In:
Circulation Research, Ovid Technologies (Wolters Kluwer Health), Vol. 119, No. suppl_1 ( 2016-07-22)
Abstract:
Background: cGMP-dependent protein kinase G I α (PKGIα) via its leucine zipper (LZ) domain prevents adverse cardiac remodeling. Identifying and characterizing PKGIα-LZ dependent substrates may reveal novel therapeutic targets in the myocardium. We previously identified the LZ-containing Mixed Lineage Kinase 3 (MLK3) as a potential PKGIα substrate. Further, MLK3 whole body knockout mice had increased LV hypertrophy and dysfunction after pressure overload. In this study we sought to further explore the PKGIα-MLK3 interaction, and to investigate MLK3 in human cardiomyopathy. Results: We first tested for a direct interaction between PKGIα and MLK3. Using affinity purified recombinant proteins we observed co-precipitation of PKGIα and MLK3 that was disrupted by mutation of the PKGIα LZ domain (LZ mutation: MLK3 binding decreased by 61.61% ±13.6, n=4). In mouse heart the interaction between native MLK3 and PKGIα was observed by co-immunoprecipitation (n=3). PKGIα phosphorylated MLK3 at the activation loop in vitro, which was attenuated by inhibiting PKGIα kinase function (n=3). We next tested if MLK3 regulates hypertrophy of cultured cardiomyocytes. Adult rat ventricular cardiomyocytes treated with the MLK3 inhibitor URMC-099 (100 nM, 48 hrs) exhibited increased cell size compared to vehicle treated cells (23.3% increase ± 3.64 SEM vs DMSO vehicle, n=3, 50 cells per treatment). Finally, we examined MLK3 expression in hearts from human patients with non-ischemic or hypertrophic cardiomyopathy. Compared to normal LV samples (NDRI, n=4), MLK3 expression was markedly elevated in both non-ischemic and hypertrophic cardiomyopathy LV samples (MLK3/GAPDH: non-ischemic: 6.26 ADU ± 0.85, n=9, hypertrophic: 6.97 ADU ± 1.42, n=8). Conclusion: These data support a model in which PKGIa directly binds and activates MLK3, leading in the cardiomyocyte to repression of cellular hypertrophy. Our findings in tissue from human failing hearts further suggest that MLK3 upregulation may act as a compensatory anti-remodeling signal in the setting of cardiac dysfunction, which ultimately becomes overwhelmed by pro-remodeling signals. More broadly our findings support that identifying PKGIa LZ-dependent substrates can reveal novel anti-remodeling molecules.
Type of Medium:
Online Resource
ISSN:
0009-7330
,
1524-4571
DOI:
10.1161/res.119.suppl_1.251
Language:
English
Publisher:
Ovid Technologies (Wolters Kluwer Health)
Publication Date:
2016
detail.hit.zdb_id:
1467838-X
