The Renin-Angiotensin-Aldosterone System Regulates Sarcoidosis Granulomatous Inflammation.

Rationale: Sarcoidosis is a granulomatous disorder of unclear cause notable for abnormal elevation of blood and tissue angiotensin converting enzyme 1 (ACE1) levels and activity. ACE1 regulates the renin-angiotensin-aldosterone system (RAAS), the terminal product of which is aldosterone, which selectively engages mineralocorticoid receptors (MR) to promote inflammation. Objectives: We sought to determine whether the RAAS promotes sarcoidosis granuloma formation and related inflammatory responses. Methods: Using an established ex vivo model, we first determined whether aldosterone was produced by sarcoidosis granulomas and verified the presence of CYP11B2, the enzyme required for its production. We then evaluated the effects of selective inhibitors of ACE1 (captopril), angiotensin type 1 receptor (losartan) and MR (spironolactone, eplerenone) on granuloma formation, reflected by computer image analysis-generated granuloma area, and selected cytokines incriminated in sarcoidosis pathogenesis. Measurements and Main Results: Aldosterone was spontaneously produced by sarcoidosis PBMCs, and both intra- and extracellular levels steadily increased during granuloma formation. In parallel, PBMCs were shown to express more CYP11B2 during granuloma formation. Significant inhibition of sarcoidosis granulomas and related cytokines (TNFα, IL-1ß, IFNγ, IL-10) was observed in response to pretreatments with captopril, losartan, spironolactone or eplerenone, comparable to that of prednisone. Conclusions: The RAAS is intact in sarcoidosis granulomas and contributes significantly to early granuloma formation and to related inflammatory mediator responses with important implications for clinical management.

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation.

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Leveraging in vitro and pharmacokinetic models to support bench to bedside investigation of XTMAB-16 as a novel pulmonary sarcoidosis treatment.

Background: Sarcoidosis is a chronic, multisystem inflammatory disorder characterized by non-caseating epithelioid granulomas; infiltration of mononuclear cells; and destruction of microarchitecture in the skin, eye, heart, and central nervous system, and the lung in >90% of cases. XTMAB-16 is a chimeric anti-tumor necrosis factor alpha (TNFα) antibody, distinct from other anti-TNF antibodies based on its molecular structure. The efficacy of XTMAB-16 has not been clinically demonstrated, and it is still undergoing clinical development as a potential treatment for sarcoidosis. The current study demonstrates the activity of XTMAB-16 in a well-established in vitro sarcoidosis granuloma model, although XTMAB-16 is not yet approved by the United States Food and Drug Administration (FDA) for treatment of sarcoidosis, or any other disease. Objective: To provide data to guide safe and efficacious dose selection for the ongoing clinical development of XTMAB-16 as a potential treatment for sarcoidosis. Methods: First, XTMAB-16 activity was evaluated in an established in vitro model of granuloma formation using peripheral blood mononuclear cells from patients with active pulmonary sarcoidosis to determine a potentially efficacious dose range. Second, data obtained from the first-in-human study of XTMAB-16 (NCT04971395) were used to develop a population pharmacokinetic (PPK) model to characterize the pharmacokinetics (PK) of XTMAB-16. Model simulations were performed to evaluate the sources of PK variability and to predict interstitial lung exposure based on concentrations in the in vitro granuloma model. Results: XTMAB-16 dose levels of 2 and 4 mg/kg, once every 2 weeks (Q2W) or once every 4 weeks (Q4W) for up to 12 weeks, were supported by data from the non-clinical, in vitro secondary pharmacology; the Phase 1 clinical study; and the PPK model developed to guide dose level and frequency assumptions. XTMAB-16 inhibited granuloma formation and suppressed interleukin-1ß (IL-1ß) secretion in the in vitro granuloma model with a half maximal inhibitory concentration (IC50) of 5.2 and 3.5 µg/mL, respectively. Interstitial lung concentrations on average, following 2 or 4 mg/kg administered Q2W or Q4W, are anticipated to exceed the in vitro IC50 concentrations. Conclusion: The data presented in this report provide a rationale for dose selection and support the continued clinical development of XTMAB-16 for patients with pulmonary sarcoidosis.

Circulating exosomal microRNA expression patterns distinguish cardiac sarcoidosis from myocardial ischemia.

