The open circulatory system of arthropods consists a dorsal heart and arteries, but there are no veins to bring the blood back to the heart. So, spider heart is simply an aorta or other blood vessel, and the hemolymph is pulsed throughout the body by muscle contractions (Wirkner et al. 2010, 2013). Nevertheless, basic principles and structural organization of cardiac muscle cells are quite similar with more complex system of vertebrates (Kim and Moon 2018; Sun et al. 2020).
Basically, spider heart is a simple muscular tube that composed of two layers of heart wall, the internal myocardium and the external epicardium. The myocardium is the muscular tissue responsible for the contraction of the heart, and intercalated discs are present in this layer (Sommer and Waugh 1978). The myocardium in L. mactans showed as a thick muscle layer which are in direct contact to the hemolymph without intima.
In vertebrates, tunicates, and some molluscs, the heart beat is initiated and regulated by specialized groups of muscle cells, myocardial conducting cells. Although they are one of specialized cardiac muscle cells, the myocardial conduction cells initiate the action potential myogenically (Gordon et al. 2000; van Weerd and Christoffels 2016). But it has been reported that some invertebrates including insects, heart contraction is initiated and regulated by external nerve (Sherman 1987). Our TEM observation clearly shows that the cardiac muscle fiber of this spider is innervated by a branch of external nerves through neuromuscular junctions. It means that this spider’s heart is not myogenically driven but neurogenically driven.
Typically, cardiac muscles are connected with neighboring cells by intercalated discs, and they are observed under light microscope as dark-staining lines running perpendicular direction of muscle fibers. In cardiac muscle of L. mactans, a unique characteristic is the presence of dark-staining transverse lines that cross the cardiac cells at irregular intervals. They occur at the Z line of the sarcomere and can be visualized easily when observing a longitudinal section of the tissue. The intercalated disc provides the electrochemical and mechanical connection between neighboring cardiac muscle cells (Bennett 2018). Since the intercalated discs were considered to be a kind of intracellular structure, there were general agreements that cardiac muscle is a single functional unit called a syncytium (Dewey 1969). This is because myocardial tissue is composed of many branched cells that are joined end to end by intercalated discs (Paniagua et al. 1996).
Our electron microscopic observation also reveals that the intercalated disc is consist of the plasma membrane of adjacent cells. It has been reported that the membranes of the intercalated discs establish specific associations with a variety of intracellular and extracellular structures (Forbes and Sperelakis 1985). The intercalated discs are known to provide a scaffold for myofibrils and allow for rapid spread of contractile stimuli between cells (Gutstein et al. 2003; Goossens et al. 2007). The rapid spread of such contraction allows cardiac muscles to act as a functional syncytium.
It has been known that the skeletal muscle are consists of multinucleated muscle fibers and exhibit no intercalated discs. But, cardiac muscle consists of individual heart muscle cells connected by intercalated discs to work as a single functional organ (Franke et al. 2006). Since the intercalated discs connecting cardiac muscle cells to the syncytium, to support the rapid spread of action potentials of the cardiac tissue (Forbes and Sperelakis 1985; Gourdie et al. 1991; Veeraraghavan et al. 2014), the intercalated discs can be support synchronized contraction of the myocardium. It has been previously reported that the membranes of the intercalated discs establish specific associations with a variety of proteins and glycoproteins (Forbes and Sperelakis 1985; Bennett 2018).
In cardiac muscles of vertebrates, it has been known that three basic types of cell junction make up an intercalated disc, fascia adherens, desmosomes and gap junctions (Ehler 2016). In L. mactans, membrane in the intercalated disc greatly increases the surface area contact between the cells, and above three types of cellular junctions are clearly identified on the intercalated discs of cardiac muscle fibers. These three membrane junctions have their own functions and capable of coordinated contraction of spider heart. Therefore, these results strengthen the premise that contractile movement of spider heart is also controlled by the sliding filament system of muscle contraction within the sarcomere.
First, an adherens junction is defined as a cell junction whose cytoplasmic face is linked to the actin cytoskeleton (Hartsock and James Nelson 2007). Thus, adherens junction in L. mactans is anchoring sites for actin, and connect to the end of adjacent sarcomere of cardiac muscle fibers. In particular, the fascia adherens of spider is found as a broad intercellular junction both of sarcolemma and intercalated disc. This type of junction is found at the peripheral region of cellular attachment where actin filaments of the I-band insert and terminate. This is consistent with results reported from other vertebrates, since it has been known that the adherens junctions are composed of N-cadherin as a transmembrane component that connects to the actin filaments (Meng and Takeichi 2009; Ehler 2016).
