Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction with Actin

Teruhiro Sakaguchi, Mikito Takefuji, Nina Wettschureck, Tomonari Hamaguchi, Mutsuki Amano, Katsuhiro Kato, Takuma Tsuda, Shunsuke Eguchi, Sohta Ishihama, Yu Mori, Yoshimitsu Yura, Tatsuya Yoshida, Kazumasa Unno, Takahiro Okumura, Hideki Ishii, Yuuki Shimizu, Yasuko K. Bando, Koji Ohashi, Noriyuki Ouchi, Atsushi EnomotoStefan Offermanns, Kozo Kaibuchi, Toyoaki Murohara

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Abstract

Background: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. Methods: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. Results: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. Conclusions: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

Original languageEnglish
Pages (from-to)1737-1752
Number of pages16
JournalCirculation
Volume140
Issue number21
DOIs
Publication statusPublished - 19-11-2019

All Science Journal Classification (ASJC) codes

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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    Sakaguchi, T., Takefuji, M., Wettschureck, N., Hamaguchi, T., Amano, M., Kato, K., Tsuda, T., Eguchi, S., Ishihama, S., Mori, Y., Yura, Y., Yoshida, T., Unno, K., Okumura, T., Ishii, H., Shimizu, Y., Bando, Y. K., Ohashi, K., Ouchi, N., ... Murohara, T. (2019). Protein Kinase N Promotes Stress-Induced Cardiac Dysfunction Through Phosphorylation of Myocardin-Related Transcription Factor A and Disruption of Its Interaction with Actin. Circulation, 140(21), 1737-1752. https://doi.org/10.1161/CIRCULATIONAHA.119.041019