Reduced Force Development Research Articles

Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension.

Background Cardiac failure is the primary cause of death in most patients with pulmonary arterial hypertension (PH). As pleiotropic cytokines, human resistin (Hresistin) and its rodent homolog, resistin-like molecule α, are mechanistically critical to pulmonary vascular remodeling in PH. However, it is still unclear whether activation of these resistin-like molecules can directly cause PH-associated cardiac dysfunction and remodeling. Methods and Results In this study, we detected Hresistin protein in right ventricular (RV) tissue of patients with PH and elevated resistin-like molecule expression in RV tissues of rodents with RV hypertrophy and failure. In a humanized mouse model, cardiac-specific Hresistin overexpression was sufficient to cause cardiac dysfunction and remodeling. Dilated hearts exhibited reduced force development and decreased intracellular Ca2+ transients. In the RV tissues overexpressing Hresistin, the impaired contractility was associated with the suppression of protein kinase A and AMP-activated protein kinase. Mechanistically, Hresistin activation triggered the inflammation mediated by signaling of the key damage-associated molecular pattern molecule high-mobility group box 1, and subsequently induced pro-proliferative Ki67 in RV tissues of the transgenic mice. Intriguingly, an anti-Hresistin human antibody that we generated protected the myocardium from hypertrophy and failure in the rodent PH models. Conclusions Our data indicate that Hresistin is expressed in heart tissues and plays a role in the development of RV dysfunction and maladaptive remodeling through its immunoregulatory activities. Targeting this signaling to modulate cardiac inflammation may offer a promising strategy to treat PH-associated RV hypertrophy and failure in humans.

Atrial cardiomyocytes from a canine model of atrial fibrillation demonstrate reduced force development and altered expression of contractile proteins.

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and associates with worse outcomes in cardiac contractile-related diseases including cardiomyopathy. As most studies surrounding AF focus on the electrophysiology of the disease, there is little known about cardiomyocyte contractile function in AF. We used atrial tissue from an established atrial tachypacing canine model of AF. Whole protein lysates were collected from the atria to conduct mass spectrometry analysis showing dysregulation of numerous molecular pathways, including contractility. Skinned atrial cardiomyocytes were isolated from the tissue for force calcium and passive tension experiments. Force calcium experiments showed a significant reduction in the maximum force of contraction of AF cardiomyocytes. To explain this, myosin heavy chain (MHC) isoform expression was evaluated because isoform switching is known to alter force of contraction. Decreased percent beta expression was found, explaining the reduction of force due to MHC alpha's capacity for reduced force of contraction. AF cardiomyocytes showed increased calcium sensitivity of force development, which may translate to prolonged contraction with slower relaxation in vivo. A significant reduction in S100A1 was seen in the AF samples via mass spectrometry, which is associated with increased calcium sensitivity. Prolonged contraction along with reduced force of each contraction could support the development of atrial clots. Atrial cardiomyocytes from the AF hearts demonstrated a significant reduction in the passive tension. Evaluation of titin isoform expression, which is known to alter passive tension, showed increased titin degradation, supporting the capacity for atrial dilation in vivo. Discerning the role of contractility in the pathology of atrial fibrillation could provide novel avenues of AF treatment, especially in instances where it is associated with contractile diseases.

Anesthetic Agents Isoflurane and Propofol Decrease Maximal Ca2+-Activated Force and Thus Contractility in the Failing Myocardium

