The Contractile Machines of the Heart.

This chapter will describe basic structural and functional features of the contractile apparatus of muscle cells of the heart, namely, cardiomyocytes and smooth muscle cells. Cardiomyocytes form the contractile myocardium of the heart, while smooth muscle cells form the contractile coronary vessels. Both muscle types have distinct properties and will be considered with respect to their cellular appearance (brick-like cross-striated versus spindle-like smooth), arrangement of contractile proteins (sarcomeric versus non-sarcomeric organization), calcium activation mechanisms (thin-filament versus thick-filament regulation), contractile features (fast and phasic versus slow and tonic), energy metabolism (high oxygen versus low oxygen demand), molecular motors (type II myosin isoenzymes with high adenosine diphosphate [ADP]-release rate versus myosin isoenzymes with low ADP-release rates), chemomechanical energy conversion (high adenosine triphosphate [ATP] consumption and short duty ratio versus low ATP consumption and high duty ratio of myosin II cross-bridges [XBs]), and excitation-contraction coupling (calcium-induced calcium release versus pharmacomechanical coupling). Part of the work has been published (Neuroscience - From Molecules to Behavior", Chap. 22, Galizia and Lledo eds 2013, Springer-Verlag; with kind permission from Springer Science + Business Media).

Modulation of Titin and Contraction-Regulating Proteins in a Rat Model of Heart Failure with Preserved Ejection Fraction: Limb vs. Diaphragmatic Muscle.

Heart failure with preserved ejection fraction (HFpEF) is characterized by biomechanically dysfunctional cardiomyocytes. Underlying cellular changes include perturbed myocardial titin expression and titin hypophosphorylation leading to titin filament stiffening. Beside these well-studied alterations at the cardiomyocyte level, exercise intolerance is another hallmark of HFpEF caused by molecular alterations in skeletal muscle (SKM). Currently, there is a lack of data regarding titin modulation in the SKM of HFpEF. Therefore, the aim of the present study was to analyze molecular alterations in limb SKM (tibialis anterior (TA)) and in the diaphragm (Dia), as a more central SKM, with a focus on titin, titin phosphorylation, and contraction-regulating proteins. This study was performed with muscle tissue, obtained from 32-week old female ZSF-1 rats, an established a HFpEF rat model. Our results showed a hyperphosphorylation of titin in limb SKM, based on enhanced phosphorylation at the PEVK region, which is known to lead to titin filament stiffening. This hyperphosphorylation could be reversed by high-intensity interval training (HIIT). Additionally, a negative correlation occurring between the phosphorylation state of titin and the muscle force in the limb SKM was evident. For the Dia, no alterations in the phosphorylation state of titin could be detected. Supported by data of previous studies, this suggests an exercise effect of the Dia in HFpEF. Regarding the expression of contraction regulating proteins, significant differences between Dia and limb SKM could be detected, supporting muscle atrophy and dysfunction in limb SKM, but not in the Dia. Altogether, these data suggest a correlation between titin stiffening and the appearance of exercise intolerance in HFpEF, as well as a differential regulation between different SKM groups.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Effects of proinflammatory cytokines on lacrimal gland myoepithelial cells contraction.

In the lacrimal gland, myoepithelial cells (MEC) express muscle contractile proteins such as alpha smooth muscle actin (SMA) and calponin and therefore can contract to help expel lacrimal fluid. In a previous study, we demonstrated that lacrimal gland MEC express the oxytocin receptor (OXTR) and they contract under oxytocin (OXT) stimulation. Using NOD and MRL/lpr mice (animal models of Sjogren's syndrome), we reported a decrease in SMA and calponin protein levels plus a decline in acini contraction after stimulation with OXT. It is known that proinflammatory cytokines, such as interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-α) or interferon gamma (IFN-γ), can affect OXTR expression and signaling capacity and inhibit MEC contraction. The aim of the current study was to investigate if proinflammatory cytokines are implicated in the loss of MEC contractile ability. Thus, lacrimal gland MEC from a SMA-GFP transgenic mouse were treated with IL-1ß (10 ng/ml) for a total of 7 days. At days 0, 2, 4 and 7, GFP intensity, cell size/area, contractile proteins amounts and MEC contraction were assessed. At day 0, control and treated cells showed no differences in GFP intensity and cell size. GFP intensity started to decrease in treated MEC at day 2 (20%; p=0.02), continuing after day 4 (25%; p=0.007) and 7 (30%; p=0.0001). Mean cell area was also reduced at day 2 (34%; p=0.0005), and after 4 (51%; p<0.0001) and 7 days (30%; p=0.0015). The contraction assay at day 2 showed a 70% decrease of contraction in treated MEC (p<0.0001), 73% (p<0.0001) at day 4 and 82% (p=0.0015) at day 7 when compared to control. Levels of contractile proteins were measured on day 7 showing a decrease in SMA and calponin amount in treated MEC compared with the control group (around 30%; p=0.0016 and p=0.0206; respectively). Similar results were observed when TNF-α and IFN-γ were added along with IL-1ß. Taken together the present data and those from our previous studies with Sjogren's syndrome mouse models, they strongly suggest that proinflammatory cytokines affect lacrimal gland MEC contractile ability that may account for the reduced tear secretion associated with Sjogren's syndrome dry eye disease.

