Background:
The most common genetic cause of dilated cardiomyopathy (DCM) is a heterozygous truncation in TTN, which codes for the cardiomyocyte protein titin. The pathogenicity of a TTN truncating variant (TTNtv) is highest for mutations in the constitutively expressed, myosin-binding, A-band titin region. I-band TTNtv are less frequent in TTN-truncation cardiomyopathy, which is explained, in part, by the extensive alternative splicing of the I-band titin segment. An alternative proposition for the lower prevalence of I-band versus A-band TTNtv in DCM is the up-regulation of a C-terminal titin isoform called Cronos, which spans across half A-bands. Cronos expression is driven by an alternative promoter in the intron upstream of exon 240 (human) or 223 (mouse). If the TTNtv is located in the I-band N-terminal to the Cronos promoter, a milder disease phenotype may occur, because Cronos could be upregulated and partially compensate for the dysfunctional (truncated) full-length titin.

Objective:
To study the role of Cronos in the pathomechanisms of TTNtv-DCM, as well as in heart development and hypertrophy.

Methods & Results:
We quantified Cronos protein expression by titin gel electrophoresis of left ventricular tissue samples from 14 nonfailing donor hearts and 113 DCM patients with end-stage heart failure, of which 22 patients had a TTNtv as detected by next-generation sequencing. Among patients with a TTNtv, we distinguished between mutations in the I-band (pre-Cronos, N=7) and the A-band titin region (post-Cronos, N=15). On Coomassie-stained titin gels, Cronos protein represented, on average, ~12% of the combined titin species (N2BA+N2B+T2+Cronos) in both patients (N=91 without and N=22 with a TTNtv) and donor hearts (N=14). No differences between these groups were also found regarding the proportion of Cronos transcript, quantified by real-time qRT-PCR. Similarly, there were no differences in relative Cronos transcript and Cronos protein expression in patients with pre-Cronos versus post-Cronos TTNtv. To elucidate the role of Cronos in development, we quantified Cronos protein at different stages of mouse heart development, using anti-Cronos immunoblot. Normalized Cronos expression was approximately two times higher in embryonic compared to neonatal mouse heart tissue, and was lowest in adult hearts. A similar difference in Cronos protein expression was apparent when comparing embryonic and adult human heart tissue samples, suggesting Cronos is highly expressed during heart development but declines in adult hearts. Surprisingly, transverse aortic constriction (TAC) surgery in mice did not promote re-expression of Cronos in hypertrophied hearts but suppressed Cronos expression at both transcript and protein levels. The downregulation of Cronos began 1 week after TAC surgery and fully manifested after 4-8 weeks.

Conclusions:
Our study shows that alterations in Cronos expression are unlikely to be part of the pathomechanisms of TTNtv-DCM. However, Cronos appears to be particularly relevant in physiological heart hypertrophy during development, probably supporting sarcomerogenesis, whereas it becomes downregulated in pathophysiological hypertrophy.