Force Generation in Eccentric Contractions of the

Skeletal Muscle:

The Influence of Contraction Velocity, Fiber Type, Activation Level and

Structural Components

Von der Fakultät für Wirtschafts- und Sozialwissenschaften der Universität

Stuttgart zur Erlangung der Würde eines Doktors der Philosophie (Dr. phil.)

genehmigte Abhandlung

Vorgelegt von

Sven Weidner

aus Kyritz

Hauptberichter: Prof. Dr. Tobias Siebert

Mitberichter: Prof. Dr. Oliver Röhrle

Tag der mündlichen Prüfung: 16.10.2025

Institut für Sport- und Bewegungswissenschaft

Abteilung für Bewegungs- und Trainingswissenschaft

2025



ii



Acknowledgements

...

Keep Ithaka always in your mind.

Arriving there is what you’re destined for.

But don’t hurry the journey at all.

Better if it lasts for years,

so you’re old by the time you reach the island,

wealthy with all you’ve gained on the way,

not expecting Ithaka to make you rich.

...

— C.P. Cavafy, Ithaka

The journey of completing this dissertation has indeed been long, winding, and at times

unpredictable — but, just as Cavafy writes, it has enriched me in countless ways. Reaching

this point is not just about the destination, but about everything I’ve learned, experienced,

and discovered along the way.

First and foremost, I would like to express my deepest gratitude to my parents Jürgen &

Sibylle and my sister Claudia for always supporting me throughout my entire life. A special

thanks goes to my partner Bianca, who had to put up with me after days of bad experiments

and helped me with the color concept of this work. Furthermore, I would like to thank my

supervisor, Prof. Dr. Tobias Siebert, for his unwavering guidance, patience, and insightful

feedback throughout all stages of this project. In this context, I would also like to mention Dr.

André Tomalka, who taught me the basics of the practical work and was a reliable constant

during my time at the University of Stuttgart. I’d also like to take this opportunity to thank

another important person who is often forgotten: Sybille Kegreiß. She is the heart and soul

of our department and always has an open ear for our problems.

I am also immensely grateful to my lab colleagues and friends, especially [Dr. Matthew

Millard, Tobias Elst, Dr. Mischa Borsdorf, Lukas Vosse and Stefan Papenkort], for the

many discussions, shared frustrations, and moments of encouragement. Your support turned

the challenges of research into something communal and worthwhile. In addition to my

colleagues in Stuttgart, I would also like to thank my project partners at other universities



ii

[Christian Rode, Matthew Araz and Daniel Häufle]. In this context, the German Research

Foundation (DFG) deserves special mention. It was thanks to their funding (under projects

405834662, 354863464, and 449912641) that this thesis was possible.

Also thanks goes to my alma mater, Bielefeld University, and its staff and students. A

special thank you goes to Dr. Christian Vobejda and Dr. Konstantinos "Kostas" Velentzas,

who often discouraged me from pursuing a career in academia, but who, through their work,

set a professional role model for me, one I have tried to follow. Therefore, it is your fault

that I wrote this thesis. I would also like to thank Kostas, my big Greek brother, for the

critical feedback on this work. Saying this I also like to thank my former classmate Laura

Buchner for her critical and detailed feedback on this thesis and all the articles she provided

me through my years in Stuttgart.

Lastly, I want to acknowledge the quiet moments, the setbacks, the doubts — they, too,

were part of the journey. I reach the end of this phase not with the expectation that Ithaka

will reward me, but with deep appreciation for all the treasures collected along the way.



Abstract

The present dissertation aims to deepen our understanding of the complex physiology of

skeletal muscle during eccentric contractions and stretch-shortening cycles. Building on

a series of in vitro experiments using skinned muscle fiber preparations, this work inves-

tigates the mechanical behavior of muscle tissue under dynamic loading conditions and

varying contraction velocities. The individual chapters describe and interpret specific force-

and power-related characteristics during active muscle stretching and shortening, which

are essential to understanding the mechanisms underlying enhanced force production and

energy efficiency in eccentric contractions and stretch-shortening cycles. Despite extensive

research in muscle physiology, key aspects of force generation during dynamic muscle

actions remain unresolved. Therefore, quantifying specific biomechanical properties under

stretch-shortening cycle conditions contributes to a more comprehensive understanding of

muscle function.

Chapter 1 serves as the conceptual and methodological foundation of this thesis and pro-

vides the context for the subsequent chapters. At the beginning it presents the motivation for

this research, emphasizing the fundamental importance of movement for humans—whether

in daily life, sports, health, or therapeutic contexts.

Section 1.1 provides the physiological background, introducing fundamental concepts

related to the structure and function of muscle tissue. It describes key mechanisms of muscle

contraction and highlights physiological effects that occur during or following dynamic

contractions, such as residual force enhancement after active stretch or force depression after

shortening.

Section 1.2 outlines the handling of biological tissue, with particular attention to the

careful preparation and manipulation of permeabilized muscle fibers. The experimental

setup used in the study is explained in detail. Additionally, the section offers an overview of

different methods for selectively inhibiting cross-bridge activity, especially through the use

of pharmacological agents such as blebbistatin.

Section 1.3 defines the overarching research objective of this dissertation: What are the

mechanisms underlying force generation in dynamic muscle contractions, and how do

fiber type, contraction velocity, and the contributions of cross-bridge and non-cross-



iv

bridge elements influence the mechanical response of muscle tissue? From this central

question, four specific research questions are derived:

• Into which phases can force generation in muscle fibers be divided during large-

amplitude eccentric contractions, and how is this phase structure affected by

stretch velocity?

• What proportion of force generation in stretch-shortening cycles can be attributed

to cross-bridge and non-cross-bridge elements?

• How do the parameters force, power, and force redevelopment change as a func-

tion of contraction velocity or activation level in stretch-shortening cycles?

• How accurately can the contractile element in a current muscle model predict the

force development of an actual muscle fiber?

To address these questions, a total of seven experiments were conducted, and their key

results are summarized in Sections 1.3.1 through 1.3.7. The key findings of the first two

contributions are that during long eccentric contractions (active stretching of more than

20% of the optimal fiber length) at a velocity exceeding 1% of the maximal contraction

velocity, we observe a drop in the force response. This force drop increases with stretch

velocity in both fast- and slow-twitch muscle fibers. The decline can be explained by the

rupture of strained cross-bridges. Interestingly, after reaching the local force minimum,

both fiber types show an almost linear increase in force until the end of the active stretch,

which is influenced by the protein titin. This increase is significantly steeper in fast-twitch

fibers compared to slow-twitch fibers. Contributions three through six present important

results regarding stretch-shortening cycles. Contribution three, for instance, shows that force

and mechanical work during the concentric phase of stretch-shortening cycles are elevated

compared to pure shortening contractions, both with and without cross-bridge inhibition.

Contribution four demonstrates that the effects of an stretch-shortening cycle are dependent

on execution velocity — power output increases significantly with increasing velocity. The

fifth contribution focuses on the phase following an stretch-shortening cycle, revealing that

force redevelopment occurs faster and to a greater extent after an stretch-shortening cycle

than after pure shortening. This effect remains detectable even after cross-bridge inhibition,

suggesting a significant contribution of titin. The sixth contribution shows that the peak force

during stretch-shortening cycles increases from cycle to cycle when performed consecutively.

The same applies to the mechanical work produced within each cycle. Notably, both findings

are observed across a wide range of activation levels (20–100%). Lastly, contribution seven



v

demonstrates that both muscle stiffness and damping properties depend on the level of

activation.

Chapter 2 presents the full scientific contributions, offering readers the opportunity to

explore the individual experiments in detail.

Finally, Chapter 3 places the findings of these experiments into a broader context. It begins

with an analysis of the relevant phases of force development during eccentric contractions in

Section 3.1, followed by a discussion of potential influencing mechanisms in Section 3.2.

The focus here is on cross-bridge and non-cross-bridge structures. The subsequent sections

examine the effects of contraction velocity and activation level. In the initial force response,

stretching of the bound cross-bridges seems to explain the differences, whereas in the second

half of the active stretch, titin seems to be mainly responsible for the differences. Section

3.4 discusses the implications of the results for improving and validating existing muscle

models.

Taken together, this work contributes to resolving open questions concerning dynamic

force development at the molecular and cellular levels. By reassessing existing models

and enhancing our understanding of contractile mechanisms, the findings support a more

holistic view of the role of muscle force in biological movement and motility. Of particular

importance are the new findings on the behavior of skinned muscle fibers during submaximal

activation.





Zusammenfassung

Die vorliegende Dissertation zielt darauf ab, unser Verständnis der komplexen Physiologie

der Skelettmuskulatur während exzentrischer Kontraktionen und Dehnungs-Verkürzungs-

Zyklen zu vertiefen. Aufbauend auf einer Reihe von in vitro-Experimenten mit gehäuteten

Muskelfasern untersucht diese Arbeit das mechanische Verhalten von Muskelgewebe unter

dynamischen Belastungsbedingungen und unterschiedlichen Kontraktionsgeschwindigkeiten.

Die einzelnen Kapitel beschreiben und interpretieren spezifische kraft- und leistungsbezoge-

ne Merkmale während aktiver Muskeldehnung und -verkürzung. Diese sind entscheidend,

um die zugrunde liegenden Mechanismen der erhöhten Kraftentwicklung und Energieeffizi-

enz in exzentrischen Kontraktionen und Dehnungs-Verkürzungszyklen zu verstehen. Trotz

umfangreicher Forschung auf dem Gebiet der Muskelphysiologie sind zentrale Aspekte der

Kraftgenerierung bei dynamischen Muskelaktionen nach wie vor ungeklärt. Daher trägt

die Quantifizierung spezifischer biomechanischer Eigenschaften zu einem umfassenderen

Verständnis der Muskelfunktion bei.

Kapitel 1 dient als einführende Grundlage dieser Arbeit und liefert die kontextuelle und

methodische Basis für die folgenden Kapitel. Zunächst wird die Motivation für diese Arbeit

dargelegt, wobei insbesondere die zentrale Bedeutung von Bewegung für den Menschen

hervorgehoben wird – sowohl im Alltag als auch in sportlichen, gesundheitlichen und

therapeutischen Kontexten.

Abschnitt 1.1 vermittelt den physiologischen Hintergrund und führt grundlegende Begrif-

fe zur Struktur und Funktion von Muskelgewebe ein. Es werden zentrale Mechanismen der

Muskelkontraktion beschrieben sowie verschiedene physiologische Effekte erläutert, die wäh-

rend oder nach dynamischen Kontraktionen auftreten, etwa Kraftsteigerung nach Dehnung

(residual force enhancement) oder Kraftminderung nach Verkürzung (force depression).