OBJECTIVE: Cardiac sarcoidosis is difficult to diagnose, often requiring expensive and inconvenient advanced imaging techniques. Circulating exosomes contain genetic material, such as microRNA (miRNA), that are derived from diseased tissues and may serve as potential disease-specific biomarkers. We thus sought to determine whether circulating exosome-derived miRNA expression patterns would distinguish cardiac sarcoidosis (CS) from acute myocardial infarction (AMI). METHODS: Plasma and serum samples conforming to CS, AMI or disease-free controls were procured from the Biologic Specimen and Data Repository Information Coordinating Center repository and National Jewish Health. Next generation sequencing (NGS) was performed on exosome-derived total RNA (n = 10 for each group), and miRNA expression levels were compared after normalization using housekeeping miRNA. Quality assurance measures excluded poor quality RNA samples. Differentially expressed (DE) miRNA patterns, based upon >2-fold change (p < 0.01), were established in CS compared to controls, and in CS compared to AMI. Relative expression of several DE-miRNA were validated by qRT-PCR. RESULTS: Despite the advanced age of the stored samples (~5-30 years), the quality of the exosome-derived miRNA was intact in ~88% of samples. Comparing plasma exosomal miRNA in CS versus controls, NGS yielded 18 DE transcripts (12 up-regulated, 6 down-regulated), including miRNA previously implicated in mechanisms of myocardial injury (miR-92, miR-21) and immune responses (miR-618, miR-27a). NGS further yielded 52 DE miRNA in serum exosomes from CS versus AMI: 5 up-regulated in CS; 47 up-regulated in AMI, including transcripts previously detected in AMI patients (miR-1-1, miR-133a, miR-208b, miR-423, miR-499). Five miRNAs with increased DE in CS included two isoforms of miR-624 and miR-144, previously reported as markers of cardiomyopathy. CONCLUSIONS: MiRNA patterns of exosomes derived from CS and AMI patients are distinct, suggesting that circulating exosomal miRNA patterns could serve as disease biomarkers. Further studies are required to establish their specificity relative to other cardiac disorders.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19, exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes that occur during cancer cachexia.NEW & NOTEWORTHY We used proteomics and metadata analysis software to identify contributors to metabolic changes in striated muscle during cancer cachexia. We found increased expression of hypoxia-inducible factor-1α in the heart and skeletal muscle, suggesting a potential target for the therapeutic treatment of cancer cachexia.

Ibuprofen ameliorates fatigue- and depressive-like behavior in tumor-bearing mice.

AIMS: Cancer-related fatigue (CRF) is often accompanied by depressed mood, both of which reduce functional status and quality of life. Research suggests that increased expression of pro-inflammatory cytokines is associated with skeletal muscle wasting and depressive- and fatigue-like behaviors in rodents and cancer patients. We have previously shown that treatment with ibuprofen, a nonsteroidal anti-inflammatory drug, preserved muscle mass in tumor-bearing mice. Therefore, the purpose of the present study was to determine the behavioral effects of ibuprofen in a mouse model of CRF. MAIN METHODS: Mice were injected with colon-26 adenocarcinoma cells and treated with ibuprofen (10mg/kg) in the drinking water. Depressive-like behavior was determined using the forced swim test (FST). Fatigue-like behaviors were determined using voluntary wheel running activity (VWRA) and grip strength. The hippocampus, gastrocnemius muscle, and serum were collected for cytokine analysis. KEY FINDINGS: Tumor-bearing mice showed depressive-like behavior in the FST, which was not observed in mice treated with ibuprofen. VWRA and grip strength declined in tumor-bearing mice, and ibuprofen attenuated this decline. Tumor-bearing mice had decreased gastrocnemius muscle mass and increased expression of IL-6, MAFBx and MuRF mRNA, biomarkers of protein degradation, in the muscle. Expression of IL-1ß and IL-6 was also increased in the hippocampus. Treatment with ibuprofen improved muscle mass and reduced cytokine expression in both the muscle and hippocampus of tumor-bearing mice. SIGNIFICANCE: Ibuprofen treatment reduced skeletal muscle wasting, inflammation in the brain, and fatigue- and depressive-like behavior in tumor-bearing mice. Therefore, ibuprofen warrants evaluation as an adjuvant treatment for CRF.

Metalloproteinase expression is altered in cardiac and skeletal muscle in cancer cachexia.

