Obscurin regulates ankyrin macromolecular complex formation.

Obscurin is a large scaffolding protein in striated muscle that maintains sarcolemmal integrity and aligns the sarcoplasmic reticulum with the underlying contractile machinery. Ankyrins are a family of adaptor proteins with some isoforms that interact with obscurin. Previous studies have examined obscurin interacting with individual ankyrins. In this study, we demonstrate that two different ankyrins interact with obscurin's carboxyl terminus via independent ankyrin-binding domains (ABDs). Using in-vitro binding assays, co-precipitation assays, and FLIM-FRET analysis, we show that obscurin interacts with small ankyrin 1.5 (sAnk1.5) and the muscle-specific ankyrin-G isoform (AnkG107). While there is no direct interaction between sAnk1.5 and AnkG107, obscurin connects the two ankyrins both in vitro and in cells. Moreover, AnkG107 recruits ß-spectrin to this macromolecular protein complex and mutating obscurin's ABDs disrupts complex formation. To further characterize AnkG107 interaction with obscurin, we measure obscurin-binding to different AnkG107 isoforms expressed in the heart and find that the first obscurin-binding domain in AnkG107 principally mediates this interaction. We also find that AnkG107 does not bind to filamin-C and displays minimal binding to plectin-1 compared to obscurin. Finally, both sAnk1.5-GFP and AnkG107-CTD-RFP are targeted to the M-lines of ventricular cardiomyocytes and mutating their obscurin-binding domains disrupts the M-line localization of these ankyrin constructs. Altogether, these findings support a model in which obscurin can interact via independent binding domains with two different ankyrin protein complexes to target them to the sarcomeric M-line of ventricular cardiomyocytes.

Identification and characterization of self-association domains on small ankyrin 1 isoforms.

In striated muscles, the large scaffolding protein obscurin and a small SR-integral membrane protein sAnk1.5 control the retention of longitudinal SR across the sarcomere. How a complex of these proteins facilitates localization of longitudinal SR has yet to be resolved, but we hypothesize that obscurin interacts with a complex of sAnk1.5 proteins. To begin to address this hypothesis, we demonstrate that sAnk1.5 interacts with itself and identify two domains mediating self-association. Specifically, we show by co-precipitation and FLIM-FRET analysis that sAnk1.5 and another small AnkR isoform (sAnk1.6) interact with themselves and each other. We demonstrate that obscurin interacts with a complex of sAnk1.5 proteins and that this complex formation is enhanced by obscurin-binding. Using FLIM-FRET analysis, we show that obscurin interacts with sAnk1.5 alone and with sAnk1.6 in the presence of sAnk1.5. We find that sAnk1.5 self-association is disrupted by mutagenesis of residues Arg64-Arg69, residues previously associated with obscurin-binding. Molecular modeling of two interacting sAnk1.5 monomers facilitated the identification of Gly31-Val36 as an additional site of interaction, which was subsequently corroborated by co-precipitation and FLIM-FRET analysis. In closing, these results support a model in which sAnk1.5 forms large oligomers that interact with obscurin to facilitate the retention of longitudinal SR throughout skeletal and cardiac myocytes.

Loss of type 9 adenylyl cyclase triggers reduced phosphorylation of Hsp20 and diastolic dysfunction.

Adenylyl cyclase type 9 (AC9) is found tightly associated with the scaffolding protein Yotiao and the IKs ion channel in heart. But apart from potential IKs regulation, physiological roles for AC9 are unknown. We show that loss of AC9 in mice reduces less than 3% of total AC activity in heart but eliminates Yotiao-associated AC activity. AC9-/- mice exhibit no structural abnormalities but show a significant bradycardia, consistent with AC9 expression in sinoatrial node. Global changes in PKA phosphorylation patterns are not altered in AC9-/- heart, however, basal phosphorylation of heat shock protein 20 (Hsp20) is significantly decreased. Hsp20 binds AC9 in a Yotiao-independent manner and deletion of AC9 decreases Hsp20-associated AC activity in heart. In addition, expression of catalytically inactive AC9 in neonatal cardiomyocytes decreases isoproterenol-stimulated Hsp20 phosphorylation, consistent with an AC9-Hsp20 complex. Phosphorylation of Hsp20 occurs largely in ventricles and is vital for the cardioprotective effects of Hsp20. Decreased Hsp20 phosphorylation suggests a potential baseline ventricular defect for AC9-/-. Doppler echocardiography of AC9-/- displays a decrease in the early ventricular filling velocity and ventricular filling ratio (E/A), indicative of grade 1 diastolic dysfunction and emphasizing the importance of local cAMP production in the context of macromolecular complexes.

Identification and characterization of two ankyrin-B isoforms in mammalian heart.

AIMS: Excitation-contraction coupling in cardiomyocytes requires the proper targeting and retention of membrane proteins to unique domains by adaptor proteins like ankyrin-B. While ankyrin-B has been shown to interact with a variety of membrane and structural proteins located at different subcellular domains in cardiomyocytes, what regulates the specificity of ankyrin-B for particular interacting proteins remains elusive. METHODS AND RESULTS: Here, we report the identification of two novel ankyrin-B isoforms AnkB-188 and AnkB-212 in human, rat, and mouse hearts. Novel cDNAs for both isoforms were isolated by long-range PCR of reverse-transcribed mRNA isolated from human ventricular tissue. The isoforms can be discriminated based on their function and subcellular distribution in cardiomyocytes. Heterologous overexpression of AnkB-188 increases sodium-calcium exchanger (NCX) membrane expression and current, while selective knockdown of AnkB-188 in cardiomyocytes reduces NCX expression and localization in addition to causing irregular contraction rhythms. Using an isoform-specific antibody, we demonstrate that the expression of AnkB-212 is restricted to striated muscles and is localized to the M-line of cardiomyocytes by interacting with obscurin. Selective knockdown of AnkB-212 significantly attenuates the expression of endogenous ankyrin-B at the M-line but does not disrupt NCX expression at transverse tubules in cardiomyocytes. CONCLUSION: The identification and characterization of two functionally distinct ankyrin-B isoforms in heart provide compelling evidence that alternative splicing of the ANK2 gene regulates the fidelity of ankyrin-B interactions with proteins.

A PKM2 signature in the failing heart.

A salient feature of the failing heart is metabolic remodeling towards predominant glucose metabolism and activation of the fetal gene program. Sunitinib is a multitargeted receptor tyrosine kinase inhibitor used for the treatment of highly vascularized tumors. In diabetic patients, sunitinib significantly decreases blood glucose. However, a considerable proportion of sunitinib-treated patients develop cardiac dysfunction or failure. We asked whether sunitinib treatment results in shift towards glycolysis in the heart. Glucose uptake by the heart was increased fivefold in mice treated with sunitinib. Transcript analysis by qPCR revealed an induction of genes associated with glycolysis and reactivation of the fetal gene program. Additionally, we observed a shift in the enzyme pyruvate kinase from the adult M1 (PKM1) isoform to the fetal M2 (PKM2) isoform, a hallmark of the Warburg Effect. This novel observation led us to examine whether a similar shift occurs in human heart failure. Examination of tissue from patients with heart failure similarly displayed an induction of PKM2. Moreover, this phenomenon was partially reversed following mechanical unloading. We propose that pyruvate kinase isoform switching represents a novel feature of the fetal gene program in the failing heart.