A Selective Inhibitor of Cardiac Troponin I Phosphorylation by Delta Protein Kinase C (δPKC) as a Treatment for Ischemia-Reperfusion Injury

Abstract

Myocardial infarction is the leading cause of cardiovascular mortality, with myocardial injury occurring during ischemia and subsequent reperfusion (IR). We previously showed that the inhibition of protein kinase C delta (δPKC) with a pan-inhibitor (δV1-1) mitigates myocardial injury and improves mitochondrial function in animal models of IR, and in humans with acute myocardial infarction, when treated at the time of opening of the occluded blood vessel, at reperfusion. Cardiac troponin I (cTnI), a key sarcomeric protein in cardiomyocyte contraction, is phosphorylated by δPKC during reperfusion. Here, we describe a rationally-designed, selective, high-affinity, eight amino acid peptide that inhibits cTnI's interaction with, and phosphorylation by, δPKC (ψTnI), and prevents tissue injury in a Langendorff model of myocardial infarction, ex vivo. Unexpectedly, we also found that this treatment attenuates IR-induced mitochondrial dysfunction. These data suggest that δPKC phosphorylation of cTnI is critical in IR injury, and that a cTnI/δPKC interaction inhibitor should be considered as a therapeutic target to reduce cardiac injury after myocardial infarction.

Keywords: cardiac ischemia-reperfusion injury; cardiac troponin I; peptides.

Figure 1

ψTnI sequence is highly conserved. Sequence alignment identifies a short sequence of homology between the C terminus of human cTnI and δPKC C2 domain (A) that is conserved in evolution (B,C), but is absent in other PKC isozymes (D). In (E), conservation is measured by the number of identical or similar residues compared to the human cTnI (top) and δPKC (bottom) gene. Blue regions represent the sequences corresponding to ψTnI in cTnI and δPKC, respectively.

Figure 2

Binding activity and selectivity of ψTnI in vitro. The ψTnI 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 δPKC. (C) A scheme describing the mechanism of an inhibitor that selectively inhibits the docking and phosphorylation of cTnI by the multi-substrate kinase δPKC. ((C); top) Intramolecular interactions in δPKC are disrupted by PKC activation, exposing the catalytic site and selective substrate docking sites on δPKC. 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 δPKC (left), the docking site interacts with a PKC sequence, e.g., ψTnI site, which mimics the kinase docking site on cTnI. ((C); bottom). ψTnI is a competitive inhibitor of docking and phosphorylation of cTnI by δPKC; it does not affect docking and phosphorylation of other δPKC substrates (e.g., substrate XX, yellow). (D) Binding curves of δPKC and εPKC, at 75 µg/mL (about 1 μM), to ψTnI (6 µM). The peptide selectivity binds to δPKC, and not to another novel PKC, εPKC. Binding assay with increasing amounts of δPKC to ψTnI and binding assay with increasing amounts of εPKC. (E) Calculated K_off, K_on, and K_d between ψTnI and δPKC, shown in ηM. Results are from three independent experiments.

Figure 3

ψTnI inhibits cTnI phosphorylation by δPKC. Male rat hearts were subjected to 90 min perfusion (Nor; normoxia) or I_30minR_60min in the presence of a selective pan-inhibitor of δPKC (δV1-1), control (vehicle; TAT), or in the presence of the selective inhibitor of cTnI, ψTnI. Phosphorylation was determined by Western blots. ψTnI selectively inhibits δPKC-mediated cTnI phosphorylation (A). ψTnI does not affect phosphorylation of other δPKC targets, such as dynamin-related protein 1 (Drp1), signal transducer and activator of transcription (STAT), insulin receptor substrate 1 (IRS1), and myristoylated alanine-rich c-kinase substrate (MARCKS) (B); n = 4 biological replicates (open circles) unless otherwise stated; 1µM δV1-1, TAT, or ψTnI used. Data were evaluated by one-way ANOVA with Tukey’s multiple comparisons between each treatment group and p < 0.05.

Figure 4

ψTnI prevents IR-induced cardiac damage and mitochondrial dysfunction. Treatment with ψTnI attenuates cardiac injury following IR in male rat hearts, similar to the pan-δPKC inhibitor δV1-1, while ψTnI^MU, an inactive analog of ψTnI, does not, as demonstrated by creatine kinase release (A) and infarct size (B); n = 6/condition. IR-induced mitochondrial dysfunction in male rat hearts was attenuated with ψTnI treatment at reperfusion, as evidenced by changes in rates of maximal ADP-stimulated (state III) oxygen consumption (C) and H₂O₂ production (D); n = 5 biological replicates unless otherwise stated; 1µM δV1-1, TAT, ψTnI, or ψTnI^MU used. Data were evaluated by one-way ANOVA with Tukey’s multiple comparisons between each treatment group and p < 0.05.