Diet-Induced Hypercholesterolemia Leads to Cardiac Dysfunction and Alterations in the Myocardial Proteome.

Elevated blood cholesterol is a major risk factor for coronary heart disease. Moreover, direct effects on the myocardium also contribute to the adverse effects of hypercholesterolemia. Here, we investigated the effect of hypercholesterolemia on the cardiac proteome. Male Wistar rats were fed with a laboratory rodent chow supplemented with 2% cholesterol for 8 weeks to induce hypercholesterolemia. The protein expression data obtained from the proteomic characterization of left ventricular samples from normo- and hypercholesterolemic animals were subjected to gene ontology (GO) and protein interaction analyses. Elevated circulating cholesterol levels were accompanied by diastolic dysfunction in cholesterol-fed rats. The proteomic characterization of left ventricular samples revealed altered expression of 45 proteins due to hypercholesterolemia. Based on the Gene Ontology analysis, hypercholesterolemia was associated with disturbed expression of cytoskeletal and contractile proteins. Beta-actin was downregulated in the hypercholesterolemic myocardium, and established a prominent hub of the protein interaction network. Analysis of the unfiltered dataset revealed concordant downregulated expression patterns in proteins associated with the arrangement of the contractile system (e.g., cardiac-specific troponins and myosin complex), and in subunits of the mitochondrial respiratory chain. We conclude that the observed changes in the cardiac proteome may contribute to the development of diastolic dysfunction in hypercholesterolemia.

Stabilizing Ryanodine Receptors Improves Left Ventricular Function in Juvenile Dogs With Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy is associated with progressive deterioration in left ventricular (LV) function. The golden retriever muscular dystrophy (GRMD) dog model recapitulates the pathology and clinical manifestations of Duchenne muscular dystrophy. Importantly, they develop progressive LV dysfunction starting at early age. OBJECTIVES: The authors tested the cardioprotective effect of chronic administration of the ARM036, a small molecule that stabilizes the closed conformation of the cardiac sarcoplasmic reticulum ryanodine receptor/calcium release channel (RyR2) in young GRMD-dogs. METHODS: Two-month-old GRMD-dogs were treated with ARM036 or placebo for 4 months. Healthy-dogs of the same genetic background served as controls. Cardiac function was evaluated by conventional and 2-dimensional speckle-tracking echocardiography. Cardiac cellular and molecular analyses were performed at 6 months old. RESULTS: Conventional echocardiography showed normal LV dimensions and ejection fraction in 6-month-old GRMD dogs. Interestingly, 2-dimensional speckle-tracking echocardiography revealed decreased global longitudinal strain and the presence of hypokinetic segments in placebo-treated GRMD dogs. Single-channel measurements revealed higher RyR2 open probability at low resting Ca2+ in GRMD cardiomyocytes than in controls. ARM036 prevented those in vivo and in vitro dysfunctions in GRMD dogs. Myofilament Ca2+-sensitivity was increased in permeabilized GRMD cardiomyocytes at short sarcomere length. ARM036 had no effect on this parameter. Cross-bridge cycling kinetics were altered in GRMD myocytes and recovered with ARM036 treatment, which coincided with the level of myosin binding protein-C-S glutathionylation. CONCLUSIONS: GRMD-dogs exhibit early LV dysfunction associated with altered myofilament contractile properties. These abnormalities were prevented pharmacologically by stabilizing RyR2 with ARM036.

UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin.

The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (KD = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1-UBE2L3 affinity, binding of MHC to MuRF1 (KD = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.

Postnatal development alters functional compartmentalization of myosin light chain kinase in ovine carotid arteries.

The rate-limiting enzyme for vascular contraction, myosin light chain kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared with adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from nonpregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 s, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded, respectively, to 43 ± 10% and 31 ± 3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in periluminal than periadventitial regions over the time course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.

