**5. Conclusions**

Heart failure is a common clinical syndrome often caused by abnormalities within sarcomeric proteins including the giant, spring like protein titin. The structure of titin, specifically Ig-like domains, impart important physiologic characteristics to cardiac tissues including length dependent activation and passive tension of the ventricle. Truncation mutations of titin are the most common genetic causes of dilated cardiomyopathy and often lead to a phenotype that presents later in life and is associated with atrial arrhythmias. The pathophysiology of TTNtv leading to DCM is likely related to increased intracellular stress associated with increased nonsense mRNA decay leading to activation of the mTOR signaling cascade which results in abnormalities in cellular metabolism and ultimate dysfunction of the cardiomyocyte. This increased metabolic stress is likely to predispose patients with TTNtv to development of DCM when combined with other cardiac risk factors such as age, chemotherapy, alcohol

use, and pregnancy. There are promising therapies for treating TTNtv including mTOR inhibitors and oligonucleotides that lead to alternative splicing that exclude mutated exons (Table 1).

Transcriptional modifications of titin are associated with both systolic and diastolic dysfunction and therefore titin represents a viable therapeutic target for treatment of cardiomyopathy. Patients with systolic heart failure have increased expression of the N2BA titin transcriptional variant that encodes a longer and softer titin protein, which correlates with decreased passive tension and increased left ventricular end diastolic volume. Therefore, transcriptional modification of titin to preference the shorter, stiffer N2B isoform may improve systolic heart failure. Conversely, in patients with abnormal ventricular relaxation, favoring the longer, softer titin isoform may improve diastolic filling and treat diastolic dysfunction. Although we do not yet have therapies to modulate transcription of titin, RBM20 is a key transcriptional regulator and therapeutic target (Figure 5).

**Figure 5.** Transcriptional and post-translational modifications of titin alter passive tension and serve as therapeutic targets for treatment of heart failure. Patients with systolic dysfunction have decreased passive stiffness of the ventricle. Modifications of titin by upregulation of RNA binding motif protein 20 (RBM20) and preferencing the N2B isoform, or phosphorylation of the PEVK element can increase passive tension of the I-band and may improve cardiac function. Conversely, patients with diastolic dysfunction have increased passive tension of the ventricle. Decreased expression of RBM20 leading to increased expression of the N2BA isoform, or phosphorylation of the N2Bus element may decrease passive tension and improve cardiac function.

Post-translational modifications of titin by phosphorylation of the spring-like I-band are capable of changing the length and stiffness of titin and are correlated with cardiomyopathy. Increased phosphorylation of the PEVK element increases titin passive tension, whereas phosphorylation of the N2Bus element decreases passive tension. Many molecules and signaling pathways affect phosphorylation of these regions and further understanding of their control may provide an opportunity to selectively modify titin and improve both systolic and diastolic heart failure (Table 1, Figure 5).

**Author Contributions:** Conceptualization, C.T., M.T. and L.M.; resources, C.T.; writing—original draft preparation, C.T.; writing—review and editing, C.T., M.T. and L.M.; funding acquisition, M.T. and L.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported in part by NIH grants R01 HL69071, R01 HL116906, R01 HL147064, NCATS/CTSA UL1 TR001082 and AHA17GRNT33670495 (L.M.); NIH 1K23HI067915 and R01HL109209 (M.T.). This work was also supported in part by the Trans-Atlantic Network of Excellence grants from the Foundation Leducq (14-CVD 03) (M.T., and L.M.).

**Acknowledgments:** Figures were created with Biorender.com.

**Conflicts of Interest:** The authors declare no conflict of interest.
