SummaryThis proposal focuses on titin the largest protein known in heart function and disease. Titin forms a novel andmultifunctional myofilament in the striated-muscle sarcomere with important roles that include regulating thediastolic stiffness of the heart. Recent breakthrough studies revealed that titin is of high clinical importance inboth heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction(HFrEF). Although significant progress has been made in understanding the basic biology of titin major gaps inour understanding still remain including a mechanistic understanding of how titin causes/contributes to heartdisease. An important focus of this proposal will be on titins role in diastolic dysfunction motivated by recentstudies on patients with HFpEF that revealed deranged phosphorylation of titins molecular spring elements anddiastolic stiffening. The full spectrum of posttranslational modifications that occur in HFpEF will be studied andhigh-resolution time-resolved spectroscopic techniques will focus on uncovering the structural changes in titinsspring elements triggered by posttranslational modification. Drug screens will focus on identifying compoundsthat mimic or block these structural changes and functional studies will test whether newly discovered andcandidate drugs ameliorate titin-based diastolic stiffening in HFpEF. Post-transcriptional mechanisms will beinvestigated as well taking advantage of our recent work that has shown that splicing of titin can be manipulatedto upregulate complaint titin isoforms and restore diastolic function. The functional efficacy of identifiedcompounds will be tested on engineered heart tissues as well as on animal models. The second major focus ofthis proposal will be on titin in HFrEF. Several recent sequencing studies in large groups of patients revealedthat mutations in the titin gene (TTN) are causative in ~20% of studied dilated cardiomyopathy (DCM) patients.Many of the mutations are truncation mutations (TTNtv) and they have a preferential location in the A-bandsegment of titin. The A-band segment is the least well-studied part of titin and an important goal of our researchis to critically examine the biology of titin in this region of the sarcomere where disease-causing mutations areprominent. These studies include a focus on the role of titin in interacting with cMyBP-C (cardiac myosin-bindingprotein C a clinically important thick filament protein). Animal models will be investigated in which TTNtv havebeen introduced in different regions of titins A-band segment. The effects of the mutations will be studied underbaseline conditions when stressed and when occurring in combination with mutations in other genes.Importantly we will also test whether excision of the mutated titin exons ameliorates titin-based DCM. Insummary capitalizing on my >20-year track record of innovative titin research and utilizing our team ofexperienced scientists and talented trainees this proposal sets ambitious goals that are expected to furtheraccelerate understanding of the biology of titin its role in heart disease and titins potential to function as atherapeutic target.