Reduction In Contractile Force Research Articles

Thrombospondin-1 promotes fibro-adipogenic stromal expansion and contractile dysfunction of the diaphragm in obesity.

Pulmonary disorders impact 40% to 80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragmatic weakness. Increased intra-diaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs) - mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing the effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 knockout (Thbs1-/-) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGF β-related expression signatures and augmentation of a Thy1-expressing sub-population previously linked to type 2 diabetes. Despite similar weight gain, Thbs1-/- mice were protected from these transcriptomic changes and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1-/- diaphragms maintained normal contractile force and motion after DIO challenge. These findings establish THBS1 as a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition and a potential therapeutic target in obesity-associated respiratory dysfunction.

Effects of Aging and Fatigue on Human Skeletal Muscle Cellular Contractile Function

Aging is associated with a reduction in skeletal muscle mass and function, known as sarcopenia. While reductions in contractile force are typically explained by loss in muscle cellular cross sectional area (CSA) loss of contractile power during high velocity contractions exceed reductions in force and cannot be solely explained by loss of muscle size and are observed at the single fiber within the same myosin heavy chain isoform (MHC). These phenomenon may be explained by myosin binding protein C (MyBP-C), which is known to modulate contractile force and velocity in a phosphorylation-dependent manner in both cardiac and skeletal muscle. Few studies have investigated the inherent contractile performance of single fibers between old and young adults, and none have explored the effects of altered MyBP-C phosphorylation on single fiber function. Therefore, the PURPOSE of this study was to investigate the effects of age and fatigue-induced MyBP-C phosphorylation on single fiber contractile performance. We HYPOTHESIZED that isometric tension will be enhanced in older adults but velocity and power will be reduced, and fatigue will enhance velocity and power in single fibers. To test this hypothesis, we recruited 4 young (18-35) and 3 older (65-80) adult males to perform a single bout of unilateral knee extensions at 30% maximal voluntary isometric contraction (MVIC) until task failure. Immediately following, a bilateral biopsy of the vastus lateralis (VL) muscle was performed on the fatigued and non-fatigued control limbs. Tissue was either flash frozen for western blot or prepared for mechanical analysis of single fiber tension (Tmax), velocity (Vmax), and power (Pmax) under optimal intracellular metabolic conditions. Our analyses were limited to MHC IIA due to differences in fiber type distribution between the groups. Our RESULTS showed that neither age nor fatigue had an effect on contractile performance in MHC IIA fibers. However, independent analysis of fatigue within age groups suggest trends towards an increase in velocity (p=0.055) in young (MHC IIA) and reduction in tension (p=0.085) in old (MHC I) adult fibers. We CONCLUDE that fatigue may modulate contractile function in the single fiber in an age- and fiber-type dependent manner. Future studies will be aimed at collecting multiple fiber types and investigating the role of biological sex. Wu Tsai Human Performance Alliance. 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.

From the Literature

From the Literature

Multi-organ system for the evaluation of efficacy and off-target toxicity of anticancer therapeutics.

A pumpless, reconfigurable, multi-organ-on-a-chip system containing recirculating serum-free medium can be used to predict preclinical on-target efficacy, metabolic conversion, and measurement of off-target toxicity of drugs using functional biological microelectromechanical systems. In the first configuration of the system, primary human hepatocytes were cultured with two cancer-derived human bone marrow cell lines for antileukemia drug analysis in which diclofenac and imatinib demonstrated a cytostatic effect on bone marrow cancer proliferation. Liver viability was not affected by imatinib; however, diclofenac reduced liver viability by 30%. The second configuration housed a multidrug-resistant vulva cancer line, a non-multidrug-resistant breast cancer line, primary hepatocytes, and induced pluripotent stem cell-derived cardiomyocytes. Tamoxifen reduced viability of the breast cancer cells only after metabolite generation but did not affect the vulva cancer cells except when coadministered with verapamil, a permeability glycoprotein inhibitor. Both tamoxifen alone and coadministration with verapamil produced off-target cardiac effects as indicated by a reduction of contractile force, beat frequency, and conduction velocity but did not affect viability. These systems demonstrate the utility of a human cell-based in vitro culture system to evaluate both on-target efficacy and off-target toxicity for parent drugs and their metabolites; these systems can augment and reduce the use of animals and increase the efficiency of drug evaluations in preclinical studies.

