-collagen interactions that may not include α1β1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

Deficiency of the sphingosine-1-phosphate (S1P) transporter Mfsd2b protects the heart against hypertension-induced cardiac remodeling by suppressing the L-type-Ca2+ channel.

The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b+/+ and Mfsd2b-/- mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b-/- mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by ~ 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b-/- ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b-/- ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b+/+ and Mfsd2b-/- ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b-/- hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.

Cardiac length-dependent activation driven by force-dependent thick-filament dynamics

The length-dependent activation (LDA) of maximum force and calcium sensitivity are established features of cardiac muscle contraction but the dominant underlying mechanisms remain to be fully clarified. Alongside the well-documented regulation of contraction via the thin filaments, experiments have identified an additional force-dependent thick-filament activation, whereby myosin heads parked in a so-called off state become available to generate force. This process produces a feedback effect that may potentially drive LDA. Using biomechanical modeling of a human left-ventricular myocyte, this study investigates the extent to which the off-state dynamics could, by itself, plausibly account for LDA, depending on the specific mathematical formulation of the feedback. We hypothesized four different models of the off-state regulatory feedback based on (A) total force, (B) active force, (C) sarcomere strain, and (D) passive force. We tested if these models could reproduce the isometric steady-state and dynamic LDA features predicted by an earlier published model of a human left-ventricle myocyte featuring purely phenomenological length dependences. The results suggest that only total-force feedback (A) is capable of reproducing the expected behaviors, but that passive tension could provide a length-dependent signal on which to initiate the feedback. Furthermore, by attributing LDA to off-state dynamics, our proposed model also qualitatively reproduces experimentally observed effects of the off-state-stabilizing drug mavacamten. Taken together, these results support off-state dynamics as a plausible primary mechanism underlying LDA.

Empagliflozin, an SGLT2i, relaxes coronary smooth muscle at pharmacological concentrations

Morbidity and mortality due to heart failure with preserved ejection fraction (HFpEF) is increasing and there are no universally recognized strategies for treatment. Sodium-glucose co-transporter type 2 inhibitors (SGLT2i), such as Empagliflozin, have been shown to reduce myocardial stiffness and improve coronary microvascular performance, but mechanisms are unclear. Previous studies in rabbit aorta and rat mesentery suggest Empagliflozin relaxes smooth muscle through activation of K+ channels. This study investigated the effects of therapeutic and pharmacological concentrations of Empagliflozin (10-300 μM) on the isometric tension of left circumflex (CFX) coronary arteries from Yorkshire swine. We tested the hypothesis that Empagliflozin would relax coronary smooth muscle via a concentration-dependent manner, involving K+ channels. CFX arteries were dissected, cleaned of adventitia, cut into 3 mm rings (n = 16), and mounted in organ baths containing Krebs-Henseleit buffer (37 °C) for isometric tension studies. Each CFX segment was adjusted to optimal preload (~3 g passive tension) as determined by <10 % change in active tension in response to 60 mM KCl. In order to determine whether Empagliflozin caused relaxation and, if so, by what mechanism, rings were contracted with either 30 mM K+ or the thromboxane mimetic U46619 (1 μM). Once tension plateaued, Empagliflozin (10-300 μM) was added in increasing concentrations. Therapeutic concentrations of Empagliflozin (10-30 μM), elicited no significant relaxation. Pharmacologic concentrations of Empagliflozin (≥100 μM) caused significant relaxation from ~15 ± 11% to ~45 ± 10% in response to 100 μM vs. 300 μM Empagliflozin, respectively. Relaxation was not different between rings contracted with K+ or U46619, suggesting membrane hyperpolarization via K+ channels is not involved. Our results represent the first study of the effects of Empagliflozin on coronary smooth muscle reactivity. Data support the idea that relaxation of coronary smooth muscle by Empagliflozin occurs at supra-pharmacologic concentrations in vitro, but K+ channel activation appears to be an unlikely mechanism. This project was supported by National Institutes of Health grant R01 HL158723. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Translating myosin-binding protein C and titin abnormalities to whole-heart function using a novel calcium-contraction coupling model

