Abstract
BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.
Highlights
Transverse tubules (TTs) are membranous invaginations primarily associated with mammalian ventricular myocytes
These observations demonstrate that all four proteins are present exclusively in the TTs, raising the possibility that an important relationship exists between Ca1.2, cardiac BIN1 (cBIN1), PIP2, and PLCβ 1
Cardiac TT remodeling and associated dysfunction of Ca2+ release have been extensively reported in heart failure (HF) (Balijepalli et al, 2003; Louch et al, 2004, 2012; Song et al, 2006), the processes underlying the formation and maintenance of TTs in general are not known, we do know that BIN1 seems to be a critical component of TT assembly
Summary
Transverse tubules (TTs) are membranous invaginations primarily associated with mammalian ventricular myocytes. These inward projections of the external sarcolemmal membrane bring voltage-gated calcium channels on the surface membrane into proximity with the sarcoplasmic reticulum (SR), the main intracellular Ca2+ store within the myocyte. Work from several investigators has demonstrated that the loss of TTs may be responsible for the development of diastolic and systolic dysfunction (Song et al, 2006; Wei et al, 2010; Shah et al, 2014) making it of far greater importance to understand the mechanisms of normal TT formation as well as of TT remodeling during HF. BIN1 knockdown produces a cardiomyopathy (Muller et al, 2003), making this protein a potential mediator for TT remodeling during the development of HF
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