SERCA is critical to control the Bowditch effect in the heart

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca2+ in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCAA617T induced positive staircase and arrhythmia, while SERCAE442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.

Resolvin E1 (Rv E1 ) attenuates LPS induced inflammation and subsequent atrophy in C2C12 myotubes.

Resolution of inflammation is now known to be an active process which in part is instigated and controlled by specialized pro-resolving lipid mediators (SPM's) derived from dietary omega-3 fatty acids. Resolvin E1 (Rv E1 ) is one of these SPM's derived from the omega-3 fatty acid eicosapentaenoic acid. Using both molecular and phenotypic functional measures we report that in a model of Lipopolysaccharide (LPS) induced inflammation, Rv E1 attenuated mRNA levels of both interlukin-6 and monocyte chemoattractant protein-1 whilst having no effect on tumor necrosis factor-α or interlukin-1β in C2C12 skeletal muscle myotubes. Findings at the molecular level were transferred into similar changes in extracellular protein levels of the corresponding genes with the greatest attenuation being noted in IL-6 protein concentrations. Rv E1 instigated beneficial morphological changes through the prevention of LPS induced skeletal muscle atrophy, in tandem with attenuation of the LPS induced reduction in contractile force in tissue engineered skeletal muscle. These findings demonstrate, in our model of endotoxin induced inflammation in skeletal muscle, that Rv E1 has pro-resolving properties in this cell type. Our data provides rationale for further investigation into the mechanistic action of Rv E1 in skeletal muscle, with the vision of having potential benefits for the prevention/resolution of in-vivo skeletal muscle atrophy.

Impaired contractile function of the supraspinatus in the acute period following a rotator cuff tear

BackgroundRotator cuff (RTC) tears are a common clinical problem resulting in adverse changes to the muscle, but there is limited information comparing histopathology to contractile function. This study assessed supraspinatus force and susceptibility to injury in the rat model of RTC tear, and compared these functional changes to histopathology of the muscle.MethodsUnilateral RTC tears were induced in male rats via tenotomy of the supraspinatus and infraspinatus. Maximal tetanic force and susceptibility to injury of the supraspinatus muscle were measured in vivo at day 2 and day 15 after tenotomy. Supraspinatus muscles were weighed and harvested for histologic analysis of the neuromuscular junction (NMJ), intramuscular lipid, and collagen.ResultsTenotomy resulted in eventual atrophy and weakness. Despite no loss in muscle mass at day 2 there was a 30% reduction in contractile force, and a decrease in NMJ continuity and size. Reduced force persisted at day 15, a time point when muscle atrophy was evident but NMJ morphology was restored. At day 15, torn muscles had decreased collagen-packing density and were also more susceptible to contraction-induced injury.ConclusionMuscle size and histopathology are not direct indicators of overall RTC contractile health. Changes in NMJ morphology and collagen organization were associated with changes in contractile function and thus may play a role in response to injury. Although our findings are limited to the acute phase after a RTC tear, the most salient finding is that RTC tenotomy results in increased susceptibility to injury of the supraspinatus.

Reducing Risk of Cardiovascular Disease: Exploring the Effects of Quercetin on the Contractile Force of Vascular Smooth Muscle

Calcium plays an important role in regulating the contraction of smooth muscles. Membrane potential of the smooth muscles regulates the smooth muscle contractility through variation in Ca2+ influx with the assistance of the voltage-operated Ca2+ channel. Using the porcine pulmonary arteries (PPA) as the model system, the effect of the antioxidant quercetin on the regulation of pulmonary arteries was investigated in the presence and absence of calcium. It was hypothesized that quercetin reduces the contractile force in PPA by limiting Ca2+ release from the sarcoplasmic reticulum (SR) without imposing any effect on the voltage-operated channel (VOC). However, the biochemical pathway of quercetin is still unknown. In this study, it is shown that PPA contracts both in the presence and absence of Ca2+. Further investigations are required to study the exact effects of KCl in 0 Ca2+ to completely break down the biochemical pathway of quercetin and reassure its effect on the SR. Since quercetin demonstrates reduction in contractile force, it has high potential to provide a solution for the side effects of hypertension, which is a complex heterogeneous disorder that can coexist with other cardiovascular abnormalities.

