Peter A. Merrifield

Blocking gap junctional intercellular communication in myoblasts inhibits myogenin and MRF4 expression

Cells rely heavily on cues from their extracellular environment and other cells to coordinate normal physiological processes, and the exchange of molecules via gap junctions has been suggested as on important avenue for cell-cell communication. Gap junctions are found in virtually all mammalian tissues with the notable exception of adult skeletal muscle. However, since functional gap junctions have been detected during the early stages of muscle development, gap junctional intercellular communication (GJIC) may play on important role in myogenesis. In this study, GJIC in normal 16 myoblasts was inhibited using the known blockers l-octanol and beta-glycyrrhetinic acid (beta-GA). Under differentiation promoting conditions, 16 cells fused to form multinucleated myotubes, but when treated with either octanol or beta-GA, no fusion was observed. The expression of two muscle regulatory factors (MRFs), myogenin and MRF4, was examined in both the blocked and control cells. As expected, the activation of both the myogenin and MRF4 genes coincided with the onset of differentiation in the control 16 cells. Neither of these genes were turned on in the blocked cells, even when grown under low serum conditions. This inhibition of differentiation by octanol and beta-GA was reversible, since the activation of both MRF genes as well as myoblast fusion were observed when the blocking medium was replaced with normal differentiating medium. These results suggest that intercellular communication via gap junctions plays an important role in skeletal muscle development and perhaps in the cell signaling events that trigger the activation of muscle-specific MRF genes.

Determining the minimum number of detectable cardiac-transplanted 111In-tropolone-labelled bone-marrow-derived mesenchymal stem cells by SPECT

In this work, we determined the minimum number of detectable 111In-tropolone-labelled bone-marrow-derived stem cells from the maximum activity per cell which did not affect viability, proliferation and differentiation, and the minimum detectable activity (MDA) of 111In by SPECT. Canine bone marrow mesenchymal cells were isolated, cultured and expanded. A number of samples, each containing 5x10(6) cells, were labelled with 111In-tropolone from 0.1 to 18 MBq, and cell viability was measured afterwards for each sample for 2 weeks. To determine the MDA, the anthropomorphic torso phantom (DataSpectrum Corporation, Hillsborough, NC) was used. A point source of 202 kBq 111In was placed on the surface of the heart compartment, and the phantom and all compartments were then filled with water. Three 111In SPECT scans (duration: 16, 32 and 64 min; parameters: 128x128 matrix with 128 projections over 360 degrees) were acquired every three days until the 111In radioactivity decayed to undetectable quantities. 111In SPECT images were reconstructed using OSEM with and without background, scatter or attenuation corrections. Contrast-to-noise ratio (CNR) in the reconstructed image was calculated, and MDA was set equal to the 111In activity corresponding to a CNR of 4. The cells had 100% viability when incubated with no more than 0.9 MBq of 111In (80% labelling efficiency), which corresponded to 0.14 Bq per cell. Background correction improved the detection limits for 111In-tropolone-labelled cells. The MDAs for 16, 32 and 64 min scans with background correction were observed to be 1.4 kBq, 700 Bq and 400 Bq, which implies that, in the case where the location of the transplantation is known and fixed, as few as 10,000, 5000 and 2900 cells respectively can be detected.

Cell tracking and therapy evaluation of bone marrow monocytes and stromal cells using SPECT and CMR in a canine model of myocardial infarction

BackgroundThe clinical application of stem cell therapy for myocardial infarction will require the development of methods to monitor treatment and pre-clinical assessment in a large animal model, to determine its effectiveness and the optimum cell population, route of delivery, timing, and flow milieu.ObjectivesTo establish a model for a) in vivo tracking to monitor cell engraftment after autologous transplantation and b) concurrent measurement of infarct evolution and remodeling.MethodsWe evaluated 22 dogs (8 sham controls, 7 treated with autologous bone marrow monocytes, and 7 with stromal cells) using both imaging of 111Indium-tropolone labeled cells and late gadolinium enhancement CMR for up to12 weeks after a 3 hour coronary occlusion. Hearts were also examined using immunohistochemistry for capillary density and presence of PKH26 labeled cells.ResultsIn vivo Indium imaging demonstrated an effective biological clearance half-life from the injection site of ~5 days. CMR demonstrated a pattern of progressive infarct shrinkage over 12 weeks, ranging from 67–88% of baseline values with monocytes producing a significant treatment effect. Relative infarct shrinkage was similar through to 6 weeks in all groups, following which the treatment effect was manifest. There was a trend towards an increase in capillary density with cell treatment.ConclusionThis multi-modality approach will allow determination of the success and persistence of engraftment, and a correlation of this with infarct size shrinkage, regional function, and left ventricular remodeling. There were overall no major treatment effects with this particular model of transplantation immediately post-infarct.