Cardiac and skeletal muscle dysfunction is a recognized effect of cancer-induced cachexia, with alterations in heart function leading to heart failure and negatively impacting patient morbidity. Cachexia is a complex and multifaceted disease state with several potential contributors to cardiac and skeletal muscle dysfunction. Matrix metalloproteinases (MMPs) are a family of enzymes capable of degrading components of the extracellular matrix (ECM). Changes to the ECM cause disruption both in the connections between cells at the basement membrane and in cell-to-cell interactions. In the present study, we used a murine model of C26 adenocarcinoma-induced cancer cachexia to determine changes in MMP gene and protein expression in cardiac and skeletal muscle. We analyzed MMP-2, MMP-3, MMP-9, and MMP-14 as they have been shown to contribute to both cardiac and skeletal muscle ECM changes and, thereby, to pathology in models of heart failure and muscular dystrophy. In our model, cardiac and skeletal muscles showed a significant increase in RNA and protein levels of several MMPs and tissue inhibitors of metalloproteinases. Cardiac muscle showed significant protein increases in MMP-2, MMP-3, MMP-9, and MMP-14, whereas skeletal muscles showed increases in MMP-2, MMP-3, and MMP-14. Furthermore, collagen deposition was increased after C26 adenocarcinoma-induced cancer cachexia as indicated by an increased left ventricular picrosirius red-positive-stained area. Increases in serum hydroxyproline suggest increased collagen turnover, implicating skeletal muscle remodeling. Our findings demonstrate that cancer cachexia-associated matrix remodeling results in cardiac fibrosis and possible skeletal muscle remodeling. With these findings, MMPs represent a possible therapeutic target for the treatment of cancer-induced cachexia.

Losartan treatment attenuates tumor-induced myocardial dysfunction.

UNLABELLED: Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin-angiotensin-aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. METHODS AND RESULTS: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8weeks of age. Simultaneously, mice were administered Losartan (10mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. CONCLUSIONS: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation.

Fluoxetine prevents the development of depressive-like behavior in a mouse model of cancer related fatigue.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with tiredness and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, escalates during treatment, and can persist for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. We have previously shown that increased pro-inflammatory cytokine expression in the brain contributes to depressive- and fatigue-like behaviors in a mouse model of CRF. Inflammatory cytokines increase the activity of indoleamine 2,3-dioxygenase (IDO) and kynurenine 3-monooxygenase (KMO), which competitively reduce serotonin synthesis. Reduced serotonin availability in the brain and increased production of alternative neuroactive metabolites of tryptophan are thought to contribute to the development of depression and fatigue. The purpose of this study was to determine the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), on brain cytokines and behavioral measures of fatigue and depression in tumor-bearing mice. Here we show that tumor growth increased brain expression of pro-inflammatory cytokines and KMO. Treatment with fluoxetine had no effect on tumor growth, muscle wasting, fatigue behavior, or cytokine expression in the brain. Fluoxetine, however, reduced depressive-like behaviors in tumor bearing mice. In conclusion, our data confirm that increased brain expression of pro-inflammatory cytokines is associated with tumor-induced fatigue- and depressive-like behaviors. However, it is possible to separate the effects of tumor growth on mood and fatigue-like behaviors using SSRIs such as fluoxetine.

Tumor growth increases neuroinflammation, fatigue and depressive-like behavior prior to alterations in muscle function.

Cancer patients frequently suffer from fatigue, a complex syndrome associated with loss of muscle mass, weakness, and depressed mood. Cancer-related fatigue (CRF) can be present at the time of diagnosis, during treatment, and persists for years after treatment. CRF negatively influences quality of life, limits functional independence, and is associated with decreased survival in patients with incurable disease. Currently there are no effective treatments to reduce CRF. The aim of this study was to use a mouse model of tumor growth and discriminate between two main components of fatigue: loss of muscle mass/function and altered mood/motivation. Here we show that tumor growth increased fatigue- and depressive-like behaviors, and reduced body and muscle mass. Decreased voluntary wheel running activity (VWRA) and increased depressive-like behavior in the forced swim and sucrose preference tests were evident in tumor-bearing mice within the first two weeks of tumor growth and preceded the loss of body and muscle mass. At three weeks, tumor-bearing mice had reduced grip strength but this was not associated with altered expression of myosin isoforms or impaired contractile properties of muscles. These increases in fatigue and depressive-like behaviors were paralleled by increased expression of IL-1ß mRNA in the cortex and hippocampus. Minocycline administration reduced tumor-induced expression of IL-1ß in the brain, reduced depressive-like behavior, and improved grip strength without altering muscle mass. Taken together, these results indicate that neuroinflammation and depressed mood, rather than muscle wasting, contribute to decreased voluntary activity and precede major changes in muscle contractile properties with tumor growth.