In Abschnitt 1.2 wird der Umgang mit dem biologischen Gewebe vorgestellt, insbesonde-

re die präzise Präparation und Handhabung permeabilisierter Muskelfasern. Zudem wird der

verwendete experimentelle Aufbau erläutert. Der Abschnitt bietet außerdem einen Überblick

über verschiedene Methoden zur gezielten Hemmung der Querbrückenbindung, insbesondere

durch den Einsatz pharmakologischer Inhibitoren wie Blebbistatin.



viii

Abschnitt 1.3 formuliert das übergeordnete Forschungsziel dieser Arbeit: Welche Me-

chanismen liegen der Krafterzeugung bei dynamischen Muskelkontraktionen zugrunde

und wie beeinflussen Fasertyp, Kontraktionsgeschwindigkeit und die Beiträge von

Querbrücken- und Nicht-Querbrückenelementen die mechanische Reaktion des Mus-

kelgewebes? Daraus leiten sich vier zentrale Forschungsfragen ab:

• In welche Phasen lässt sich die Kraftgenerierung von Muskelfasern während

exzentrischer Kontraktionen großer Dehnungsamplitude unterteilen und wie

beeinflusst die Dehnungsgeschwindigkeit diese Phasen?

• Welchen Anteil leisten Querbrücken- und Nicht-Querbrücken-Strukturen zur

Kraftgenerierung in Dehnungs-Verkürzungs-Zyklen?

• Wie verändern sich die Parameter Kraft, Leistung und Kraft-Redevelopment in

Abhängigkeit von der Kontraktionsgeschwindigkeit oder dem Aktivierungsniveau

in Dehnungs-Verkürzungs-Zyklen?

• Wie genau kann das kontraktile Element eines aktuellen Muskelmodells die reale

Kraftentwicklung einer Muskelfaser vorhersagen?

Zur Beantwortung dieser Fragen wurden insgesamt sieben Experimente durchgeführt,

deren zentrale Ergebnisse in den Abschnitten 1.3.1 bis 1.3.7 kurz zusammengefasst werden.

Die wichtigsten Ergebnisse der ersten zwei Artikel sind, dass wir bei langen exzentrischen

Kontraktionen (aktive Dehnung von mehr als 20% der optimalen Faserlänge) bei einer Ge-

schwindigkeit von mehr als 1% der maximalen Kontraktionsgeschwindigkeit ein Einbruch

der Kraftantwort finden. Dieser nimmt sowohl bei schnell als auch bei langsam zuckenden

Muskelfasern mit zunehmender Dehnungsgeschwindigkeit zu. Dieser Einbruch lässt sich

durch das Abreißen von gedehnten Querbrücken erklären. Interessanterweise folgt bei beiden

Fasertypen nach erreichen des lokalen Kraftminimums eine beinahe lineare Zunahme der

Kraft bis zum Ende der aktiven Dehnung, welche durch das Protein Titin beeinflusst wird. Die

Zunahme erfolgt bei schnell zuckenden Fasern signifikant steiler als bei langsam zuckenden.

In Artikel drei bis sechs werden wichtige Ergebnisse zu Dehnungs-Verkürzungs-Zyklen

gemacht. So zeigt Artikel drei unter anderem, dass Kräfte und mechanische Arbeit während

der konzentrischen Phase von Dehnungs-Verkürzungs-Zyklen im Vergleich zu Verkürzungs-

kontraktionen mit und ohne Querbrückenhemmung-Hemmung erhöht waren. In Artikel

vier wird gezeigt, dass der Effekt eines Dehnungs-Verkürzungs-Zyklus von der Durchfüh-

rungsgeschwindigkeit abhängig ist. So erhöht sich die erbrachte Leistung mit zunehmender

Geschwindigkeit signifikant. Im fünften Artikel wird die Phase nach Beendigung eines



ix

Zyklus betrachtet. Dort kann beobachtet werden, dass der Wiederaufbau der Kraft schneller

und höher verläuft nach einem Dehnungs-Verkürzungs-Zyklus im Vergleich zu einer reinen

Verkürzung. Dieser Effekt lässt sich noch nach einer Hemmung der Querbrücken finden, was

auf einen signifikanten Anteil von Titin spricht. Der sechste Artikel dieser Thesis zeigt, dass

sich die Spitzenkraft von Dehnungs-Verkürzungs-Zyklus zu Dehnungs-Verkürzungs-Zyklus

erhöht, sofern diese konsekutiv aufeinander folgen. Gleiches gilt für die geleistete Arbeit

innerhalb der Zyklen. Besonders hervorzuheben ist die Tatsache, dass beide Ergebnisse über

einen großen Bereich von Aktivierungslevel (20-100%) zu finden sind. Schließlich wird in Ar-

tikel sieben aufgezeigt, dass die Steifheit des Muskels sowie seine Dämpfungseigenschaften

von der Aktivierung abhängig sind.

Kapitel 2 enthält schließlich die vollständigen wissenschaftlichen Beiträge (Contributions)

und bietet interessierten Leser*innen die Möglichkeit, sich vertiefend mit den einzelnen

Experimenten auseinanderzusetzen.

Kapitel 3 diskutiert die Ergebnisse im gemeinsamen Kontext. Zunächst wird auf die

relevanten Phasen der Kraftgenerierung bei exzentrischen Kontraktionen eingegangen, ge-

folgt von einer Diskussion möglicher struktureller Einflussmechanismen in Abschnitt 3.2.

Hierbei stehen Querbrücken und nicht-Querbrücken Strukturen im Vordergrund. In den

anschließenden Abschnitten werden die Effekte von Kontraktionsgeschwindigkeit und Akti-

vierungsniveau beleuchtet. Bei der initialen Kraftantwort scheint eine Dehnung der gebunde-

nen Querbrücken die Unterschiede zu erklären, während im der zweiten Hälfte der aktiven

Dehnung hauptsächlich Titin für die Unterschiede verantwortlich zu sein scheint. Abschnitt

3.4 widmet sich der Frage, inwiefern die gewonnenen Erkenntnisse zur Verbesserung und

Validierung existierender Muskelmodelle beitragen können.

Insgesamt trägt diese Arbeit zur Beantwortung offener Fragen zur dynamischen Kraftent-

wicklung auf molekularer und zellulärer Ebene bei. Durch die Neubewertung bestehender

Modelle sowie das bessere Verständnis kontraktiler Mechanismen fördert sie eine ganzheitli-

che Sichtweise auf die Rolle der Muskelkraft in biologischer Bewegung und Motilität. Von

besonderer Bedeutung sind dabei die neuen Erkenntnisse zum Verhalten von gehäuteten

Muskelfasern bei submaximaler Aktivierung.





Contents

List of Figures xiii

Nomenclature xv

1 Introduction 1

1.1 Physiological Background . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.1.1 Muscle Tissue Types . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.1.2 Skeletal Muscle Structure . . . . . . . . . . . . . . . . . . . . . . 7

1.1.3 Skeletal Muscle Contraction . . . . . . . . . . . . . . . . . . . . . 9

1.1.4 Fundamental Muscle Properties . . . . . . . . . . . . . . . . . . . 11

1.1.5 Dynamic Contraction Effects . . . . . . . . . . . . . . . . . . . . . 18

1.1.6 History Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

1.1.7 Stretch-Shortening Cycle . . . . . . . . . . . . . . . . . . . . . . . 21

1.1.8 Give . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1.2.1 Muscle Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 24

1.2.2 Experimental Apparatus . . . . . . . . . . . . . . . . . . . . . . . 26

1.2.3 Cross-Bridge Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 28

1.3 Objectives and Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

1.3.1 Contribution I: How velocity impacts eccentric force generation of

fully activated skinned skeletal muscle fibers in long stretches . . . 31

1.3.2 Contribution II: Impact of lengthening velocity on the generation of

eccentric force by slow-twitch muscle fibers in long stretches . . . . 32

1.3.3 Contribution III: Cross-bridges and sarcomeric non-cross-bridge

structures contribute to increased work in stretch-shortening cycles 32

1.3.4 Contribution IV: Power amplification increases with contraction

velocity during stretch-shortening cycles of skinned muscle fibers . 33



xii Contents

1.3.5 Contribution V: Force re-development after shortening reveals a role

for titin in stretch–shortening performance enhancement in skinned

muscle fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

1.3.6 Contribution VI: Consecutive SSCs increase the SSC effect in single

skinned rat muscle fibres . . . . . . . . . . . . . . . . . . . . . . . 34

1.3.7 Contribution VII: Muscle preflex response to perturbations in loco-

motion: In vitro experiments and simulations with realistic boundary

conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2 Contributions 37

2.1 Contribution I: How velocity impacts eccentric force generation of fully

activated skinned skeletal muscle fibers in long stretches . . . . . . . . . . 38

2.2 Contribution II: Impact of lengthening velocity on the generation of eccentric

force by slow-twitch muscle fibers in long stretches . . . . . . . . . . . . . 50

2.3 Contribution III: Cross-bridges and sarcomeric non-cross-bridge structures

contribute to increased work in stretch-shortening cycles . . . . . . . . . . 62

2.4 Contribution IV: Power amplification increases with contraction velocity

during stretch-shortening cycles of skinned muscle fibers . . . . . . . . . . 77

2.5 Contribution V: Force re-development after shortening reveals a role for titin

in stretch–shortening performance enhancement in skinned muscle fibres . . 94

2.6 Contribution VI: Consecutive SSCs increase the SSC effect in single skinned

rat muscle fibres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

2.7 Contribution VII: Muscle preflex response to perturbations in locomotion:

In vitro experiments and simulations with realistic boundary conditions . . 128

3 Discussion 143

3.1 Key Phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

3.2 Mechanisms of Force Generation in Skinned Muscle Fibers . . . . . . . . . 147

3.3 Influence of Velocity on Force Generation in Eccentric Contractions . . . . 148

3.4 Influence of Activation on Force Generation in Eccentric Contractions . . . 151

3.5 Challenges in Muscle Modelling . . . . . . . . . . . . . . . . . . . . . . . 153

3.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Bibliography 157

4 Apendix 177



List of Figures

1.1 Overview of the Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Overview over different Tissue Types . . . . . . . . . . . . . . . . . . . . 6

1.3 Hierarchical Structure of Skeletal Muscles . . . . . . . . . . . . . . . . . . 8

1.4 Schematic of Huxley Model . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.5 Force-Length Relation and Filament Overlap . . . . . . . . . . . . . . . . 12