Cardiac myosin-binding protein C interaction with actin is inhibited by compounds identified in a high-throughput fluorescence lifetime screen.

Cardiac myosin-binding protein C (cMyBP-C) interacts with actin and myosin to modulate cardiac muscle contractility. These interactions are disfavored by cMyBP-C phosphorylation. Heart failure patients often display decreased cMyBP-C phosphorylation, and phosphorylation in model systems has been shown to be cardioprotective against heart failure. Therefore, cMyBP-C is a potential target for heart failure drugs that mimic phosphorylation or perturb its interactions with actin/myosin. Here we have used a novel fluorescence lifetime-based assay to identify small-molecule inhibitors of actin-cMyBP-C binding. Actin was labeled with a fluorescent dye (Alexa Fluor 568, AF568) near its cMyBP-C binding sites; when combined with the cMyBP-C N-terminal fragment, C0-C2, the fluorescence lifetime of AF568-actin decreases. Using this reduction in lifetime as a readout of actin binding, a high-throughput screen of a 1280-compound library identified three reproducible hit compounds (suramin, NF023, and aurintricarboxylic acid) that reduced C0-C2 binding to actin in the micromolar range. Binding of phosphorylated C0-C2 was also blocked by these compounds. That they specifically block binding was confirmed by an actin-C0-C2 time-resolved FRET (TR-FRET) binding assay. Isothermal titration calorimetry (ITC) and transient phosphorescence anisotropy (TPA) confirmed that these compounds bind to cMyBP-C, but not to actin. TPA results were also consistent with these compounds inhibiting C0-C2 binding to actin. We conclude that the actin-cMyBP-C fluorescence lifetime assay permits detection of pharmacologically active compounds that affect cMyBP-C-actin binding. We now have, for the first time, a validated high-throughput screen focused on cMyBP-C, a regulator of cardiac muscle contractility and known key factor in heart failure.

Post-Translational Modification Networks of Contractile and Cellular Stress Response Proteins in Bladder Ischemia.

Molecular mechanisms underlying bladder dysfunction in ischemia, particularly at the protein and protein modification levels and downstream pathways, remain largely unknown. Here we describe a comparison of protein sequence variations in the ischemic and normal bladder tissues by measuring the mass differences of the coding amino acids and actual residues crossing the proteome. A large number of nonzero delta masses (11,056) were detected, spanning over 1295 protein residues. Clustering analysis identified 12 delta mass clusters that were significantly dysregulated, involving 30 upregulated (R2 > 0.5, ratio > 2, p < 0.05) and 33 downregulated (R2 > 0.5, ratio < -2, p < 0.05) proteins in bladder ischemia. These protein residues had different mass weights from those of the standard coding amino acids, suggesting the formation of non-coded amino acid (ncAA) residues in bladder ischemia. Pathway, gene ontology, and protein-protein interaction network analyses of these ischemia-associated delta-mass containing proteins indicated that ischemia provoked several amino acid variations, potentially post-translational modifications, in the contractile proteins and stress response molecules in the bladder. Accumulation of ncAAs may be a novel biomarker of smooth muscle dysfunction, with diagnostic potential for bladder dysfunction. Our data suggest that systematic assessment of global protein modifications may be crucial to the characterization of ischemic conditions in general and the pathomechanism of bladder dysfunction in ischemia.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. METHODS: Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. RESULTS: Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. CONCLUSION AND INFERENCES: The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

MuRF1/TRIM63, Master Regulator of Muscle Mass.

The E3 ubiquitin ligase MuRF1/TRIM63 was identified 20 years ago and suspected to play important roles during skeletal muscle atrophy. Since then, numerous studies have been conducted to decipher the roles, molecular mechanisms and regulation of this enzyme. This revealed that MuRF1 is an important player in the skeletal muscle atrophy process occurring during catabolic states, making MuRF1 a prime candidate for pharmacological treatments against muscle wasting. Indeed, muscle wasting is an associated event of several diseases (e.g., cancer, sepsis, diabetes, renal failure, etc.) and negatively impacts the prognosis of patients, which has stimulated the search for MuRF1 inhibitory molecules. However, studies on MuRF1 cardiac functions revealed that MuRF1 is also cardioprotective, revealing a yin and yang role of MuRF1, being detrimental in skeletal muscle and beneficial in the heart. This review discusses data obtained on MuRF1, both in skeletal and cardiac muscles, over the past 20 years, regarding the structure, the regulation, the location and the different functions identified, and the first inhibitors reported, and aim to draw the picture of what is known about MuRF1. The review also discusses important MuRF1 characteristics to consider for the design of future drugs to maintain skeletal muscle mass in patients with different pathologies.