Electrophoretic separation of reptilian skeletal and cardiac muscle myosin heavy chain isoforms: dependence on gel format.

This report provides a comparison of multiple gel formats to study myosin heavy chain (MHC) isoforms that are expressed in reptilian skeletal and cardiac muscles of five turtle species, water monitor, and prehensile tailed skink. Three gel formats were tested. The results identify one format that is superior, for the overall extent of electrophoretic separation and for the assessment of the number of MHC isoforms in reptilian striated muscles. The same format was shown previously to separate MHC isoforms that are expressed in American alligator. The results also show that another gel format reveals the distinct electrophoretic mobility of MHC isoforms in atrial, ventricular, and jaw adductor samples, compared to those expressed in skeletal muscles in the limbs and elsewhere in the body. In addition, the results reveal that the electrophoretic mobility of specific MHC isoforms, relative to other isoforms, depends on the gel format, as shown previously for mammalian and avian species. The discovery of the expression of masticatory MHC, which is abundantly expressed in jaw adductors of members of Carnivora and several other vertebrate orders, in the homologous muscles of prehensile tailed skink, an herbivore, and the carnivorous water monitor, was made during the course of this study.

Complex tropomyosin and troponin T isoform expression patterns in orbital and global fibers of adult dog and rat extraocular muscles.

We reported marked differences in the myosin heavy and light chain (MHC and MLC) isoform composition of fast and slow fibers between the global and orbital layers of dog extraocular muscles. Many dog extraocular fibers, especially orbital fibers, have MHC and MLC isoform patterns that are distinct from those in limb skeletal muscles. Additional observations suggested possible differences in the tropomyosin (Tm) and troponin T (TnT) isoform composition of global and orbital fibers. Therefore, we tested, using SDS-PAGE and immunoblotting, whether differences in Tm and TnT isoform expression do, in fact, exist between global and orbital layers of dog and rat EOMs and to compare expression patterns among identified fast and slow single fibers from both muscle layers. The Tm isoforms expressed in global fast and slow fibers are the same as in limb fast (α-Tm and ß-Tm) and slow (γ-Tm and ß-Tm) fibers, respectively. Orbital slow orbital fibers, on the other hand, each co-express all three sarcomeric Tm isoforms (α, ß and γ). The results indicate that fast global and orbital fibers express only fast isoforms of TnT, but the relative amounts of the individual isoforms are different from those in limb fast muscle fibers and an abundant fast TnT isoform in the orbital layer was not detected in fast limb muscles. Slow fibers in both layers express slow TnT isoforms and the relative amounts also differ from those in limb slow fibers. Unexpectedly, significant amounts of cardiac TnT isoforms were also detected in slow fibers, especially in the orbital layer in both species. TnI and TnC isoform patterns are the same as in fast and slow fibers in limb muscles. These results expand the understanding of the elaborate diversity in contractile protein isoform expression in mammalian extraocular muscle fibers and suggest that major differences in calcium-activation properties exist among these fibers, based upon Tm and TnT isoform expression patterns.

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin.

We recently reported that masticatory ('superfast') myosin is expressed in jaw-closing muscles of some rodent species. Most mammalian limb muscle fibers express tropomyosin-ß (Tm-ß), along with fast-type or slow-type tropomyosin-ß (Tm-ß), but jaw-closing muscle fibers in members of Carnivora express a unique isoform of Tm [Tm-masticatory (Tm-M)] and little or no Tm-ß. The goal of this study was to determine patterns of Tm and troponin-T (TnT) isoform expression in the jaw-closing muscles of rodents and other vertebrate species that express masticatory myosin, and compare the results to those from members of Carnivora. Comparisons of electrophoretic mobility, immunoblotting and mass spectrometry were used to probe the Tm and fast-type TnT isoform composition of jaw-closing and limb muscles of six species of Carnivora, eight species of Rodentia, five species of Marsupialia, big brown bat, long-tailed macaque and six species of Reptilia. Extensive heterogeneity exists in Tm and TnT isoform expression in jaw-closing muscles between phylogenetic groups, but there are fairly consistent patterns within each group. We propose that the differences in Tm and TnT isoform expression patterns between phylogenetic groups, which share the expression of masticatory myosin, may impart fundamental differences in thin-filament-mediated muscle activation to accommodate markedly different feeding styles that may require high force generation in some species (e.g. many members of Carnivora) and high speed in others (e.g. Rodentia).

