Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium.

BACKGROUND: Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. METHODS AND RESULTS: Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1+/-0.3 gold particles/micrometer A-band 3.3+/-0.2 gold particles/micrometer mean+/-SE, P=0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. CONCLUSIONS: Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.

Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.

Downregulation of immunodetectable connexin43 and decreased gap junction size in the pathogenesis of chronic hibernation in the human left ventricle.

BACKGROUND: The regional wall motion impairment and predisposition to arrhythmias in human ventricular hibernation may plausibly result from abnormal intercellular propagation of the depolarizing wave front. This study investigated the hypothesis that altered patterns of expression of connexin43, the principal gap junctional protein responsible for passive conduction of the cardiac action potential, contribute to the pathogenesis of hibernation. METHODS AND RESULTS: Patients with poor ventricular function and severe coronary artery disease underwent thallium scanning and MRI to predict regions of normally perfused, reversibly ischemic, or hibernating myocardium. Twenty-one patients went on to coronary artery bypass graft surgery, during which biopsies representative of each of the above classes were taken. Hibernation was confirmed by improvement in segmental wall motion at reassessment 6 months after surgery. Connexin43 was studied by quantitative immunoconfocal laser scanning microscopy and PC image software. Analysis of en face projection views of intercalated disks revealed a significant reduction in relative connexin43 content per unit area in reversibly ischemic (76.7+/-34.6%, P<.001) and hibernating (67.4+/-24.3%, P<.001) tissue compared with normal (100+/-30.3%); ANOVA P<.001. The hibernating regions were further characterized by loss of the larger gap junctions normally seen at the disk periphery, reflected by a significant reduction in mean junctional plaque size in the hibernating tissues (69.5+/-20.8%) compared with reversibly ischemic (87.4+/-31.2%, P=.012) and normal (100+/-31.5%, P<.001) segments; ANOVA P<.001. CONCLUSIONS: These results indicate progressive reduction and disruption of connexin43 gap junctions in reversible ischemia and hibernation. Abnormal impulse propagation resulting from such changes may contribute to the electromechanical dysfunction associated with hibernation.

Ultrastructural analysis of skeletal muscle. Microvascular dimensions and basement membrane thickness in chronic heart failure.

Chronic heart failure (CHF) is characterized by increased systemic vascular resistance and diminished blood flow to exercising skeletal muscle. The pathogenesis of the increased resistance is not known, and may be due to muscle atrophy, functional abnormalities of resistance vessels or to structural changes in the microcirculation such as endothelial cell swelling. We have compared the ultrastructure of the microvasculature in needle biopsies of the quadriceps muscle from seven control subjects with normal left ventricular function to 10 patients with moderate or severe heart failure, optimally treated and without evidence of fluid overload. Samples were processed for ultrathin sectioning using ruthenium red as a specific basement membrane (BM) stain. Electron micrographs were taken of 10 transversely cut capillaries from each specimen. The total cross-sectional area of the vessels and the area of the endothelium was determined, and the short axis diameter was measured as an index of vessel diameter. The BM thickness was calculated from the mean of six readings around the periphery of the vessel. The short axis diameter in the two groups was not significantly different (controls 3.37 +/- 0.21 microns, CHF 3.56 +/- 0.37 microns, mean +/- 1SD). No difference in total cross-sectional area (controls 11.64 +/- 1.86 microns 2, CHF 13.56 +/- 2.78 microns 2) or area of the endothelium (controls 4.90 +/- 1.18 microns 2, CHF 6.00 +/- 1.58 microns 2) was observed. The thickness of the BM was marginally increased in subjects with CHF when compared to control subjects (0.31 +/- 0.077 microns vs 0.246 +/- 0.047 microns, P = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Spatiotemporal relation between gap junctions and fascia adherens junctions during postnatal development of human ventricular myocardium.

BACKGROUND: The growing postnatal human heart maintains electromechanical function while undergoing substantial changes of cellular topology and myocardial architecture. The capacity for growth and remodeling of ventricular myocardium in adaptation to the hemodynamic changes of early infancy later declines. This decline is associated with changes in electromechanical properties of the myocardium, which suggest that the electrical and mechanical interactions between the myocytes may change in an age-dependent manner. Thus, reduction in the capacity for myocardial growth and adaptability may relate to age-dependent alterations in the patterns of the intercellular junctions that mediate electrical and mechanical coupling. We therefore examined the hypotheses that (1) age-dependent changes in the distribution patterns of gap junctions and fasciae adherentes, the intercellular junctions responsible, respectively, for electrical and mechanical coupling, accompany postnatal development in the human heart and that (2) such changes continue into the first few years of childhood. Further, the spatial relation between the two types of junction, for which a close association has been hypothesized as necessary, was explored. METHODS AND RESULTS: Ventricular myocardial gap-junction distribution was investigated in 23 pediatric surgical patients (4 weeks to 15 years old) by quantitative immunohistochemical localization of the principal cardiac gap-junctional protein, connexin43, using confocal microscopy. Immunolocalization of fascia adherens junctions by labeling N-cadherin, and correlative immunogold and standard electron microscopy, were performed in parallel. In the neonate, connexin43 gap junctions have a punctate distribution over the entire surface of the ventricular myocytes. With advancing age, gap junctions become progressively confined to the transverse terminals of the cell, ie, toward the distribution within the intercalated disk characteristic of the adult ventricle. The transversely arrayed proportion of gap-junctional label showed a linear increase with age (R = .88, P < .001), reaching the adult pattern at about 6 years, and the fascia adherens junctions showed a similar progression. Electron microscopy confirmed the changing pattern of junctional contacts and demonstrated that initially gap junctions and adhering junctions are frequently not closely adjacent but become increasingly so with maturation of the intercalated disk. CONCLUSIONS: Changes in the spatiotemporal patterns of the intercellular junctions responsible for electrical and mechanical coupling are closely coordinated in postnatal human ventricular myocardium and continue to about 6 years of age. Over this period there is a close and increasing association between the gap junctions and fascia adherens junctions. These changes in the distribution of intercellular electrical and adhering junctions may parallel the changing functional requirements of the ventricle, from a distribution that facilitates the remodeling necessitated by rapid growth and changing hemodynamics to that of the relatively stable and rapidly conducting adult myocardium. These age-related changes may also diminish the ability for appropriate myocardial remodeling in response to physiological, pathological, or surgical hemodynamic alterations.