In the normal heart, frequently used anesthetics such as isoflurane and propofol can reduce inotropy. However, the impact of these agents on the failing myocardium is unclear. Here, we examined whether and how isoflurane and propofol influence cardiac contractility in intact cardiac muscles from rats treated with monocrotaline to induce heart failure. We measured force and intracellular Ca2+ ([Ca2 +]i) in trabeculae from the right ventricles of the rats in the absence or presence of propofol or isoflurane. At low to moderate concentrations, both propofol and isoflurane dose-dependently depressed cardiac force generation in failing trabeculae without altering [Ca2+]i At high doses, propofol (but not isoflurane) also decreased amplitude of [Ca2+]i transients. During steady-state activation, both propofol and isoflurane impaired maximal Ca2+-activated force (Fmax) while increasing the amount of [Ca2+]i required for 50% of maximal activation (Ca50). These events occurred without apparent change in the Hill coefficient, suggesting no impairment of cooperativity. Exposing these same muscles to the anesthetics after fiber skinning resulted in a similar decrement in Fmax and rise in Ca50 but no change in the myofibrillar ATPase-Ca2+ relationship. Thus, our study demonstrates that challenging the failing myocardium with commonly used anesthetic agents such as propofol and isoflurane leads to reduced force development as a result of lowered myofilament responsiveness to Ca2+ SIGNIFICANCE STATEMENT: Commonly used anesthetics such as isoflurane and propofol can impair myocardial contractility in subjects with heart failure by lowering myofilament responsiveness to Ca2+. High doses of propofol can also reduce the overall amplitude of the intracellular Ca2+ transient. These findings may have important implications for the safety and quality of intra- and perioperative care of patients with heart failure and other cardiac disorders.

Ca2+ ] changes in sympathetic varicosities and Schwann cells in rat mesenteric arteries-Relation to noradrenaline release and contraction.

This study aimed to assess intracellular Ca2+ dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development. Ca2+ in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca2+ in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells. Schwann cells but not axons responded with a Ca2+ increase to ATP. Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca2+ ] ([Ca2+ ]v ). ω-conotoxin-GVIA (100nmol/L) reduced the [Ca2+ ]v transient to 2 and 16Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16Hz. The KV channel blocker, 4-aminopyridine (10µmol/L), increased [Ca2+ ]v , noradrenaline release and force development both in the absence and presence of ω-conotoxin-GVIA. Yohimbine (1µmol/L) increased both [Ca2+ ]v and noradrenaline release but reduced force development. Acetylcholine (10µmol/L) caused atropine-sensitive inhibition of [Ca2+ ]v , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters. Modification of [Ca2+ ] in arterial sympathetic axons and Schwann cells was assessed separately. KV 3.1 channels may be important regulators of [Ca2+ ]v , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca2+ ]v .

Genetic approaches to identify pathological limitations in aortic smooth muscle contraction.

Aortic smooth muscle contains limiting amounts of myosin light chain kinase (MLCK) for myosin regulatory light chain (RLC) phosphorylation and contraction that predisposes to thoracic aortic disease in humans containing heterozygous loss-of-function mutations in MYLK. We tested the hypothesis that thoracic aortic smooth muscle contraction may also be susceptible to variations in the smooth muscle-specific isoform of the motor protein myosin where inactivation of one Myh11 allele or the presence of one Myh11 missense variant associated with an increased risk of human aortic disease may result in a reduced force development response. Additionally, other kinds of smooth muscles may be less sensitive to the effects of mutations in one smooth muscle myosin allele, similar to results obtained with Mylk. Force development responses were reduced in aortic tissue from a conditional knockout of smooth muscle myosin heavy chain in adult mice (Myh11+/- or Myh11-/-) with a greater reduction with homozygous vs heterozygous tissues. Similar reductions in force responses were obtained with tissues containing either a heterozygous or homozygous knockin mutation in smooth muscle myosin heavy chain (Myh11+/R247C or Myh11R247C/R247C mutations that cause human aortic disease) with no significant changes in RLC phosphorylation. Agonist-dependent force responses were not reduced significantly in urinary bladder, ileal, or tracheal tissues from Myh11+/- mice while only ileal tissue showed a reduced force response in Myh11R247C/R247C mice. Thus, heterozygous mutations in Myh11 associated with reduced myosin function result in compromised contractile function primarily in aortic smooth muscle.

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Exercise training can prevent cardiac hypertrophy induced by sympathetic hyperactivity with modulation of kallikrein-kinin pathway and angiogenesis.