Host reaction to vaginally inserted collagen containing polypropylene implants in sheep

We aimed to characterize the effect of vaginal or abdominal mesh insertion and of different collagen augmentation of polypropylene mesh in a sheep model. Outcome measures were passive and active biomechanical properties and semiquantitative morphometry. Forty-two Texel sheep were used: 6 were nonimplanted controls (n= 6), the rest were implanted with polypropylene mesh (n= 12; Avaulta Solo; Bard Medical, Covington, GA) or collagen-coated meshes: Avaulta Plus (n= 12; Bard Medical) and Ugytex (n= 12; Sofradim International, Trevoux, France). Through a single incision, the rectovaginal septum was dissected and a 35× 35-mm mesh was sutured to the underlying tissues. Abdominally, a 50× 50-mm mesh was laid over a primarily sutured full thickness 40-mm longitudinal incisional defect. Animals were explanted after 60 or 180 days (n= 6 per group). Outcome measures were passive biomechanics by biaxial tensiometry, active contractility of vaginal explants, and histologic evidence. Vaginal explants were 2.4 times stiffer compared with native vaginal tissue (P < .001), but without differences in comfort zone stiffness or slope of the load-elongation in the physiologic range between the products that were tested. Collagen coating was associated with a 16-fold reduction in contractile force at 180 days, compared with native vaginal tissue, both for Avaulta Plus (P= .032) and Ugytex (P= .015). Abdominal explants were 1.3-times stiffer compared with native abdominal wall tissue (P < .001) and were 1.9-times stiffer compared with vaginal explants. Vaginal mesh implantation yields less stiff explants compared with abdominal explants. Vaginal mesh implantation also alters the passive and active biomechanical properties compared with native vaginal tissues. Collagen matrices did not reduce the number of graft-related complications.

B. anthracisedema toxin increases cAMP levels and inhibits phenylephrine-stimulated contraction in a rat aortic ring model

B. anthracis edema toxin (ET) and lethal toxin (LT) are each composed of protective antigen (PA), necessary for toxin uptake by host cells, and their respective toxic moieties, edema factor (EF) and lethal factor (LF). Although both toxins likely contribute to shock during infection, their mechanisms are unclear. To test whether ET and LT produce arterial relaxation, their effects on phenylephrine (PE)-stimulated contraction in a Sprague-Dawley rat aortic ring model were measured. Rings were prepared and connected to pressure transducers. Their viability was confirmed, and peak contraction with 60 mM KCl was determined. Compared with PA pretreatment (control, 60 min), ET pretreatment at concentrations similar to those noted in vivo decreased the mean (±SE) maximum contractile force (MCF; percent peak contraction) in rings generated during stimulation with increasing PE concentrations (96.2 ± 7.0 vs. 57.3 ± 9.1) and increased the estimated PE concentration producing half the MCF (EC50; 10(-7) M, 1.1 ± 0.3 vs. 3.7 ± 0.8, P ≤ 0.002). ET inhibition with PA-directed monoclonal antibodies, selective EF inhibition with adefovir, or removal of the ring endothelium inhibited the effects of ET on MCF and EC50 (P ≤ 0.02). Consistent with its adenyl cyclase activity, ET increased tissue cAMP in endothelium-intact but not endothelium-denuded rings (P < 0.0001 and 0.25, respectively). LT pretreatment, even in high concentrations, did not significantly decrease MCF or increase EC50 (all P > 0.05). In rings precontracted with PE compared with posttreatment with PA (90 min), ET posttreatment produced progressive reductions in contractile force and increases in relaxation in endothelium-intact rings (P < 0.0001) but not endothelium-denuded rings (P = 0.51). Thus, ET may contribute to shock by producing arterial relaxation.