Mutations in cardiac myosin-binding protein C (cMyBP-C) or titin may respectively lead to hypertrophic (HCM) or dilated (DCM) cardiomyopathies. The mechanisms leading to these phenotypes remain unclear because of the challenge of translating cellular abnormalities to whole-heart and system function.We developed and validated a novel computer model of calcium-contraction coupling incorporating the role of cMyBP-C and titin based on the key assumptions: 1) tension in the thick filament promotes cross-bridge attachment mechanochemically, 2) with increasing titin tension, more myosin heads are unlocked for attachment, and 3) cMyBP-C suppresses cross-bridge attachment.Simulated stationary calcium-tension curves, isotonic and isometric contractions, and quick release agreed with experimental data. The model predicted that a loss of cMyBP-C function decreases the steepness of the calcium-tension curve, and that more compliant titin decreases the level of passive and active tension and its dependency on sarcomere length. Integrating this cellular model in the CircAdapt model of the human heart and circulation showed that a loss of cMyBP-C function resulted in HCM-like hemodynamics with higher left ventricular end-diastolic pressures and smaller volumes. More compliant titin led to higher diastolic pressures and ventricular dilation, suggesting DCM-like hemodynamics.The novel model of calcium-contraction coupling incorporates the role of cMyBP-C and titin. Its coupling to whole-heart mechanics translates changes in cellular calcium-contraction coupling to changes in cardiac pump and circulatory function and identifies potential mechanisms by which cMyBP-C and titin abnormalities may develop into HCM and DCM phenotypes. This modeling platform may help identify distinct mechanisms underlying clinical phenotypes in cardiac diseases.

Rho-Kinase Inhibition of Active Force and Passive Tension in Airway Smooth Muscle: A Strategy for Treating Airway Hyperresponsiveness in Asthma.

Rho-kinase inhibitors have been identified as a class of potential drugs for treating asthma because of their ability to reduce airway inflammation and active force in airway smooth muscle (ASM). Past research has revealed that, besides the effect on the ASM's force generation, rho-kinase (ROCK) also regulates actin filament formation and filament network architecture and integrity, thus affecting ASM's cytoskeletal stiffness. The present review is not a comprehensive examination of the roles played by ROCK in regulating ASM function but is specifically focused on passive tension, which is partially determined by the cytoskeletal stiffness of ASM. Understanding the molecular basis for maintaining active force and passive tension in ASM by ROCK will allow us to determine the suitability of ROCK inhibitors and its downstream enzymes as a class of drugs in treating airway hyperresponsiveness seen in asthma. Because clinical trials using ROCK inhibitors in the treatment of asthma have yet to be conducted, the present review focuses on the in vitro effects of ROCK inhibitors on ASM's mechanical properties which include active force generation, relaxation, and passive stiffness. The review provides justification for future clinical trials in the treatment of asthma using ROCK inhibitors alone and in combination with other pharmacological and mechanical interventions.

Effect of breast milk intake volume on early behavioral neurodevelopment of extremely preterm infants

