Skeletal muscles drive voluntary movements. Striated muscles allow fish to swim, birds to fly and our heart to beat. Skeletal muscles are built of multiple fascicles, which are bundles containing many muscle fibers. Looking at these structures under a microscope, smaller structures of muscle fibers, so-called myofibrils, become visible. These structures are highly organized and show regular patterns of specific units. These specific periodic subunits are the sarcomeres. Sarcomeres self-assemble as the smallest unit of skeletal muscles. Mature sarcomeres are crystal-like structures with a specific size of 2-3 micrometer. Sarcomeres are bounded by the so-called Z-disc, which contains more than thirty different proteins. Polar actin filaments are cross-linked to the Z-disc. Myosin motor filaments are anchored, facing the center of the sarcomere. The giant protein titin links myosin and actin filaments and stabilizes the sarcomere. Sarcomeres shorten in length during muscle contraction, by relative sliding of myosin through actin filaments. The myosin motor filaments walk through the polar actin filament, under energy conversion. While this sliding mechanism is known, it is unclear how sarcomeres form during the multi-stage developmental process of skeletal muscles. How to build a sarcomere? Despite many years of research, it is poorly understood how sarcomeres self-assemble into regular patterns. In this context, the main questions are: which sarcomere components regularly align first, and how are actin filaments orientated with the correct polarity? To answer these questions, we observe early stages of myofibrillogenesis in the fruit fly Drosophila melanogaster and quantify the regular alignment of selected proteins using a new algorithm. Our result: Data of early stages of myofibrillogenesis display a temporal order during sarcomere assembly. Myosin, titin (Sallimus in fruit fly) and the Z-disc proteins alpha-actinin pattern first, while actin filaments only follow later. With these experimental observations, we postulate a new theoretical framework of sarcomere assembly. We establish a minimal mathematical model, including possible molecular interactions between myosin and Z-disc proteins. In particular, we show that a non-local interaction with the protein titin is sufficient to drive the pattern formation process. With this non-local interaction, we take into account that myosin and titin are extended filaments of a specific size, setting the sarcomere length. With agent-based simulations we demonstrate that the model is robust to stochastic small number fluctuations. In addition, it is known that mechanical tension is induced during myofibrillogenesis. Local tension, produced by myosin motor activity can guide the assembly of sarcomeres, too. Thus, we formulate a second minimal mathematical model accounting for local tension. Specifically, we set the focus on the non-covalent binding of alpha-actinin (catch-bond behavior). In the presence of local tension, the lifetime of alpha-actinin increases. We demonstrate for this second model that such an alternative non-local interaction can give rise to periodic patterns of a specific length under presence of mechanical tension, even though it is less robust. We discuss similarities and differences of both models and propose the possibility that myofibrillogenesis in biological systems is a combination of both models.

To analyse the large amounts of image data that are generated by biologists with modern microscopes, machine learning algorithms became increasingly popular. In collaboration with Frank Schnorrer and Cl ́ement Rodier at Institut de Biologie du Developpement de Marseille, as well as Ian Estabrook at Physics of Life, TU Dresden, this thesis applies the supervised machine learning algorithms ‘Support Vector Machine’ and ‘Random Forest’ to data obtained from fluorescence microscope images of myofibrillogenesis in Drosophila pupae with the aim to identify sarcomeres, the structures that makeup the highly regular myofibrils. For the implementation in MATLAB, methods such as ‘feature engineering’ are used to increase the performance by reinterpreting the input data and using physical characteristics of the sample system. The project also identifies the problem of class imbalance between positive and negative examples in the input data and counters it with a redefined learning cost. In conclusion, the use of machine learning algorithms for image analysis in biophysics is a very promising way to reduce manual labour. The choice of the best learning algorithm depends on the purpose the obtained output data should serve.

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The left ventricle of the heart relaxes when it fills with blood and contracts to eject blood into circulation to meet the body’s metabolic demands. Dysfunction in either relaxation or contraction of the left ventricle can lead to heart failure. Transmural heterogeneity is thought to contribute to normal ventricular wall motion but it is not well understood how transmural modifications affect the failing left ventricle. The overall hypothesis of this dissertation is that normal left ventricles exhibit transmural heterogeneity in cellular level contractile properties and with aging and heart failure there are region-specific changes in cellular level contractile mechanisms. Age is the biggest risk factor associated with heart failure and therefore we investigated transmural changes in Ca2+ handling and contractile proteins in aging F344 rats before the onset of heart failure. We found that in 22-month old F344 rats there is a region-specific decrease in cardiac troponin I phosphorylation in the sub-epicardium that may contribute to slowed myocyte relaxation in the sub-epicardial cells of the same age. We then investigated the transmural patterns of contractile properties in myocardial tissue samples from patients with heart failure. Force and power output reduced most significantly in the samples from the mid-myocardial region when compared to sub-epicardium and sub-endocardium of the failing hearts. There was a region-specific increase in fibrosis is the mid-myocardium of the failing hearts. Myocardial power output was correlated with key sarcomeric proteins including cardiac troponin I, desmin and myosin light chain-1. The results in this dissertation reveal novel region-specific modifications in contractile properties in aging and heart failure. These transmural effects can potentially contribute to disruption in normal wall motion and lead to ventricular dysfunction.

