Brenda Russell

CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function

MyoD, a master regulator of myogenesis, exhibits a circadian rhythm in its mRNA and protein levels, suggesting a possible role in the daily maintenance of muscle phenotype and function. We report that MyoD is a direct target of the circadian transcriptional activators CLOCK and BMAL1, which bind in a rhythmic manner to the core enhancer of the MyoD promoter. Skeletal muscle of ClockΔ19 and Bmal1−/− mutant mice exhibited ∼30% reductions in normalized maximal force. A similar reduction in force was observed at the single-fiber level. Electron microscopy (EM) showed that the myofilament architecture was disrupted in skeletal muscle of ClockΔ19, Bmal1−/−, and MyoD−/− mice. The alteration in myofilament organization was associated with decreased expression of actin, myosins, titin, and several MyoD target genes. EM analysis also demonstrated that muscle from both ClockΔ19 and Bmal1−/− mice had a 40% reduction in mitochondrial volume. The remaining mitochondria in these mutant mice displayed aberrant morphology and increased uncoupling of respiration. This mitochondrial pathology was not seen in muscle of MyoD−/− mice. We suggest that altered expression of both Pgc-1α and Pgc-1β in ClockΔ19 and Bmal1−/− mice may underlie this pathology. Taken together, our results demonstrate that disruption of CLOCK or BMAL1 leads to structural and functional alterations at the cellular level in skeletal muscle. The identification of MyoD as a clock-controlled gene provides a mechanism by which the circadian clock may generate a muscle-specific circadian transcriptome in an adaptive role for the daily maintenance of adult skeletal muscle.

Fabrication of microtextured membranes for cardiac myocyte attachment and orientation

To understand the role of tissue adaptation to altered physiological states, a more physiologically and dimensionally relevant in vitro model of cardiac myocyte organization has been developed. A microtextured polymeric membrane with micron range dimensions promotes myocyte adhesion through substrate/cell interlocking and, thus, provides a more suitable stretchable matrix for studying overlying cell populations. These microtextured membranes are created using photolithography and microfabrication techniques. Biologically, mechanically, and optically compatible interfaces with specified microarchitecture and surface chemistry have been designed, microfabricated, and characterized for this purpose. Cardiac myocytes plated on these membranes display greater attachment and cell height compared to conventional culture substrates. Advantages of the microtextured membranes include the high degree of reproducibility and the ability to create features on the micron and submicron size scale. Because of the flexibility of substrate material and the ease of creating micron size structures, this technique can be applied to many other physiological and biological systems.

Microtextured substrata alter gene expression, protein localization and the shape of cardiac myocytes.

Many of the experiments designed to understand fundamental principles in cardiac physiology are performed in vitro using myocytes isolated from adult or neonatal hearts. However, these cells have probably lost some of their original properties in culture prior to study. Our objective is to recapitulate cardiac myocyte structure and function by growing cells on microtextured silicone substrata produced by photolithography and microfabrication techniques. Myocytes are plated on nontextured, micropegged (5 microm high), microgrooved (parallel grooves with a depth of 5 microm) or combination (micropegged and grooved) substrata. Myocytes plated on microtextured surfaces display a change in cell shape with an increase in myofibrillar height and a decrease in cell area. This shape change did not affect the stoichiometry of the myofibrillar proteins but did elicit microenvironmental remodeling of proteins that mechanically attach the cell to its surroundings. Cells terminate in a sarcomeric striation on the vertical interface of the peg whereas on nontextured surfaces they end in long nonstriated cables. Vinculin, a focal adhesion protein, was found to decrease in expression on combination surfaces as compared to nontextured substrata. A three-dimensional microtextured substratum appears to reintroduce a more physiological microarchitecture for tissue culture that may have potential uses in biological research as well as in tissue engineering and diagnostic applications.

Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width

A ventricular myocyte experiences changes in length and load during every beat of the heart and has the ability to remodel cell shape to maintain cardiac performance. Specifically, myocytes elongate in response to increased diastolic strain by adding sarcomeres in series, and they thicken in response to continued systolic stress by adding filaments in parallel. Myocytes do this while still keeping the resting sarcomere length close to its optimal value at the peak of the length-tension curve. This review focuses on the little understood mechanisms by which direction of growth is matched in a physiologically appropriate direction. We propose that the direction of strain is detected by differential phosphorylation of proteins in the costamere, which then transmit signaling to the Z-disc for parallel or series addition of thin filaments regulated via the actin capping processes. In this review, we link mechanotransduction to the molecular mechanisms for regulation of myocyte length and width.

Restoration of Resting Sarcomere Length After Uniaxial Static Strain Is Regulated by Protein Kinase Cε and Focal Adhesion Kinase

Abstract— Physiological or pathological stresses and strains produce longer or wider muscle cells, but resting sarcomere length remains constant. Our goal was to investigate the cellular mechanisms for controlling this optimal, resting sarcomere length. To do so, we cultured neonatal rat cardiomyocytes on microfabricated peg-and-groove, laminin-coated silicone surfaces and applied a uniaxial static strain of 10%. Sarcomere length was accurately measured by fast Fourier transform analysis of images before, within 5 minutes of, and 4 to 6 hours after imposition of the strain. Sarcomere length of aligned cardiomyocytes (1.94±0.07 &mgr;m) was lengthened acutely (2.06±0.06 &mgr;m), and recovered (1.95±0.07 &mgr;m) by 4 hours. Puromycin, an mRNA translational inhibitor, prevented recovery of resting sarcomere length by 4 hours, thus indicating a requirement for new protein synthesis in the recovery process. Furthermore, activation of protein kinase C&egr; (PKC&egr;) was necessary for length recovery, as nonselective PKC inhibitors [staurosporine (5 &mgr;mol/L) and chelerythrine chloride (10 &mgr;mol/L)], and a replication-defective adenovirus (Adv) encoding a dominant-negative mutant of PKC&egr; prevented the restoration of sarcomere length. To assess the importance of focal adhesion complexes, cardiomyocytes were infected with an Adv encoding a dominant-negative inhibitor of focal adhesion kinase (FAK) (Adv-GFP-FRNK). Adv-GFP-FRNK also prevented resting sarcomere length recovery, whereas a control Adv encoding only GFP did not. In conclusion, using our novel culture system, we provide evidence indicating that the length remodeling process requires new protein synthesis, PKC&egr; and FAK.

Myocyte remodeling in response to hypertrophic stimuli requires nucleocytoplasmic shuttling of muscle LIM protein

CSRP3 or muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein and a mechanosensor in cardiac myocytes. MLP regulation and function was studied in cultured neonatal rat myocytes treated with pharmacological or mechanical stimuli. Either verapamil or BDM decreased nuclear MLP while phenylephrine and cyclic strain increased it. These results suggest that myocyte contractility regulates MLP subcellular localization. When RNA polymerase II was inhibited with alpha-amanitin, nuclear MLP was reduced by 30%. However, when both RNA polymerase I and II were inhibited with actinomycin D, there was a 90% decrease in nuclear MLP suggesting that its nuclear translocation is regulated by both nuclear and nucleolar transcriptional activity. Using cell permeable synthetic peptides containing the putative nuclear localization signal (NLS) of MLP, nuclear import of the protein in cultured rat neonatal myocytes was inhibited. The NLS of MLP also localizes to the nucleolus. Inhibition of nuclear translocation prevented the increased protein accumulation in response to phenylephrine. Furthermore, cyclic strain of myocytes after prior NLS treatment to remove nuclear MLP resulted in disarrayed sarcomeres. Increased protein synthesis and brain natriuretic peptide expression were also prevented suggesting that MLP is required for remodeling of the myofilaments and gene expression. These findings suggest that nucleocytoplasmic shuttling MLP plays an important role in the regulation of the myocyte remodeling and hypertrophy and is required for adaptation to hypertrophic stimuli.

