Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes

Ji Hyun Jang; Min Jueng Kang; Gwang Pyo Ko; Sung Joon Kim; Eugene C Yi; Yin Hua Zhang

doi:10.1016/j.niox.2015.07.005

Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes

Nitric Oxide. 2015 Nov 15:50:20-27. doi: 10.1016/j.niox.2015.07.005. Epub 2015 Aug 11.

Authors

Ji Hyun Jang¹, Min Jueng Kang², Gwang Pyo Ko³, Sung Joon Kim¹, Eugene C Yi⁴, Yin Hua Zhang⁵

Affiliations

¹ Department of Physiology & Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University, College of Medicine, Seoul, Republic of Korea.
² Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Medicine and College of Pharmacy, Seoul National University, Republic of Korea.
³ Department of Environmental Health, Graduate School of Public Health, Seoul National University, Kwanak-ro 1, Kwanak-gu, Seoul, Republic of Korea.
⁴ Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, College of Medicine and College of Pharmacy, Seoul National University, Republic of Korea. Electronic address: euyi@snu.ac.kr.
⁵ Department of Physiology & Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University, College of Medicine, Seoul, Republic of Korea; Yanbian University Hospital, Yanji, Jilin Province, China; Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK. Electronic address: yinzhang@snu.ac.kr.

PMID: 26271450
DOI: 10.1016/j.niox.2015.07.005

Abstract

Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSμ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095-1289 and Santa Cruz Biotechnology aa 2-300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was ∼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSβ. RT-PCR results confirmed the expressions of both nNOSα and nNOSβ mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. ∼140 kDa), confirming nNOSβ. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS^-/- mice (which lacks nNOSα/μ). Furthermore, nNOSβ expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of ∼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.

Keywords: Cardiomyocytes; Hypertension; Myofilament; nNOS splice variants; nNOSβ.