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

Expression of Smooth Muscle Myosin Heavy Chains and Unloaded Shortening in Single Smooth Muscle Cells

The functional significance of the variable expression of the smooth muscle myosin heavy chain (SM-MHC) tail isoforms, SM1 and SM2, was examined at the mRNA level (which correlates with the protein level) in individual permeabilized rabbit arterial smooth muscle cells (SMCs). The length of untethered single permeabilized SMCs was monitored during unloaded shortening in response to increased Ca2+ (pCa 6.0), histamine (1 μM), and phenylephrine (1 μM). Subsequent to contraction, the relative expression of SM1 and SM2 mRNAs from the same individual SMCs was determined by reverse transcription-polymerase chain reaction amplification and densitometric analysis. Correlational analyses between the SM2-to-SM1 ratio and unloaded shortening in saponin- and α-toxin-permeabilized SMCs (n = 28) reveal no significant relationship between the SM-MHC tail isoform ratio and unloaded shortening velocity. The best correlations between SM2/SM1 and the contraction characteristics of untethered vascular SMCs were with the minimum length attained following contraction (n = 20 andr = 0.72 for α-toxin,n = 8 andr = 0.78 for saponin). These results suggest that the primary effect of variable expression of the SM1 and SM2 SM-MHC tail isoforms is on the cell final length and not on shortening velocity

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

Writing Assignments with a Metacognitive Component Enhance Learning in a Large Introductory Biology Course

Writing assignments, including note taking and written recall, should enhance retention of knowledge, whereas analytical writing tasks with metacognitive aspects should enhance higher-order thinking. In this study, we assessed how certain writing-intensive “interventions,” such as written exam corrections and peer-reviewed writing assignments using Calibrated Peer Review and including a metacognitive component, improve student learning. We designed and tested the possible benefits of these approaches using control and experimental variables across and between our three-section introductory biology course. Based on assessment, students who corrected exam questions showed significant improvement on postexam assessment compared with their nonparticipating peers. Differences were also observed between students participating in written and discussion-based exercises. Students with low ACT scores benefited equally from written and discussion-based exam corrections, whereas students with midrange to high ACT scores benefited more from written than discussion-based exam corrections. Students scored higher on topics learned via peer-reviewed writing assignments relative to learning in an active classroom discussion or traditional lecture. However, students with low ACT scores (17–23) did not show the same benefit from peer-reviewed written essays as the other students. These changes offer significant student learning benefits with minimal additional effort by the instructors

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

Preferential Myosin Heavy Chain Isoform B Expression May Contribute to the Faster Velocity of Contraction in Veins versus Arteries

Smooth muscle myosin heavy chains occur in 2 isoforms, SMA (slow) and SMB (fast). We hypothesized that the SMB isoform is predominant in the faster-contracting rat vena cava compared to thoracic aorta. We compared the time to half maximal contraction in response to a maximal concentration of endothelin-1 (ET-1; 100 nM), potassium chloride (KCl; 100 mM) and norepinephrine (NE; 10 µM). The time to half maximal contraction was shorter in the vena cava compared to aorta (aorta: ET-1 = 235.8 ± 13.8 s, KCl = 140.0 ± 33.3 s, NE = 19.8 ± 2.7 s; vena cava: ET-1 = 121.8 ± 15.6 s, KCl = 49.5 ± 6.7 s, NE = 9.0 ± 3.3 s). Reverse-transcription polymerase chain reaction supported the greater expression of SMB in the vena cava compared to aorta. SMB was expressed to a greater extent than SMA in the vessel wall of the vena cava. Western analysis determined that expression of SMB, relative to total smooth muscle myosin heavy chains, was 12.5 ± 4.9-fold higher in the vena cava compared to aorta, while SMA was 4.9 ± 1.2-fold higher in the aorta than vena cava. Thus, the SMB isoform is the predominant form expressed in rat veins, providing one possible mechanism for the faster response of veins to vasoconstrictors