Fig 1: 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 2: 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.
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: Myocardial injury in the trauma hemorrhage induced CD murine model. TEM shows severe myocardial injury in the heart of THS mice, with interstitial oedema, widespread disorganization of the myoc ardium with relaxation of the sarcomere and poorly circumscribed mitochondria distributed in a disorderly fashion (A). Significant amounts of mitochondrial oedema (B) Nuclei shows margination of chromatin, rarefaction of the nucleoplasm (C) and glycogen depletion (D); and some cardiomyocytes dead cells with margination of chromatin (E). TEM of sham mice heart tissue shows a well-organized myofibrils, with mitochondria evenly organized along the cristae sarcomere (F) with intact nucleus (G) and mitochondria (H). WB analysis of Troponin-I (cTnI) (I) and H-FABP (J) expression shows elevated levels in the myocardium of THS mice (p = 0.06 NS; *p = 0.04, respectively; two-tailed unpaired t-test; means ± SD). (K,L) Low caspase 8 expression is observed in heart of THS mice (IHC ***p < 0.0001; WB p > 0.05 compared to sham animals).
Fig 5: 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.
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