Embryonic and fetal rat myoblasts form different muscle fiber types in an ectopic in vivo environment

Limb muscle development is characterized by the migration of muscle precursor cells from the somite followed by myoblast differentiation and the maturation of myotubes into distinct muscle fiber types. Previous in vitro experiments have suggested that rat limb myoblasts are composed of at least two distinct myoblast subpopulations that appear in the developing hindlimb at different developmental stages. These embryonic and fetal myoblast subpopulations are believed to generate primary and secondary myotubes, respectively. To test this hypothesis, cells obtained from embryonic day 14 (ED 14) and ED 20 rat hindlimbs were analyzed for myosin heavy chain expression after long‐term differentiation in adult rat brains. Fetal myoblasts from ED 20 hindlimbs produced muscle fibers with a phenotype similar to that seen in tissue culture—predominantly fast myosin with a small proportion also coexpressing slow myosin. However, injection sites populated by embryonic myoblasts from ED 14 hindlimbs produced a different phenotype from that previously reported in culture, with fibers expressing an entire array of myosin isoforms. In addition, a subpopulation of fibers expressing exclusively slow myosin was found only in the embryonic injection sites. Our results support the existence of at least three myogenic subpopulations in early rat limb buds with only one exhibiting the capability to differentiate in vitro. These findings are consistent with a model of muscle fiber type development in which the fiber type potential of myoblast populations is established before differentiation into myotubes. This process establishes myogenic subpopulations that have restricted adaptive ranges regulated by both intrinsic and extrinsic factors.

Regionalized expression of myosin isoforms in heterotypic myotubes formed from embryonic and fetal rat myoblasts in vitro

The development of mammalian limb muscles involves the appearance and fusion of at least two separate populations of muscle precursor cells. These two populations, termed embryonic and fetal myoblasts, are first detected within the limb bud at different stages of development. We have previously demonstrated that, in the rat, each myoblast population expresses a unique pattern of myosin heavy chains (MyHCs) during differentiation in vitro (Pin and Merrifield [1993] Dev. Genet. 14:356–368). Embryonic myoblasts accumulate embryonic and slow MyHCs, whereas fetal myoblasts accumulate embryonic, neonatal, and adult fast MyHCs but not slow MyHC. To determine if the two populations can fuse with each other and whether the pattern of MyHC expression is altered in the resulting heterokaryons, embryonic and fetal myoblasts were labelled with the lipophilic dye PKH26, [3H]‐thymidine, or 5‐bromodeoxyuridine (BRDU) and cocultured for 24–48 hr. Our results demonstrate that fusion occurs between embryonic and fetal myoblasts in vitro. Moreover, analysis of the resulting heterokaryons revealed regionalized accumulations of MyHC around individual nuclei. Interestingly, these accumulations were typical of the default pattern of expression that individual nuclei would have normally expressed in single culture. Nuclei contributed by embryonic myoblasts were surrounded by localized accumulations of slow MyHC, whereas nuclei from fetal myoblasts were surrounded by neonatal/fast MyHC. The occurrence of such nuclear domains indicates that the myoblast‐specific expression of MyHC isoforms is dictated by cis‐acting factors established prior to fusion. Dev. Dyn. 208:420–431, 1997.

DHA Supplemented in Peptamen Diet Offers No Advantage in Pathways to Amyloidosis: Is It Time to Evaluate Composite Lipid Diet?