GRGDSP peptide-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion.

Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture. Peptides covalently bound to silicone surfaces are examined here for the enhancement of cellular adhesion during in vitro dynamic flexing. A procedure is described for the chemical modification of medical grade silicone membranes with covalently bound GRGDSP peptides. The conditions for mechanical studies of cardiac cell cultures are then duplicated and it is demonstrated that the peptide layers survive 48 h of mechanical flexing in vitro. Specifically, mechanical flexing in vitro of the 30 pmol/cm2 peptide-modified silicone membranes has no significant effect on the amount of peptides that remains bound to the surface. Cardiac fibroblasts display enhanced adhesion to these peptide-bound silicone membranes for at least 24 h of growth, compared with native silicone or tissue culture polystyrene. The effects of serum versus serum-free media on fibroblast growth are also examined.

Cardiac Tissue Engineering

The first 2 reviews in this series have described the defining properties of stem cells, their possible sources, and some initial attempts at their clinical use for tissue regeneration and repair. This third and final article in the series describes bioengineering methods for building physical structures to contain and organize implanted cells. The relevant theory is that appropriate physical supporting structures will help implanted cardiac stem cell populations organize themselves into functioning cardiac tissue that integrates physically and functionally with the receiving heart. The purpose of cardiac tissue engineering is to replace or repair injured heart muscle effectively. Supporting materials to create habitable spaces can provide the basic requirements of cardiac muscle cells. The design of such supporting materials influences the behavior of cells; the shape, dimensions, and chemistry of substrates affect such processes as attachment, cell signaling, and differentiation. As cardiac muscle cells flourish in artificial environments, they may become functional tissue with clinical value. This review summarizes the major bioengineering approaches for containing and organizing cardiac muscle cells and their potential to ameliorate total heart failure.

Pudendal denervation affects the structure and function of the striated, urethral sphincter in female rats

Our aim was to examine the effects of denervation on urethral anatomy and urine voiding pattern. Rats usually void at one end of their cage, which gives a behavioral index of continence. The voiding preference for denervated rats was decreased to 88.8+4.7%,n=32,P<0.001, compared to improvements with time for unoperated (117±10%,n=16) or sham-operated rats (105±8%,n=5). The volume of urine or the frequency of voidings between denervated, unoperated or sham-operated rats did not differ significantly. Urethral sections were analyzed immunochemically and quantified morphometrically. Smooth muscle volume remained constant but skeletal muscle volume decreased after denervation, from 43±2% to 36±3% (P<0.05,n=5). Fiber diameter decreased from 14.3±1.4 μm to 8.5±0.7 μm (P<0.005). We concluded that pudendal nerve transection in female rats causes behavioral alterations in voiding and muscular atrophy of the striated sphincter.

Cardiomyocyte remodeling and sarcomere addition after uniaxial static strain in vitro

Individual cardiomyocytes are lengthened in dilated cardiomyopathy. However, it is not known how the new sarcomeres are added to preexisting myofibrils. Using a three-dimensional microtextured culturing system, a 10% mechanical static strain was applied to aligned, well-attached cardiomyocytes from neonatal rat. The morphology of the myofibrils and the ends of the myocytes were examined. Disruptions of the sarcomeric pattern for actin showed a progression from weak to intense staining over 4 hr. The lightly stained sarcomeres were common at 1 hr after being strained, peaked at 2 hr, and then subsided. In contrast, the numbers of intensely stained sarcomeres were initially low, peaked at 3 hr, and then began to decline when compared with control values. The myocyte ends showed elongations and convolutions after 3 hr and 4 hr of mechanical strain when observed with α-actinin and N-cadherin staining. We suggest that myocytes from neonatal rat hearts remodel by insertion of new sarcomeres throughout the cell length and also by enhancement at the intercalated discs.