1.6 Force-Velocity Relation Data Plot . . . . . . . . . . . . . . . . . . . . . . 14

1.7 Representative Force-Velocity Relationship and Power-Velocity Relationship

for Sol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

1.8 A Schematic Force-pCa Relationship . . . . . . . . . . . . . . . . . . . . 17

1.9 Representative Plots of History Effects . . . . . . . . . . . . . . . . . . . . 20

1.10 Representative Plots of a Stretch-Shortening Cycle . . . . . . . . . . . . . 22

1.11 Mean Plot of Stretch Contractions Showing Give . . . . . . . . . . . . . . 24

1.12 The Aurora 1400A Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . 27





Nomenclature

Roman Symbols

Ca2+ Calcium Ion

∆Ls Difference in Sarcomere Length

F Force of Muscle Fiber

Fim Isometric Force at Optimal Fiber Length

Fmin Force at the End of Stretch-Shortening Cycle

Fonset Force Plateau Before Start of Stretch-Shortening Cycle

Fpeak Force at the End of Stretch during Stretch-Shortening Cycle

Give Difference between First Local Maximum and Minimum in Force during Active

Lengthening

lopt Optimal Fiber Length

LS0 Optimal Sarcomere Length

P Power of Muscle Fiber

pCa Negative Logarithm of the Calcium Ion Concentration

s2 First Peak in Force during Active Stretches

se Force at the End of Active Stretch

sg Local Minimum in Force during Active Stretches

slope1 First Linear Increase in Force after Onset of Stretch

slope2 Second Linear Increase in Force during Second Half of Active Stretch



xvi Nomenclature

v Velocity of Shortening or Lengthening

vmax Proportion of Maximal Shortening Velocity

WorkSHO Work done during Shortening Phase of Stretch-Shortening Cycle

WorkSSC Work done during whole Stretch-Shortening Cycle

Acronyms / Abbreviations

ADP Adenosine Diphosphate

AT P Adenosine Triphosphate

BDM 2,3-Butanedione Monoxime

BT S N-Benzyl-p-Toluene Sulphonamide

CE Contractile Element

EDL M. Extensor Digitorum Longus of the Rat

FLR Force-Length Relationship

FR Force Ramp Method

FT Fast-Twitch Muscle

FV R Force-Velocity Relation

PEV K Stands for Proline (P), Glutamate (E), Valine (V), and Lysine (K)

Pi Inorganic Phosphate

PV R Power-Velocity Relation

rFD Residual Force Depression

rFE Residual Force Enhancement

Sol M. Soleus of the Rat

SSC Stretch-Shortening Cycle

ST Slow-Twitch Muscle

XB Cross-Bridge



Chapter 1

Introduction

Human movement is an intrinsic part of our existence, serving as a fundamental mode of

expression, communication, and interaction with the world. From the moment we are born,

movement becomes a critical aspect of our personal development, shaping our physical

abilities, social skills, and emotional well-being. Both, in everyday life and in the realm

of sports, movement holds profound significance, acting as a bridge between our physical

body and our experiences, goals, and identities. From this point of view, human movement

encompasses a wide array of daily life activities, including basic functions as walking and

running or more complex behaviors such as dancing and exercising. Beyond the physical

benefits, movement also has considerable implications for mental health. Engaging in

regular physical activity has been shown to reduce symptoms of anxiety and depression,

improve mood, and boost overall feelings of well-being. Activities such as walking in

nature or participating in community sports foster a sense of connectedness and belonging,

reinforcing social bonds [159, 253, 41]. Additionally, movement plays a role in cognitive

development, particularly in children. Active play promotes motor skills, coordination,

and spatial awareness, laying the foundation for future learning and development. Several

past-investigations have shown that, for instance, there is a direct link between movement

and cognition development during infancy [242, 91, 217]. In addition, movement allows us

to maintain physical fitness, improve cardiovascular health, and enhance muscular strength

and endurance [127, 132, 183].

Furthermore, the act of moving can symbolize freedom and autonomy. The ability to

navigate one’s environment and engage in various activities enhances an individual’s sense of

agency. In contrast, limited mobility, wether due to physical disabilities or other constraints,

can lead to feelings of isolation and dependence [76, 204]. Thus, recognizing the value

of movement is essential for fostering inclusive communities that accommodate varying

abilities and encourage participation.



2 Introduction

When we transition to the world of sports, the significance of movement takes on a

different dimension. Sports provide a structured environment through which individuals

can channel their innate desire to move, compete, and excel [173]. Athletes often push

the boundaries of human potential, demonstrating the incredible capabilities of the human

body through exceptional displays of speed, strength, and agility as demonstrated by e.g.

Armand Duplantis crossing 6.27 m in pole vault or Tigist Assefa running a marathon under

2 h and 12 min. In order to achieve complex athletic performances in sports, movement

is facilitated by an intricate system of muscles, bones, joints, and nerves. The muscular

system, which is subdivided in the literature into smooth, heart and skeletal muscles, allows

for movement through contraction and relaxation, affecting posture and supporting various

physical activities. The skeletal system provides structure and protection [222], while the

nervous system coordinates and controls movement [245], sending signals from the brain

to the muscles to initiate and to precisely perform sport specific movements [93]. From the

fluid grace of a ballet dancer to the explosive energy of a sprinter, muscles are used both for

acts of brute force and fine control.

The ability to perform complex and highly coordinated movements, such as sprinting or

ballet dancing, relies on the precise control of skeletal muscle contractions. These movements

require not only the activation of muscles but also the ability to generate and regulate

force dynamically across a range of contraction velocities and movement patterns. The

interplay between concentric (active shortening), isometric (active without length change),

and eccentric (active lengthening) contractions allows for the production of the necessary

forces to accelerate, decelerate, stabilize, and optimize movement efficiency.

Among these contraction types, eccentric contractions play a particularly crucial role due

to their unique mechanical and metabolic properties. Compared to isometric or concentric

contractions, eccentric contractions enable greater force production at a lower metabolic

cost [53, 149, 185]. This efficiency arises from the combined contributions of cross-bridge

(XB) cycling and passive elastic elements, such as titin, which provides resistance against

stretch and contribute to force generation without additional adenosine triphosphate (AT P)

consumption [1, 10, 109, 165, 212]. Furthermore, eccentric contractions are fundamental to

the stretch-shortening cycle (SSC), a movement strategy in which muscles are first stretched

eccentrically before immediately transitioning to a concentric contraction. The SSC enhances

movement efficiency and power output by utilizing stored elastic energy and pre-activation

mechanisms [14, 85]. This principle is widely observed in human locomotion, such as

during running and jumping, where rapid transitions between eccentric and concentric phases

improve performance [27, 130, 87].



3

A key aspect of muscle function during dynamic contractions is the velocity dependence

of force production. Studies have shown that force generation during eccentric contractions

is significantly influenced by the velocity of stretch, with higher velocities leading to greater

forces [239, 188]. This higher force is thought to result from both XB mechanics and non-

XB elements such as titin, whose stiffness increases with stretch velocity, contributing to

additional force production [96, 151, 168]. Moreover, fiber type plays an essential role

in these dynamics, as fast-twitch (FT ) fibers exhibit different mechanical and metabolic

properties compared to slow-twitch (ST ) fibers, influencing their capacity to generate force

under varying contraction conditions [33, 8, 9].

Understanding the mechanisms underlying force generation during dynamic contractions

is critical for multiple applications, including muscle injury prevention, biomechanical mod-

eling, and the development of muscle-like actuators. To fully comprehend these mechanisms,

it is necessary to investigate the influence of contraction velocity, fiber type, and struc-

tural components—including actin, myosin, and titin—on force production during dynamic

contractions in skeletal muscle.

This thesis addresses this important goal and is structured around seven key contributions

(Figure 1.1), which were grant-aided by the German Research Foundation under projects

405834662, 354863464, and 449912641. First, Contribution I (Section 2.1) explores the

different phases of force development during long eccentric stretches and examines how

velocity influences these phases. The second contribution (Section 2.2) investigates fiber type-

dependent differences in force trajectories at comparable contraction velocities, providing

insight into how slow- and fast-twitch muscles respond under similar mechanical conditions.

The third contribution (Section 2.3) distinguishes between XB and non-XB forces in eccentric

contractions that are immediately followed by a concentric contraction, commonly referred

to as a SSC. Building on this, the fourth contribution (Section 2.4) aims to further dissect

the velocity dependence of XB and non-XB structures, providing insight on how these

elements interact to modulate force production. The fifth contribution (Section 2.5) examines

how an SSC affects force redevelopment, offering a deeper understanding of the transient

changes in muscle force following rapid stretch-shortening transitions. The sixth contribution

(Section 2.6) extends this analysis by investigating the effect of multiple SSCs at different

activation levels and their role in force amplification. Finally, the seventh contribution

(Section 2.7) compares experimental force data from muscle fibers with model predictions to

assess the ability of current biomechanical models to accurately replicate force production in

realistic conditions.



4 Introduction

Figure 1.1 Schematic overview of the interdisciplinary connections between published articles
in this thesis, illustrating the synergistic relationships between contraction velocity, muscle
fiber type, and fiber architecture in modulating force production during eccentric contractions.
Overlap of the dark green circles denotes interactions explored in the thesis. The abbreviation
XB stands for cross-bridge.



1.1 Physiological Background 5

1.1 Physiological Background

At the beginning it is important to clarify the actual scientific state of the art of muscle

and emerging questions. In order to provide those information, a concise overview of key

physiological muscle properties as a foundation for understanding skeletal muscle mechanics

is given. It begins by distinguishing among the three major muscle tissue types—skeletal,

cardiac, and smooth—highlighting their functional roles and control mechanisms. Atten-

tion then turns to skeletal muscle’s hierarchical organization, from whole muscle down to

sarcomeres, and to the molecular level of contraction, emphasizing actin–myosin XBs and

the structural contributions of titin. The core sections explore how these contractile and

structural elements interact to produce force under varying lengths (force–length relation-

ship), velocities (force–velocity and power–velocity relationships), and levels of Calcium

Ion (Ca2+) dependent activation. Finally, history effects of contractions—residual force

enhancement, residual force depression, stretch–shortening cycles, and Give—are briefly

introduced, underscoring the complexity of muscle behavior during dynamic movements.

1.1.1 Muscle Tissue Types

Like all organisms, humans need to convert chemical energy into mechanical energy in order

to move. This mechanical energy acts on specific proteins that are part of the cytoskeleton

and serve as motor proteins within the cell. In addition to enabling intracellular motion, these

proteins also facilitate all forms of movement outside the cell, such as the heartbeat or the

contraction of the soleus muscle (Sol). In this context, the proteins actin and myosin play

an essential role in muscle cells. This chapter describes the structure and function of the

different muscle types. As in all higher animals (such as mammals or amphibians), human

musculature is generally divided into three types: striated (skeletal) muscle, smooth (visceral)

muscle, and cardiac muscle, the latter being a modified specialized form of striated muscle

(c.f. Figure 1.2).

Skeletal muscle (Figure 1.2,a) contraction is a highly coordinated process that trans-

forms electrical signals into mechanical force. This process, known as excitation-contraction

coupling, begins when an action potential is generated at the neuromuscular junction. Depo-

larization propagates along the sarcolemma and enters the muscle fiber through the transverse

(T)-tubule system, triggering calcium release from the sarcoplasmic reticulum via ryanodine

receptors [32, 153]. The subsequent increase in cytosolic calcium concentration facilitates

actin-myosin interaction, leading to force production through XB cycling. Relaxation occurs

when calcium is actively pumped back into the sarcoplasmic reticulum, reducing cytosolic

calcium levels and allowing the detachment of XBs.