BackgroundThis study aimed to explore the effects of breast milk feeding volume on the early behavioral neurodevelopment of extremely preterm infants (gestational age < 28 weeks).MethodsThe study was conducted from 1 January 2021 to 31 March 2023. A total of 187 preterm infants from a neonatal intensive care unit (NICU) in a Grade III Class A hospital in Zhejiang, China, were divided based on the proportion of breast milk in their total enteral nutrition: high proportion (≥ 80%, including exclusive breast milk feeding), medium proportion (20% ~ < 80%), and low proportion (< 20%). The study investigated motor performance and behavioral neurodevelopment at 37 weeks of corrected gestational age, as well as the total incidence of intracranial hemorrhage within the first four weeks postpartum.ResultsThe low breast milk feeding group had significantly lower scores in infant motor performance (31.34 ± 5.85) and elicited item scores (19.89 ± 5.55) compared to the medium and high groups (33.52 ± 4.33, 22.13 ± 4.22; and 35.86 ± 5.27, 23.91 ± 4.98), p < 0.05, respectively. Despite no significant difference in behavioral ability, the low proportion group exhibited lower passive muscle tension and primitive reflex scores than the medium and high proportion groups. The high proportion group showed higher active muscle tension scores. Ultrasound results revealed varying incidences of intracranial hemorrhage: 72.9% in low, 52.5% in medium, and 19.6% in the high proportion groups.ConclusionsMedium to high levels of breast milk feeding contribute positively to motor and behavioral neurological development in extremely preterm infants and decrease the likelihood of ventricular hemorrhage. However, it does not have a significant effect on the development of behavioral abilities. Due to the limited sample size, the next step will be to expand the sample size and further investigate the extent of the impact on various aspects of the nervous system.

Relative motion splints versus metacarpophalangeal joint blocking splints in the management of trigger finger: Study protocol for a randomized comparative trial.

Evidence supports the use of hand-based metacarpophalangeal joint (MCPJ) blocking splints as an intervention for trigger finger (TF). In practice, finger-based relative motion (RM) splints are also implemented without evidence. This randomized comparative trial (RCT) aims to evaluate implementation of MCPJ blocking and RM splints for effectiveness, function, occupational performance and wearability after 6 weeks of TF management. Priori analysis determined 36 individuals were needed for random assignment to the RM or MCPJ blocking splint groups. Individuals must be aged ≥21 years, and diagnosed with TF involving ≥1 finger. For blinding purposes, the primary author screens for eligibility, fabricates the splints and educates. Therapist A administers the primary outcome measures Week-1 and Week-6-stage of stenosing tenosynovitis and secondary outcome measures- number of triggering events in 10 active fists, visual analog scales (VAS) for pain, splint comfort and satisfaction, Disabilities of the Arm, Shoulder and Hand, and Canadian Occupational Performance Measure. Therapist B in Week-3 instructs participants in deep tissue massage and administers splint wearability VASs. The RM pencil test is used to determine the affected finger(s) MCPJ splint position i.e., more extension or flexion based on participant response. The MCPJ blocking splint holds the MCPJ in a neutral position. Analysis involves a mixed-effects ANOVA to compare Week-1 and Week-6 primary and secondary outcomes. Recruitment and data collection are ongoing. Biomechanically RM splints control tendon excursion and reduce passive tendon tension while allowing unencumbered finger motion and hand function. Hence clinicians use RM splints as an intervention for TF, despite the lack of implementation evidence. This RCT implements a function-focused as well as patient-centered approach with partial blinding of assessors and participants. We anticipate that this study will provide evidence for the implementation of RM splints to manage adults with TF. Clinical trial registration This trial is registered with ClinicalTrials.gov (NCT05763017).

Unraveling the pathogenesis of novel ANKRD1 missense mutation causing familial dilated cardiomyopathy using human-induced pluripotent stem cells derived cardiomyocytes