The myosin light chain 1 isoform associated with masticatory myosin heavy chain in mammals and reptiles is embryonic/atrial MLC1.

We recently reported that masticatory myosin heavy chain (MHC-M) is expressed as the exclusive or predominant MHC isoform in masseter and temporalis muscles of several rodent species, contrary to the prevailing dogma that rodents express almost exclusively MHC isoforms that are typically found in fast limb muscles and not masticatory myosin. We also reported that the same rodent species express the embryonic/atrial isoform of myosin light chain 1 (MLC1E/A) in jaw-closing muscles and not a unique masticatory MLC1 isoform that others have reported as being expressed in jaw-closing muscles of carnivores that express MHC-M. The objective of this study was to test the hypothesis that MLC1E/A is consistently expressed in jaw-closing muscles whenever MHC-M is expressed as the predominant or exclusive MHC isoform. Jaw-closing muscles, fast and slow limb muscles, and cardiac atria and ventricles of 19 species (six Carnivora species, one Primates species, one Chiroptera species, five marsupial species, an alligator and five turtle species) were analyzed using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA sequencing. Gel electrophoresis and immunoblotting indicate that MHC-M is the exclusive or predominant MHC isoform in the jaw-closing muscles of each of the studied species. The results from all of the approaches collectively show that MLC1E/A is exclusively or predominantly expressed in jaw-closing muscles of the same species. We conclude that MLC1E/A is the exclusive or predominant MLC1 isoform that is expressed in jaw-closing muscles of vertebrates that express MHC-M, and that a unique masticatory isoform of MLC1 probably does not exist.

Masticatory (;superfast') myosin heavy chain and embryonic/atrial myosin light chain 1 in rodent jaw-closing muscles.

Masticatory myosin is widely expressed among several vertebrate classes. Generally, the expression of masticatory myosin has been associated with high bite force for a carnivorous feeding style (including capturing/restraining live prey), breaking down tough plant material and defensive biting in different species. Masticatory myosin expression in the largest mammalian order, Rodentia, has not been reported. Several members of Rodentia consume large numbers of tree nuts that are encased in very hard shells, presumably requiring large forces to access the nutmeat. We, therefore, tested whether some rodent species express masticatory myosin in jaw-closing muscles. Myosin isoform expression in six Sciuridae species was examined, using protein gel electrophoresis, immunoblotting, mass spectrometry and RNA analysis. The results indicate that masticatory myosin is expressed in some Sciuridae species but not in other closely related species with similar diets but having different nut-opening strategies. We also discovered that the myosin light chain 1 isoform associated with masticatory myosin heavy chain, in the same four Sciuridae species, is the embryonic/atrial isoform. We conclude that rodent speciation did not completely eliminate masticatory myosin and that its persistent expression in some rodent species might be related to not only diet but also to feeding style.

Myosin isoform expression in dog rectus muscles: patterns in global and orbital layers and among single fibers.

PURPOSE: To quantitate the distribution of myosin heavy chain (MyHC) isoforms along the global and orbital layers of dog rectus muscles and determine MyHC and myosin light chain (MLC) isoform patterns among single fibers from both layers. METHODS: Serial samples of both layers of rectus muscles were prepared for gel electrophoresis. Relative amounts of each MyHC isoform in each sample were determined with scanning densitometry. Single fibers were isolated from each layer for analyses of MyHC and MLC isoforms. RESULTS: Nine MyHC isoforms were detected. Four prominent MyHC isoforms, and an additional MyHC isoform at very low levels, are expressed in the global layer. Evidence suggests that all nine MyHC isoforms are expressed in the orbital layer. There are marked gradients in the levels of some MyHC isoforms along the length of both layers. Complex patterns of coexpression of multiple MyHC isoforms exist in single fibers from both layers. Most fibers express conventional slow or fast MLC isoforms, in accordance with the type (slow or fast) of MyHC isoform(s) in a given fiber, with the exception that slow fibers in the orbital layer express the atrial/embryonic isoform of MLC1. CONCLUSIONS: MyHC isoform expression patterns differ markedly between and along global and orbital layers of dog rectus muscles, with greater complexity in the orbital layer. Heterogeneity in MyHC isoform expression in rectus muscles is much greater than in limb muscles and presumably is the basis for the broad spectrum of extraocular muscle (EOM) contractile properties in driving oculomotor functions.