Numerous reports have documented the beneficial effects of dietary docosahexaenoic acid (DHA) on beta-amyloid production and Alzheimers disease (AD). However, none of these studies have examined and compared DHA, in combination with other dietary nutrients, for its effects on plaque pathogenesis. Potential interactions of DHA with other dietary nutrients and fatty acids are conventionally ignored. Here we investigated DHA with two dietary regimes; peptamen (pep+DHA) and low fat diet (low fat+DHA). Peptamen base liquid diet is a standard sole-source nutrition for patients with gastrointestinal dysfunction. Here we demonstrate that a robust AD transgenic mouse model shows an increased tendency to produce beta-amyloid peptides and amyloid plaques when fed a pep+DHA diet. The increase in beta-amyloid peptides was due to an elevated trend in the levels of beta-secretase amyloid precursor protein (APP) cleaving enzyme (BACE), the proteolytic C-terminal fragment beta of APP and reduced levels of insulin degrading enzyme that endoproteolyse beta-amyloid. On the contrary, TgCRND8 mice on low fat+DHA diet (based on an approximately 18% reduction of fat intake) ameliorate the production of abeta peptides and consequently amyloid plaques. Our work not only demonstrates that DHA when taken with peptamen may have a tendency to confer a detrimental affect on the amyloid plaque build up but also reinforces the importance of studying composite lipids or nutrients rather than single lipids or nutrients for their effects on pathways important to plaque development.

Expression of the myosin heavy chain genes in the tail muscle of thyroid hormone-induced metamorphosing Rana catesbeiana tadpoles.

In tadpoles of the North American bullfrog, Rana catesbeiana, spontaneous and thyroid hormone (T3)-induced metamorphosis is characterized by regression of the tail, which is preceded by a decrease in total protein synthesis in tail tissues. We have demonstrated that thyroid hormone treatment of a tadpole does not affect the synthesis of all proteins equally in the tadpole tail muscle. For example, the synthesis of myosin heavy chains (MHCs) is depressed within 1 day and decreases to 45% of control values after 5 days of T3 treatment, whereas the decreased synthesis of soluble muscle proteins is transient and returns to above control levels by day 5. To determine whether the hormone-induced decrease in MHC synthesis is the result of changes in the transcription of translation of MHC mRNAs, we isolated cDNAs complementary to five different MHC mRNAs from a tail muscle cDNA library and used them to examine the levels of each MHC mRNA in the tail muscle of T3-treated tadpoles. mRNAs that recognize the cDNAs for these five different MHCs are all expressed in the tadpole tail and limb muscles, as well as in the adult leg muscles. MHC mRNAs unique to tadpole tail were not detected. Interestingly, the relative amounts of mRNA for four of the five MHCs increase in tail muscle after T3 treatment of the tadpole, suggesting that repression of MHC gene expression at the protein level does not result from a decrease in the amount of MHC mRNAs. Rather, these results support the contention that the decreased synthesis of MHCs in the tail muscle of T3-treated tadpoles is caused by this hormone, either directly or indirectly, depressing the translation of the MHC mRNAs in this tissue. These results, coupled with the observation that the synthesis of soluble muscle proteins is depressed only in a transient fashion, suggest that T3 may be initiating the expression of a gene(s) that encodes a protein(s) responsible for inhibiting the translation of the MHCs and, perhaps, other structural proteins in the tadpole tail muscle. Whatever the case, the translational regulation of MHC synthesis occurs well before any degradation of the tail tissue is evident and appears to be one of the earliest events in the hormone-induced cell death program of the tadpole tail muscle.

Connexins in skeletal muscle development and disease

Gap junctions consist of clusters of intercellular channels composed of connexins that connect adjacent cells and allow the exchange of small molecules. While the 21 member multi-gene family of connexins are ubiquitously found in humans, only Cx39, Cx40, Cx43 and Cx45 have been documented in developing myoblasts and injured adult skeletal muscle while healthy adult skeletal muscle is devoid of connexins. The use of gap junctional blockers and cultured myoblast cell lines have suggested that these connexins play a critical role in myotube formation and muscle regeneration. More recent genetically-modified mouse models where Cx43 function is greatly compromized or ablated have further supported a role for Cx43 in regulating skeletal muscle development. In the last decade, we have become aware of a cohort of patients that have a development disorder known as oculodentodigital dysplasia (ODDD). These patients harbor either gain or loss of Cx43 function gene mutations that result in many organ anomalies raising questions as to whether they suffer from defects in skeletal muscle formation or regeneration upon injury. Interesting, some ODDD patients report muscle weakness and loss of limb control but it is not clear if this is neurogenic or myogenic in origin. This review will focus on the role connexins play in muscle development and repair and discuss the impact of Cx43 mutants on muscle function.