Efficiency of skeletal muscle decellularization methods and their effects on the extracellular matrix.

Extracellular matrix (ECM) is widely considered to be integral to the function of skeletal muscle, providing mechanical support, transmitting force, and contributing to passive stiffness. Many functions and dysfunctions attributed to ECM are thought to stem from its mechanical properties, yet there are few data describing the mechanics of intact ECM. Such measurements require isolating intact ECM from the muscle cells it surrounds. The objectives of this study were to quantify the efficiency of three techniques for this purpose: Triton, Triton with sodium dodecyl sulfate, and latrunculin B; and to determine their impact on properties of the remaining ECM. Efficiency was quantified by DNA content and evaluation of western blot intensities for myosin and actin. The properties of ECM were quantified by collagen content and uniaxial tensile testing. We found that latrunculin B was the most efficient method for removing skeletal muscle cells, reducing DNA content to less than 10% of that seen in control muscles, and substantially reducing the myosin and actin to 15% and 23%, respectively; these changes were larger than for the competing methods. Collagen content after decellularization was not significantly different from control muscles for all methods. Only the stiffness of the muscles decellularized with latrunculin B differed significantly from control, having a Young's modulus reduced by 47% compared to the other methods at matched stresses. Our results suggest that latrunculin B is the most efficient method for decellularizing skeletal muscle and that the remaining ECM accounts for approximately half of the stiffness in passive muscle.

Effects of intrauterine growth restriction on Ca2+-activated force and contractile protein expression in the mesenteric artery of 1-year-old Wistar-Kyoto rats.

Intrauterine growth restriction (IUGR) affects vascular reactivity in older rats, but at present the causative factors for this change are unknown. Therefore, we investigated downstream events associated with vascular reactivity, specifically, Ca2+-regulated force production and shifts in contractile protein content. The mesenteric artery from male and female 1-year-old Wistar-Kyoto rats was examined using two distinct experimental growth restriction models. Uterine ligation surgery restriction or a sham surgery was conducted at day 18 of pregnancy, whilst a food restriction diet (40% control diet) began on gestational day 15. Extracellular vascular reactivity was studied using intact mesenteric arteries, which were subsequently chemically permeabilized using 50 µM ß-escin to examine Ca2+-activated force. Peak contractile responses to a K+-induced depolarization and phenylephrine were significantly elevated due to an increase in maximum Ca2+-activated force in the male surgery restricted group. No changes in contractile forces were reported between female experimental groups. Sections of mesenteric artery were examined using western blotting, revealing IUGR increased the relative abundance of the voltage-gated Ca2+ channel, inositol-1,4,5-trisphosphate receptor and myosin light chain kinase, in both male growth restricted groups, whereas no changes were seen in females. These findings demonstrate for the first time in 1-year-old rats that changes in vascular reactivity due to IUGR are caused by a change in Ca2+-activated force and shifts in important contractile protein content. These changes affect the Wistar-Kyoto rat in a sex-specific and maternal insult-dependent manner.

Rats genetically selected for low and high aerobic capacity exhibit altered soleus muscle myofilament functions.

Aerobic exercise capacity is critical to bodily health. As a model to investigate the mechanisms that determine health and disease, we employed low (LCR) and high (HCR) capacity running rat models selectively bred to concentrate the genes responsible for divergent aerobic running capacity. To investigate the skeletal muscle contribution to this innate difference in running capacity we employed an approach combining examination of the myofilament protein composition and contractile properties of the fast fiber extensor digitorum longus (EDL) and slow fiber soleus (SOL) muscles from LCR and HCR rats. Intact muscle force experiments demonstrate that SOL, but not EDL, muscles from LCR rats exhibit a three times greater decrease in fatigued force. To investigate the mechanism of this increased fatigability in the LCR SOL muscle, we determined the myofilament protein composition and functional properties. Force-Ca2+ measurements demonstrate decreased Ca2+ sensitivity of single skinned SOL muscle fibers from LCR compared with that of HCR rats. Segregating SOL fibers into fast and slow types demonstrates that the decreased Ca2+ sensitivity in LCR SOL results from a specific decrease in slow-type SOL fiber Ca2+ sensitivity such that it was similar to that of fast-type fibers. These results identify that the altered myofilament contractile properties of LCR SOL slow-type fibers result in a fast muscle type Ca2+ sensitivity and the LCR muscle phenotype. Overall our findings demonstrate alterations of the myofilament proteins could contribute to fatigability of the SOL muscle and the decreased innate aerobic running performance of LCR compared with HCR rats.

