G-protein-coupled receptor (GPCR) agonists are well-known inducers of cardiac hypertrophy. We found that the shedding of heparin-binding epidermal growth factor (HB-EGF) resulting from metalloproteinase activation and subsequent transactivation of the epidermal growth factor receptor occurred when cardiomyocytes were stimulated by GPCR agonists, leading to cardiac hypertrophy. A new inhibitor of HB-EGF shedding, KB-R7785, blocked this signaling. We cloned a disintegrin and metalloprotease 12 (ADAM12) as a specific enzyme to shed HB-EGF in the heart and found that dominant-negative expression of ADAM12 abrogated this signaling. KB-R7785 bound directly to ADAM12, suggesting that inhibition of ADAM12 blocked the shedding of HB-EGF. In mice with cardiac hypertrophy, KB-R7785 inhibited the shedding of HB-EGF and attenuated hypertrophic changes. These data suggest that shedding of HB-EGF by ADAM12 plays an important role in cardiac hypertrophy, and that inhibition of HB-EGF shedding could be a potent therapeutic strategy for cardiac hypertrophy.

Phospholamban (PLN), a homopentameric, integral membrane protein, reversibly inhibits cardiac sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA2a) activity through intramembrane interactions. Here, alaninescanning mutagenesis of the PLN transmembrane sequence was used to identify two functional domains on opposite faces of the transmembrane helix. Mutations in one face diminish inhibitory interactions with transmembrane sequences of SERCA2a, but have relatively little effect on the pentameric state, while mutations in the other face activate inhibitory interactions and enhance monomer formation. Double mutants are monomeric, but loss of inhibitory function is dominant over activation of inhibitory function. These observations support the proposal that the SERCA2a interaction site lies on the helical face which is not involved in pentamer formation. Four highly inhibitory mutants are effectively devoid of pentamer, suggesting that pentameric PLN represents a less active or inactive reservoir that dissociates to provide inhibitory monomeric PLN subunits. A model is presented in which the degree of PLN inhibition of SERCA2a activity is ultimately determined by the concentration of the inhibited PLN monomer⅐SERCA2a heterodimeric complex. The concentration of this inhibited complex is determined by the dissociation constant for the PLN pentamer (which is mutation-sensitive) and by the dissociation constant for the PLN/SERCA2a heterodimer (which is likely to be mutation-sensitive). Phospholamban (PLN)1 is a 52-amino acid, integral membrane protein (1) that interacts with and reversibly inhibits the activity of the cardiac sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA2a). In this role, it is a major regulator of the kinetics of cardiac contractility (2). PLN has the mobility of a homopentamer in SDS gels, but the pentamer is dissociated to monomers by boiling in SDS (3). It is an open question whether the functional inhibitory unit is a pentamer or a monomer and whether pentamers and monomers are in dynamic equilibrium in the sarcoplasmic reticulum membrane.Much attention has been directed toward the phosphorylation sites on Ser 16 and Thr 17 in cytoplasmic domain Ia of PLN and their role in regulating the inhibitory function of PLN (4,5). In earlier studies we showed that the PLN cytoplasmic interaction site is formed by charged and hydrophobic amino acids 1-20 (6), while the complementary SERCA2a interaction site consists of amino acids Lys-Asp-Asp-Lys-Pro-Val 402 (7). In a later evaluation of potential transmembrane interaction sites (8), we coexpressed SERCA2a with PLN transmembrane sequences 31-52 (PLN domain II) or with PLN domain II constructs to which NH 2 -terminal, cytoplasmic epitopes such as PLN 1-20 or hemagglutinin were fused. We found that the inhibitory interaction site lies entirely in the transmembrane sequences of PLN and SERCA2a, but can be modulated, through long range interactions, by the noninhibitory cytoplasmic interaction site. We also discovered the phenomenon of "supershifting," in which the apparen...

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and ␣-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitopetagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with ␣-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the Nterminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and ␣-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.Gap junctions are aggregates of channels at cell-cell interfaces (1-3). Each channel is formed through the docking of two hemichannels located in opposing cell membranes, and each hemichannel is composed of a connexin homohexamer. By permitting the direct exchange of ions and small molecules between cells, these channels play a major role in a wide variety of cellular processes, including embryogenesis, cellular differentiation and development, and electrical coupling. In heart, gap junctions are a prominent feature of intercalated discs, which connect myocytes in an end-to-end orientation; the coupling provided by gap junctions serves to synchronize the activity of cells, thus providing an isochronous front for the wave of excitation that sweeps through ventricular muscle (4, 5).How gap junctions are localized at the intercalated discs in cardiac myocytes is unknown, but a component of gap junctions, connexin-43, may interact with specific elements of the cytoskeleton that restrict its diffusion in the plane of the membrane. Recent studies indicate that a number of membrane proteins are anchored by cytoskeletal elements such as PSD-95/SAP90 (6), ankyrin (7), and ␣-spectrin (8). ZO-1, which has been identified at vertebrate tight junctions (9, 10), is thought to play a role in tissue compartmentalization and in maintaining the apical-basolateral polarity of epithelial cells. In cardiac myocytes, ZO-1 appears at the intercalated discs in the i...

The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.

The infarct-limiting effect of ischemic preconditioning is believed to be a transient phenomenon. We examined the delayed effects of repetitive brief ischemia on limiting infarct size in an open-chest dog model by an occlusion (90 minutes) of the left anterior descending coronary artery (LAD) followed by reperfusion (5 hours). The dogs were preconditioned with four brief repeated ischemic episodes induced by 5-minute LAD occlusions with subsequent reperfusion. The size of infarcts initiated by a sustained occlusion immediately or 24 hours after preconditioning was significantly smaller when compared with infarcts in sham-operated dogs (for the immediate occlusion, 14.4 +/- 2.0% versus 39.0 +/- 3.7%, respectively [p < 0.01]; and for the delayed occlusion, 18.8 +/- 3.4% versus 35.1 +/- 4.6%, respectively [p < 0.05]); however, when the infarction was induced 3 hours (31.2 +/- 3.7% versus 37.5 +/- 4.2%, respectively) or 12 hours (25.4 +/- 4.8% versus 35.0 +/- 5.3%, respectively) after repetitive ischemia, the infarct size did not differ. No differences were seen in regional myocardial blood flow or rate-pressure products between the two groups. These results indicate that an infarct-limiting effect of brief repeated ischemia can be observed 24 hours after sublethal preconditioning.