6 Introduction

Figure 1.2 Schematic representation of the structure and innervation of the various muscle
types. The contractile elements (CE) are shown in gray, the nerve control and excitation
coupling is shown in green. a) Skeletal muscle cells are organized in long individual fibers
in contrast to b) heart muscle which has shorter fibers that have connections between fibers.
c) Smooth muscle differs greatly as its cells are organized into large unconnected groups of
cells. The figure is adapted from [32].

The strength of contraction is regulated by action potential frequency and motor unit

recruitment. Low-frequency stimulation results in individual twitches, while higher frequen-

cies cause summation of twitches, leading to a sustained contraction (tetanus). Additionally,

small motor units with slow-twitch fibers are recruited first for low-force tasks, while larger

motor units with fast-twitch fibers are activated as force demand increases. This hierarchical

recruitment mechanism optimizes energy efficiency and adaptability in voluntary movement

[64, 215, 227].

Cardiac muscle (Figure 1.2,b) shares structural similarities with skeletal muscle, par-

ticularly in the arrangement of overlapping actin and myosin filaments that generate force.

However, cardiac myocytes differ significantly in function and regulation. Each cardiac cell

contains a single nucleus and connects to adjacent cells through intercalated discs, which

include gap junctions that facilitate electrical coupling. This connectivity ensures rapid

signal transmission and synchronizes contraction, producing the rhythmic beating of the

heart [32, 129, 60]. Unlike skeletal muscle, where neural input initiates contraction, cardiac

muscle is intrinsically rhythmic, with autonomic innervation modulating rather than trig-

gering contraction. A defining feature of cardiac muscle is its inability to sustain tetanic

contractions, which results from the prolonged plateau phase of the cardiac action poten-

tial. This phase is maintained by calcium influx from both the extracellular space and the

sarcoplasmic reticulum. Contraction strength in cardiac muscle is directly influenced by the

amount of cytosolic calcium, which is regulated by autonomic neurotransmitters acting via a-



1.1 Physiological Background 7

and b-adrenergic receptors. Additionally, temperature has a pronounced effect on cardiac

muscle contractility (through changes in Ca2+ sensitivity [90]), highlighting the delicate

interplay between electrophysiological signals and biochemical processes in maintaining

heart function [172, 153, 187].

Smooth muscle (Figure 1.2,c) tissue forms the contractile layers of various internal

organs, including the stomach, intestines, bronchi, bladder, and uterus. Unlike skeletal

muscle, smooth muscle fibers are spindle-shaped and contain actin-myosin filaments that are

not organized into sarcomeres. Instead, they have a more irregular, non-striated arrangement

of filaments. Furthermore, calcium, for contraction primarily, enters through membrane-

bound Ca2+ channels rather than being released from an internal reservoir [5, 32, 16].

Many smooth muscle cells exhibit spontaneous oscillations in membrane potential driven

by pacemaker cells and are electrically coupled via gap junctions. In single-unit smooth

muscle cells, such as in the gastrointestinal tract and blood vessels, this coupling enables

synchronous and coordinated contractions. In contrast, multi-unit smooth muscle cells, as

found in the iris, rely on direct autonomic innervation, leading to independently controlled

contractions. The contraction in smooth muscle is regulated by neural, chemical, and

mechanical stimuli. Instead of troponin (as in cardiac and skeletal muscles), Ca2+ binds to

calmodulin, activating myosin light-chain kinase, which enables XB cycling. Additionally,

mechanical stretch can trigger contraction, but prolonged or gradual stretching can reduce

stress over time, demonstrating the tissue’s adaptability. This complex regulatory system

allows smooth muscle to maintain and modulate contractions essential for vital physiological

functions [172, 215].

1.1.2 Skeletal Muscle Structure

Skeletal muscle is organized in a strict hierarchy (Figure 1.3), beginning with the whole

muscle and breaking down to the molecular scale of its contractile apparatus. At the macro-

scopic level, each entire muscle is encased by a layer of connective tissue (fascia) and,

just beneath that, by the epimysium. Internally, the muscle is subdivided into fascicles

that can contain anywhere from 10 to 100 fibers [52]. Each fascicle is surrounded by an-

other connective tissue layer called the perimysium. Within each fascicle, the individual

muscle fibers—multinucleated and typically 10 µm to 80 µm in diameter—are wrapped in

endomysium and oriented primarily along the muscle’s force-generating axis.

When examined at higher magnification, each muscle fiber contains numerous parallel

myofibrils, which in turn comprise many serially arranged sarcomeres [82]. A sarcomere

extends from one Z-disc to the adjacent Z-disc, housing the overlapping thick filaments

(primarily myosin II) and thin filaments (primarily actin). In skeletal muscle, the thick



8 Introduction

Figure 1.3 A schematic representation of skeletal muscle structure is shown, progressing
from the whole muscle (top) to the molecular level (bottom). The sarcomere is identified as
the fundamental contractile unit of striated muscle tissue. Notably, the optimal sarcomere
length (LS0) differs between species due to variations in thin filament length (see text for
details). In the schematic at the bottom, the depicted sarcomere length of 2.5 µm corresponds
to LS0 for peak isometric force (Fim) generation in rat muscles [229]. Adapted from Schmidt
et al. [215].



1.1 Physiological Background 9

filaments are roughly 1.6 µm in length, whereas the thin filaments range from approximately

0.9 µm to 1.3 µm [250, 97]. The alignment of these filaments gives muscle fibers their

distinctive striated appearance: the A-band where thick and thin filaments overlap, the lighter

I-band containing only actin, and the H-zone in the center when no thin filament overlap is

present.

In addition to the contractile proteins, structural and regulatory proteins further stabilize

and tune force production. Particularly titin, often described as the third most abundant

protein in skeletal muscle [137], contributes to this process. Spanning from the Z-disc to the

M-line, titin anchors the thick filaments and contributes elastic recoil, preventing overstretch

and maintaining proper sarcomere alignment [150, 102]. Titin’s long molecule includes

immunoglobulin-like domains, a PEV K region (PEV K means rich in proline, glutamate,

valine, and lysine), and other elastic segments that allow it to behave as a molecular spring

[92, 152].

Slow-twitch fibers exhibit lower myosin AT Pase activity, reduced XB cycling frequencies,

and correspondingly lower maximal shortening velocities than their fast-twitch counterparts

[8, 26, 209]. In addition to these differences in contractile protein kinetics, various non-XB

structures further distinguish slow- and fast-twitch muscles. For instance, the extracellular

matrix (comprising the endomysium, perimysium, and epimysium) and different titin iso-

forms may alter passive stiffness and thus influence the capacity for eccentric force generation

[33, 83, 254, 190, 251]. Together, these divergent contractile properties, shape how each

fiber type—and consequently each muscle —performs under various mechanical demands

ranging from steady endurance to explosive power output.

1.1.3 Skeletal Muscle Contraction

Skeletal muscle contraction is widely understood through the lens of the sliding filament

theory, in which thin (actin) and thick (myosin) filaments slide past one another without

altering their own lengths [114, 112]. Central to this mechanism is the XB cycle, introduced

mathematically by Huxley [111], where myosin heads are viewed as force generators con-

necting to actin binding sites Figure 1.4. In the simplest formulation, each XB exists in either

an attached or detached state. When attached, it pulls on the actin filament via an elastic link

to the myosin backbone, generating force. Huxley [111] showed that appropriately chosen

attachment and detachment rates could reproduce the classical force–velocity behavior of

frog skeletal muscle [108], thus solidifying the XB concept as a unifying concept for under-

standing contraction. Subsequent refinements recognized that myosin heads can rotate to

the thick filament, forming lever arms that pivot through power strokes [115]. In addition,

more complex multi-state models were developed, as experiments indicated that XBs might



10 Introduction

Figure 1.4 A schematic representation of the cross-bridge interaction between myosin (M) and
actin filaments (A), as described by Huxley [111]. The ‘x-distance’ refers to the displacement
from the cross-bridge equilibrium position (0) to the nearest attachment site on the actin
filament. Adapted from Huxley [111].

shift among multiple attached conformations, each with distinct energy levels [113]. These

rotating and multi-state models help explain rapid force transients observed in single muscle

fibers following quick length changes, as well as the near-instantaneous drop in force that

recovers partially within milliseconds when fibers are suddenly shortened. Furthermore,

a recently published multi-state model can explain history effects using a thermodynamic

approach [7].

Despite these successes, purely actin–myosin XB models have struggled to account for

certain phenomena, particularly those linked to stretch and residual force enhancement. One

solution is the addition of titin, a giant, elastic filament spanning each half-sarcomere from

the Z-disc to the M-line [101, 161, 252]. Contrary to the traditional assumption, that titin is

only viewed as a source of passive stress, titin is now recognized to play a potentially active

role, particularly in eccentrically stretched fibers. Evidence suggests that titin can bind to

actin in a force- and activation-dependent manner, effectively increasing titin’s stiffness when

sarcomeres are both activated and elongated [47, 150]. Under such conditions, titin may bear

additional load and supplement the XB-derived force, offering a compelling explanation for

heightened force levels during active stretches that cannot be explained by XB theories alone.

By incorporating titin into what is often called a three-filament model, the fundamental

XB mechanism remains intact, but new pathways emerge for force modulation. For instance,

if titin stiffens in response to calcium or attaches to actin upon muscle activation, the

sarcomere’s effective spring properties change [203, 181, 180]. This action can elevate overall



1.1 Physiological Background 11

force, stabilize the thick filaments, and even alter energy usage under certain lengthening

conditions. Such a model resolves many of the contradictions of two-filament theories,

especially regarding history-dependent effects like residual force enhancement and the

complex mechanical responses observed when muscle fibers are forcibly stretched.

1.1.4 Fundamental Muscle Properties

The aforementioned processes cause muscles to display a wide array of fundamental prop-

erties that determine how they produce force and movement. The upcoming sections will

explore the classic force–length, force–velocity, and power–velocity relationships that de-

scribe muscle behavior under varied mechanical conditions, followed by an overview of

how calcium concentration modulates contraction on the fiber level. Finally, we examine

dynamic effects—including residual force enhancement, residual force depression, SSC, and

Give—which add complexity to dynamic muscle performance.

Force-Length-Relation

The pioneering work by Blix [11] and Ramsey and Street [197] led to the first description

of the length dependence of muscle force in striated skeletal muscles. Their investigations

revealed that active isometric muscle force rises approximately linearly with increasing

muscle length up to an optimal point, then decreases linearly once the muscle is stretched

beyond this optimum. Despite this early insight into the fundamental importance of length

on force generation, the underlying mechanisms of skeletal muscle function remained only

partially understood for decades thereafter.

