Analysis of Myofibrillar Organization and Degeneration by Fluorescence Confocal Microscopy

Abstract

Myofibrillar degeneration is an important pathological process in progressive cardiomyopathy leading to heart failure. Loss of myofibrils in vivo has been observed in both adaptive cardiac responses (i.e. hypertrophy) as well as in chemotherapeutic use of antitumor drugs with cardiotoxic side effects (i.e. doxorubicin). The molecular mechanism(s) of myofibrillar degeneration are poorly understood in comparison with the sequence of events involved in myofibrillar assembly and organization. Maintenance of myofibril integrity is dependent upon a variety of factors, including contractile protein stoichiometry and protein kinase activity.The repeating sarcomeric architecture of myofibrils is well suited to structural analysis, since disruption of normal organization is easily visualized by fluorescence microscopy. Many antibodies are available for use in observation of myofibril structure (see Table at right). Confocal microscopy provides advantages in studies of myofilament organization by allowing for direct and accurate measurement of distances to within 0.2 βm and the ability to perform vertical sectioning through individual cells. This vertical (Z-axis) sectioning can be used to select the focal plane within the cell for observation, resulting in higher resolution images by reducing fluorescent signals from above and below the plane. Image analysis software enables the user to create projections of optically sectioned cardiomyocytes which can be rotated to reveal interior structural relationships previously unobserved with single sections or conventional epifluorescence microscopy. Examples of image analysis will highlight useful features such as distance measurement, periodicity, pixel intensity, colocalization of dual labels, and three dimensional reconstruction.