Abstract Introduction Familial dilated cardiomyopathy(f-DCM) can be caused by mutations in 30 different genes encoding sarcomeric, cytoskeletal, mechanotransducers and transcription factors. One of the genes ANKRD1 encode Cardiac ankyrin repeat protein (CARP) which is involved in the mechanosensory unit located on the I-band of the sarcomere. Here we demonstrate a novel mutation in ANKRD1 gene causing f-DCM, and the usage of human-induced pluripotent(hiPSC) technology unraveling the pathogenesis of the disease. Purpose of the study: 1) Establishing patient specific hiPSC-derived cardiomyocytes from a family harboring a mutation in ANKRD1 gene causing f-DCM; 2) Phenotypical assessment of the morphological and ultrastructural properties of the diseased cells upon CARP activation; 3) Functional assessment of the 3 component of excitation-contraction coupling at the single-cell and tissue level. Methods and Results Patient-specific hiPSC were generated from a family harboring missense mutation in the ANKRD1 gene, with a substitution of the amino acid arginine to cysteine at position 182 of the Cardiac ankyrin repeat protein (CARP). ANKRD1-mut hiPSC were differentiated into cardiomyocytes and later matured using hormonal treatment. Morphological assessment using immunofluorescence(IF) techniques revealed that ANKRD1-mut hiPSC-CMs demonstrate an attenuated response to hypertrophy (upon Angiotensin-2 and Phenylephrine treatment). In addition ANKRD1-mut hiPSC-CMs displayed a higher cellular death rate upon increasing substrate stiffness and starvation. Electrophysiological assessment of the ANKRD1-mut cardiac tissues exhibit no alternations in action potential durations and conduction velocities while assessment of calcium transients demonstrated slower kinetics. In addition forces were quantified at the single-cell and tissue level , and demonstrated reduced active tension values and abnormal post-extrasystolic potentiation, while only single-cell experiments have demonstrated decreased passive tensions. These phenotypical alternations might be attributed to decreased nuclear translocation of CARP upon phenylephrine treatment as detected in IF experiments. Conclusion Novel mutation in ANKRD1 were found and validated to be causing f-DCM, by revealing phenotypical alternation using hiPSC-CM technology. Increased cellular death rate and impaired nuclear translocation of CARP might be the underlying causes of the morphological and contractile abnormalities detected in-vitro, leading to systolic dysfunction in the affected family.

Isometric myography is a feasible method to identify and quantify endothelial dysfunction in dogs with myxomatous mitral valve disease.

To assess the feasibility of isometric myography in pet dogs with myxomatous mitral valve disease (MMVD) to determine its use in quantifying endothelial dysfunction. 9 dogs euthanized for medical reasons. Femoral, renal, and mesenteric arteries were collected postmortem and stored in physiological saline solution at 4 °C for myography. Mitral valves were scored for myxomatous degeneration (grades 1 to 4). Sections of arteries were mounted in wells, immersed in physiological saline solution perfused with 95% O2 and 5% CO2 at 37 °C, and stretched to an internal circumference (IC) that generated the maximal difference between active and passive wall tension (IC1). Normalization factors were calculated by dividing the IC1 by the IC at which the passive wall tension was 100 mm Hg (IC100). Vasoconstriction to phenylephrine and vasodilation to acetylcholine (endothelial dependent) and sodium nitroprusside (endothelial independent) were assessed by cumulative dose-response curves. Median MMVD grade was 3. Mean values of normalization factors were 1.00 ± 0.14 (renal, n = 15), 1.00 ± 0.10 (femoral, 8), and 1.05 ± 0.12 (mesenteric, 6). Responses to phenylephrine were similar between dogs (P = .14). Reduced responses to acetylcholine compared with sodium nitroprusside were identified in 15 arteries, suggesting endothelial dysfunction. Isometric myography of arteries from pet dogs is feasible and can identify loss of endothelial-dependent relaxation in dogs with MMVD postmortem. Its use in further research can lead to a better understanding of the pathophysiology mechanisms of this disease.

Right Atrial Adaptation to Precapillary Pulmonary Hypertension: Pressure-Volume, Cardiomyocyte, and Histological Analysis

