Andy Wessels

Demonstration of 'cardiac-specific' myosin heavy chain in masticatory muscles of human and rabbit

SummaryHuman and rabbit masticatory muscles were analyzed immuno-and enzyme-histochemically using antibodies specific to ‘cardiac’ α, slow and fast myosin heavy chain isoforms. In human masseter, temporalis, and lateral pterygoid muscle ‘cardiac’ α myosin heavy chain is found in fibres that contain either fast, or fast and slow myosin heavy chain. In rabbit masseter, temporalis and digastric muscles, fibres are present that express ‘cardiac’ α myosin heavy chain either exclusively, or concomitantly with slow myosin heavy chain or fast myosin heavy chain. Our results demonstrate a much broader distribution of ‘cardiac’ α myosin heavy chain than hitherto recognized and these might explain in part the specific characteristics of masticatory muscles. The ‘cardiac’ α myosin heavy chain is only found in skeletal muscles originating from the cranial part of the embryo (including the heart muscle) suggesting that its expression might be determined by the developmental history of these muscles.

New findings concerning ventricular septation in the human heart. Implications for maldevelopment.

BackgroundThe mechanics involved in development of the inlet component of the morphologically right ventricle are, as yet, undecided. Some argue that this component is derived from the descending limb of the ventricular loop, and that the inlet and apical trabecular components of the muscular ventricular septum have separate developmental origins. Others state that the entirety of the right ventricle grows from the ascending limb of the loop, and that the muscular septum, apart from its outer component, has a unitary origin. We now have material from human embryos at our disposal, which, we believe, solves this conundrum Methods and ResultsWe used a monoclonal antibody against an antigen to neural tissue from the chick to demarcate a ring of cells separating the descending (inlet) and ascending (outlet) limbs of the developing ventricular loop of the human heart. Preparation of serial sections of graded human embryos enabled us to trace the fate of this ring, and hence the formation of the inlet of the right ventricle, to the completion of cardiac septation. Eight embryos were studied, encompassing stages 14–23 of the Carnegie classification. The ring of cells initially separating the ascending and descending limbs of the ventricular loop were, at the conclusion of ventricular septation, located within the atrioventricular junction, sequestrated for the most part in the terminal segment of atrial myocardium ConclusionsOur study conclusively shows that the inlet component of the morphologically right ventricle is derived from the ascending limb of the embryonic ventricular loop, and that the inlet and apical trabecular components of the muscular septum are derived from the same primary ventricular septum.

New method for the accurate characterization of single human skeletal muscle fibres demonstrates a relation between mATPase and MyHC expression in pure and hybrid fibre types.

SummaryIn the present study we have developed a method which, by combining histochemical, immunohistochemical, electrophoretic and immunoblotting analyses on a single fibre, enables a sensitive characterization of human skeletal muscle fibres dissected from freeze-dried biopsy samples. For histochemical (and immunohistochemical) analysis fibre fragments (500 μm) of individual fibres were mounted in an embedding medium to allow cryostat sections of normalized thickness to be reproducibly obtained. The specificity of the myofibrillar Ca2+ ATPase (mATPase) staining profiles in gelatin-embedded single fibre sections was tested by immunohistochemical reactions with anti-myosin heavy chain (MyHC) monoclonal antibodies specific to human MyHC I, IIA, IIB and IIA+IIB and by gel electrophoresis.The combined methodologies demonstrated the specificity of the mATPase staining patterns which correlated to the expression of distinct MyHC isoforms. In addition the results provide evidence that many fibres co-expressed different MyHC isoforms in variable relative amounts, forming a continuum. Staining intensities for mATPase, converted into optical density values by image analysis revealed that a relationship between mATPase and MyHC expression holds for hybrid fibres even when displaying one MyHC type with overwhelming dominance. The results also revealed that three MyHC isoforms I, IIA and IIB can be co-expressed on a single muscle fibre. In such a case mATPase alone, with the current protocols, does not allow an accurate characterization of the specific MyHC-based fibre type(s). although some hybrid fibres may have displayed a non-uniform expression of myosins along their lengths, most fibres from the IIA/B group (type) remained very stable with respect to the relative amounts of the MyHCs expressed. Finally, a second slow MyHC isoform was recognized on immunoblots of a mixed muscle sample.

