Fig 2: The cardiac benefit of SS-31 treatment is not additive to that of mCAT expression but the two interventions differentially regulate myofilament protein phosphorylation.(a) Diastolic function (Ea/Aa) improved at both 8 and 12 weeks after AAV9-mCAT administration. n = 3 (saline) and n = 5 (AAV-mCAT) female mice were analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points and Sidak post hoc analysis between treatment groups. (b) 8-week SS-31 improved diastolic function in old WT but did not further improve the function of old mCAT mice; n = 5 (for SS-WT and SS-mCAT) and n = 6 (for Saline-WT) mixed-sex mice were analyzed by repeated measure ANOVA with Tukey’s multiple comparison test between time points. (c) Late-life mCAT expression reduced Ser282 phosphorylation of MyBP-C. (d–e) Late-life mCAT expression increased phosphorylation of cTnI at Ser23/24 (d) and Ser150 (e). For panel c-e, n = 3 and 5 female mice were used for saline and AAV-mCAT, respectively, and were analyzed by unpaired T-test.

Fig 3: PKA and PKC activities, and cMyBP-C and cTnI phosphorylation of myocardial biopsies of human HF patients. (A) Protein kinase A (PKA) activity. (B) PKA monomer levels. (C) Cardiac myosin binding protein-C (cMyBP-C) levels. (D) Total cMyBP-C phosphorylation. (E) Protein kinase C (PKC) activity. (F) PKCa monomer levels. (G) Cardiac troponin I (cTnI) levels. (H) Phospho(P)-site-specific within cTnI at Ser23/24. (I) P-site-specific within cTnI at Ser42. (J) P-site-specific within cTnI at Thr 143. Data are shown as mean ± SEM. * p < 0.05/*** p < 0.001 donor vs. HF by Student t-test (n = 8–10, hearts per group).

Fig 4: cTnI phosphorylation. A, Typical example of donor and PAH samples immunostained against the unphosphorylated form of cTnI and normalized for Ponceau-S stained actin. cTnI total phosphorylation was determined by Western blot analysis in donor and PAH samples (nDon=8, nPAH=9). B, Typical example of donor and PAH samples immunostained against cTnI serine 22/23 residue and normalized for Ponceau-S stained actin. cTnI serine 22/23 phosphorylation was determined by Western blot analysis in donor and PAH samples (nDon=9, nPAH=11). C, Typical example of donor and PAH samples immunostained against unphosphorylated (P0) cTnI, mono-phosphorylated (P1) cTnI and bis-phosphorylated (P2) cTnI. cTnI relative phosphorylation distribution was determined by Phos-Tag analysis in donor and PAH samples (nDon=5, nPAH=9). Data presented as mean±SEM. cTnI indicates cardiac troponin I; cTnT, cardiac troponin T; PAH, pulmonary arterial hypertension.

Fig 5: Binding activity and selectivity of ?TnI in vitro. The ?TnI sequences (blue surface) are exposed in the C terminus unstructured region of cTnI (A), as observed in the AlphaFold predicted structure of cTnI (AF-P19429-F1) [14] shown in the green surface superimposed with troponin I in the crystal structure of the complex of troponin T (cyan surface), C (orange surface), and I (red ribbon) (PDB ID: 4Y99) [15]; they are also exposed in the C2 domain (highlighted in pink; (B)) in dPKC. (C) A scheme describing the mechanism of an inhibitor that selectively inhibits the docking and phosphorylation of cTnI by the multi-substrate kinase dPKC. ((C); top) Intramolecular interactions in dPKC are disrupted by PKC activation, exposing the catalytic site and selective substrate docking sites on dPKC. Docking of these substrates to the kinase, concomitantly or one substrate at a time, increases the access of the catalytic site for the substrates, leading to their phosphorylation (red P circles). In the inactive dPKC (left), the docking site interacts with a PKC sequence, e.g., ?TnI site, which mimics the kinase docking site on cTnI. ((C); bottom). ?TnI is a competitive inhibitor of docking and phosphorylation of cTnI by dPKC; it does not affect docking and phosphorylation of other dPKC substrates (e.g., substrate XX, yellow). (D) Binding curves of dPKC and ePKC, at 75 µg/mL (about 1 µM), to ?TnI (6 µM). The peptide selectivity binds to dPKC, and not to another novel PKC, ePKC. Binding assay with increasing amounts of dPKC to ?TnI and binding assay with increasing amounts of ePKC. (E) Calculated Koff, Kon, and Kd between ?TnI and dPKC, shown in ?M. Results are from three independent experiments.