BackgroundPrecapillary pulmonary hypertension (precPH) patients have altered right atrial (RA) function and right ventricular (RV) diastolic stiffness. ObjectivesThis study aimed to investigate RA function using pressure-volume (PV) loops, isolated cardiomyocyte, and histological analyses. MethodsRA PV loops were constructed in control subjects (n = 9) and precPH patients (n = 27) using magnetic resonance and catheterization data. RA stiffness (pressure rise during atrial filling) and right atrioventricular coupling index (RA minimal volume / RV end-diastolic volume) were compared in a larger cohort of patients with moderate (n = 39) or severe (n = 41) RV diastolic stiffness. Cardiomyocytes were isolated from RA tissue collected from control subjects (n = 6) and precPH patients (n = 9) undergoing surgery. Autopsy material was collected from control subjects (n = 6) and precPH patients (n = 4) to study RA hypertrophy, capillarization, and fibrosis. ResultsRA PV loops showed 3 RA cardiac phases (reservoir, passive emptying, and contraction) with dilatation and elevated pressure in precPH. PrecPH patients with severe RV diastolic stiffness had increased RA stiffness and worse right atrioventricular coupling index. Cardiomyocyte cross-sectional area was increased 2- to 3-fold in precPH, but active tension generated by the sarcomeres was unaltered. There was no increase in passive tension of the cardiomyocytes, but end-stage precPH showed reduced number of capillaries per mm2 accompanied by interstitial and perivascular fibrosis. ConclusionsRA PV loops show increased RA stiffness and suggest atrioventricular uncoupling in patients with severe RV diastolic stiffness. Isolated RA cardiomyocytes of precPH patients are hypertrophied, without intrinsic sarcomeric changes. In end-stage precPH, reduced capillary density is accompanied by interstitial and perivascular fibrosis.

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues. This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECTsuspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process. The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Cyclophosphamide enfeebles myocardial isometric contraction force via RIP1/RIP3/MLKL/TRPM7-mediated necroptosis

This study explores the negative impact of cyclophosphamide (CP) on cardiac contractility by specifically examining its effect on the active and passive tension of the cardiac muscle in-vitro and revealing the mechanism through which CP induces myocardial insult in-vivo. In young male Sprague-Dawley rats, cardiac toxicity was induced by a single intraperitoneal injection of CP (150 mg/kg body weight). Axial heart tissue slices were electrically stimulated, and the total isometric contraction force was measured at varying pretension levels. Blood and tissue biochemical assays, and histological/ immuno-histological assessments were conducted to evaluate the underlying molecular mechanisms. Statistical analysis shows that there is a significant difference between the drugged and the control groups in terms of the active tension values. Moreover, the pre-tension stress significantly affects both the active and passive tension values. CP altered heart, body, and heart-to-body weight, desolated cardiac muscle architecture, surged cardiac enzymes (CK-MB, LDH, and cTn l), augmented myocardial oxidative stressors (MDA), and weakened myocardial antioxidant status (SOD and GSH). Mechanistically, cyclophosphamide prompted the necroptotic trajectory evidenced by the activation of RIPK1, RIPK3, MLKL and TRPM7, the inhibition of caspase 8 and BCL2 and the upregulation of the protein/mRNA expression of TNF-α and TNFR1. This study identifies necroptosis as a key factor in cyclophosphamide-evoked myocardial contractility impairment, highlighting its potential as a target for alleviating antitumor-related myocardial damage. This innovative approach to investigating the underlying mechanisms of CP-induced cardiac toxicity offers valuable insights into the potential of developing new therapies to mitigate cyclophosphamide’s negative impact.

Antisenescence Therapy Improves Function in a Human Model of Cardiac Fibrosis-on-a-Chip

Cardiac fibrosis is a significant contributor to heart failure and is characterized by abnormal ECM deposition and impaired contractile function. We have previously developed a model of cardiac fibrosis via TGF-β treatment of engineered microtissues using heart-on-a-chip technology which incorporates human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts. Here, we describe that these cardiac fibrotic tissues expressed markers associated with cellular senescence via transcriptomic analysis. Treatment of fibrotic tissues with the senolytic drugs dasatinib and quercetin (D+Q) led to an improvement of contractile function, reduced passive tension, and downregulated senescence-related gene expression, an outcome we were previously unable to achieve using standard-of-care drugs. The improvement in functional parameters was also associated with a reduction in fibroblast density, though no changes in absolute collagen deposition were observed. This study demonstrates the benefit of senolytic treatment for cardiac fibrosis in a human-relevant model, supporting data in animal models, and will enable the further elucidation of cell-specific effects of senolytics and how they impact cardiac fibrosis and senescence.