Sympathetic hyperactivity induces adverse effects in myocardial. Recent studies have shown that exercise training induces cardioprotection against sympathetic overload; however, relevant mechanisms of this issue remain unclear. We analyzed whether exercise can prevent pathological hypertrophy induced by sympathetic hyperactivity with modulation of the kallikrein-kinin and angiogenesis pathways. Male Wistar rats were assigned to non-trained group that received vehicle; non-trained isoproterenol treated group (Iso, 0.3 mg kg−1 day−1); and trained group (Iso+Exe) which was subjected to sympathetic hyperactivity with isoproterenol. The Iso rats showed hypertrophy and myocardial dysfunction with reduced force development and relaxation of muscle. The isoproterenol induced severe fibrosis, apoptosis and reduced myocardial capillary. Interestingly, exercise blunted hypertrophy, myocardial dysfunction, fibrosis, apoptosis and capillary decreases. The sympathetic hyperactivity was associated with high abundance of ANF mRNA and β-MHC mRNA, which was significantly attenuated by exercise. The tissue kallikrein was augmented in the Iso+Exe group, and kinin B1 receptor mRNA was increased in the Iso group. Moreover, exercise induced an increase of kinin B2 receptor mRNA in myocardial. The myocardial content of eNOS, VEGF, VEGF receptor 2, pAkt and Bcl-2 were increased in the Iso+Exe group. Likewise, increased expression of pro-apoptotic Bad in the Iso rats was prevented by prior exercise. Our results represent the first demonstration that exercise can modulate kallikrein-kinin and angiogenesis pathways in the myocardial on sympathetic hyperactivity. These findings suggest that kallikrein-kinin and angiogenesis may have a key role in protecting the heart.

Effect of Intermittent Phrenic Nerve Stimulation During Cardiothoracic Surgery on Mitochondrial Respiration in the Human Diaphragm*

Recent studies have shown that brief periods of mechanical ventilation in animals and humans can lead to ventilator-induced diaphragmatic dysfunction, which includes muscle atrophy, reduced force development, and impaired mitochondrial function. Studies in animal models have shown that short periods of increased diaphragm activity during mechanical ventilation support can attenuate ventilator-induced diaphragmatic dysfunction but corresponding human data are lacking. The purpose of this study was to examine the effect of intermittent diaphragm contractions during cardiothoracic surgery, including controlled mechanical ventilation, on mitochondrial respiration in the human diaphragm. Within subjects repeated measures study. Operating room in an academic health center. Five subjects undergoing elective cardiothoracic surgery. In patients (age 65.6 ± 6.3 yr) undergoing cardiothoracic surgery, one phrenic nerve was stimulated hourly (30 pulses/min, 1.5 msec duration, 17.0 ± 4.4 mA) during the surgery. Subjects received 3.4 ± 0.6 stimulation bouts during surgery. Thirty minutes following the last stimulation bout, samples of diaphragm muscle were obtained from the anterolateral costal regions of the stimulated and inactive hemidiaphragms. Mitochondrial respiration was measured in permeabilized muscle fibers with high-resolution respirometry. State III mitochondrial respiration rates (pmol O2/s/mg wet weight) were 15.05 ± 3.92 and 11.42 ± 2.66 for the stimulated and unstimulated samples, respectively (p < 0.05). State IV mitochondrial respiration rates were 3.59 ± 1.25 and 2.11 ± 0.97 in the stimulated samples and controls samples, respectively (p < 0.05). These are the first data examining the effect of intermittent contractions on mitochondrial respiration rates in the human diaphragm following surgery/mechanical ventilation. Our results indicate that very brief periods (duty cycle ~1.7%) of activity can improve mitochondrial function in the human diaphragm following surgery/mechanical ventilation.

