The muscle fibre cytoskeleton of skeletal and heart muscle cells is composed mainly of intermediate filaments (IFs), that surround the myofibrils and connect the peripheral myofibrils with the sarcolemma and the nuclear membrane. Desmin is the first muscle specific IF protein to be produced in developing muscles and is the main IF protein in mature muscles. In skeletal muscle, desmin is particularly abundant at myotendinous and neuromuscular junctions. In the heart an increased amount of desmin is found at intercalated discs and in Purkinje fibres of the conduction system. Interactions between the IFs themselves, and between IFs and other structures such as Z-discs and the sarcolemma, are mediated by intermediate filament associated proteins (IFAPs). A transgenic mice model, which lacks the desmin gene have been developed to study the function of desmin. In these mice, morphological abnormalities are observed in both heart and skeletal muscles. Similar defects have been observed in human myopathies, caused by different mutations in the desmin gene. In the present thesis, skeletal and heart muscles of both wild type and desmin knock-out (K/O) mice have been investigated. Furthermore the cytoskeletal organisation in skeletal muscles from human controls and from a patient with desmin myopathy was examined. In the desmin K/O mice, no morphological alterations were observed during embryogenesis. These mice postnatally developed a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. Ruptures of the sarcolemma appear to be the primary event leading to muscle degeneration and fibrosis both in cardiac and affected skeletal muscles. In the heart the muscle degeneration gave rise to calcifications, whereas in skeletal muscles regeneration of affected muscle was seen. In mature wild type mice, the IF proteins synemin and paranemin, and the IFAP plectin were present together with desmin at the myofibrillar Z-discs, the sarcolemma, the neuromuscular junctions and the myotendinous junctions. Nestin was only found in these junctional regions. In desmin K/O mice, all four proteins were detected at neuromuscular and myotendinous junctions. The normal network of synemin and paranemin were not observed, whereas the distribution of plectin was preserved. In normal human muscles, synemin, paranemin, plectin and αB-crystallin were colocalised with desmin in between the myofibrils, at the sarcolemma and at myotendinous and neuromuscular junctions. In the human desmin myopathy, the distribution of desmin varied considerably. A normal pattern was seen in some fibres areas, whereas other regions either contained large subsarcolemmal and intermyofibrillar accumulations of desmin or totally lacked desmin. Nestin, synemin, paranemin, plectin and αB-crystallin also exhibited an abnormal distribution. They were often aggregated in the areas that contained accumulations of desmin. In cultured satellite cells from the patient, a normal network of desmin was present in early passages, whereas aggragates of desmin occurred upon further culturing. In the latter, also the nestin network was disrupted, whereas vimentin showed a normal pattern. αB-crystallin was only present in cells with a disrupted desmin network. Plectin was present in a subset of cells, irrespective of whether desmin was aggregated or showed a normal network. From the present study it can be concluded that an intact desmin network is needed to maintain the integrity of muscle fibres. Desmin may be an important component in the assembly of proteins, which connect the extrasarcomeric cytoskeleton with the extracellular matrix.

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The human heart has poor endogenous regeneration. If myocytes are lost due to injury, the myocardium is unable to restore its myocyte content and instead undergoes compensatory hypertrophy and remodeling. Cardiac tissue engineering aims to recreate and provide functional myocardium that replaces the injured myocardium. In this study, human engineered heart muscle (EHM) from cardiomyogenically differentiated human embryonic stem cells was generated. EHMs consisted of elongated, anisotropically organized cardiomyocyte bundles and responded “physiologically” to increasing calcium concentrations. To generate large myocardium capable of encompassing the ventricles, a novel process to systematically upscale the dimensions of engineered myocardium to a humanized Biological Ventricular Assisted Device (hBioVAD) was introduced. The hBioVADs formed a “pouch-like” myocardium at rabbit heart dimensions and were beating spontaneously. Further enhancement by biomimetic pulsatile loading generated “more mature” myocardium. Additional paracrine functionality was integrated by generating insulin-like growth factor-1 (IGF-1) secreting fibroblasts for tissue engineering applications. IGF-1 release induced higher levels of Akt phosphorylation and hypertrophy in cardiomyocytes resulting in increased force generation of EHM. Finally, feasibility of “paraBioVAD” (IGF-1 cell line and cardiomyocytes) implantation was demonstrated in a healthy rat model. Histological observations demonstrated engraftment on the heart and the presence of vascular structures. In conclusion, a humanized “paraBioVAD” technology for mechanic and paracrine heart support was developed. Future studies will assess its therapeutic utility in heart failure