Postembryonic expression of the myosin heavy chain genes in the limb, tail, and heart muscles of metamorphosing amphibian tadpoles

Thyroid hormone is presumed to play a role in initiating and/or orchestrating the postembryonic expression of the genes encoding isoforms of the myosin heavy chains (MHCs) that characterize the muscle fibres in an adult organism. The fact that the postembryonic development of a free‐living amphibian tadpole takes place during its thyroid hormone‐dependent metamorphosis has made the metamorphosing tadpole an ideal system for elucidating the molecular mechanism(s) by which this hormone affects these postembryonic changes. In this review, we summarize the results from recent studies focused on the postembryonic expression of the MHC genes in the skeletal muscles and hearts of metamorphosing anuran (Rana catesbeiana) tadpoles. The demonstration that mRNAs encoding at least five of the MHC isoforms present in the tadpole tail muscles are also present in the adult hind‐limb muscles and that an mRNA encoding a cardiac‐specific MHC isoform is present in the heart of both the tadpole and adult organism, rules out the possibility that thyroid hormone initiates the expression of these MHC genes. Instead, it seems more likely that this hormone acts by modulating the expression of one or more of the genes encoding these particular MHC isoforms. Whatever the case, the fact that sequence homology suggests that the five distinct skeletal muscle‐specific MHCs are all “fast” isoforms raises the question of how these MHCs are distributed among the three different fibre types described for Rana. On the other hand, the possibility exists that the mRNAs for one or more of these fast MHC isoforms encode developmental isoforms that are present but not translated in the muscles of the tadpole and/or adult frog. Finally, an evaluation of the evolutionary relatedness of the R. catesbeiana MHCs to the MHCs in another species of Rana and to the MHCs in other vertebrates discloses, among other things, that the nucleotide sequence in the R. catesbeiana cardiac MHC isoform is more closely related to the chicken ventricular MHC isoform than it is to any of the other MHC isoforms examined. Microsc. Res. Tech. 50:458–472, 2000.

Ultrasound-Guided Greater Palatine Nerve Block: A Case Series of Anatomical Descriptions and Clinical Evaluations

BACKGROUND:Greater palatine nerve (GPN) block is commonly performed for maxillary and palatal anesthesia by using bony landmarks. Ultrasound (US) can be used to consistently identify greater palatine foramen (GPF) as a defect in the bony palate enabling US-guided injections near the foramen. METHODS:We scanned and injected 16 undissected well-embalmed hemisectioned cadaveric heads after excluding major anatomical malformations. A linear high-frequency hockey stick probe (7–13 MHz) positioned in long axis to the hard palate visualized GPF as a discontinuity in the hard palate. US-guided injections of 0.1 mL India ink were made in an oblique plane. Specimens were dissected immediately after injection, and dye distribution was noted. The success rate of identification of GPF, number of attempts, and number of successful injections were recorded. The technique was evaluated clinically in 7 patients undergoing dental procedures. Five patients had US-guided injections, and 2 patients received US-assisted greater palatine canal blocks. RESULTS:GPF was successfully identified in 16 hemisectioned heads (n = 16). In 7 of 16 hemisectioned cadaveric specimens (n = 7/16), needle pass was seen on the US and traces of India ink were found within the greater palatine canal and pterygopalatine fossa. In the remaining heads (n = 9/16), the dye was observed in the mucosal tissue of the hard palate anterior to the GPF or in the soft palate. Clinical evaluation reconfirmed successful identification of GPF by US in 6 of 7 patients (n = 6/7). US-guided injections were successful in 6 of the 8 attempted blocks (n = 6/8) with median number (range) of attempts being 2 (1–4). US-assisted injections were successful in 2 patients (n = 2/2). CONCLUSIONS:US has the potential to successfully locate and characterize GPF in normal and edentulous maxilla. US-guided GPN blocks can be technically challenging. The clinical applicability of US guidance or assistance for GPN block needs further evaluation in a larger sample of patients.