Isoform-specific roles of protein phosphatase 1 catalytic subunits in sarcoplasmic reticulum-mediated Ca(2+) cycling.
OBJECTIVE
protein phosphatase 1 (PP1) is the major isotype of serine/threonine phosphatase in cardiomyocytes, and its activity has been thought to be important for heart failure progression. The PP1 catalytic subunits consist of three distinct genes, PP1α, PP1β/δ, and PP1γ. To date, the function of each PP1 isoform is not well characterized in cardiomyocytes. We sought to determine the functional contribution of each PP1 isoform to sarcoplasmic reticulum (SR)-mediated Ca(2+) cycling in isolated adult rat cardiomyocytes.


RESULTS
adenoviral vectors encoding short hairpin RNA for each PP1 isoform were transfected into isolated rat cardiomyocytes, and this was followed by analysis of cell shortening, Ca(2+) transients, and the phosphorylation levels of Ca(2+) regulatory proteins. Physical interactions between each PP1 isoform and SR Ca(2+) regulatory proteins were characterized in isolated cardiomyocytes expressing green fluorescent protein (GFP)-tagged PP1 catalytic subunits, and also in canine junctional and longitudinal SR preparations. Successful PP1 isoform knockdown was achieved for each isoform without affecting the expression of the other isoforms. PP1β knockdown most significantly enhanced the Ca(2+) transient and cell shortening by augmenting phospholamban (PLN) phosphorylation at baseline and with low-dose isoproterenol stimulation (10 nM). Interestingly, PP1β was preferentially associated with sarco-endoplasmic ATPase and PLN in GFP-PP1-transfected cardiomyocytes, as well as in canine longitudinal SR preparations.


CONCLUSIONS
these findings indicate that PP1β is the most significant PP1 isoform involved in regulating SR Ca(2+) cycling in rat cardiomyocytes.
Aoyama
Hidekazu
H
Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Ikeda
Yasuhiro
Y
Miyazaki
Yosuke
Y
Yoshimura
Koichi
K
Nishino
Shizuka
S
Yamamoto
Takeshi
T
Yano
Masafumi
M
Inui
Makoto
M
Aoki
Hiroki
H
Matsuzaki
Masunori
M
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004285:Dogs; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D049452:Green Fluorescent Proteins; D007527:Isoenzymes; D054645:Protein Phosphatase 1; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban