Abstract 885 Truncated titin-protein species due to truncating mutations in the TTN gene are expressed in human DCM hearts and can be modulated by manipulating the cellular protein quality control systems

Background. Among patients diagnosed with dilated cardiomyopathy (DCM), ~20% carry a heterozygous truncating mutation in TTN (TTNtv). Whereas some TTNtv do not cause DCM, others lead to mild or severe disease manifestation. The chance to get DCM from TTNtv depends on the position of the mutation in TTN. Evidence for nonsense-mediated decay of TTNtv mRNA is sparse; ribosome profiling showed that TTNtv mRNA is actively translated in cardiomyocytes. However, previous biochemical tests of TTNtv-DCM heart tissues or cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) differentiated from TTNtv-DCM patients largely failed to detect TTNtv-protein species.

Objective. To elucidate whether TTNtv protein species can be detected in LV samples of >20 different endstage failing DCM patients with a heterozygous TTNtv. A second goal was to test whether the ubiquitin-proteasome and autophagy-lysosome systems contribute to the elimination of TTNtv protein species in DCM patient-derived hiPSC-CMs with a TTNtv.

Methods & Results. Deep sequencing confirmed the presence of TTNtv in I-, A- or M-band titin in 22 out of 112 endstage failing hearts; most TTNtv were in A-band titin. By immunoblot, we quantified the expression of full-length titin (N2BA and N2B isoforms), titin-degradation products (T2) and Cronos, which lacks the titin N-terminus and is driven by an alternative promoter, using antibodies to both titin and Cronos. Truncated titin protein was occasionally observed at low levels in TTNtv hearts using antibodies to titin at the I/A-band junction, notably for truncations in the largest TTN exon 327. Interestingly, Cronos antibody consistently detected truncated Cronos protein in samples carrying a TTNtv in A-band titin. The highest tv-Cronos levels were found in hearts with a truncation in TTN exon 327 (tv:WT Cronos, 1:1); truncations in other A-band exons revealed lower and variable tv:WT Cronos ratios. To test whether this heterogeneity in TTNtv protein levels could be due to variability in protein quality control activities, we cultured hiPSC-CMs derived from healthy humans (WT) and a DCM patient with TTNtv in exon 327, for up to 140 days. Western blot detected fetal N2BA, N2B, T2 and Cronos in all cell lines, and additionally tv-titin and tv-Cronos in TTNtv hiPSC-CMs. Cells were treated with either blockers of proteolytic activity of the 26S proteasome (MG132 and bortezomib) or late-stage autophagy inhibitor (bafilomycin A1). In WT hiPSC-CMs, expression of N2BA+N2B titin increased by 9.0±7.5% with MG132-treatment and by 13.0±9.9% with bafilomycin-treatment, relative to vehicle-treated controls (n=10). In TTNtv hiPSC-CMs, proteasome-blockade caused accumulation of TTNtv species, which increased by 11.8±3.5% (MG132), 17.6±6.6% (MG132 combined with bafilomycin) and 25.2±9.8% (bortezomib) relative to controls (n=5). The ratio between Cronos and tv-Cronos protein remained unaffected by treatment.

Conclusions. Our data show the regular presence of truncated “poison peptide” in TTNtv hearts. Findings suggest that the expression levels of truncated Cronos (and TTNtv?) protein depend on the position of the truncating mutation within the TTN gene. Our results also demonstrate that a variable activity of the protein quality control machinery may contribute to the variability in poison peptide expression levels in TTNtv DCM patient hearts. Collectively, these observations can explain the phenotypic variety of TTNtv DCM patients.

Clin Res Cardiol 108, Suppl 1, April 2019
Truncated titin-protein species due to truncating mutations in the TTN gene are expressed in human DCM hearts and can be modulated by manipulating the cellular protein quality control systems
A. Fomin¹, A. Gärtner-Rommel², L. Cyganek³, M. von Frieling-Salewsky⁴, A. Goedel⁵, A. Moretti⁵, H. Milting⁶, W. A. Linke⁴
¹Kardiologie, Universitätsmedizin Göttingen, Göttingen; ²Klinik für Thorax- und Kardiovaskularchirurgie, Herz- und Diabeteszentrum NRW, Bad Oeynhausen; ³Herzzentrum Göttingen - Stem Cell Unit, Universitätsmedizin Göttingen, Göttingen; ⁴Institut für Physiologie II, Universitätsklinikum Münster, Münster; ⁵Klinik und Poliklinik für Innere Medizin I, Kardiologie, Klinikum rechts der Isar, München; ⁶E.& H. Klessmann-Institut f. kardiovask. Forschung, Herz- und Diabeteszentrum NRW, Bad Oeynhausen;
Background. Among patients diagnosed with dilated cardiomyopathy (DCM), ~20% carry a heterozygous truncating mutation in TTN (TTNtv). Whereas some TTNtv do not cause DCM, others lead to mild or severe disease manifestation. The chance to get DCM from TTNtv depends on the position of the mutation in TTN. Evidence for nonsense-mediated decay of TTNtv mRNA is sparse; ribosome profiling showed that TTNtv mRNA is actively translated in cardiomyocytes. However, previous biochemical tests of TTNtv-DCM heart tissues or cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) differentiated from TTNtv-DCM patients largely failed to detect TTNtv-protein species. Objective. To elucidate whether TTNtv protein species can be detected in LV samples of >20 different endstage failing DCM patients with a heterozygous TTNtv. A second goal was to test whether the ubiquitin-proteasome and autophagy-lysosome systems contribute to the elimination of TTNtv protein species in DCM patient-derived hiPSC-CMs with a TTNtv. Methods & Results. Deep sequencing confirmed the presence of TTNtv in I-, A- or M-band titin in 22 out of 112 endstage failing hearts; most TTNtv were in A-band titin. By immunoblot, we quantified the expression of full-length titin (N2BA and N2B isoforms), titin-degradation products (T2) and Cronos, which lacks the titin N-terminus and is driven by an alternative promoter, using antibodies to both titin and Cronos. Truncated titin protein was occasionally observed at low levels in TTNtv hearts using antibodies to titin at the I/A-band junction, notably for truncations in the largest TTN exon 327. Interestingly, Cronos antibody consistently detected truncated Cronos protein in samples carrying a TTNtv in A-band titin. The highest tv-Cronos levels were found in hearts with a truncation in TTN exon 327 (tv:WT Cronos, 1:1); truncations in other A-band exons revealed lower and variable tv:WT Cronos ratios. To test whether this heterogeneity in TTNtv protein levels could be due to variability in protein quality control activities, we cultured hiPSC-CMs derived from healthy humans (WT) and a DCM patient with TTNtv in exon 327, for up to 140 days. Western blot detected fetal N2BA, N2B, T2 and Cronos in all cell lines, and additionally tv-titin and tv-Cronos in TTNtv hiPSC-CMs. Cells were treated with either blockers of proteolytic activity of the 26S proteasome (MG132 and bortezomib) or late-stage autophagy inhibitor (bafilomycin A1). In WT hiPSC-CMs, expression of N2BA+N2B titin increased by 9.0±7.5% with MG132-treatment and by 13.0±9.9% with bafilomycin-treatment, relative to vehicle-treated controls (n=10). In TTNtv hiPSC-CMs, proteasome-blockade caused accumulation of TTNtv species, which increased by 11.8±3.5% (MG132), 17.6±6.6% (MG132 combined with bafilomycin) and 25.2±9.8% (bortezomib) relative to controls (n=5). The ratio between Cronos and tv-Cronos protein remained unaffected by treatment. Conclusions. Our data show the regular presence of truncated “poison peptide” in TTNtv hearts. Findings suggest that the expression levels of truncated Cronos (and TTNtv?) protein depend on the position of the truncating mutation within the TTN gene. Our results also demonstrate that a variable activity of the protein quality control machinery may contribute to the variability in poison peptide expression levels in TTNtv DCM patient hearts. Collectively, these observations can explain the phenotypic variety of TTNtv DCM patients.
https://www.abstractserver.com/dgk2019/jt/abstracts//V1180.htm