Familial hypertrophic cardiomyopathy (FHC) is the most frequent hereditary heart disease. Because FHC is in most cases associated to genes encoding sarcomeric proteins, it has been termed a `disease of the sarcomere'. One leading symptom of FHC is diastolic dysfunction. Here, we use isolated myofibrils to investigate the mechanisms by which the FHC-mutant DK183 in the inhibitory subunit (TnI) of the regulatory troponin complex (Tn) induces diastolic dysfunction. Tn is a Ca²⁺-dependent molecular switch that regulates the force generation of cross-bridges. The initial sarcomeric process of diastolic relaxation is the switch-off of Tn. To measure the effect of the TnI^DK183 mutation on this switch-off, fluorescently labelled mutant or wildtype Tn was incorporated into myofibrils and the kinetics of the fluorescence changes induced by a rapid decrease in the [Ca²⁺] was recorded by stopped-flow technique. This reveald that the switch-off kinetics is slowed down by the mutation. The altered switch-off kinetics correlates with slower kinetics of force relaxation, which was observed in myofibrils isolated from transgenic mice overexpressing the TnI^DK183 protein. Since Troponin C (TnC), the Ca²⁺-binding subunit of Tn, is a major Ca²⁺-buffer, we hypothesize, that slowed down switch kinetics and relaxation may delay the Ca²⁺-dissociation from TnC, which would also delay the intracellular Ca²⁺-decay. To verify this hypothesis, we presently investigate intracellular Ca²⁺-transients in cardiomyocytes isolated from TnI^DK183 mice. For Ca²⁺-imaging, the cells are loaded with Fura-2, a Ca²⁺-sensitive dye, and analyzed by fluorescence video microscopy. Preliminary results show that the Ca²⁺-decay is only slightly slowed down, indicating that there is either no major feedback of the slowed down switch-off of Tn on the Ca²⁺-release of TnC or that the slower Ca²⁺-release is compensated by faster reuptake of Ca²⁺. Using various biochemical and pharmacological tool we will discriminate between different possible compensatory mechanisms. This will give us detailed information about the consequences of the impaired inhibitory activity of TnI on the Ca²⁺-homeostasis.