Limits...
Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schötterl S, Goedel A, Metzger K, Brade T, Parrotta E, Schaller M, Gerull B, Thierfelder L, Aartsma-Rus A, Labeit S, Atherton JJ, McGaughran J, Harvey RP, Sinnecker D, Mann M, Laugwitz KL, Gawaz MP, Moretti A - EMBO Mol Med (2015)

Bottom Line: Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326.Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression.AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia michael.gramlich@med.uni-tuebingen.de amoretti@med1.med.tum.de.

No MeSH data available.


Related in: MedlinePlus

Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytesExperimental design.Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492817&req=5

fig03: Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytesExperimental design.Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.

Mentions: We next investigated the impact of the TTN A-band truncating mutation on sarcomere organization of iPSC-derived cardiomyocytes and evaluated the effects of reframing TTN transcripts by exon skipping in patient-specific DCM cells (Fig3A). We dissociated single cardiomyocytes from spontaneously beating foci and analyzed them 7 days later, to provide the time necessary for reorganization of the myofibrils that typically undergo disarray during the dissociation process (Atherton et al, 1986). Initial immunocytochemical analysis for titin and various proteins marking different portions of the sarcomere—α-actinin (Z-disk), cardiac troponin T (cTNT, A-band), and myosin heavy chain (MHC, M-line)—revealed, in the DCM group, a higher percentage of cardiomyocytes in which organized myofibrils occupied only half of the whole cytoplasm or less (Fig3B). Moreover, the immunofluorescence signal of Z-disk titin in striated myofibrils appeared more diffuse in patient cells (Fig3C). These results suggested that truncated titin mutants could alter assembly and/or stability of the sarcomeric units in human embryonic myocytes. To gain a better understanding of sarcomere remodeling in iPSC-derived cardiomyocytes after dissociation, we stably overexpressed actin as a fusion with a red fluorescent protein (RFP) in single cells by baculovirus technology (Supplementary Fig S7A), which has been recently successfully applied to mammalian cells (Sung et al, 2014), and performed live-cell imaging over time (Supplementary Fig S7B). This analysis showed that, similarly to neonatal cardiomyocytes (Atherton et al, 1986), there is a considerable heterogeneity in the degree of myofibril organization at day 1–2 after dissociation, but most of the cells presented striated RFP+ sarcomeres mainly around the nucleus, suggesting this as starting site of myofibril reassembly. With the time, the myofibril organization extended to the whole cell and this associated with the onset of rhythmic contraction. However, around 7–10 days of culture as single cells, few of the ‘fully organized' cardiomyocytes began to lose their sarcomeric striated pattern, starting at the perinuclear region and eventually throughout the whole cytoplasm till the cell periphery (Supplementary Fig S7B). On the basis of these observations, we hypothesized that sarcomere remodeling in iPSC-derived cardiomyocytes after dissociation is a radially occurring process that mimics the one earlier observed in neonatal cardiomyocyte (Atherton et al, 1986).


Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy.

Gramlich M, Pane LS, Zhou Q, Chen Z, Murgia M, Schötterl S, Goedel A, Metzger K, Brade T, Parrotta E, Schaller M, Gerull B, Thierfelder L, Aartsma-Rus A, Labeit S, Atherton JJ, McGaughran J, Harvey RP, Sinnecker D, Mann M, Laugwitz KL, Gawaz MP, Moretti A - EMBO Mol Med (2015)

Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytesExperimental design.Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492817&req=5

fig03: Exon skipping-based rescue of sarcomeric defects in TTN Ser14450fsX4 iPSC-derived cardiomyocytesExperimental design.Percentage of iPSC-derived cardiomyocytes with cross-striated myofibrils occupying a fraction (partially organized, dark gray) or the whole cytoplasm (fully organized, light gray) from two control and two patient clones. Statistical difference was tested using the two-sided chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236; ***P = 1.93E−16).Immunofluorescence images of titin (Z-disk portion) and cardiac troponin T (cTNT, A-band) in well-organized sarcomeres from CTR and DCM single cardiomyocytes under basal conditions. Scale bars, 5 μm.Immunofluorescence images (left) of α-actinin and cTNT in CTR and DCM single cardiomyocytes, illustrating 3 different levels of sarcomeric organization (perinuclear, fully and peripherally organized). Percentage of cells with different levels of sarcomeric organization (right) under basal and stress conditions. Statistical difference was tested using the chi-squared test (CTR1 basal: n = 283, CTR2 basal: n = 250, DCM1 basal: n = 255, DCM2 basal: n = 236, CTR1 Iso: n = 245, CTR2 Iso: n = 230; DCM1 Iso: n = 242 and DCM2 Iso: n = 269; ***P = 1.93E−16 CTR basal versus DCM basal; ***P = 9.61E−34 CTR Iso versus DCM Iso, **P = 0.001 DCM basal versus DCM Iso). No significant differences were observed comparing CTR basal and CTR Iso groups. Scale bars, 25 μm.Percentage of perinuclear, fully, and peripherally organized single cardiomyocytes from two CTR and two DCM iPSC clones after infection with the U7snRNA-ScrAONs-IRES-GFP and U7snRNA-TTNAONs-IRES-GFP lentiviruses. Statistical difference was tested using the two-sided chi-squared test (CTR1 Scr-AON: n = 190, CTR2 Scr-AON: n = 200, DCM1 Scr-AON: n = 221, DCM2 Scr-AON: n = 115, CTR1 TTN-AON: n = 223, CTR2 TTN-AON: n = 187, DCM1 TTN-AON: n = 171, DCM2 TTN-AON: n = 243; ***P = 4.22E−15 CTR Scr-AON versus DCM Scr-AON; ***P = 4.61E−02 DCM Scr-AON versus DCM TTN-AON). No significant differences were observed comparing CTR Scr-AON and CTR TTN-AON groups.
Mentions: We next investigated the impact of the TTN A-band truncating mutation on sarcomere organization of iPSC-derived cardiomyocytes and evaluated the effects of reframing TTN transcripts by exon skipping in patient-specific DCM cells (Fig3A). We dissociated single cardiomyocytes from spontaneously beating foci and analyzed them 7 days later, to provide the time necessary for reorganization of the myofibrils that typically undergo disarray during the dissociation process (Atherton et al, 1986). Initial immunocytochemical analysis for titin and various proteins marking different portions of the sarcomere—α-actinin (Z-disk), cardiac troponin T (cTNT, A-band), and myosin heavy chain (MHC, M-line)—revealed, in the DCM group, a higher percentage of cardiomyocytes in which organized myofibrils occupied only half of the whole cytoplasm or less (Fig3B). Moreover, the immunofluorescence signal of Z-disk titin in striated myofibrils appeared more diffuse in patient cells (Fig3C). These results suggested that truncated titin mutants could alter assembly and/or stability of the sarcomeric units in human embryonic myocytes. To gain a better understanding of sarcomere remodeling in iPSC-derived cardiomyocytes after dissociation, we stably overexpressed actin as a fusion with a red fluorescent protein (RFP) in single cells by baculovirus technology (Supplementary Fig S7A), which has been recently successfully applied to mammalian cells (Sung et al, 2014), and performed live-cell imaging over time (Supplementary Fig S7B). This analysis showed that, similarly to neonatal cardiomyocytes (Atherton et al, 1986), there is a considerable heterogeneity in the degree of myofibril organization at day 1–2 after dissociation, but most of the cells presented striated RFP+ sarcomeres mainly around the nucleus, suggesting this as starting site of myofibril reassembly. With the time, the myofibril organization extended to the whole cell and this associated with the onset of rhythmic contraction. However, around 7–10 days of culture as single cells, few of the ‘fully organized' cardiomyocytes began to lose their sarcomeric striated pattern, starting at the perinuclear region and eventually throughout the whole cytoplasm till the cell periphery (Supplementary Fig S7B). On the basis of these observations, we hypothesized that sarcomere remodeling in iPSC-derived cardiomyocytes after dissociation is a radially occurring process that mimics the one earlier observed in neonatal cardiomyocyte (Atherton et al, 1986).

Bottom Line: Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326.Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression.AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Cardiovascular Diseases, Eberhard Karls University, Tübingen, Germany Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia michael.gramlich@med.uni-tuebingen.de amoretti@med1.med.tum.de.

No MeSH data available.


Related in: MedlinePlus