Actin Carbonylation is Higher in Human Hypertrophic Cardiomyopathy Due to MYH7 Mutations

Introduction: Hypertrophic cardiomyopathy(HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Decreased force development was found in human HCM due to mutations in genes encoding myosin binding protein-C(MYBPC3), myosin heavy chain(MYH7) and in sarcomere mutation-negative HCM(HCMsmn). This is partially caused by the mutation (especially MYH7 mutations), increased cell size and reduced myofibrillar density. However, 10% force reduction could not be explained by the mutation or cellular remodeling. In end-stage human heart failure increased actin-carbonylation, induced by oxidative stress, negatively correlated with myocardial function. Therefore, we investigated whether actin is carbonylated in human HCM.Methods and results: Left ventricular tissue of human HCMsmn, MYBPC3mut and MYH7mut patients was obtained. Non-failing donors were used as control. Actin-carbonylation was analyzed using Oxyblot. Carbonyl groups of myofilament proteins were derivatized by 2,4-dinitrophenyl-hydrazine(DNPH). Protein lysates were processed via SDS-page, western blot and chemiluminescence using appropriate antibodies. Actin-carbonylation was higher in HCM patients compared to donors. It was highest in MYH7mut(Figure).Conclusion: Increased actin-carbonylation could partially underlie reduced force development in human HCM. Increased oxidative stress may contribute to impaired contractile function during disease development in sarcomere mutation-positive and mutation-negative HCM patients.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Muscle capillary supply takes the load

Muscle capillary supply takes the load

Abstract 3789: Induction of Murf-1 is Required for Tnf-alpha Induced Loss of Skeletal Muscle Function

Background: Inflammatory cytokines like tumor necrosis factor alpha (TNF-α) are known to impair skeletal muscle (SM) function. Furthermore, TNF-α induces the expression of atrogin-like muscle specific ubiquitin E3-ligases, presumed to mediate muscle atrophy. The relative contributions of respective ubiquitin ligases, like MuRF1 for the TNF-α induced reduction in muscle function are not known. Methods: TNF-α or saline was injected either into C57Bl6 or MuRF1 −/− mice. After 16 –24h the expression of MuRF1 in the SM was quantified by qRT-PCR and western blot. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, 2D-gel electrophoresis was performed. Results: WT animals injected with TNF-α had higher MuRF1 mRNA (saline: 56.6±12.1 vs. TNF-α: 133.6±30.3 arb. Units; p&lt;0.05) and protein expression (saline: 0.38±0.11 vs. TNF-α: 1.07±0.25 arb. Units; p&lt;0.05) as compared to saline injected littermates. However, TNF- α was unable to induce MurRF1 expression in MuRF1 −/− mice. Furthermore, TNF- α reduced force development at 150Hz by 25% in C57Bl6 animals (saline: 2412±120 vs. TNF-α: 1799±114 g/cm2; p&lt;0.05), but not in MuRF1 −/− mice (saline: 2424±198 vs. TNF-α: 2431±180 g/cm2; p=NS). The proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T (TNNT3) in WT animals treated with TNF- α as compared to MuRF1 −/− mice receiving TNF-α . In addition, TNF-α injection into C57Bl6 animals resulted in a down-regulation of eEF1γ ( WT: 0.60±0.02 vs. WT+TNF-α: 0.39±0.05 arb. Units; p&lt;0.05). This reduction was not seen in MuRF1 −/− mice receiving TNF- α (KO: 0.59±0.03 vs. KO+TNF-α: 0.68±0.01 arb. Units; p&lt;0.05) Conclusion: The results of this study demonstrate for the first time, that the TNF- α induced reduction in SM force development depends on the induction of the atrophy related E3-ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α-MuRF1-mediated down-regulation of TNNT3 and the elongation factor eEF1γ.

