Abstract
RationaleIntegrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype. However, ILK has been implicated as both a cardioprotective and oncogenic target, which imposes therapeutic constraints that are generally relevant to the translational potential of many kinases.ObjectiveTo study the cardioprotective properties of the activation-resistant, non-oncogenic, mutation of ILK (ILKR211A) against experimental MI in vivo and Doxorubicin induced apoptosis in vitro and it’s relationships to stress induced heat shock proteins.Methods/ResultsThe transgenic mouse heart over-expressing a point mutation in the ILK pleckstrin homology (PH) domain (TgR211A) exhibits a highly cardioprotective phenotype based on LAD-ligation-induced MI reduction in vivo, and on protection against doxorubicin (DOX)-induced cardiomyocyte apoptosis when overexpressed in human induced pluripotent stem cell (iPS)-derived cardiomyocytes in vitro. Intriguingly, the degree of cardioprotection seen with the ILKR211A mutation exceeded that with the ILKS343D mutation. Microarray and immunoprecipitation analyses revealed upregulation of expression levels and specific binding of ILKWT, ILKS343D and ILKR211A to both constitutively active heat-shock protein 70 (Hsc70) and inducible Hsp70 in response to MI, and to acute ILK overexpression in iPSC-cardiomyocytes. ILK-mediated cardioprotection was shown to depend upon Hsp70 ATPase activity.ConclusionsThese findings indicate that wild type ILK and the non-oncogenic ILKR211A mutation comprise a cardioprotective module with Hsp/c70. These results advance a novel target discovery theme in which kinase mutations can be safely engineered to enhance cardioprotective effects.
Highlights
Little progress has been made in the past decade on mitigating the increasingly large clinical and economic impacts of heart failure (HF)
We show that both wild type and R211A Integrin-linked kinase (ILK) mutations are client molecules of the constitutively active and stress-inducible heat-shock protein 70 (Hsc/p70), and that both ILK and heat shock protein 70 (Hsp70) are required for the broadly cardioprotective phenotype induced by ILK upregulation
Our results support a model in which protection against myocardial infarction and DOX cardiotoxicity depends upon a functional module minimally comprised by ILK (WT and R211A) and Hsp70
Summary
Little progress has been made in the past decade on mitigating the increasingly large clinical and economic impacts of heart failure (HF). Integrin-linked kinase (ILK) has been proposed as a novel molecular target that has translational potential in diverse cardiac diseases, since its upregulation promotes a broadly cardioprotective phenotype[2,3,4]. ILK activation by growth factor stimulation is normally regulated downstream of phosphatidylinositol 3-kinase (PI3K) by phosphatidylinositol (3–5)-trisphosphate (PIP3), which binds with the central pleckstrin homology (PH)-like domain of ILK[5]. ILKR211A carries a synthetic arginine (R) to alanine (A) point mutation in its PH domain which makes it deficient in membrane PIP3 binding and renders it resistant to PI3Kdependent activation[2,6], [7]. The ILKR211A mutation is of translational interest since wild type ILK, as is the case with other cytoprotective Ser/Thr kinases such as PKB/Akt[8], has been implicated as an oncogene[7,9,10,11]. To explore the cardioprotective properties of ILK, we generated and performed LAD ligation in two distinct transgenic mouse models: one using a constitutively-active serine (S) to aspartate (D) substitution in the putative autophosphorylation site of the human ILK gene (TgS343D)[12]; the other expressing the activation-resistant TgR211A variant in the myocardium[2,6,7]
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