Unique Contractile and Structural Protein Expression in Dog Ileal Inner Circular Smooth Muscle

This study was designed to test the hypothesis that there is heterogeneous expression of contractile and structural proteins between the smooth muscle cells (SMCs) in the inner and outer circular muscle (ICM and OCM) layers of the ileum. Immunohistochemical staining and quantitation of fresh frozen sections of the dog ileum was performed using protein specific antibodies. Smooth muscle (SM) SMA myosin heavy chain (MHC), α- and γ-SM actin, and vinculin all show greater expression in the ICM relative to the OCM. SMB MHC and fibronectin show the opposite pattern, with greater expression in the OCM relative to the ICM. Differences in expression of these proteins are consistent with proposed differences in function of these muscle layers. Hypotheses regarding muscle tone and the coordination and regulation of peristalsis via these different muscle layers based on this data can now be made and tested

Smooth Muscle Myosin Heavy Chain Isoform Distribution in the Swine Stomach

To evaluate the distribution of smooth muscle myosin heavy chain isoforms (SMB, with head insert), we examined frozen sections from the various regions of swine stomachs using isoform-specific antibodies. We previously reported variable SMB myosin heavy chain (MHC) expression in stomach cells that correlates with unloaded shortening velocities. This is consistent with the generalization of tonic fundic muscle having low expression and phasic antral muscle having high expression of the SMB MHC isoform. Using immunohistochemistry (IHC), we show a progression of the SMB MHC from very low immunoreactivity in the fundus to very intense immunoreactivity in the antrum. In the body, the average level of SMB MHC immunoreactivity lies between that of the antrum and fundus. Intercellular heterogeneity was observed in all stomach regions to a similar extent. However, the intercellular range in SMB MHC immunoreactivity decreases from fundus to antrum. All stomach regions show isolated pockets or clusters of cells with similar SMB MHC immunoreactivity. There is a non-uniform intracellular immunoreactivity in SMB MHC, with many cells showing greater-intensity staining of SMB MHC in their cell peripheries. This information may prove useful in helping to elucidate possible unique physiological roles of SMB MHC

Myosin Heavy Chain Isoforms and Dynamic Contractile Properties: Skeletal Versus Smooth Muscle

Myosin, one of the primary contractile muscle proteins, displays molecular, enzymatic, structural, functional and regulatory variability. This variability has been shown to account for a significant amount of the functional uniqueness of skeletal and smooth muscle. However, the universal generation of force and/or shortening by these two muscle types belies the ever-increasing number of known distinct differences that bring this about. Thus, the notion that the functional roles of skeletal and smooth muscle, their development and regulation, all appear to be uniquely applicable for their physiological purpose no longer appears heretical. This manuscript presents a cursory overview of the numerous ways in which these two types of muscle use a host of myosin molecules to bring about a common result, force generation and/or shortening

Myosin Isoform Heterogeneity in Single Smooth Muscle Cells

We review the current understanding of the myosin heavy chain (MHC) isoforms and show that the mRNA levels of smooth muscle (SM)1 and SM2 mimic the expressed levels of SM1 and SM2 protein. The reverse transcriptase-polymerase chain reaction technique has been shown to be sufficiently sensitive to examine SM-MHC expression at the single cell level. Most single smooth muscle cells isolated from adult rabbit carotid express both SM1 and SM2. However, expression of these SM-MHC isoforms at the cellular level is nonuniform and highly variable. This work provides a foundation for future investigations as to the possible unique functional characteristics of the SM-MHC isoforms, SM1 and SM2. This methodology may also prove useful when used with mechanical studies to determine the physiological significance of the alternatively spliced myosin isoforms, including the SM-MHC-head and LC17 isoforms

Fiber Number and Type Composition in Extensor Digitorum Longus, Soleus, and Diaphragm Muscles with Aging in Fisher 344 Rats

Histochemical (M-ATPase) fiber typing was done on extensor digitorum longus, (EDL), soleus (SOL), and diaphragm (DIA) muscles of barrier-reared Fisher 344 rats obtained at four different ages (3, 9, 28, and 30 months) from the colonies of the National Institute of Aging. In the EDL there are no differences in the percent of type I fibers among the four age groups. The percent of type IIa and IIb fibers also showed no difference between the 3 and 30 month age groups. There was no apparent trend for an increase or decrease in the percent of type IIa or IIb fibers between the four age groups. In both the SOL and DIA muscles the percent of type I fibers was greater in the aged than in the young groups. The percent of type IIa fibers was lower in the 30 month group than in the younger groups for both muscles. The percent of type IIb (DIA) and IIc (SOL) fibers did not change between groups. Total fiber number per cross section of muscle showed no change in the EDL over this age range or in the SOL after 9 months of age. These findings bring into question published results that imply that decreasing fiber number and preferential loss of type II (a and b) fibers are typical aging phenomena

Gene Expression in Experimental Aortic Coarctation and Repair: Candidate Genes for Therapeutic Intervention?