Induction of MuRF1 Is Essential for TNF-α-Induced Loss of Muscle Function in Mice

BackgroundHumoral circulating inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) can impair skeletal muscle contractility. Furthermore, TNF-α expression correlates with elevated levels of atrogin-like muscle-specific ubiquitin E3 ligases, which are presumed to mediate muscle protein breakdown and atrophy. However, the casual relationships between MuRF1 and TNF-α and their relative contributions to muscle function impairment are not known. MethodsTNF-α or saline was injected into either C57Bl6 or MuRF1−/− mice. After 16–24 h, the expression of MuRF1 in skeletal muscle was quantified by quantitative reverse transcription–PCR and Western blot analysis. Muscle function was measured in an organ bath. To obtain a broader overview on potential alterations, two-dimensional gel electrophoresis was performed. ResultsWild-type animals injected with TNF-α had higher MuRF1 mRNA expression (saline versus TNF-α: 56.6±12.1 versus 133.6±30.3 arbitrary units; p<0.05) and protein expression (saline versus TNF-α: 0.38±0.11 versus 1.07±0.25 arbitrary units; p<0.05) as compared to saline-injected littermates. Furthermore, TNF-α reduced force development at 150 Hz by 25% in C57Bl6 animals (saline versus TNF-α: 2412±120 versus 1799±114 g/cm2; p<0.05), but not in MuRF1−/− mice (saline versus TNF-α: 2424±198 versus 2431±180 g/cm2; p=NS). Proteome analysis revealed a significant down-regulation of fast skeletal muscle troponin T in wild-type animals treated with TNF-α as compared to MuRF1−/− mice that received TNF-α. ConclusionThe results of this study demonstrate for the first time that TNF-α-induced reduction in skeletal muscle force development depends on the induction of the atrophy-related E3 ubiquitin ligase MuRF1. A link for the reduction in muscle force may be the TNF-α/MuRF1-mediated down-regulation of fast skeletal muscle troponin T.

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development.

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.

Functional genomics of pressure-loaded cardiomyocytes: etomoxir in heart failure?

Drugs for counteracting the neuroendocrine activation in heart failure can reduce the adverse remodelling of the extracellular matrix of the heart. Progression of heart failure can, however, often not be prevented and the question arises whether important pharmacological targets remain unidentified. Promising are drugs targeted at ventricular diastolic dysfunction which is a marker of early progression of heart failure. Left ventricular dysfunction is characteristic of overloaded hypertrophied hearts with molecular structures that are not adapted to the increased Ca2+ diffusion distances. Thus, the Ca(2+)-pump (SERCA2) of sarcoplasmic reticulum is inadequately expressed leading to a reduced force development and relaxation of hypertrophied cardiomyocytes. Drugs in development (CPT-1 inhibitor/PPARalpha activator) that increase glucose oxidation can enhance SERCA2 expression. The lead compound etomoxir had a selective influence on the contraction and relaxation rate of pressure-overloaded hearts. The functional parameters were correlated with the proportion of alpha-myosin heavy chains. Since viral or inflammatory injury of the heart can also induce a fetal phenotype, metabolic modulators such as etomoxir represent a promising therapeutic approach also for cardiomyopathies with inadequate SERCA2 expression.

Role of sarcoplasmic reticulum in regulation of tonic contraction of rabbit basilar artery.

Superficial sarcoplasmic reticulum (SR) regulates smooth muscle force development directly by Ca(2+) release and removal to and from the cytoplasm (Somlyo and Somlyo. J Cardiovasc Pharmacol 8, Suppl 8: S42-S47, 1986) by buffering Ca(2+) influx and contributing to Ca(2+) extrusion (Mueller and van Breemen. Nature 281: 682-683, 1979) and indirectly by releasing Ca(2+) near Ca(2+)-activated K(+) channels (K(Ca)) to hyperpolarize the plasma membrane (Bolton and Imaizumi. Cell Calcium 20: 141-152, 1996 and Nelson et al. Science 270: 633-637, 1995). In the rabbit basilar artery, relative contributions of direct effects and those mediated through activation of K(Ca) were evaluated by measuring force and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to the SR-depleting agents thapsigargin and ryanodine and the large conductance K(Ca) (BK(Ca)) blockers iberiotoxin (IbTX) and tetraethylammonium ion (TEA). A large contraction was observed in response to K(Ca) blockade with either 3 mM TEA or 100 nM IbTX and also after addition of 10 microM ryanodine or 2 microM thapsigargin. When K(Ca) was blocked first with TEA or IbTX, subsequent addition of thapsigargin or ryanodine also increased force. Measurements of fura 2 fluorescence showed parallel increases in [Ca(2+)](i) in response to sequential blockade of sarco(endo)plasmic reticulum Ca(2+)-ATPase and K(Ca) regardless of the order of application. It appears that a significant fraction of K(Ca) remains activated in the absence of SR function and that SR contributes to relaxation after blockade of K(Ca). We found that depletion of SR before stimulating Ca(2+) influx through voltage-gated Ca(2+) channels markedly reduced force development rate and that thapsigargin abolished this effect. We conclude that the SR of rabbit cerebral arteries modulates constriction by direct and indirect mechanisms.

