The correlation of the physiological activity with the structural integrity of the building blocks has recently been documented by the discovery of several mutations in contractile proteins which lead ultimately to myofibrillar disarray and severe myopathies especially in the human heart. All of these observations indicate that primary sequence of contractile proteins is intimately linked to sarcomere structure and function. Thus, the interaction of contractile muscle proteins sustaining the assembly of sarcomeres are important maintaining fully functional muscle. This is especially important in heart where regeneration does not allow repair of impaired or lost functions.
The assembly of new sarcomeres is studied in cultured cells of skeletal and cardiac origin. The non-sarcomeric cytoskeleton was thought to form a scaffold leading to the final structure of the myofibril. There are, however, also adaptive cytoskeletal responses to the culture conditions of cells which may overshadow the factual contribution of the cytoskeletal structures to myofibrillogenesis. In addition, most of the studies carried out with cultured muscle cells concluded that there is a sequential expression of contractile proteins during the differentiation. It has been postulated that such a timely expression sequence may be a most important prerequisites for the assembly of new sarcomeres. With the improved confocal microscopy technology developed also in our laboratory, we are currently investigating the occurrence of the first sarcomeres in the nascent heart tube and the relation of such "pioneer" sarcomeres to the cytoskeletal structures present in the same cell.
Even if the myofibrils are established, the contracting cardiomyocytes can still proliferate until they become post mitotic and enter a phase of physiological hypertrophy, when the cells meet the increased demand on work load upon birth by cell growth and myofibrillogenesis rather than by proliferation of the cardiomyocytes. To date a lot of research has been devoted to the elucidation of the mechanisms of induction of heart hypertrophy under pathological conditions. Comparatively little research has been dedicated to the study of regulation of myofibril accumulation during early postnatal development leading to cell growth also known as the physiological hypertrophy of cardiomyocytes. The factors regulating this type of hypertrophy are possibly crucial for the understanding of pathological hypertrophy.
It has been found that gene inactivation of contractile proteins genes lead to severe impairment of heart function in mouse embryos or neonatal animals. Recently a collaboration was established with the group of Dr. Pico Caroni from the FMI in Basel concerning the MLP knock out mice that have been generated in the Caroni laboratory. MLP is a small molecule interacting with actin. The phenotype of the MLP deficient mice was perceived by a marked cardiomyopathy resembling the human disease of "dilated cardiomyopathy". The myofibrils in the tissue are in disarray and very likely lead to a deficiency in cardiac performance. The investigation of cytoarchitecture of developing cardiomyocytes should allow the characterization of the lesion and indicate the role of the MLP protein in normal heart.
The mechanisms that guide macromolecular assembly of contractile proteins into sarcomeres at specific sites are not clear at all. Epitope labeling techniques have been introduced allowing to study the spatial organization within living cells of any component of the contractile apparatus even against the homologous cellular background. It has been shown that several viral epitopes can be introduced into contractile proteins without interfering with their assembly. Most recently, green fluorescent labeled proteins have been expressed in cardiomyocytes and were shown in living cells to interact correctly with existing myofibrils and not to interfere with beating activity. Such cells will be used as indicator cells to test other proteins and mutants for interference with myofibril integrity. The sorting specificity was determined for members of several isoprotein families like the alkali MLC, the actins, most of the tropomyosin isoproteins (collaboration with Dr. D. Helfman), several myosin heavy chains (collaboration with Drs. K. Chien and D. Becker, UC San Diego), a-actinins and others. In addition to the behavior in cardiomyocytes other cell types have been investigated like smooth muscle cells (collaboration with Drs. Nicole Mounier, Christine Chaponnier, Giulio Gabbiani, University of Geneva, Switzerland). So far in all studies the types of isoprotein was shown to play an important role for the cytoarchitecture studied.
The mechanism of direction of myofibrillar proteins like myomesin to their site of incorporation into sarcomeres is investigated at molecular level. Full length and fragments corresponding to single domains or groups of domains of myomesin heart cDNA were constructed as expression plasmids with an epitope tag and were expressed in neonatal cardiomyocytes. We have been able to localize the domain 2 responsible for specific interaction at the M-line. The mechanisms of how this specificity is achieved is now being analyzed.
The biological importance of the isoprotein-specific C-terminal extensions generated by alternative splicing in chicken striated muscle is investigated in more detail because it may also help to understand the tissue specific isoforms of chicken myomesin. The C-terminal extensions have been cloned and expressed in E. coli with the aim to test the resulting peptides for binding to the myofibrils and for the generation of isoprotein-specific antibodies using the proteins expressed in bacteria. The mouse genes of M-protein and myomesin have been cloned and are analyzed (collaboration with Dr. D. Furst , University of Potsdam, Germany). Although the coding sequences are quite distinct the structure of the genes are highly conserved. Both genes will be targeted in mice in order to inactivate them using ES cell lines. The construction of the targeting vectors using standard elements is under way and mutant ES cells will be constructed soon. These mutant cells will be used to create mice with ablated M-protein or myomesin genes. The analysis of the mice if the homozygotes produce offspring will be analyzed for their myofibrillar structures and physiological performance in heart and skeletal muscle. In case the KO phenotype is lethal for one or both proteins, double allelic KO ES cells will be created. Under selected culture conditions such cells can be brought to differentiate to cardiomyocytes and such cells can then be analyzed in detail for the structure of the myofibril. From these experiments we hope to learn more on the function of the M-band proteins and their importance for myofibrillogenesis.
It is well established that muscle and non-muscle cells contain many different contractile proteins. These proteins are expressed in a temporal manner and their spatial organization within the cells gives rise to cytoskeletal structures and highly specialized myofibrils resulting in a complex cytoarchitecture. The mechanisms governing cytoplasmic organization is a major question in cell biology and concern developmental processes as well as the investigation of the steady state of cells. The goal of the projects in the Perriard group is to understand macromolecular assembly of cytoskeletal structures and organelle functions associated with these assembly processes. Myofibrillogenesis is in the center of our attention, because sarcomere structures are unique to cross-striated muscle, have features which are easy to characterize at the structural and as well as at the functional level. The contractile activity is a peculiarity of muscle and a sign of correct assembly of the numerous species of molecules into sarcomeres. This is especially obvious for heart muscle cells with their autonomous beating activity.