Fig 2: TNNT2p.K217del increases passive tension A, F pass remained significantly increased (P < 0.0001) in TNNT2p.K217del cardiomyocytes (N = 1, n = 9) compared to controls (N = 4, n = 13) after incubation with exogenous PKA. B, phos-tag gel analysis showed high phosphorylation of native troponin complex prior to exchange (NE) and incorporation of unphosphorylated recombinant protein after exchange. C, after exchange 43% of present troponin complex in controls was recombinant WT cTnI while in the TNNT2p.K217del sample this was 59% and in control exchanged with TNNT2p.K217del mutant troponin complex this was 34%. D, upon exchange with WT troponin complex, cardiomyocytes of TNNT2p.K217del (N = 1, n = 11) showed restoration of F pass compared to controls exchanged with WT troponin complex (N = 2, n = 8) while F pass was significantly increased (P = 0.001) in control cardiomyocytes exchanged with mutant TNNT2p.K217del troponin complex (N = 2, n = 7). E, after incubation with exogenous PKA, cardiomyocytes of TNNT2p.K217del exchanged with recombinant WT troponin complex (N = 1, n = 13) showed normalization of F pass compared to control cardiomyocytes exchanged with WT troponin complex (N = 2, n = 9) while F pass was significantly increased (P < 0.0001) in control cardiomyocytes exchanged with mutant TNNT2p.K217del troponin complex (N = 2, n = 7). N, number of samples; n, number of total cardiomyocytes measured.

Fig 3: Schematic representation of the troponin complexcTnT is shown in yellow, cTnI in blue and cTnC in red. The letters N and C indicate the N- and C-terminus, respectively. The upper diagram shows the troponin complex in the presence of Ca2+ while the lower diagram shows the troponin complex without Ca2+. Location of the studied mutations are indicated with stars and an arrow in the upper panel. The letter H indicates a helix structure. IR, inhibitory region.

Fig 4: Expression of troponin in troponin mutants A and B, cTnI levels measured with an antibody directed to the N-terminal of cTnI and normalized to GAPDH were decreased to 46% in the TNNI3p.98trunc (0.28) and to 40% in TNNT2p.K217del (0.24) samples compared to controls (N = 8, mean = 0.60, CI = 0.43–0.77). A, corresponding gel image showed no additional bands indicative of a truncated cTnI protein. C and D, cTnI levels were decreased to 39% in TNNI3p.98trunc (0.34) and to 51% in TNNT2p.K217del (0.44) samples compared to controls (N = 8, mean = 0.87, CI = 0.59–1.15) when normalized for a-actinin. E and F, cTnT levels normalized to a-actinin were also decreased to 64% in TNNI3p.98trunc (0.70) and to 53% in TNNT2p.K217del (0.59) samples compared to controls (N = 8, mean = 1.11, CI = 0.83–1.38). G and H, cTnC levels normalized to a-actinin were slightly decreased to 73% in TNNI3p.98trunc sample (0.66) but still within the 95% CI of controls (N = 8, mean = 0.91, CI = 0.67–1.15). TNNT2p.K217del showed normal (0.88, 97% of controls) cTnC levels. [Color figure can be viewed at wileyonlinelibrary.com]

Fig 5: Secondary disease remodelling and direct mutation effects A, phos-tag analysis showed separation of non- (0P), mono- (1P) and bis- (2P) phosphorylated cTnI. B, phosphorylation of cTnI was increased in TNNI3p.98trunc and TNNT2p.K217del samples compared to controls (N = 7) while cTnI phosphorylation in LMNAp.R331Q (N = 3) and IDCM (N = 3) was decreased compared to controls. C, Ca2+-sensitivity was normalized in IDCM cardiomyocytes (N = 5, n = 12) compared to control cardiomyocytes (N = 6, n = 14) after incubation with exogenous PKA. D, Ca2+-sensitivity was normalized in LMNAp.R331Q cardiomyocytes (N = 3, n = 7) compared to control cardiomyocytes (N = 6, n = 14) after incubation with exogenous PKA. E, after incubation with exogenous PKA, Ca2+-sensitivity in TNNI3p.98trunc cardiomyocytes (N = 1, n = 7) remained significantly increased (P < 0.01) compared to control cardiomyocytes (N = 6, n = 14). F, exchange with WT troponin complex restored cTnI levels in the TNNI3p.98trunc sample to 83% of that of controls exchanged with WT troponin complex (H). G, phos-tag gel analysis showed high phosphorylation of native troponin complex prior to exchange (NE) and incorporation of unphosphorylated recombinant protein after exchange. H, the 83% was composed of 46% recombinant troponin and 37% native troponin in the TNNI3p.98trunc sample compared with 43% recombinant troponin in the control exchanged with WT troponin complex. I, Ca2+-sensitivity and LDA were restored in TNNI3p.98trunc cardiomyocytes (N = 1, n = 9) compared to control (N = 2, n = 11) after exchange with WT troponin complex and incubation with exogenous PKA. N, number of samples; n, number of total cardiomyocytes measured.