Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes.
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.
Department of Physiology & Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University, College of Medicine, Seoul, Republic of Korea.
Ji Hyun
JH
Jang
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.
Min Jueng
MJ
Kang
Department of Environmental Health, Graduate School of Public Health, Seoul National University, Kwanak-ro 1, Kwanak-gu, Seoul, Republic of Korea.
Gwang Pyo
GP
Ko
Department of Physiology & Biomedical Sciences, Ischemic/Hypoxic Disease Institute, Seoul National University, College of Medicine, Seoul, Republic of Korea.
Sung Joon
SJ
Kim
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.
Eugene C
EC
Yi
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.
Yin Hua
YH
Zhang
Cardiomyocytes; Hypertension; Myofilament; nNOS splice variants; nNOSβ