Right ventricular contractile protein function in rats with left ventricular myocardial infarction.

We studied contractile function in cardiac trabeculae isolated from the right ventricles (RV) of rats with experimental heart failure (HF) induced by left ventricular (LV) myocardial infarction (24 wk post-MI; n = 6) and from sham-operated rats (n = 7). Sarcomere length (SL) was measured by laser diffraction techniques, and force (F) was measured by silicon strain gauge. SL was kept constant at all times by computer feedback control. HF was associated with marked LV dilation and pulmonary congestion. In intact, RV twitching trabeculae, HF was associated with a depression of the F-SL relation at extracellular Ca2+ concentration ([Ca2+]o) = 1.5 mM and a depression of the F-[Ca2+]o relation at SL = 2.0 microns. HF was also associated with a significant depression of the F-intracellular [Ca2+] relation at SL = 2.0 microns measured after chemical permeabilization of these RV trabeculae (skinned fibers). Our results suggest that reduced force development in this model of HF is due, in part, to depressed function of the contractile filaments.

Myosin light chain phosphorylation in contraction of gastric antral smooth muscle from neonate and adult rabbits.

The decreased contractility of gastric antral smooth muscle in the neonate has been attributed to reduced levels of activator calcium. It is generally accepted that calcium-dependent myosin light chain phosphorylation (MLCP) is the key step in the initiation of force development in smooth muscle. In this study, we investigated the relationship between MLCP and force development in gastric antral smooth muscle from neonatal (4-6 d old) and adult rabbits. We tested the hypothesis that the reduced force development of circular smooth muscle from the neonate would be accompanied by decreased levels of MLCP, as compared with data from adult animals. Full thickness muscle strips oriented parallel to the circular muscle layer were examined for their contractile response to acetylcholine (ACh) (10(-8) M to 10(-3) M) or 10(-4) M ACh only. In the latter study, tissues were rapidly frozen in a dry ice-acetone slurry for subsequent MLCP determination. MLCP was determined at times corresponding to 5, 10, 15, 30, and 60 s of stimulation. For each age group, maximal active force developed at an ACh concentration of 10(-4) M and was significantly greater in tissues from adults (1.86 +/- 0.24 N/m2, adult; 0.95 +/- 0.05 N/m2, neonate; p < 0.05). In contrast, no significant differences were observed with respect to basal or agonist-stimulated levels of MLCP. The data suggest that factors other than levels of MLCP contribute to the reduced force-generating capacity of antral smooth muscle from the neonate.