Direct measurement of human skeletal muscle specific tension and fiber length

Skeletal muscle’s isometric contractile properties are one of the classic structure-function relationships in all of physiology allowing for extrapolation of single fiber mechanical properties to whole muscle properties based on the muscle’s optimal fiber length and physiological cross-sectional area (PCSA). However, this relationship has only been validated in small animals and then extrapolated to human muscles which are much larger in terms of length and PCSA. We leveraged a unique surgical technique in which an intact, living, human gracilis muscle is transferred from the thigh to the arm, restoring elbow flexion after brachial plexus injury. During this surgery we directly measured subject specific gracilis muscle force-length relationship in situ and properties ex vivo. We hypothesized that the human gracilis would have a functional fiber length of about 23 cm, based on the anatomical literature, and a specific tension of 225 kPa, based on the physiological literature. Data are provided as mean±standard deviation of the mean. Twelve subjects (7 males, average age 54±12 years). In situ passive and active tension were directly measured using a buckle force transducer (BFT) placed on the distal gracilis tendon. Passive tension was measured in each of four joint configurations (JC) prior to muscle stimulation, and, to produce active tension, the anterior branch of the obturator nerve was stimulated. The muscle was progressively lengthened by extending the knee and abducting the hip. Since we could not measure optimal fiber length directly in our subjects during isometric testing we used a functional surrogate for optimal fiber length, the full-width at half-maximum (FWHM) defined from their force vs. length relationship. The FWHM is the width of the active force vs. muscle length curve at half the maximum force. A linear relationship between FWHM and optimal fiber length has been established across several mammalian species and muscles. Thus, to calculate FWHM, we fit each patient’s calculated fully active force vs. muscle length data to a quadratic curve and then calculated width at half the calculated maximum force from the equation. Optimal fiber length was then calculated from the previously established linear relationship between FWHM and optimal fiber length. Each subject’s PCSA was calculated from their muscle volume and optimal fiber length.From these experimental data we established a human muscle fiber-specific tension of 171±84 kPa (n=12). We also determined the average gracilis optimal fiber length was 12.9±3.3 cm (n=12). Surprisingly, these fiber lengths were about half of the previously reported optimal fascicle lengths of 23 cm. Thus, the long gracilis muscle appears to be composed of relatively short fibers acting in parallel that may not have been appreciated based on traditional anatomical methods. We thus suggest that the value of 171 kPa be considered the best current estimate of human muscle specific tension. This work was supported the Department of Veterans Affairs grant 1 I01 RX002462 and, in part, by Research Career Scientist Award Number IK6 RX003351 from the United States (U.S.) Department of Veterans Affairs Rehabilitation R&amp;D (Rehab RD) Service. Pilot projects leading to this protocol were supported by the Shirley Ryan AbilityLab Catalyst Grant Program, National Science Foundation Graduate Research Fellowship under Grant No. 1255833 and the Mayo Clinic Graduate School. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Difference in functional constituents of myocardial passive compliance renders BalbC/J mice sensitive to pulmonary edema compared to C57BL6/J mice