Impact of angiotensin II on skeletal muscle metabolism and function in mice: Contribution of IGF-1, Sirtuin-1 and PGC-1α

Activation of the renin–angiotensin–aldosterone system and increased levels of angiotensin II (Ang-II) occurs in numerous cardiovascular diseases such as chronic heart failure (CHF). Another hallmark in CHF is a reduced exercise tolerance with impaired skeletal muscle function. The aim of this study was to investigate in an animal model the impact of Ang-II on skeletal muscle function and concomitant molecular alterations. Mice were infused with Ang-II for 4 weeks. Subsequently, skeletal muscle function of the soleus muscle was assessed. Expression of selected proteins was quantified by qRT-PCR and Western blot. Infusion of Ang-II resulted in a 33% reduction of contractile force, despite a lack of changes in muscle weight. At the molecular level an increased expression of NAD(P)H oxidase and a reduced expression of Sirt1, PGC-1α and IGF-1 were noticed. No change was evident for the ubiquitin E3-ligases MuRF1 and MafBx and α-sarcomeric actin expression. Cytophotometrical analysis of the soleus muscle revealed a metabolic shift toward a glycolytic profile. This study provides direct evidence of Ang-II-mediated, metabolic deterioration of skeletal muscle function despite preserved muscle mass. One may speculate that the Ang-II-mediated loss of muscle force is due to an activation of NAD(P)H oxidase expression and a subsequent ROS-induced down regulation of IGF-1, PGC-1α and Sirt1.

Effects of MnDPDP and ICRF-187 on Doxorubicin-Induced Cardiotoxicity and Anticancer Activity

Oxidative stress participates in doxorubicin (Dx)-induced cardiotoxicity. The metal complex MnDPDP and its metabolite MnPLED possess SOD-mimetic activity, DPDP and PLED have, in addition, high affinity for iron. Mice were injected intravenously with MnDPDP, DPDP, or dexrazoxane (ICRF-187). Thirty minutes later, mice were killed, the left atria were hung in organ baths and electrically stimulated, saline or Dx was added, and the contractility was measured for 60 minutes. In parallel experiments, 10 µM MnDPDP or MnPLED was added directly into the organ bath. The effect of MnDPDP on antitumor activity of Dx against two human tumor xenografts (MX-1 and A2780) was investigated. The in vitro cytotoxic activity was studied by co-incubating A2780 cells with MnDPDP, DPDP, and/or Dx. Dx caused a marked reduction in contractile force. In vivo treatment with MnDPDP and ICRF-187 attenuated the negative effect of Dx. When added directly into the bath, MnDPDP did not protect, whereas MnPLED attenuated the Dx effect by approximately 50%. MnDPDP or ICRF-187 did not interfere negatively with the anti-tumor activity of Dx, either in vivo or in vitro. Micromolar concentrations of DPDP but not MnDPDP displayed an in vitro cytotoxic activity against A2780 cells. The present results show that MnDPDP, after being metabolized to MnPLED, protects against acute Dx cardiotoxicity. Both in vivo and in vitro experiments show that cardioprotection takes place without interfering negatively with the anticancer activity of Dx. Furthermore, the results suggest that the previously described cytotoxic in vivo activity of MnDPDP is an inherent property of DPDP.

Vascular-selective effect of lercanidipine and other 1,4-dihydropyridines in isolated rabbit tissues.

The aim of this study was to characterize the in-vitro vasoselectivity of lercanidipine (in comparison with lacidipine, amlodipine, nitrendipine and felodipine) by evaluating its functional calcium antagonistic activity on rabbit vascular (aorta) and cardiac tissues (heart ventricle). Although incubation with all the compounds tested elicited a concentration-dependent relaxant effect on vascular tissue precontracted with KCl (80 mM), 50% relaxation was reached at different times for each concentration and drug tested. At 10 nM concentration 50% relaxation was reached after 210 min with lercanidipine, 278 min with amlodipine, 135 min with lacidipine, 75 min with nitrendipine and 70 min with felodipine. The onset of the effect was, therefore, similar for lercanidipine, amlodipine and lacidipine, but faster for nitrendipine and felodipine. Similarly, all the compounds tested concentration-dependently reduced the force of cardiac contraction (negative inotropic activity). In this model, the time needed to reach 50% reduction in contractile force was also concentration-dependent, and the ranking order of the speed of onset of the effect (evaluated as the ratio of the IC50 values (the concentrations inhibiting contraction by 50%) calculated after 1 and 4 h incubation) was lacidipine (3.8) > amlodipine (9.6) > felodipine (39) > lercanidipine (68) = nitrendipine (89). The vasoselectivity, expressed as the ratio of the IC50 values obtained on cardiac and vascular tissue, were (for 4 h incubation) 730, 193, 95, 6 and 3 for lercanidipine, lacidipine, amlodipine, felodipine and nitrendipine, respectively, showing that lercanidipine is the most vasoselective of the calcium-antagonists tested. The results show that lercanidipine reduces the inotropic force of the rabbit heart to a lesser extent than do other calcium antagonists, and that this drug had the best heart/vessel selectivity index among the compounds tested at all the times tested.

