Universiẗat, Diss.--Jena, 2007.

In the first half of this work, titin's role in cardiac function was studied using intact cardiac myocytes. The development of a carbon fiber based cell-attachment system allowed diastolic and systolic function of the isolated intact myocyte to be investigated. Addition of actomyosin inhibitor to the intact myocyte revealed that the majority of the cell's diastolic stiffness is due to titin but that actomyosin interaction exists as well and contributes ~ 30% of total diastolic stiffness. The details of this study are provided in chapter 1. Heart failure with preserved ejection fraction (HFpEF) accounts for up to 50% of total heart failure cases and is characterized by increased diastolic stiffness. An effective treatment for HFpEF does not exist. Reducing titin stiffness as a therapeutic strategy for lowering left ventricular (LV) chamber stiffness in HFpEF is currently under consideration. To understand the functional consequence of reduced titin stiffness on global cardiac function a Rbm20 Δᴿᴿᴹ mouse model was created. The Rbm20 Δᴿᴿᴹ model has deficiency in titin splicing that results in expression of very large and compliant titin isoforms in the sarcomeres. Study of Rbm20 Δᴿᴿᴹ cells revealed that cellular diastolic stiffness was inversely related to the size of titin and was reduced in a graded manner in Rbm20 Δᴿᴿᴹ heterozygous (+/-) and homozygous (-/-) cells. Importantly, reduced titin-based stiffness manifested in vivo as reduced LV chamber stiffness, which could be observed by echocardiography and pressure volume (PV) analysis. The systolic function of Rbm20 Δᴿᴿᴹ was studied by measuring the Frank-Starling mechanism (FSM), first at the intact myocyte level. The FSM was reduced in Rbm20 Δᴿᴿᴹ +/- and -/- with the largest reduction in -/- cells. PV analysis demonstrated a reduced FSM at the LV chamber level, consistent with the result at the cellular level. Surprisingly, exercise testing showed an enhanced exercise performance in cardiac specific Rbm20 Δᴿᴿᴹ +/- mice (relative to wild-type mice). Thus, this work indicates that increasing titin compliance improves diastolic function but negatively impacts systolic function. Importantly, findings suggest that the beneficial effect of improving diastolic function is a dominant effect. This work is described in Chapter 2.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20(Delta RRm)) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20(Delta RRm) animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20(Delta RRm) on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbrn20<^>R'Rm and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20(Delta RRm) vs. WT mice. As sarcomere length increased from 1.9 to 2.2 mu m, length-dependent activation of contraction was eliminated in the Rbrn20<^>R'Rm myocardium, even though myosin MgADP release rate decreased similar to 20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Die vorliegende Studie zeigt erstmals, dass eine erhöhte Ca2+-Empfindlichkeit der kardialen Myofilamente im Tiermodell einen selbstständigen Risikofaktor bei der Entstehung von Herzrhythmusstörungen darstellt. Dies konnte sowohl für chronische Erhöhung der Ca2+-Empfindlichkeit im Rahmen einer Familiären Hypertrophen Kardiomyopathie (FHK) als auch für eine akute Erhöhung mit Hilfe eines Ca2+-Sensitizers gezeigt werden. Die Ergebnisse der Arbeit bieten so eine mögliche Erklärung für plötzlichen Herztod bei bestimmten Patienten mit FHK. Sie schränken weiterhin den Einsatz von Ca2+-Sensitizern ein. Schließlich beleuchten sie einen bisher kaum untersuchten Aspekt in der Arrhythmogenese von Herzinsuffizienz und nach einem Myokard-Infarkt. Auf zellulärer Ebene findet sich ein veränderter Ca2+-Zyklus mit erniedrigten und verlangsamten Transienten. Diese Veränderungen sind wahrscheinlich eine direkte Konsequenz der erhöhten Bindungsaffinität für Ca2+. Die Myofilamente sind „klebriger“ für Ca2+, während der Systole wird mehr Ca2+ gebunden, während der Diastole hingegen dissoziiert es langsamer. Bei adrenerger Stimulation und schnellen Herzfrequenzen mit entsprechender Verkürzung der Diastole kommt es zu erhöhtem diastolischem [Ca2+]i und zu erhöhten Ca2+-Inhalt des Sarkoplasmatischen Retikulums. Der so veränderte Ca2+-Zyklus führt wahrscheinlich mit Hilfe des Na+/Ca2+-Austauschers zu Veränderungen der Repolarisation des Aktionspotentials. Bei schnellen Herzfrequenzen treten Aktionspotential-Verlängerung, Ca2+-abhängige Nachdepolarisationen und getriggerte Schläge auf. Auf Organ-Ebene findet sich eine verkürzte Refraktärzeit. Damit sind sowohl ein Trigger als auch ein arrhythmogenes Substrat für die beobachteten ventrikulären Arrhythmien gegeben. / The present study shows for the first time that increased Ca2+-sensitity of the cardiac myofilaments is an indepent mechanism in the development of cardiac arrhythmias. This could be shown both for chronically increased Ca2+-sensitity in Familial Hypertrophic Cardiomyopathy (FHC) and for acute drug-induced Ca2+-sensitization. The results provide an explanation for sudden cardiac death in certain patients with FHC. Moreover they limit the use of Ca2+-sensitizers. Furthermore they elucidate new aspects in the arrhythmogenesis of important aquired hearts diseases such as heart failure and myocardial infarction. On the cellular level the Ca2+-cycle is altered, Ca2+-transients show a smaller amplitude and slower rate of decay. These changes are probably a direct consequence of the increased Ca2+-puffering capacity. The myofilaments are “sticky” for Ca2+, durig systole more Ca2+ is bound while the dissociation during diastole is decelerated. With adrenergic stimulation and faster frequencies there is an increased diastolic [Ca2+]i and subsequently an Ca2+-overloading of the Sarcoplasmatic Reticulum. These changes in the Ca2+-cycle cause remodelling of the action potential repolarisation via the Na+-Ca2+-exchanger. At fast frequencies one finds action potential prolongation, Ca2+-dependent afterdepolarisations and triggered activity. On the organ level Ca+-sensitized hearts show a shortened refractory period. Thus there is both trigger and substrate for the observed ventricular arrhytrhmia.