Study objective: We have found different survival rates in response to a 4-day angiotensin II (AngII) and high salt diet - treatment in two mouse strains, BalbC/J and C57BL6/J. The mortality is increased in BalbC/J mice alongside increased lung weight but the reason is unknown. Hypothesis: We hypothesize the increased mortality is due to differences in myocardial compliance. Methodology: Male mice were treated with AngII by osmotic mini-pump infusion and fed a high salt-diet (3%) for 4 days. At the end of the treatment the lungs were weighed. Left ventricle (LV) passive compliance was assessed by Langendorff, and collagen and non-collagen functional fractions using demembranated trabecular strips. A commercial assay was used to determine total collagen of the myocardium. All procedures were approved by the local ethics committee. Comparisons were carried out using unpaired t-test. All intervals reflect standard error of the mean. Data: Lung weights of animals before treatment were similar in BalbC/J (148 ± 8.8 mg (n=5)) and C57BL6/J (155 ± 12 mg (n=5)) mice (p&gt;0.05). After treatment they were higher in BalbC/J (255 ± 24 mg (n=7)) than C57BL6/J (166 ± 5.1 mg (n=6)) (p&lt;0.01). LV passive strain was higher in BalbC/J (62.4 ± 11.9 mN/mm2 (n=9)) than C57BL6/J (28.2 ± 2.3 mN/mm2 (n=7)) (p&lt;0.05), at 36 μl LV inflation past 5 mmHg end-diastolic pressure. In demembranated trabeculae, passive stiffness was higher in BalbC/J (38.7 ± 5.5 mN/mm2 (n=6)) than C57BL6/J (22.5 ± 2.0 mN/mm2 (n=6)) (p&lt;0.05) at baseline, as measured by passive tension at 15 % stretch from sarcomere length 2.0 μm. This was derived from the non-collagen fraction which was higher in BalbC (23.4 ± 2.8 mN/mm2 (n=6)) than C57BL6/J (12.4 ± 1.3 mN/mm2 (n=6)) (p&lt;0.01), unlike the collagen fraction that was the same (BalbC/J 15.3 ± 3.8 mN/mm2 (n=6), C57BL6/J 10.1 ± 1.5 mN/mm2 (n=6) (p&gt;0.05)). There was no difference in myocardial collagen content (BalbC/J 0.20 ± 0.01 μg/μl (n=7), C57BL6/J 0.18 ± 0.01 μg/μl (n=9) (p&gt;0.05)). Summary of results: Gross organ evaluation by Langendorff indicates higher myocardial stiffness in BalbC/J than C57BL6/. This renders the heart susceptible to backward failure. This is supported by findings in demembranated tissue bundles. The origin of this appears to lie in the non-collagen fraction of passive stiffness, as indicated by functional and biochemical assays. Conclusion: An acute lung weight increase indicates pulmonary edema. The most common reason for this is backwards failure of the LV. Our investigations into myocardial function suggest that BalbC/J mice has a propensity for this due to lower compliance secondary to increased non-collagen fraction, compared to C57BL6/J. This is likely caused by titin-derived stiffness. Therapies to modulate titin-derived stiffness may be useful to avoid backward failure. These findings also represent important knowledge for the research community modeling human heart disease on the backbone of two common mouse strains. The study was funded with grants from the Swedish Society of Medical Research and Åke Wiberg Foundation to HI, Swedish Heart-Lung Foundation to MH, and from Uppsala University Hospital to HI and MH. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Perfusable Biohybrid Designs for Bioprinted Skeletal Muscle Tissue.

Engineered, centimeter-scale skeletal muscle tissue (SMT) can mimic muscle pathophysiology to study development, disease, regeneration, drug response, and motion. Macroscale SMT requires perfusable channels to guarantee cell survival, and support elements to enable mechanical cell stimulation and uniaxial myofiber formation. Here, stable biohybrid designs of centimeter-scale SMT are realized via extrusion-based bioprinting of an optimized polymeric blend based on gelatin methacryloyl and sodium alginate, which can be accurately coprinted with other inks. A perfusable microchannel network is designed to functionally integrate with perfusable anchors for insertion into a maturation culture template. The results demonstrate that i) coprinted synthetic structures display highly coherent interfaces with the living tissue, ii) perfusable designs preserve cells from hypoxia all over the scaffold volume, iii) constructs can undergo passive mechanical tension during matrix remodeling, and iv) the constructs can be used to study the distribution of drugs. Extrusion-based multimaterial bioprinting with the inks and design realizes in vitro matured biohybrid SMT for biomedical applications.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.