Chronic Alcohol Consumption, but not Acute Intoxication, Decreases In Vitro Skeletal Muscle Contractile Function.

BACKGROUND: Skeletal muscle myopathy accompanying chronic alcohol misuse results in part from a decrease in protein synthesis typically observed in type II-rich muscles that leads to muscle weakness. However, there is a paucity of studies investigating whether the alcohol-induced weakness is intrinsic to the muscle or results primarily from the loss of muscle mass. The present study determines whether acute alcohol (ethanol) intoxication or chronic alcohol consumption decreases the intrinsic contractile function of muscle. METHODS: Adult male mice were randomly assigned to the chronic alcohol group or given a binge dose of alcohol, and contractile characteristics of the extensor digitorum longus (EDL) were determined in vitro. RESULTS: The weight and physiological cross-sectional area (PCSA) of the EDL were decreased in alcohol-fed mice. Maximum twitch and tetanic tension were also reduced, and there was a downward shift of the absolute force-frequency curve in alcohol-fed mice. However, no alcohol-induced changes were noted when these contractile parameters were normalized for the lower PCSA. Alcohol-fed mice demonstrated greater fatigability, and alcohol-induced decreases in postfatigue specific twitch and tetanic force were independent of a decreased PCSA. Furthermore, postfatigue recovery of muscle force over time was reduced. While alcohol did not alter the content of high-energy phosphates or oxidative phosphorylation complexes I-V, it did reduce myosin heavy chain and troponin-T content. In contrast, contractile properties were not altered when examined 2 hours after binge alcohol. CONCLUSIONS: These data demonstrate chronic alcohol consumption decreases isometric and tetanic tension development due to a reduction in muscle CSA, whereas the increased fatigability observed was independent of muscle mass. As none of the functional changes were produced by acute alcohol, which produced higher blood alcohol levels than chronic ingestion, our data suggest defects in intrinsic muscle contractility require sustained intake and appear independent of defects in basal energy production.

Quantitative in situ proximity ligation assays examining protein interactions and phosphorylation during smooth muscle contractions.

Antibody-based in situ proximity ligation assays (isPLA) have the potential to study protein phosphorylation and protein interactions with spatial resolution in intact tissues. However, the application of isPLA at the tissue level is limited by a lack of appropriate positive and negative controls and the difficulty in accounting for changes in tissue shape. Here we demonstrate a set of experimental and computational approaches using gastric fundus smooth muscles to improve the validity of quantitative isPLA. Appropriate positive and negative biological controls and PLA technical controls were selected to ensure experimental rigor. To account for changes in morphology between relaxed and contracted smooth muscles, target PLA spots were normalized to smooth muscle myosin light chain 20 PLA spots or the cellular cross-sectional areas. We describe the computational steps necessary to filter out false-positive improperly sized spots and set the thresholds for counting true positive PLA spots to quantify the PLA signals. We tested our approach by examining protein phosphorylation and protein interactions in smooth muscle myofilament Ca2+ sensitization pathways from resting and contracted gastric fundus smooth muscles. In conclusion, our tissue-level isPLA method enables unbiased quantitation of protein phosphorylation and protein-protein interactions in intact smooth muscle tissues, suggesting the potential for quantitative isPLA applications in other types of intact tissues.

Inhibitory effects of rubratoxin A, a potent inhibitor of protein phosphatase 2, on the Ca2+-dependent contraction of skinned carotid artery from guinea pig.

Rubratoxin A, a potent inhibitor of PP2A, is known to suppress smooth muscle contraction. The inhibitory role of PP2A in smooth muscle contraction is still unclear. In order to clarify the regulatory mechanisms of PP2A on vascular smooth muscle contractility, we examined the effects of rubratoxin A on the Ca2+-induced contraction of ß-escin skinned carotid artery preparations from guinea pigs. Rubratoxin A at 1 µM and 10 µM significantly inhibited skinned carotid artery contraction at any Ca2+ concentration. The data fitting to the Hill equation in [Ca2+]-contraction relationship indicated that rubratoxin A decreased Fmax-Ca2+ and increased [Ca2+]50, indices of Ca2+ sensitivity for the force and myosin-actin interaction, respectively. These results suggest that PP2A inhibition causes downregulation of the myosin light chain phosphorylation and direct interference with myosin-actin interaction.