*2.2. TTNtv Cause DCM through Haploinsu*ffi*ciency, Increased Metabolic Stress, and Activation of the mTOR Signalling Cascade*

The exact mechanism for how TTNtv lead to a cardiac phenotype has not been clearly demonstrated. There are studies that suggest specific mutations cause direct damage to the sarcomere and cardiomyocyte. This was observed in a cell culture experiment using human induced pluripotent stem-cell derived cardiomyocytes (hIPSC-CM) isolated from a symptomatic patient with an A-band TTNtv. In the derived cardiomyocytes, a shortened titin protein was isolated and associated with disorganized sarcomere formation and impaired contractility [28]. Similarly, hiPSC-CM derived from a patient with unique A-band TTNtv demonstrated abnormal sarcomerogenesis and cardiomyocyte force generation due to loss of a β-cardiac myosin binding site on the mutated titin protein [29]. Despite these findings

in cell culture experiments, abnormal titin proteins leading to direct sarcomere damage in a "poison peptide" model have not been shown in human studies. Biopsied cardiomyocytes from symptomatic patients with TTNtv did not demonstrate similar alterations in maximum force in myofibril contraction studies [30].

An alternative to the dominant negative model of TTNtv is a model of haploinsufficiency. In this more likely explanation for pathogenicity, TTNtv do not directly damage the sarcomere but instead lead to increased metabolic stress that has chronic effects on cardiomyocyte and cardiac function. Metabolic stress is generated by abnormal mRNA transcripts from the truncation variant leading to increased non-sense mRNA decay (NMD). This leads to long term compensatory changes and development of a common DCM phenotype that is independent of *TTN* mutation location [31–33] (Figure 3). Rat models with A-band and I-band TTNtv do not alter the amount of TTN expressed, but there is increased NMD and a shift in cardiac metabolism to preference branched chain amino acids and glycolytic intermediates instead of fatty acids that are typically utilized in healthy cardiomyocytes [34,35]. Increased metabolic stress associated with NMD activates mammalian target of rapamycin (mTOR) complex-1 signaling resulting in pathologic protein synthesis and autophagy leading to cardiac phenotypes [31,36]. This was demonstrated in a rat model with TTNtv where there was elevated mTOR complex activation and decreased antiphagocytic degradation in cells. This led to increased reactive oxygen species and mitochondrial dysfunction. Interestingly, treatment of rats with the mTOR inhibitor, rapamycin, rescued the cardiac phenotype [31]. Activation of the mTOR complex appears to be a common downstream pathway of heart failure as its activation is associated with other causes of DCM [37,38]. Further elucidation of these pathways is required to better understand how TTNtv lead to DCM phenotypes which will provide additional opportunities to develop therapies. Animal models are essential for understanding molecular pathways, and several have been utilized to study TTNtv including a mouse model with an A-band truncation mutation, and zebrafish models that have several different truncation variations [39–42]. A review of these important models has been recently carried out [43].

**Figure 3.** Titin truncation variations (TTNtv) lead to dilated cardiomyopathy related to haploinsufficiency and increased metabolic stress. TTNtv is likely to lead to dilated cardiomyopathy phenotype due to increased non-sense mRNA decay leading to increased metabolic stress and activation of the mammalian target of rapamycin (mTOR) complex signaling pathway. mTOR complex activation is associated with development of dilated cardiomyopathy as a downstream signaling cascade.