Distribution of Atrial and Nodal Cells Within the Rabbit Sinoatrial Node Models of Sinoatrial Transition

BACKGROUNDnIn the sinoatrial node (SAN) the course of the action potential gradually changes from the primary pacemaker region toward the atrium. It is not known whether this gradient results from different intrinsic characteristics of the nodal cells, from an increasing electrotonic interaction with the atrium, or from both. Therefore we have characterized the immunohistochemical, morphological, and electrophysiological correlates of this functional gradient.nnnMETHODS AND RESULTSnThe distribution of rabbit nodal myocytes in the SAN has been studied by immunohistochemistry. After cell isolation, the electrophysiological characteristics of different nodal cell types were measured. (1) The staining pattern of a neurofilament protein coincides with the electrophysiologically mapped pacemaker region in the SAN. (2) Enzymatic digestion of the SAN reveals three morphologically different nodal cell types and one atrial type. Of each nodal cell type, neurofilament-positive as well as neurofilament-negative myocytes are found. Atrial cells are all neurofilament-negative. (3) In contrast to previous findings, we observed atrial cells in the very center of the SAN. The relative number of atrial cells gradually increases from the central pacemaker area toward the atrium. (4) Differences in electrophysiological characteristics between individual nodal cells are not associated with differences in cell type.nnnCONCLUSIONSn(1) The expression of neurofilaments can be used to delineate the nodal area in the intact SAN but is not sufficiently sensitive for characterizing all individual isolated nodal cells. (2) A fundamentally different organization of the SAN is presented: The gradual increase in density of atrial cells from the dominant area toward the crista terminalis in the SAN causes a gradual increase of atrial electrotonic influence that may be an important cause of the gradual transition of the nodal to the atrial type of action potential.

THE MATPASE HISTOCHEMICAL PROFILE OF RAT TYPE IIX FIBRES : CORRELATION WITH MYOSIN HEAVY CHAIN IMMUNOLABELLING

SummaryIn the present study we report a novel histochemical method which, by sequential pre-incubations in alkaline and acidic media, selectively differentiates muscle fibres expressing myosin heavy chain IIX, on the basis of a specific profile for myofibrillar actomyosin ATPase (mATPase) activity. The enzyme reactions were tested for specificity by means of anti-myosin heavy chain monoclonal antibodies, which were characterized on Western blots of muscle homogenates. Enzyme histochemical reactions with the traditional pH buffers were compared to those of the new method and, in conjunction with the immunoreactions, used to confirm the relationship between MyHC expression and the distinct profiles for mATPase. Imrnunohistochemical reactions demonstrated that the new method only differentiates those fibres expressing myosin heavy chain IIX. The method revealed a continuum in which the intermediate staining intensities corresponded to hybrid fibres expressing myosin heavy chain IIX in combination with either the IIA or IIB forms. Quantitative histochemistry and immunohistochemistry (by image analysis), used to examine the relationship between staining intensities for mATPase and amounts of myosin heavy chain IIX expression, revealed that the new method discriminates well between hybrid fibres expressing variable amounts of the IIX isoform (r2 = 0.93).

Immunohistochemical studies show truncated dystrophins in the myotubes of three fetuses at risk for Duchenne muscular dystrophy.

We have performed immunohistochemical studies on muscle tissue of three 12 week old fetuses at risk for DMD, using antisera directed against regions located NH2-proximally and centrally in the rod shaped spectrin-like domain and against the COOH-terminus of dystrophin. All three fetuses had a family history of DMD. Truncated dystrophins were identified in all three cases by a positive reaction with the NH2-proximal antibody, different reactions with the central antibody, and a negative reaction with the COOH-terminal antibody. These data indicate that a panel of antibodies would, in principle, permit immunological mapping of dystrophin mutations. This is diagnostically important in the 35% of families where no mutation is detectable at the DNA level. Secondly, by using this mapping technique it may also become possible to identify the at risk haplotype when DNA analysis is not informative. This may be of great value in DMD carrier detection.