Increased myosin heavy chain-beta with atrial expression of ventricular light chain-2 in canine cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy is a naturally occurring disease in humans and dogs. Human studies have shown increased levels of myosin heavy chain (MHC)-beta in failing ventricles and the left atria (LA) and of ventricular light chain (VLC)-2 in the right atria in dilated cardiomyopathy. METHODS AND RESULTS: This study evaluates the levels of MHC-beta in all heart chambers in prolonged canine right ventricular pacing. In addition, we determined whether levels of VLC2 were altered in these hearts. Failing hearts demonstrated significantly increased levels of MHC-beta in the right atria, right atrial appendage, LA, left atrial appendage (LAA), and right ventricle compared with controls. Significant levels of VLC2 were detected in the right atria of paced hearts. Differences in MHC-beta expression were observed between the LA and the LAA of paced and control dogs. MHC-beta expression was significantly greater in the LA of paced and control dogs compared with their respective LAA. CONCLUSIONS: The cardiac myosin isoform shifts in this study were similar to those observed in end-stage human heart failure and more severe than those reported in less prolonged pacing models, supporting the use of this model for further study of end-stage human heart failure. The observation of consistent differences between sampling sites, especially LA versus LAA, indicates the need for rigorous sampling consistency in future studies.

Variations in apparent mass of mammalian fast-type myosin light chains correlate with species body size, from shrew to elephant.

A recent study (Bicer S and Reiser PJ. J Muscle Res Cell Motil 25: 623-633, 2004) suggested considerable variation in the apparent molecular mass (M(a)), deduced from electrophoretic mobility, in fast-type myosin light chains (MLCF), especially MLC1F, among mammalian species. Furthermore, there was an indication that MLC1F M(a) generally correlates with species body mass, over an approximately 4,000-fold range in body mass. The results also suggested that M(a) of other low-molecular-weight myofibrillar proteins is less variable and not as strongly correlated with body mass among the same species. The objective of this study was to test the hypotheses that the M(a) of MLCs does, in fact, vary and correlate with species body mass. The electrophoretic mobilities of MLCF isoforms from 19 species, varying in size approximately 500,000-fold, were quantitated. The results confirm that the M(a) of MLC1F and MLC2F vary significantly among mammals, spanning a very broad range in body mass; the MLC1F M(a) varies more than that of other low-molecular-weight myofibrillar proteins; and there is a significant correlation between species body mass and MLC1F M(a). Differences in MLC1F M(a) among five species can be accounted for by differences in the reported amino acid sequence, especially the length of a common polyalanine region near the NH(2)-terminal actin-binding site. The possibility that the differences in MLC1F sequence among mammalian species, in and adjacent to the actin-binding region, are related to differences in modulation of cross-bridge kinetics in species with diverse locomotion kinetics is discussed.

Multiple isoforms of myosin light chain 1 in pig diaphragm slow fibers: correlation with maximal shortening velocity and force generation.

Pig diaphragm slow fibers exhibit heterogeneity in myosin light chain 1 (MLC1) isoform expression, with many expressing fast-type MLC1 (MLC1F), as well as two isoforms of slow-type MLC1 (MLC1Sa and MLC1Sb). The goal of this study was to test if there is a relationship between MLC1 isoform expression and contractile properties among these fibers. Maximal shortening velocity (V(max)) and maximal isometric force generation, normalized with fiber cross-sectional area (P(o)/CSA), were measured in single fibers. V(max) was inversely related to the relative level of MLC1Sa. The level of MLC1Sa was reciprocally related to the levels of MLC1Sb and of MLC1F among individual fibers. Fibers expressing MLC1Sa and in which MLC1Sb was not detected generated greater P(o)/CSA, compared to fibers expressing MLC1Sb and not MLC1Sa. The results indicate a complex pattern of MLC1 isoform expression among pig diaphragm slow fibers and suggest that shortening velocity and force generation are modulated, in these fibers, by the MLC1 isoform composition.