A major advance came with the experiments by Gordon et al. [82], who examined single

frog muscle fibers at different lengths and established a more comprehensive force–length

relationship (FLR). They observed a linear ascending limb, a plateau region near the optimal

length, and a linear descending limb. Their geometrical model attributed much of this

behavior to the overlap of actin and myosin filaments: at intermediate sarcomere lengths

(roughly 2.0 µm to 2.2 µm), the overlap—and thus XB potential—is maximized, producing

peak force. As the sarcomere is either shortened below or stretched beyond this range, fewer

XBs can form, causing force generation to drop (c.f. Figure 1.5).

Although this concept proved enormously influential for muscle physiology, it does not

fully explain every detail of the force–length curve. Specifically, accounting for the steep part

of the ascending limb (part below approximately 1.6 µm) often requires additional, still un-

verified assumptions, such as possible myosin compression or filament misalignment at very

short lengths [82, 156, 244]. Furthermore, multiple studies have demonstrated appreciable



12 Introduction
F

o
rc

e
 [
F

/F
im

]

Sarcomere Length [µm]

Figure 1.5 Relationship between sarcomere length, filament overlap, and contractile force.
On the left, the active FLR is shown as a dashed black line. The individual measurements
from n = 20 m. Extensor Digitorum Longus (EDL) fibers at 12 ◦C are indicated by the small
green diamonds. The purple line shows the fit through measured passive force data. On
the right, the corresponding overlap of actin and myosin filaments at prominent points of
the FLR (at a)1.6 µm; b) 2.8 µm; c) 3.5 µm and d) 4.2 µm) is shown (which is adapted from
Schmidt et al. [215]).

force production at short fiber lengths [197, 235, 216, 208, 233, 240], challenging traditional

views that presume minimal active force generation in these ranges. Consequently, a struc-

turally complete explanation of the ascending limb— especially its steep section—remains

the focus of ongoing research [240].

Beyond the active contractile components, passive elements also contribute to the absolute

force–length relationship [215]. Elastic structures such as titin and intramuscular connective

tissue resist stretching, causing a progressively steeper rise in passive stress as the muscle

is elongated. This interplay between passive elasticity and active XB mechanics shapes the

total/absolute length–stress curve. Differences in passive stiffness can vary greatly between

muscles, further complicating the general understanding of the FLR but also allowing for

functional adaptations to specific tasks [138, 191, 55]. The main working range of most

mammalian muscles covers the ascending limb and the plateau region of the FLR [21, 135].

Force-Velocity-Relation

In addition to the dependence of force on muscle length contraction, velocity serves as a

second key determinant of active muscle force production [108, 177]. This force–velocity



1.1 Physiological Background 13

relation (FV R) was initially described for concentric contractions by Hill [108] and mathe-

matically relates the maximal force a muscle can exert as it is shortened. Specifically, the

classic hyperbolic function indicates that muscle force decreases with increasing velocity: the

faster the muscle shortens, the lower the force it can sustain. In Hill’s original formulation, the

maximal isometric force at optimal length steadily declines toward zero force as shortening

speed (v) increases, eventually reaching a critical velocity at which external force can no

longer be produced. Although the force–velocity equation for concentric muscle action is

often stated as:

F =
(Fim ×b−a× v)

b+ v

The term F denotes the maximum force a muscle can generate at its optimal length, Fim

is the muscle’s maximal isometric force at that length, v represents the shortening velocity,

and a and b are constants measured in units of force (Newton) and speed (meter per second),

respectively. This equation remains widely used nearly 90 years later [54, 108], so far no

similarly accepted general equation exists for the eccentric portion of the curve [255].

A practical way to assess the force–velocity relationship in skinned muscle fibers is

through the force ramp (FR) method (Figure 1.6). To ensure consistency in contractile condi-

tions, initial fiber lengths and length change limit for both concentric and eccentric contrac-

tions are chosen to maintain sarcomere length between 2.4 µm and 2.8 µm—corresponding

to the plateau region of the force–length relationship, where maximal isometric force remains

high and relatively constant in skinned rat EDL fibers [229]. The FR method enables the

measurement of both concentric and eccentric FV Rs within the same fiber in only two

activations. By calculating velocity from changes in fiber length and plotting this against the

imposed force, the FV R is constructed. The resulting concentric FV R typically follows a

hyperbolic pattern, as originally described by Hill [108]. Compared to traditional isotonic

methods, the FR approach has two key distinctions. First, a velocity offset appears in the

FV R due to short-range stiffness [170] or in-series elastic-like responses [116, 144, 81].

Second, parameters such as maximum shortening velocity and curvature may slightly differ

from those derived using isotonic techniques [198, 116, 144]. Despite these differences,

the FR method is considered more robust for estimating model parameters across the full

range of physiological forces and velocities, and its advantages generally outweigh the noted

limitations [144, 207].

Muscle force can exceed the isometric contraction force during eccentric contractions,

often reaching between 1.5 and 2.0 times Fim [126, 177]. Interestingly, slow lengthening

yields relatively lower forces than rapid lengthening, which is in contrast to the concentric

side of the relationship. Classic Hill-type models attribute this excess force during eccentric



14 Introduction
F

o
rc

e
 [
F

/F
im

]

Velocity [lopt/s]

eccentric

concentric

0

1

-1

2

0 1 2 3

Figure 1.6 A representative sarcomere force-velocity relationship (purple: eccentric and
green line: concentric) is illustrated using a maximally Ca2+-activated fast-twitch single
skinned fiber (L = 1.09 mm) from a rat EDL muscle (n = 1). Experiments were conducted
at a constant temperature of 12 ◦C. Force-velocity characteristics were determined using
force ramp perturbations, which impose a constant change in force over time at ±2.5 Fim/s,
following the protocols established by Iwamoto et al. [116] and Lin and Nichols [144].

contractions to XB dynamics [88, 108], but more recent studies indicate that additional

non-XB elements, particularly titin and other parallel-elastic components, can contribute

substantially to total force under these conditions [188, 203, 213, 239]. Thus, current

understanding suggests that both XB and non-XB mechanisms operate in concert during

eccentric contractions, which helps explain the high force capacities observed when an active

muscle is forcibly stretched.

Moreover, the shape of the force–velocity curve is strongly influenced by fiber-type

composition. Whereas slow-twitch and fast-twitch fibers can produce roughly the same peak

isometric stress per cross-sectional area, their maximal shortening speeds differ by about

a factor higher than two, allowing fast-twitch muscles to generate higher forces at a given

submaximal shortening velocity [44, 177, 237, 238].



1.1 Physiological Background 15

Power-Velocity-Relation

The power–velocity relationship (PV R, Figure 1.7), derived from the force–velocity curve,

describes how rapidly a muscle can perform mechanical work during shortening contractions.

In its simplest mathematical form, muscle power P is the product of muscle force F and

shortening velocity v:

P = F × v

Consequently, power is zero when muscle shortening velocity is zero (isometric contraction)

or when velocity reaches its maximum (because external force is then zero). Experimental and

theoretical investigations consistently show that peak power output occurs at an intermediate

velocity—often around 25–30% of the maximal shortening speed—rather than at the extremes

of force or velocity [43, 177, 206, 205].

0 0.25 0.5
0

1

2

0.02

0.04

0

Velocity [              ]
ΔLS

LS0
x s-1

ΔL
S

L
S

0
x

x
s

-1
F F
im

(
)

F
o

rc
e

 [
F

/F
im

]

P
o

w
e

r 
[ 
  
  
  
  
  
  
  
  
  
  
  
  
 ]

Figure 1.7 A representative force-velocity relationship and power-velocity relationship for
Sol are illustrated. The green solid line represents the mean FV R for shortening contractions
(positive velocities), showing a decline in force as a function of increasing shortening
velocity. Data was taken from Section 2.4. The purple solid line represents the mean PV R for
shortening contractions, where power reaches its peak at intermediate shortening velocities,
approximately 0.09LS0

s
( 20%vmax).Here, Fim represents the maximum isometric muscle force,

velocity is expressed in relative units (LS

s
), and power is reported as relative values (F×LS

s
).

Studies of human and animal muscle function highlight this principle: frogs, for instance,

appear to time their jumps such that the primary muscles operate near 30% of their maximal

shortening velocity [154, 155]. This strategy allows the amphibian to achieve near-maximum

power for rapid escape responses. Similarly, in cycling, optimal gear selection lets athletes



16 Introduction

match their pedaling cadence to the velocity region where power production is highest.

When cycling at a set speed, the choice of an appropriate gear ratio (and thus pedaling

rate) can critically influence performance by placing muscle fibers closer to their optimal

power–velocity zone [210, 45].

The shape of the power–velocity relationship can also reflect fiber-type distribution.

Although slow-twitch (Type I) and fast-twitch (Type II) fibers have similar maximal force

capacity per cross-sectional area under isometric conditions, Type II fibers shorten faster

and typically yield higher power at a given submaximal load and speed [238, 237, 58,

133]. Consequently, muscles with higher proportions of fast-twitch fibers support explosive

movements such as sprinting, jumping, and throwing [255].

pCa-Force-Relation

A fundamental aspect of skeletal muscle contraction is the calcium-dependence of XB

activation. Even if ample AT P is available, myosin heads do not continually cycle in

resting muscle; rather, the presence of regulatory proteins on the thin filament—troponin

and tropomyosin—prevents stable binding of actin and myosin when intracellular Ca2+

concentration is low (on the order of 10×10−7 molL−1). As soon as Ca2+ rises (to about

10× 10−6 molL−1 to 10× 10−5 molL−1), it binds to troponin C, causing conformational

changes in troponin I and troponin T that shift tropomyosin away from the myosin-binding

sites on actin [215]. This exposes the sites required for strong XB attachments, allowing

the repetitive power strokes characteristic of active contractions. Such Ca2+-regulated

activation underlies the experimentally observed force–pCa relationship(i.e., normalized

force vs. -log[Ca2+], Figure 1.8) measured in isolated muscle fibers [74, 163]. Notably, ST

fibers generally require lower free Ca2+ levels to initiate contraction and exhibit a less-steep

force–pCa curve, indicating reduced cooperativity in Ca2+ activation. Conversely, FT fibers

typically display a pronounced rightward shift, needing a higher Ca2+ concentration to reach

a given submaximal force and reflecting greater cooperativity. These differences likely stem

from variations in troponin isoforms and the influence of nearby XB attachments on Ca2+

binding within the thin filament [17, 84, 163, 162].

Moreover, skeletal muscle exhibits plasticity in these Ca2+-activation characteristics.

Under conditions such as hindlimb suspension in animal models, slow-twitch fibers may

develop a more “fast-like” force–pCa profile, shifting to higher Ca2+ thresholds and steeper

activation slopes [74]. Regular endurance exercise in humans also shifts the slow-twitch

force–pCa curve, albeit without always altering the threshold for activation [57]. Such adapt-

ability indicates that physiological and training-induced changes in troponin composition, or

in other regulatory proteins, can alter how muscle responds to Ca2+.



1.1 Physiological Background 17

pCa [-]

A
c
ti
v
a

io
n

 L
e

v
e

l 
[%

]

100

80

60

40

20

0
4 4.5 5 5.5 6 6.5 7 7.5

Figure 1.8 An example of the relationship between activation level and calcium ion con-
centration. The activation level is expressed relative to a supramaximal activated isometric
contraction at optimal length. The Ca2+ concentration is expressed as a negative logarithm.
Data from n = 7 rat EDLs are shown. The green dots represent individual measurements,
while the purple line represents the relationship between concentration and activation level at
12 ◦C using the fit function (with smoothingspline) of matlab.



18 Introduction

In addition to fiber-type distinctions, changing the sarcomere length affects the force–pCa

relationship [50, 51, 229, 230]. Extending sarcomeres into the longer range (roughly 2.2 µm

to 3.6 µm) tends to increase the apparent sensitivity of force production to Ca2+ across the

entire force–pCa curve. The mechanistic basis of this length-dependent Ca2+ sensitivity is

still debated; proposed mechanisms include increased affinity of regulatory sites for Ca2+

at longer lengths or length-dependent transitions in troponin and tropomyosin positioning

[201, 202].

Together, these findings underscore that, in skeletal muscle, force generation is not purely

a function of filament overlap. Instead, it also crucially depends on intracellular Ca2+ han-

dling and on the complex interplay of regulatory proteins whose structure and activity can be

modified by fiber type, training status, and muscle length. Understanding these dimensions

of Ca2+-mediated regulation is important for interpreting muscle performance under physio-

logical, pathological, and athletic conditions alike, which motivates Contributions VI and

VII (Section 2.6 and Section 2.7).

1.1.5 Dynamic Contraction Effects

Dynamic muscle contractions are central to nearly all everyday activities, from walking and

running to lifting and reaching. In addition to the well-known steady-state properties of

muscle presented in the chapters before, several distinct phenomena arise when muscle under-

goes length changes under load. The following sections highlight four key dynamic effects:

residual force enhancement after stretch, residual force depression following shortening, the

power-boosting SSC, and the phenomenon known as Give. Understanding these properties

is essential for interpreting how muscle behavior in real locomotion often transcends the

simpler predictions of static contraction models.

1.1.6 History Effects

Muscle force production is influenced not only by instantaneous length, velocity and acti-

vation level, but also by the contractile history of the muscle. This phenomenon is termed

history dependence [1]. Two prominent examples of this history dependence are residual

force depression (rFD) and residual force enhancement (rFE) (see Figure 1.9).

rFD arises when a muscle is actively shortened from a given initial length to a final

length before being held isometric. Under these conditions, the steady-state isometric force

at the final length falls below the force measured in an equivalent, purely isometric reference

contraction. Studies consistently show that rFD becomes more pronounced with:



1.1 Physiological Background 19

• larger shortening amplitudes: The magnitude of force depression increases in parallel

with the distance or extent of muscle shortening [158].

• higher force during shortening: Contractions performed at higher force levels (e.g.,

slower shortening speeds or greater activation) tend to cause more pronounced rFD

[194, 98].

• mechanical work performed: Because both shortening amplitude and force during

shortening contribute to mechanical work, rFD often correlates closely with the total

work done by the muscle prior to the final isometric phase [100, 140, 169, 40].

• longer muscle lengths: A recent study showed a higher amount of rFD in shortening

contractions at longer lengths. [69]

Whether rFD depends directly on the speed of shortening is greater subject of debate,

since faster shortening inherently lowers force [108]. Some investigations suggest the

essential determinant is the force during shortening rather than velocity per se [121, 119].

Additionally, once induced, rFD can persist for tens of seconds unless the muscle is briefly

relaxed [1], indicating a relatively long-lasting effect.

rFE occurs when a muscle is actively lengthened and then held isometric. In this scenario,

the steady-state isometric force at the new, longer length exceeds that of an equivalent

isometric reference contraction at the same length. Key findings about rFE include:

• amplitude dependence: The larger the stretch amplitude, the greater the rFE—at

least within physiological operating ranges [48, 49, 232, 35].

• mild or uncertain velocity influence: While transient force behavior during stretching

is velocity-dependent, the steady-state force enhancement after stretch tends to show

little or only minor velocity dependence [48, 234].

• occurrence on different force–length regions: Contrary to earlier assumptions, rFE

can be observed not only on the descending limb but also on the ascending limb of the

force–length curve [186, 223], suggesting additional mechanisms beyond sarcomere-

length nonuniformities.

Scientific evidence as for instance Herzog and Leonard [101] points to potential roles for

titin (through calcium-dependent stiffness changes or actin-binding interactions) or altered

XB kinetics during or after muscle stretch [101, 199]. In practice, rFE can sometimes exceed

the maximum isometric force that would otherwise be expected at a given muscle length, and

may reduce the metabolic cost per unit force during the post-stretch steady state [120].



20 Introduction

isometric reference
isometric-stretch-isometric
isometric-shortening-isometric

0

0.5

1

1.5

0.8

0.9

1

0 20 40 60 0 20 40 60

L
e

n
g

th
 [
l o

p
t]

Time [s] Time [s]

F
o

rc
e

 [
F

/F
im

]

a) b)

RFE RFD

Figure 1.9 Representative force-time (upper graphs) and length-time traces (lower graphs)
recorded during length-controlled contractions of a single skinned Sol muscle fiber (rat, n =
1, raw, unfiltered data, 12 ◦C). The fiber is fully Ca2+-activated (pCa = 4.5). The purple line
represents the corresponding isometric reference contraction. To examine history-dependent
effects, both a concentric contraction (b, bright green line) and an eccentric contraction (a,
dark green line) are applied. Following active stretch, force is enhanced (rFE), whereas after
active shortening, force is depressed (rFD) relative to the isometric reference force. The
contraction velocity for both experimental conditions is set at 0.4 vmax.



1.1 Physiological Background 21

Although many of these experiments have used frog [48, 49], rodent [196, 28], and human

[89, 219, 218] muscle, the extent to which rFD or rFE vary with fiber type (slow-twitch vs.

fast-twitch) remains under investigation. Some evidence suggests that slower fiber types,

with longer XB lifetimes, might exhibit different magnitudes of history-dependent force,

particularly at varying stretch or shortening speeds. However, direct, controlled comparisons

of fiber-type-specific responses to rFD or rFE are relatively limited and do not permit a firm

consensus.

Velocity may also play indirect roles, since faster or slower stretches and shortenings can

change the instantaneous force levels, ultimately shaping the size of any history-dependent

effect. Finally, other factors such as sarcomere length nonuniformities, cytoskeletal proteins

like titin, and potential neural control differences (in voluntary versus electrically evoked

contractions) further complicate the explanation of the underlying mechanisms of history

effect.

1.1.7 Stretch-Shortening Cycle

A stretch–shortening cycle consists of an active muscle lengthening (eccentric) phase fol-

lowed immediately by an active muscle shortening (concentric) phase (Figure 1.10). This

pattern is characteristic of most terrestrial locomotion whether it is cyclical (e.g., running,

hopping) or non-cyclical (e.g., jumping, throwing). It often enables a higher level of force

and work in the concentric phase than it would be possible if the muscle was only shortened

[130, 27, 13, 15]. Observed both at the muscle–tendon unit level [2, 174] and within individ-

ual muscle measures [77, 178], this SSC-effect can boost mechanical power output by more

than 30–50% compared to concentric-only muscle actions [70, 72, 66]. Typically, the result

is often enhanced efficiency and reduced metabolic cost [27, 120].

Several mechanisms are commonly proposed to explain why the SSC-effect occurs.

Traditionally, these include enhanced activation dynamics, possible contributions from stretch

reflexes, and storage and release of elastic energy in tendinous or aponeurotic structures [214,

36, 220]. However, recent in vitro experiments on skinned muscle fibers suggest that part of

the SSC-effect also arises from within the sarcomere itself, where residual force enhancement

may persist into the shortening phase [66]. Because fiber-level studies eliminate reflexes and

large external tendons, they highlight an internal sarcomeric contribution to SSC mechanics.

In particular, the giant protein titin is increasingly recognized to have a velocity-dependent

viscoelastic behavior that may amplify force during and after stretch, complementing the XB

dynamics [94, 63]. For this reason, the contribution of non-sarcomeric influences on force

generation during SSCs will be investigated in Contribution III (Section 2.3).



22 Introduction

0 20 40 60

0.8

0.9

1

0

2

1

Time [s]

F
o

rc
e

 [
F

/F
im

]
L

e
n

g
th

 [
l o

p
t]

Figure 1.10 Representative force-time (upper graph) and length-time (lower graph) traces
of a permeabilized single fiber from a rat Sol (n = 1, raw, unfiltered data) recorded at an
experimental temperature of 12 ◦C. The solid green line represents the SSC, while the solid
purple line denotes the isometric reference contraction. Each experiment consists of an initial
isometric phase, followed by a ramp transient, and a subsequent isometric phase. After
maximal isometric activation (pCa 4.5) was maintained until a force plateau was reached
(defined as a change in force of less than 1% over 5 s), isokinetic ramp perturbations were
applied at 0.4 vmax.



1.1 Physiological Background 23

Though the SSC-effect is robustly observed, its dependence on contraction velocity, fiber

type, or stretch amplitude remains an active research area. Some data indicate that SSC

performance gains are accentuated at moderate velocities and with larger stretch amplitudes,

while extremely high speeds or minimal displacements yield smaller effects [70]. We attempt

to clarify the question of the velocity influence with Contribution IV (Section 2.4) of this

work. Fiber-type differences may also matter—fast-twitch fibers typically exhibit higher

force and power output but also show more pronounced force loss if the eccentric phase is too

rapid or too large [126, 31]. Meanwhile, slow-twitch fibers, with different XB kinetics and

titin isoforms, might produce somewhat smaller yet more fatigue-resistant SSC enhancements

[66].

Overall, stretch–shortening cycles underpin many functional movements by enabling

muscles to achieve heightened force, power, and efficiency. While classic explanations

centered around tendon recoil, neural reflexes, and preactivation, emerging evidence points

to an intricate interplay of XB and titin-based mechanisms within the sarcomere itself.

Understanding these velocity-, fiber-type-, and amplitude-dependent factors will be crucial for

advancing both fundamental muscle research and applied fields such as sports performance,

rehabilitation, and robotics.

1.1.8 Give

A transient drop in force during an active stretch, called Give (Figure 1.11), was first

characterized by Flitney and Hirst [59] in frog skeletal muscle. These authors observed that

force initially rose to a peak but then dipped abruptly before stabilizing again. Although this

phenomenon has been described in only a few subsequent studies, hints of a similar force dip

appear in a couple of published studies [31, 30, 86, 239, 34]. Researchers, however, seldom

highlight it in their analyses, possibly because it can be difficult to detect or isolated amid the

complexities of muscle stretch mechanics.

One hypothesized cause of Give is XB detachment: when the sarcomeres are stretched

beyond a critical overlap range faster than myosin heads can adapt, XBs “pop off ” momen-

tarily lowering force [59]. The effect has been reported in different animal models (e.g., frog,

rat) and at various organizational levels, including single fibers, small bundles, and entire

muscle–tendon units [31, 48, 59, 66, 239, 241]. Nevertheless, it is not universally reported,

which raises questions about its underlying conditions and prominence.

For instance, some data suggest that Give can emerge when the amplitude of the stretch is

in the range of 0.01–0.02 of the muscle’s optimal length [31, 86]. Furthermore, the force dip

is more prominent at higher stretch velocities, implying a velocity dependence, though the

precise relationship remains unclear. Likewise, whether certain fiber types (e.g., faster XB



24 Introduction

Length [lopt]

F
o

rc
e

 [
F
/F
im

]

 

Give

Figure 1.11 The graph shows the mean force curves (solid green line) for an active fiber
stretch with a stretch rate of 1 vmax of n = 27 Sol fibers. The force is plotted against the
measured relative fiber length. The experiments were conducted at 12 ◦C. For comparability,
the active isometric force-sarcomere length curve (dashed black line, [229]) of Sol fibers is
shown. The difference between the first local maximum and the local minimum defines the
height of the Give (represented by the arrow).

kinetics in fast-twitch fibers) accentuate muscle give is still undetermined. For this reason,

Contributions I and II (Section 2.1 and Section 2.2) of this work will first provide a precise

phase definition and then investigate the influence of stretch velocity and fiber type on these

phases.

1.2 Methods

This chapter provides an overview of the experimental and analytical methods used through-

out this thesis. First, key laboratory protocols like sample preparation (e.g., permeabilized

single-fiber techniques) to mechanical testing and force measurements are introduced. Next,

the data acquisition process is detailed, including instrumentation setups and preparation

procedures. Additionally, the inhibitor usage is presented, which is a relevant technique

in muscle physiology. Overall, the aim of this chapter is to illustrate the methodological

background upon which the subsequent results and interpretations are built.

1.2.1 Muscle Preparation

For the experiments described in this thesis, EDL and Sol were obtained from Wistar rats

(Rattus norvegicus). Overall, the Sol, which predominantly expresses slow type I myosin



1.2 Methods 25

heavy chain ( 96%), is generally specialized for continuous, moderate force production and

is resilient against fatigue. By contrast, the EDL—which predominantly contains fast type

IIA ( 19%) and type IIB ( 76%) myosin heavy chain isoforms—exhibits rapid, powerful

contractions but fatigues more readily [228, 33, 38]. Immediately after death of the animals,

each hind limb was shaved if necessary (to facilitate the procedure), and an incision was

made at the thigh level. The skin was carefully peeled back toward the paw, and the fascia

was opened from proximal to distal. Any additional superficial tissue (for instance, parts

of the gastrocnemius or flexor superficialis) was removed to expose the EDL or Sol. The

target muscle was then gently separated from surrounding connective tissue and excised.

Throughout dissection, a small volume of AT P-containing solution was occasionally dropped

onto the exposed tissue to maintain fiber viability.

Freshly isolated muscles were placed in a chilled relaxation solution (see [79]) to preserve

their contractile integrity. Under a stereomicroscope, several small bundles of fibers were

trimmed in the longitudinal direction. Any areas showing damage from the dissection (e.g.,

where pins or scissors had touched the tissue) were removed. To chemically permeabilize

the fiber bundles, they were first placed in containers (for example, small beakers or vials)

maintained on ice and filled with the same relaxation solution. After a brief rest period, the

bundles were transferred to a skinning solution containing Triton X-100 at low temperature

(4 ◦C). The permeabilization protocol overall followed Linari et al. [146]. Following Triton

treatment, the bundles were returned to relaxation solution for a short wash. They were then

stored at −20 ◦C in a solution containing glycerol (50% v/v) until use, generally within a few

weeks. During this period, muscle fibers exhibit stable mechanical properties when stored in

this conditions [125].

On the day of the mechanical experiments, fiber bundle were placed in a Petri dish filled

with relaxation solution on a temperature-controlled stage (about 4 ◦C to 6 ◦C). Single fibers

were carefully pulled out under a stereomicroscope, preserving as much of the sarcomere

integrity as possible. Each end of a chosen fiber segment was cautiously clamped using

aluminum foil “T-clips” [61] to avoid crushing the extremities. Fibers were then briefly treated

(60 s to 120 s) with a relaxing solution containing 1% Triton X-100 (v/v) to ensure thorough

removal of internal membranes (sarcolemma and sarcoplasmic reticulum) while leaving the

contractile apparatus intact [65, 146]. Depending on the experimental requirements, the

single fibers were either used immediately or stored again at −20 ◦C in the glycerol-based

storage solution.

For final mounting into the mechanical setup, individual fibers with T-clips at each end

were attached to the force transducer and length controller respectively in relaxing solution.

In certain procedures, a small droplet of glutaraldehyde mixed in rigor solution was applied



26 Introduction

to stabilize the fiber ends [124, 123, 200, 107]. Furthermore, nail polish was used to fix

the T-clip-hook junction in air, preventing the fiber from slipping from the hook during

activation [236]. The central fiber segment was monitored for sarcomere length changes,

ensuring minimal inhomogeneity of sarcomere lengths [80]. This combined mechanical

and chemical preparation maintained viability while permitting direct manipulation of the

fiber’s contractile properties. It also allowed systematic comparisons of EDL and Sol fiber

mechanics under identical experimental conditions, leveraging their distinct physiological

properties.

1.2.2 Experimental Apparatus

A permeabilized muscle fiber test system (1400A, Aurora Scientific, ON, Canada) was used

to investigate the mechanical properties of single skinned fibers (Figure 1.12). Each prepared

fiber–clip unit was transferred into the experimental chamber via a modified pipette tip. For

force and length measurements, the fiber–clip unit was attached to a force transducer (model

403A, Aurora Scientific, range 5 mN, resolution 0.1 µN) and a high-speed length controller

(model 322CI, Aurora Scientific, max. force 100 mN, resolution 0.5 µm). Steel hooks linked

the clips to the transducer lever, thereby preventing unwanted movement of the fiber during

solution exchanges or rapid length changes [22]. Finally the apparatus was placed on the

x–y moving stage of an inverted microscope (Nikon Eclipse Ti-S). The test apparatus has

a temperature-controlled platform that accommodates seven small chambers (160 µL each)

and a larger solution chamber (200 µL), all movable horizontally by a stepper motor.

Sarcomere length was monitored in the central segment of the fiber [241] and set to

2.5±0.05 µm initially, representing the length at which maximal force is produced [229].

The fiber width (w) and height (h; measured through a prisma in the side wall of well

one) were then measured at approximately 0.1 mm intervals along its length through a 10x

extra-long working distance objective (NA 0.60/0.40, Nikon). The cross-sectional area was

computed by approximating an elliptical cross section:

A =
π ×h×ω

4

Dynamic sarcomere-length changes were recorded with a high-speed video system

(901B, Aurora Scientific) combined with a 2.5 x accessory lens (Nikon), capturing images

at up to 300 Hz. All buffer solutions (relaxing, preactivating, activating, skinning, and

storage) were prepared to have a pH 7.1 at 12 ◦C. The exact composition of acativation

solutions can be found in the Apendix A (Chapter 4). The composition of all non-activation

solutions can be found in Tomalka et al. [241]. A spatula tip of creatine kinase was freshly



1.2 Methods 27

Figure 1.12 On the left hand side: Overview of the experimental setup mounted on a vibration-
isolated laboratory table (TMC-63-534). On the right hand side: Close-up view of the test
apparatus for permeabilised muscle fibres. a) shows the 403A force transducer, while the
322CI length controller is marked with b). c) shows the bathing wells of the plate. The
fibre–clip assembly (highlighted by d) and zoomed into in the green circle) is connected to a
length and a force transducer using two custom-made hooks fashioned from 29G hypodermic
needle tubing. For a secure clip-hook connection a drop of red nail polish was used.



28 Introduction

added to relaxing, pre-activating and activating solution to maintain AT P buffering capacity.

Activation solutions covered a range of Ca2+ concentrations (pCa values of 6.73, 6.34, 6.30

and 4.5) to match the boundary conditions relevant to the measurements. This setup of the

fiber test apparatus and associated chemical solutions ensured reliable manipulation of single

skeletal muscle fibers, permitting precise investigation of their contractile function under a

variety of experimental conditions.

1.2.3 Cross-Bridge Inhibitors

Several small-molecule inhibitors specifically targeting myosin II AT Pase activity were

tested in pilot experiments, including Blebbistatin, N-benzyl-p-toluene sulphonamide (BT S),

and 2,3-butanedione monoxime (BDM). Blebbistatin is a highly specific, reversible inhibitor

that binds within a hydrophobic pocket on myosin II, preventing the release of inorganic

phosphate and thereby blocking the force-generating step [3, 131, 143, 211]. By stabilizing

an actin-detached ADP/Pi-bound state, Blebbistatin avoids the formation of strongly bound

nonfunctional actomyosin complexes [247, 195, 231]. It has proven to be particularly useful

for inhibiting both non-muscle (where it is important for cell migration [248]) and striated

myosin II without affecting many other myosin classes [143]. Moreover, its inhibition is

readily reversed by washout, enabling fine temporal control of XB activity. While Blebbistatin

does exhibit some light sensitivity [211], careful shielding or illumination settings can

mitigate cytotoxic photoinactivation effects.

BT S selectively inhibits fast-twitch skeletal muscle myosin II [29]. Its half-maximal

inhibitory concentration (IC50) of around 5 µmol is significantly higher than that of Bleb-

bistatin, yet it shows much lower potency against slow-twitch skeletal or cardiac myosins

[221]. Although BTS has reversible inhibition and does not affect nonmuscle myosin II

[29], its marked preference for fast skeletal isoforms limits its suitability in experiments

involving varied fiber types or mixed populations. Moreover, comprehensive data on its

broader specificity, especially against myosins in other classes, remain scarce.

BDM was first developed for applications unrelated to myosin, and it requires millimolar

concentrations (10 mmol or more) to inhibit skeletal muscle AT Pase activity effectively [106].

Its low binding affinity and broad off-target effects—including inhibition of various kinases,

ion channels, and additional protein systems—render BDM considerably less selective

[62, 184]. Conflicting data persist regarding whether BDM inhibits non-muscle myosin II and

other myosin classes [37, 184]. These uncertainties, coupled with multiple auxiliary actions

not directly linked to XB inhibition, have dissuaded its routine use in precise contractile

studies. For a more detailed explanation of the effects on myosin, a look at Bond et al. [12]

is recommended.



1.3 Objectives and Results 29

Given the drawbacks of BT S (narrow specificity) and BDM (lack of specificity, extensive

off-target effects, and low affinity), Blebbistatin proved to be the most suitable inhibitor

for our experiments. Its ability to arrest the XB cycle in an actin-detached state minimizes

unwanted residual force or stiffness artifacts, and the efficacy of Blebbistatin in a micromolar

range confers practical advantages. Despite the need to manage light-exposure conditions,

Blebbistatin’s specificity, reversibility, and well-characterized mode of action made it a good

choice for investigating myosin II functions in isolated muscle fibers of the Contributions III,

IV and V (Section 2.3 to Section 2.5).

1.3 Objectives and Results

The primary objective of this thesis is to investigate force generation during dynamic muscle

fiber contractions, with a particular focus on the influence of fiber type and contraction

velocity on the produced forces. A key aspect of this research is the differentiation between

XB and non-XB force contributions, which was achieved through the use of the XB inhibitor

Blebbestatin. The insights gained from this work help to enhance our understanding of muscle

injury mechanisms, improve existing muscle models, and contribute to the development of

muscle-like actuators by refining the understanding of CE function.

In order to clarify the functions and relationship behind muscle contraction and injury

a series of experiments was conducted. In these experiments pure eccentric contractions

as well as eccentric contractions in combination with an immediately following concentric

contraction were analyzed. Through a systematically examination of these contraction

types, this thesis provides new insights into the mechanical behavior of muscle fibers under

dynamic loading conditions, contributing to both fundamental muscle physiology and applied

biomechanics.

Thus, the overarching research question of this thesis is: What are the mechanisms

underlying force generation in dynamic muscle contractions, and how do fiber type,

contraction velocity, and the contributions of XB and non-XB elements influence the

mechanical response of muscle tissue?

We aimed to generate and analyze a data set on the mechanisms of force generation

in dynamic muscle contractions, focusing on the influence of fiber type, a broad range of

contraction velocities, and the distinction between XB and non-XB contributions. To achieve

this, we conducted experiments analyzing eccentric contractions over a large stretch ampli-

tude based on the protocol of Tomalka et al. [241]. This served to obtain a comprehensive

characterization of the whole force response. Furthermore, we conducted experiments in

which a concentric contraction followed directly after the eccentric contraction (SSC). One of



30 Introduction

the aims of this was to investigate the influence of history effects. The SSC experiments were

carried out with a XB inhibitor in order to be able to draw conclusions about the mechanisms

of force generation. Finally, length trajectories and activation levels were derived from

the CE of a muscle model. The force responses of single skinned muscle fibers were then

compared with the model prediction to investigate any differences.

Characterizing and understanding these force-generating mechanisms is essential for

multiple applications, including improving muscle models, enhancing muscle-like actuators,

and providing insights into injury mechanisms in muscle tissue. Given the complexity of

muscle contraction dynamics and the varying mechanical properties of different fiber types,

a key challenge of this research was to systematically isolate and quantify the contribu-

tions of different contractile and non-contractile components under controlled experimental

conditions.

Defining the general methodology for this thesis led to the following research questions:

• What phases can the force generation of different muscle fibers be divided into

during large amplitude eccentric contractions and how does stretch velocity affect

these phases?

• What proportion do XBs and non-XB structures contribute to the force generation

in SSCs?

• How do the parameters force, power and force redevelopment change depending

on the contraction velocity or activation level of SSCs?

• How accurately can the contractile element in a current model predict the force

development of a real muscle fiber?

Consequently, the objectives below were identified, each of which contributes indepen-

dently to the overarching research question:

• Definition of different phases and points of eccentric force generation and com-

parison of these over a wide velocity range.

• A comparison of the behaviors exhibited by the two types of fibers under condi-

tions of comparable stretch velocities.

• Investigation of the force output and work produced by single skinned fibers of

rat soleus muscles during and after ramp contractions at a constant velocity as

well as the differentiation of XB and non-XB contributions.



1.3 Objectives and Results 31

• Investigation of the mechanisms contributing to power amplification, and how do

they depend on stretch velocity.

• Analysis of the mechanisms of force redevelopment at different contraction veloc-

ities.

• Quantification of the SSC-effect in consecutive SSCs.

• Comparison of the force response of skinned muscle fibers with the behavior of

the contractile element from current muscle models.

1.3.1 Contribution I: How velocity impacts eccentric force generation

of fully activated skinned skeletal muscle fibers in long stretches

The first contribution addresses how eccentric force generation in fully activated skeletal

muscle fibers unfolds across a wide range of stretch velocities by focusing on several key

phases and characteristic points that define the force profile during a single, long stretch.

First, we introduce the phase definitions: the initial force slope (slope1) captures how force

rises immediately after the fiber begins to lengthen from 0.85 to 1.3 of its optimal length

(lopt), culminating in a local force peak (s2). Beyond s2, the force trace may exhibit a sudden

drop known as Give, defined by the difference between s2 and its subsequent local minimum

(sg). After this Give dip, the force often redevelops in a near-linear way, represented by a

second slope (slope2), before reaching the final force at the end of the stretch (se).

By stretching fibers at slow, moderate, and high velocities (0.01, 0.1, and 1 times the

maximum shortening velocity of fast twitch muscle fibers), the data show that each force

characteristic depends on stretch speed in distinct ways. At very slow velocity, the force

rises relatively smoothly and does not exhibit any Give. As the stretch velocity increases,

s2 grows larger, while a more pronounced Give emerges. The force then rises anew with an

even steeper slope2 at higher stretch velocities, culminating in a higher final force. These

findings confirm that each of the identified phases—slope1, s2, Give, sg, slope2, and se—is

shaped by the velocity at which the muscle is lengthened. The fact that Give disappears at

low velocities underscores its velocity-dependent origins, presumably tied to the sudden

strain on XBs, while the steadily increasing slope2 suggests a viscoelastic contribution from

titin that becomes more influential with higher velocity and greater stretch distance.

We extended existing work on eccentric muscle contractions by applying long ramp

stretches to muscle fibers at different velocities, which allowed us to identify specific

phases of force development. A novel contribution of this study is the introduction of a



32 Introduction

nomenclature for these phases and the observation that slope2 is velocity-dependent—a

finding that was not described in prior literature.

1.3.2 Contribution II: Impact of lengthening velocity on the generation

of eccentric force by slow-twitch muscle fibers in long stretches

In the second contribution the main goal was to investigate how force develops during large-

amplitude eccentric contractions in fast- and slow-twitch muscle fibers at different stretch

velocities, focusing on the six specific phases or points identified in the first contribution.

When comparing slow-twitch (Sol) and fast-twitch (EDL) fibers under matched relative

stretch velocities —that is, 0.01 vmax, 0.1 vmax and 1 vmax, normalized to each fiber type’s

maximum shortening velocity— both exhibited similar overall patterns but with notable

quantitative differences. In both types, velocity strongly influenced each of these phases. For

instance, slope1 rose more steeply at higher speeds, reflecting a more rapid initial force rise.

The local maximum s2 also increased, while the position of s2 (in terms of sarcomere or fiber

length) shifted to larger elongations. Importantly, Give became far more pronounced at higher

velocities. However, it is important to note that there is no Give in the EDL at 0.01 vmax

while the Sol shows a clear one. Finally, slope2 and se grew with increasing velocity in all

trials. Nevertheless, distinct differences emerged between slow- and fast-twitch fibers in how

sharply slope1 rose. In this phase the values for ST fibers were significantly higher compared

to FT fibers. In contrast to this result the variables of the latter stretch (slope2 and se) were

significantly higher in fast-twitch fibers.

By analyzing the velocity dependencies in both ST and FT fibers during long ramp

stretches at comparable relative stretch velocities, we improved the knowledge about

fiber type specific differences in force generation during the early and latter phase of

long ramp stretches.

1.3.3 Contribution III: Cross-bridges and sarcomeric non-cross-bridge

structures contribute to increased work in stretch-shortening cy-

cles

In the third contribution to this thesis, permeabilized rat Sol fibers were subjected to SSCs

under physiological conditions, and force as well as mechanical work were measured. To

distinguish the role of XB and non-XB structures in SSC force generation, a XB inhibitor was

used. During SSCs in standard (control) conditions, the force during the eccentric phase rose

sharply, then partially declined, before redeveloping toward the end of stretch, and ultimately



1.3 Objectives and Results 33

exceeded the steady-state isometric reference. By contrast, in the presence of Blebbistatin (a

myosin AT Pase inhibitor that suppresses XB-based force), fibers still produced significant

force during SSCs, yet total force and mechanical work were markedly lower. Despite the

loss of most XB contribution in Blebbistatin-treated fibers, work remained higher in SSCs

than in purely shortening contractions. These findings indicate that, while XBs are crucial

for the maximal SSC-effect (i.e., fully enhanced force and work), non-XB structures play a

significant role in storing and releasing elastic energy during fast SSCs. Consequently, both

XB and non-XB components jointly elevate force and work in SSCs, and their combined

contributions explain how muscle can perform these dynamic contractions with enhanced

performance. This answers the research question by showing that, although XBs remain

central to the overall effect, non-XB titin-based forces substantially increase force and

work in SSCs, especially when XB attachments are partially inhibited.

1.3.4 Contribution IV: Power amplification increases with contraction

velocity during stretch-shortening cycles of skinned muscle fibers

In this study, we investigated how key mechanical parameters change with increasing stretch-

shortening velocity of single skinned rat Sol fibers. The specific goal was to pinpoint the

mechanisms that drive power amplification and examine whether they vary systematically

with contraction speed. To this end, we conducted SSC experiments across a range of stretch-

ing/shortening velocities (30%, 60%, and 85% vmax) and compared total force responses with

those obtained under inhibition of XB force using Blebbistatin.

The findings showed that, at higher stretch-shortening velocities, the negative work

performed during the eccentric phase increased, whereas the positive work produced in

the concentric phase decreased. Despite this reduction in concentric work, the overall

power output of the muscle fibers rose with increasing SSC velocity. This increase in power

during rapid SSCs implies the presence of velocity-dependent mechanisms related to the

stretch phase that carry over into the subsequent shortening phase. Moreover, we found an

amplified energy recovery in Blebbistatin condition. We suspect that titin, which is unaffected

by Blebbistatin, is both storing and releasing elastic energy during rapid SSCs. Overall,

this study underscores that titin-based elasticity and XB kinetics act in concert to

enhance fiber power at faster SSCs, thereby offering novel insight into how sarcomeric

mechanisms underlie velocity-dependent power amplification.



34 Introduction

1.3.5 Contribution V: Force re-development after shortening reveals a

role for titin in stretch–shortening performance enhancement in

skinned muscle fibres

In the fifth contribution, we investigated how the rate and magnitude of force re-development

(quantified by the maximum rate of force re-development and the time taken to reach that rate)