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Cardiac myocytes are the contractile cells that enable the heart to function as a pump. They are, thus, responsible for ensuring that sufficient oxygenated blood and metabolites reach the tissues to meet the body’s needs, whether at rest or during exercise. Each myocyte beats over three billion times in a typical human lifespan; to work as an effective pump, the activities of over three billion myocytes must be synchronized. To fulfill these functions, the cardiac myocyte is equipped with highly specialized subcellular machinery. With the aid of a range of complementary high resolution imaging techniques, this chapter illustrates and explains how the key structural components of the myocyte are organized to enable the coupling of electrical excitation to contraction of the myofibrils, and how the contractile activities of individual cells are coordinated to achieve organ function.

Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood. This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45. Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction. These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart.

The term freeze-fracture cytochemistry embraces a series of techniques which share the goal of chemical identification of the structural components viewed in freeze-fracture replicas. As one of the major features of freeze fracture is its ability to provide planar views of membranes, a major emphasis in freeze-fracture cytochemistry is to identify integral membrane proteins, study their spatial organization in the membrane plane, and examine their role in dynamic cellular processes. Effective techniques in freeze-fracture cytochemistry, of wide application in cell biology, are now available. These include fracture-label, label fracture, and the freeze-fracture replica immunolabeling technique (FRIL). In fracture-label, samples are frozen and fractured, thawed for labeling, and finally processed for viewing either by critical-point drying and platinum-carbon replication or by thin-section electron microscopy. Label-fracture involves immunogold labeling a cell suspension, processing as for standard freeze-fracture replication, and then examining the replica without removal of the cellular components. Of greatest versatility, however, is the FRIL technique, in which samples are frozen, fractured, and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate (SDS) to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits the distribution of identified components to be viewed superimposed upon high resolution planar views of replicated membrane structure, for both the plasma membrane and intracellular membranes in cells and tissues. Examples of how these techniques have contributed to our understanding of cardiovascular cell function in health and disease are discussed.

Publisher Summary Stabilization of the biological structure by the physical process of freezing (cryofixation) forms the starting point for freeze fracture and deep etching and for freeze substitution, cryoultramicrotomy, and cryoelectron microscopy. For freeze fracture and deep etching, the requirement for a larger specimen size precludes true vitrification throughout the specimen because the maximal cooling rate possible within the sample is limited by the rate of heat conduction through it. Ice crystal damage can be avoided at relatively slow cooling rates if the specimen is first infiltrated with a buffered cryoprotectant. To enable processing through the various subsequent steps of freeze fracture or deep etching, specimens are first mounted on specially designed supports. Standard specimen supports are made from metals of high thermal conductivity and are as small as is compatible with ease of handling. Various techniques have been developed for mounting cultured cells for freeze fracture. The traditional alternative method for freezing specimens is to immerse them manually into a secondary cryogenic liquid cooled to near its freezing point using liquid nitrogen as the primary cryogen.