Skeletal muscle contractile properties and proinflammatory cytokine gene expression in human endotoxaemia

Muscle dysfunction associated with sepsis contributes to morbidity and mortality but the underlying mechanisms are unclear. This study examined whether muscle weakness relates to an intrinsic defect in contraction, or to central mechanisms associated with acute illness, and whether systemic endotoxaemia induces changes in gene expression for proinflammatory cytokines within human muscle in vivo. In this experimental study, 12 healthy men received intravenous Escherichia coli lipopolysaccharide (LPS, 4 ng/kg) or saline (control). Voluntary and electrically stimulated quadriceps contraction, and tumour necrosis factor (TNF) alpha mRNA expression in quadriceps muscle biopsies were studied before and after the infusion. Endotoxaemia induced transient weakness of voluntary quadriceps contraction, equivalent to a 7.8 (95 per cent confidence interval 2.1 to 13.5) per cent reduction in contractile force at 180 min (P = 0.027) and a 9.0 (5.2 to 12.8) per cent reduction at 300 min (P = 0.008). Electrically stimulated contraction was unaffected. LPS administration resulted in an apparent fibre-specific induction of TNF-alpha mRNA. Endotoxaemia results in a reduction in voluntary muscle contractile force without an apparent defect in stimulated muscle contraction. Loss of volition may be a more important factor than intrinsic dysfunction in acute sepsis-associated human muscle weakness.

Found in translation: metoprolol improves survival more than carvedilol in a mouse model of inherited dilated cardiomyopathy

Over the past two decades, many mutations responsible for inherited forms of dilated cardiomyopathy (DCM) have been identified, providing important pathogenic insights. The first paradigm for explaining the role of genes in the mechanisms of ventricular dilation and dysfunction was elaborated after the identification of mutant proteins of the cytoskeleton that were implicated in defects of transmission of contractile force.1 In the year 2000, a mutation of a sarcomeric protein causally linked to DCM, a deletion (ΔLys210 or ΔK210 ) of the gene TNNT2 resulting in the loss of a lysine residue within a portion of the Ca2+-sensitive binding domain of troponin T (TnT), was described for the first time.2 Two years latter, in isolated cardiac muscle fibres,3 it was demonstrated that the functional consequence of this mutation is a decrease in the Ca2+ sensitivity of cardiac muscle contraction, suggesting a deficiency of force generation by the sarcomere as the primary mechanism of this type of DCM. The idea was proposed that both mechanisms, cytoskeletal and sarcomeric, may be implicated in the origin of DCM. Either the reduction of contractile force or its transmission leads to ventricular dilation as a compensatory mechanism for the decrease in stroke volume. In recent years, new evidence about clinical characteristics, prevalence, and risks of different mutations in patients with inherited DCM has been collected.4–7 TnT mutations (e.g. ΔLys210) seem to be particularly prevalent in cases with early-onset ventricular dilatation and dysfunction, leading to severe heart failure (HF) with poor outcome and, … *Corresponding author. Tel: +34 93 2746212; fax: +34 93 2746244. E-mail address : rioaguilar{at}pulso.com

Nitric oxide‐induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI‐17 and MYPT1

Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

Induction of muscle weakness by local inflammation: an experimental animal model

The objective of this study was to characterize the response of skeletal muscle to a localized inflammation induced by the inflammatory agent casein. An inflammatory agent, casein, was injected into the right hindlimb and saline was injected into the left hindlimb of normal adult mice, once daily for six consecutive days. Inflammatory response was monitored by immunohistochemical labeling of leukocytes. Muscle protein levels were determined by electrophoresis and muscle function was determined by isometric force measurements. Local inflammation was induced by casein in association with the accumulation of extensive neutrophils and macrophages in the soleus muscle. This local inflammation resulted in a shift in myosin heavy chain (MHC) isoform expression and a significant reduction in total MHC concentration in the soleus. Maximal twitch and tetanic forces were significantly reduced in the inflamed soleus. Contractile function in soleus was fully restored after two weeks of recovery, along with the restoration of protein concentration and the disappearance of inflammatory cells. This study establishes a unique and robust model in which mechanisms of local inflammation induced muscle protein degradation, reduction of contractile force, and subsequent recovery from this condition can be further studied.