Second, the intercalated discs are irregular transverse thickenings of the sarcolemma that contain another type of adherens junctions called macula adherens, or desmosome (Zhao et al. 2019). Desmosomes hold adjacent cardiac muscle fibers together during contraction by binding intermediate filaments (Delva et al. 2009). In L. mactans, a macula adherens is developed in areas of the scalloped border of the sarcolemma between the sites of filament insertion. Desmosomes are known to be composed of desmosome-intermediate filament complexes (Franke et al. 2006), which is a scaffolding of cadherin proteins, linker proteins and keratin intermediate filaments (Garrod and Martyn 2008). Thus, this macular adherens junction becomes of particular importance in intercalated discs, where the heart is exposed to increased mechanical load and needs to adapt to sustain its contractile function (Pruna and Ehler 2020).
Third, the intercalated discs also act as anchorage points for the contractile proteins, and they contain important channels called gap junctions (Forbes and Sperelakis 1985; Gourdie et al. 1991; Veeraraghavan et al. 2014). These cytoplasmic connections of adjacent cardiac muscle fibers permit the rapid spread of action potentials from one cell to another (Goodenough and Paul 2009). When gap junctions are abundant, membranes stain darker and histologists named these areas intercalated discs. Therefore, intercalated discs are gap junctions that link adjacent cardiac muscles so that electrical impulses can travel between cells and causes to contract almost simultaneously (Severs 1989). In L. mactans, intercalated discs are part of the cardiac muscle sarcolemma, so they also contain gap junctions for impulse conduction between muscle fibers. This is particularly true, because the gap junctions provide ion channels for intercellular communication between cardiac cells (Gutstein et al. 2003), producing depolarization of the heart muscle (Franke et al. 2006). Thus, cardiomyocytes are capable of coordinated contraction, controlled through the gap junctions of intercalated discs (Ehler 2016).
Recently, classification of cell junctions has been challenged by observations that classical desmosome proteins also identified in adherens junctions by immuno electron microscopy. Molecular studies have shown that intercalated discs consist for the most part of mixed type adherens junctions. Therefore, the terminology of ‘composite junction’ or ‘area composita’ was introduced to describe plaque-bearing cell-cell contacts at the intercalated disc (Franke et al. 2006). These represent an amalgamation of typical desmosomal and fascia adherens proteins in contrast to various epithelia. Thus adherens junctions in cardiac muscle differ from epithelial adherens junctions and desmosomes (Shimada et al. 2004; Borrmann et al. 2006).
Sarcolemma is used in electron microscopic studies to describe the unit membrane that encloses the cytoplasm of the muscle cell (McNutt 1975). In L. mactans, the sarcolemma of cardiac muscle cell has a basic unit membrane which composed of a plasma membrane and two boundary lamina. Previous studies have shown that the boundary lamina is a specialized surface coat which composed of fibrillar glycoprotein material (Forbes and Sperelakis 1985). The surface coat or glycocalyx covers most of the external aspect of the sarcolemma, so called as sarcolemma-glycocalyx complex (Lee and Hsu 1987). The glycocalyx may be a site of calcium binding and exchange across the cell membrane in cardiac muscle (Adams and Schwartz 1980), because depolarization of the sarcolemma is associated with an influx of calcium into the cell.
Neural control of muscle systems in arachnids is poorly understood in comparison to the other large classes of arthropods such as the crustaceans and insects (Sherman 1987). It has been reported that spiders have the neurogenic heart from the findings of a cardiac ganglion on the heart (Wilson 1967), recording electrical impulses of heartbeat (Sherman and Pax 1968) and detailed histological examination of the cardiac ganglion (Bursey and Sherman 1970). The neurogenic heartbeat in lobsters and horseshoe crab generates periodic bursts of action potentials by the neural elements associated with the myocardium, however the myogenic heartbeat in molluscs and vertebrates initiates rhythmic contractions of the myocardium by the muscle cells itself in the heart (Sherman 1987).
Our transmission electron microscopic observation clearly shows that neuronal axons from cardiac ganglion extend into the myocardium. In addition, neuromuscular synapses are also present along the surface of the myocardial cells. This is consistent with the physiological evidence for multiple neuronal input to each myocardial cell (Sherman and Pax 1968; Ude and Richter 1974), which suggests that the pacemaker cells in spiders are modified neurons that are attached to the heart. This is an important difference comparing to vertebrates since heart contraction is not self or myogenically driven but neuronally driven. Although vertebrate hearts are innervated similarly by neurons from the autonomic nervous systems but these neurons act in only a modulatory function.