Tendon transfer surgery is used to improve the hand function of patients with nerve injuries, spinal cord lesions, cerebral palsy (CP), stroke, or muscle injuries. The tendon of a muscle, usually with function opposite that of the lost muscle function, is transferred to the tendon of the deficient muscle. The aim is to balance the wrist and fingers to achieve better hand function. The position, function, and length at which the donor muscle is sutured is essential for the outcome for the procedure. In these studies the significance of the transferred muscle’s morphology, length and apillarization was investigated using both animal and human models. Immunohistochemical, biochemical, and laser diffraction techniques were used to examine muscle structure. In animal studies (rabbit), the effects of immobilization and of tendon transfers at different muscle lengths were analyzed. Immobilization of highly stretched muscles resulted in fibrosis and aberrant regeneration. A greater pull on the tendon while suturing a tendon transfer resulted in larger sarcomere lengths as measured in vivo. On examination of the number of sarcomeres per muscle fiber and the sarcomere lengths after 3 weeks of immobilization and healing time, we found a cut-off point up to which the sarcomerogenesis was optimal. Transfer at too long sarcomere lengths inhibited adaptation of the muscle to its new length, probably resulting in diminished function. In human studies we defined the sarcomere lengths of a normal human flexor carpi ulnaris muscle through the range of motion, and then again after a routinely performed tendon transfer to the finger extensor. A calculated model illustrated that after a transfer the largest force was predicted to occur with the wrist in extension. Morphological studies of spastic biceps brachii muscle showed, compared with control muscle, smaller fiber areas and higher variability in fiber size. Similar changes were also found in the more spastic wrist flexors comparing with wrist extensors in children with CP. In flexors, more type 2B fibers were found. These observations could all be due to the decreased use in the spastic limb, but might also represent a specific effect of the spasticity. In children and adults with spasticity very small fibers containing developmental myosin were present in all specimens, while none were found in controls. These fibers probably represent newly formed fibers originating from activated satellite cells. Impaired supraspinal control of active motion as well as of spinal reflexes, both typical of upper motor syndrome, could result in minor eccentric injuries of the muscle, causing activation of satellite cells. Spastic biceps muscles had fewer capillaries per cross-sectional area compared to age-matched controls, and also a smaller number of capillaries around each fiber. Nevertheless, the number of capillaries related to the specific fiber area was normal, and hence the spastic fibers are sufficiently supplied with capillaries. This study shows that the length of the muscle during tendon transfer is crucial for optimization of force output. Laser diffraction can be used for accurate measurement of sarcomere length during tendon transfer surgery. Wrist flexor muscles have more morphological alterations typical of spasticity compared to extensors.

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Cette thèse est consacrée à la modélisation de la réponse transitoire d'une fibre musculaire squelettique soumise à des sollicitations mécaniques rapides. A l'échelle du nanomètre, la fibre musculaire contient des filaments d'actine et de myosine regroupés en unités contractiles appelées "sarcomères". Le filament de myosine est un assemblage de moteurs mol ́eculaires qui, en présence d'ATP, s'attachent et se d ́etachent p ́eriodiquement au filament d'actine. Au cours de ce processus d'attachement-détachement, la myosine génère une force lors d'un changement de conformation appelé "power-stroke". Ses caractéristiques peuvent être étudiées lors de la réponse transitoire de la fibre soumise à des sollicitations mécaniques rapides. Nous proposons un modèle mécanique innovant du demi-sarcomere permettant de relier les caractéristiques de la myosine à la réponse de la fibre complète. A la différence des modèles existants, privilégiant une approche discrète, ce modèle s'appuie sur la définition d'un potentiel d'énergie continu qui prend en compte une interaction de champ moyen entre les moteurs moléculaires. Ce système présente des réponses radicallement différentes à longueur imposée et à force imposée. Nous proposons en particulier une explication à la différence de cinétique observée expérimentalement. Nous montrons également que le demi-sarcomere est m ́ecaniquement instable ce qui explique les inhomogénéités de longueurs observées dans une myofibrille.

Hypertrophic cardiomyopathy is a heart disease that is characterized by an enlarged heart muscle. Mutations to sarcomere proteins in the muscle fibers give rise to the disease, and this review aims to compile the mechanisms by which the mutations cause the disease phenotype. β-myosin heavy chain mutants affect the thick filament structure and contraction velocity of the muscle. Mutations to the myosin-binding protein C produces truncated proteins with decreased expression in the cells. Troponin T mutants cause myofibrillar disarray, alters affinity to α-tropomyosin, and are linked to a higher risk of sudden death. Troponin I is an unpredictable mutant that needs to be further researched but is thought to cause regulatory problems. Mutations to α-tropomyosin and the regulatory myosin light chain both affect the Ca2+-affinity of the proteins and leads to contractile problems. Hypercontractility as a result of the mutations seems to be the primary cause of the disease. Hypertrophic cardiomyopathy is linked to sudden death, and factors such as a family history of sudden death, multiple simultaneous mutations, unexplained syncope, non-sustained ventricular tachycardia, abnormal blood pressure response and extreme hypertrophy (>30 mm) heightens the risk of a sudden death. An increased knowledge about the disease will aid in the mission to better the treatments for the affected, but further investigation of pathological pathways needs to be performed.

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