Integrin Response To Altered Actin-Myosin Mechanochemistry In Cardiac Myocytes

Altered contractility in cardiac myocytes associated with motor protein mutations has been implicated in several pathologies such as familial hypertrophic (FHC) and dilated (DCM) cardiomyopathies. The mechanism(s) by which these altered contractile forces result in cell phenotype changes are poorly understood. However, they are generally believed to involve mechanotransduction of force by cell adhesion molecules such as the integrin class of proteins. Here we investigate the use of blebbistatin and other small molecule effectors of actin-myosin mechanochemistry as a chemical model for the reduced contractile force associated with the pathogenesis of DCM. In order to establish intercalated-disk cell associations in vitro that are representative of in vivo conditions, we employ micropatterned cardiac myocyte cell culture, where the extracellular matrix proteins collagen and laminin are printed onto the culture vessel surface in 10μm wide lines. Fluorescent polymer microspheres (0.25μm dia.) are embedded in the collagen layer. As the beads move from myocyte contraction, the movement amplitude is measured by nanometer-resolution position analysis, both in the presence of blebbistatin and in the rescue state where blebbistatin is removed. Relative force generation is calculated from the position analysis data and matrix modulus. Cells are then fixed and prepared with fluorescent antibodies for observation of connexin-43 and β1 integrin expression and localization by confocal microscopy. By employing micropatterned cardiac myocyte cell culture, optical contractile force measurement, and subsequent observation of the expression and localization of connexin-43 and β1 integrin, we assess the changes in integrin activity due to blebbistatin-induced reduction in contractile force.

The Intercostal NMJ Assay — A New Alternative to the Conventional LD50 Assay for the Determination of the Therapeutic Potency of Botulinum Toxin Preparations

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

Spatial differences of cellular origins andin vivo hypoxia modify contractile properties of pulmonary artery smooth muscle cells: lessons for arterial tissue engineering

Tissue engineering of functional arteries is challenging. Within the pulmonary artery wall, smooth muscle cells (PASMCs) have site-specific developmental and functional phenotypes, reflecting differing contractile roles. The force generated by PASMCs isolated from the inner 25% and outer 50% of the media of intrapulmonary elastic arteries from five normal and eight chronically hypoxic (hypertensive) 14 day-old piglets was quantified in a three-dimensional (3D) collagen construct, using a culture force monitor. Outer medial PASMCs from normal piglets exerted more force (528 +/- 50 dynes) than those of hypoxic piglets (177 +/- 42 dynes; p < 0.01). Force generation by inner medial PASMCs from normal and hypoxic piglets was similar (349 +/- 35 and 239 +/- 60 dynes). In response to agonist (thromboxane) stimulation, all PASMCs from normal and hypoxic piglets contracted, but the increase in force generated by outer and inner hypoxic PASMCs (ranges 13-72 and 14-56 dynes) was less than by normal PASMCs (ranges 27-154 and 34-159 dynes, respectively; p < 0.05 for both). All hypoxic PASMCs were unresponsive to antagonist (sodium nitroprusside) stimulation, all normal PASMCs relaxed (range - 87 to - 494 dynes). Myosin heavy chain expression by both hypoxic PASMC phenotypes was less than normal (p < 0.05 for both), as was the activity of focal adhesion kinase, regulating contraction, in hypoxic inner PASMCs (p < 0.01). Chronic hypoxia resulted in the development of abnormal PASMC phenotypes, which in collagen constructs exhibited a reduction in contractile force and reactivity to agonists. Characterization of the mechanical response of spatially distinct cells and modification of their behaviour by hypoxia is critical for successful tissue engineering of major blood vessels.