Coarctation of the aorta (CoA) is a constriction of the proximal descending thoracic aorta and is one of the most common congenital cardiovascular defects. Treatments for CoA improve life expectancy, but morbidity persists, particularly due to the development of chronic hypertension (HTN). Identifying the mechanisms of morbidity is difficult in humans due to confounding variables such as age at repair, follow-up duration, coarctation severity and concurrent anomalies. We previously developed an experimental model that replicates aortic pathology in humans with CoA without these confounding variables, and mimics correction at various times using dissolvable suture. Here we present the most comprehensive description of differentially expressed genes (DEGs) to date from the pathology of CoA, which were obtained using this model. Aortic samples (n=4/group) from the ascending aorta that experiences elevated blood pressure (BP) from induction of CoA, and restoration of normal BP after its correction, were analyzed by gene expression microarray, and enriched genes were converted to human orthologues. 51 DEGs with \u3e6 fold-change (FC) were used to determine enriched Gene Ontology terms, altered pathways, and association with National Library of Medicine Medical Subject Headers (MeSH) IDs for HTN, cardiovascular disease (CVD) and CoA. The results generated 18 pathways, 4 of which (cell cycle, immune system, hemostasis and metabolism) were shared with MeSH ID’s for HTN and CVD, and individual genes were associated with the CoA MeSH ID. A thorough literature search further uncovered association with contractile, cytoskeletal and regulatory proteins related to excitation-contraction coupling and metabolism that may explain the structural and functional changes observed in our experimental model, and ultimately help to unravel the mechanisms responsible for persistent morbidity after treatment for CoA

A Coupled Experimental and Computational Approach to Quantify Deleterious Hemodynamics, Vascular Alterations, and Mechanisms of Long-Term Morbidity in Response to Aortic Coarctati

Introduction Coarctation of the aorta (CoA) is associated with morbidity despite treatment. Although mechanisms remain elusive, abnormal hemodynamics and vascular biomechanics are implicated. We present a novel approach that facilitates quantification of coarctation-induced mechanical alterations and their impact on vascular structure and function, without genetic or confounding factors. Methods Rabbits underwent thoracic CoA at 10 weeks of age (~ 9 human years) to induce a 20 mm Hg blood pressure (BP) gradient using permanent or dissolvable suture thereby replicating untreated and corrected CoA. Computational fluid dynamics (CFD) was performed using imaging and BP data at 32 weeks to quantify velocity, strain and wall shear stress (WSS) for comparison to vascular structure and function as revealed by histology and myograph results. Results Systolic and mean BP was elevated in CoA compared to corrected and control rabbits leading to vascular thickening, disorganization and endothelial dysfunction proximally and distally. Corrected rabbits had less severe medial thickening, endothelial dysfunction, and stiffening limited to the proximal region despite 12 weeks of normal BP (~ 4 human years) after the suture dissolved. WSS was elevated distally for CoA rabbits, but reduced for corrected rabbits. Discussion These findings are consistent with alterations in humans. We are now poised to investigate mechanical contributions to mechanisms of morbidity in CoA using these methods

Human Genotyping and An Experimental Model Reveal NPR-C as A Possible Contributor to Morbidity In Coarctation Of The Aorta

Coarctation of the aorta (CoA) is a common congenital cardiovascular (CV) defect characterized by a stenosis of the descending thoracic aorta. Treatment exists, but many patients develop hypertension (HTN). Identifying the cause of HTN is challenging because of patient variability (e.g., age, follow-up duration, severity) and concurrent CV abnormalities. Our objective was to conduct RNA sequencing of aortic tissue from humans with CoA to identify a candidate gene for mechanistic studies of arterial dysfunction in a rabbit model of CoA devoid of the variability seen with humans. We present the first known evidence of natriuretic peptide receptor C (NPR-C; aka NPR3) downregulation in human aortic sections subjected to high blood pressure (BP) from CoA versus normal BP regions (validated to PCR). These changes in NPR-C, a gene associated with BP and proliferation, were replicated in the rabbit model of CoA. Artery segments from this model were used with human aortic endothelial cells to reveal the functional relevance of altered NPR-C activity. Results showed decreased intracellular calcium ([Ca2+]i) activity to C-type natriuretic peptide (CNP). Normal relaxation induced by CNP and atrial natriuretic peptide was impaired for aortic segments exposed to elevated BP from CoA. Inhibition of NPR-C (M372049) also impaired aortic relaxation and [Ca2+]i activity. Genotyping of NPR-Cvariants predicted to be damaging revealed that rs146301345 was enriched in our CoA patients, but sample size limited association with HTN. These results may ultimately be used to tailor treatment for CoA based on mechanical stimuli, genotyping, and/or changes in arterial function

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion