*1.1. Titin Contains Four Structural Domains That Impart Important Molecular Properties to the Sarcomere during Cardiac Function*

The essential function of the heart is generation of circulatory blood flow to organs and tissues. This circulatory flow or cardiac output is created by coordinated contraction and relaxation of the heart muscle, termed systole and diastole, respectively. During systole, the heart muscle contracts and ejects blood from the heart delivering oxygen and vital nutrients to the body. During diastole, the heart muscle relaxes allowing blood to fill the ventricles in preparation for systole. Dysfunction of systole and diastole can lead to the development of heart failure. Heart failure is a clinical syndrome where dysfunction affecting systole, diastole or both leads to significant symptoms and mortality. Heart failure is one of the most morbid health conditions affecting an estimated 5.1 million people in the United States and 23 million people worldwide [1,2]. As a basic principle, heart failure results from dysfunction of the cardiomyocyte and sarcomeric structures. Cardiomyocytes are specialized muscle cells that contain

sarcomeres which are the fundamental sub-cellular structure that convert chemical energy to muscular contraction. The sarcomere is a complex protein structure with rigid vertical M-lines and Z-lines that anchor overlapping longitudinal filaments. During cardiac contraction, the myosin heads pull actin filaments in one direction bringing the vertical M-line and Z-lines together resulting in contraction and mechanical force generation [3].

One of the most important proteins that compose the sarcomere is titin. Titin is a filamentous protein anchored in the Z-line and extending to the M-line thereby providing structural support to the sarcomere [4,5]. Titin is the largest known protein with a measured length of up to 1.2 μM and comprised of 27,000–33,000 amino acids with a molecular mass of 3800 kDa [6,7]. Titin is encoded by the titin gene *TTN*, which is 294 kb and contains 364 exons [8]. Although titin has many complex molecular properties, its mechanical role supporting the sarcomere and providing passive tension and force modulation during contraction and relaxation has been the most well described and are the focus of this paper [5,9]. The molecular properties of titin as it contributes to normal function are imparted by the four structural domains (Figure 1). The N-terminal domain embeds and anchors it to the Z-disk, while the C-terminal domain similarly anchors titin to the M-band [4,5,10]. Titin filaments in the Z-disk and M-line are embedded to domains of adjacent titin proteins from the opposing sarcomere to form overlapping connections that create a continuous filamentous connection spanning the myofibril [11]. The I-band is adjacent to the Z-disk and is composed of repetitive immunoglobulin-like domains, a region rich in proline (P), glutamate (E), valine (V), and lysine (K) termed the PEVK region, as well as the N2B element [5,11]. The I-band is highly elastic and provides a spring-like function that imparts passive tension during relaxation, elongates during contraction, and returns the sarcomere to its previous length after contraction [12,13]. The A-band serves as a rigid region that binds to the thick filament and interacts with myosin to translate force during contraction to the sarcomere [14].

**Figure 1.** Titin structural domains serve essential functional roles as part of the sarcomere, myofibril, and cardiomyocyte. Cardiac tissue is composed of cardiomyocytes that impart contractile function. The contractile subunit of the cardiomyocyte is the myofibril, which is made of sarcomeres. Titin is an essential component of the sarcomere composed of four domains. The N-terminal domain embeds titin to the Z-disk. The I-band contains immunoglobulin-like (Ig) domains that impart extensibility and provide titin "spring-like" characteristics. The A-band binds to myosin and serves as a rigid region during contraction. The C-terminal domains embeds titin to the M-band.