Fibrin structure and concentration alter clot elastic modulus but do not alter platelet mediated force development

During clot retraction, platelets interact with fibrin resulting in marked reduction of clot volume. Altered fibrin structure has been reported to affect clot retraction as measured by serum expression. This study was performed to test whether such altered retraction was the result of increased resistance to network collapse or due to decreased force development by platelets. Altered fibrin structure was documented as variation of fibre mass/length ratios (mu) and shifts in clot elastic modulus. The force developed by platelets during clotting was measured directly. Increasing the fibrinogen concentration led to thinner fibre formation (decreased mu), and a linear increase in gel elastic modulus. Over a fibrinogen concentration range of 100 to 400 mg/dl, force development was minimally affected. Force development and clot elastic modulus increased in a linear fashion with increasing platelet concentration. Increasing the calcium concentration from 5 to 20 mM caused a 160% increase in fibrin fibre size (mu), and a 52% decline in clot modulus. Force developed at 1200 s declined by 17%. At 15 mg/ml, dextran and hydroxyethyl starch (HES) also increased mu, and decreased clot modulus; however, both agents markedly reduced force development. Increasing ionic strength or the addition of IgG decreased mu and increased gel elastic modulus. Force development increased modestly with increased ionic strength, did not change with addition of IgG in saline and declined with addition of IgG in maltose. This study indicates that force development is primarily dependent on platelet function while clot modulus depends on both fibrin structure and platelet function.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypoxia on force, intracellular pH and Ca2+ concentration in rat cerebral and mesenteric small arteries.

1. The effect of severe hypoxia on force, intracellular Ca2+ concentration ([Ca2+]i) and pHi was studied in isolated small arteries from rat brain and rat mesenterium. The arteries were mounted for isometric force recording while [Ca2+]i was measured with fura-2 or pHi was measured with bis-carboxyethylcarboxyfluorescein (BCECF). 2. Hypoxia reduced the force development in response to arginine vasopressin (AVP) while [Ca2+]i was unchanged or only slightly reduced. Inhibition of acid extrusion by omission of sodium caused no force development in mesenteric arteries, but the fall in pHi was enhanced during hypoxia. In cerebral arteries, hypoxia reduced the force development associated with omission of sodium, and the fall in pHi was less than during normoxic conditions. When acid extrusion was intact, pHi was not affected by hypoxia and the changes in pHi during activation with AVP were similar during hypoxia and in the control situation. 3. Although a decrease in smooth muscle [Ca2+]i may be partly responsible for the reduced force development during hypoxia, [Ca2+]i-independent mechanism(s) may play an even more important role. Furthermore, although hypoxia and force development are associated with enhanced acid production, acid extrusion maintains pHi near the control level and it is unlikely that a decrease in smooth muscle pHi plays any role in the reduced force development during hypoxia.

Glycoprotein IIb/IIIa Blockade Inhibits Platelet-mediated Force Development and Reduces Gel Elastic Modulus

The effects of GPIIb/IIIa blockade on clot retraction were studied utilizing an instrument which directly measures force produced by platelets. GPIIb/IIIa disruption by calcium chelation, and GPIIb/IIIa blockade by peptides and anti-GPIIb/IIIa antibodies were investigated. One mM EDTA suppressed ADP-induced platelet aggregation by 72% and reduced force developed at 1200 s by 33%. At 234 microM, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) suppressed platelet aggregation by 74%, reduced force at 1200 s by 45% and reduced gel elastic modulus by 19%. At 10 microM, the peptide D-Arg-Gly-L-Asp-L-Try (D-RGDW) completely suppressed platelet aggregation, reduced force development by 38% and reduced gel elastic modulus by 29%. At 0.133 microM, monoclonal anti-GPIIIa antibody (AP-3) reduced force development by 74% and reduced gel modulus by 60%. Murine antiGPIIb/IIIa antibodies 10E5 and 7E3 markedly suppressed force development. At 0.133 microM, 10E5 reduced force by 89% and reduced gel modulus by 67%. At 0.053 microM, 7E3 completely stopped force development and reduced gel modulus by 46%. Platelet aggregation was blocked by 0.027 microM 7E3. Selective GPIIb blockade by antibodies did not affect force development. None of the agents studied altered fibrin structure as monitored by effects of fibrin mass/length ratios. Suppression of platelet aggregation occurred at inhibitor concentrations substantially lower than those required to suppress force development. Complete suppression of platelet aggregation did not assure inhibition of clot retraction probably due to profound platelet activation by thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)