Key Words: plakophilin-2 Ⅲ intercalated disc Ⅲ arrhythmogenic right ventricular cardiomyopathy Ⅲ cardiac desmosomes A high-resolution image of the site of end-end contact between cardiomyocytes reveals an electron-dense organization called "the intercalated disc." Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the intercalated disc. Among them is Na V 1.5, the major ␣ subunit of the cardiac sodium channel. 1 Here, we ask whether Na v 1.5 and the desmosomal protein plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a functional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte.

Rationale The early description of the intercalated disc defined three structures, all of them involved in cell-cell communication: desmosomes, gap junctions and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells, also populate the intercalated disc. Key among them is the voltage-gated sodium channel (VGSC) complex. An important component of this complex is the cytoskeletal adaptor protein ankyrin-G (AnkG). Objective To test the hypothesis that AnkG partners with desmosome and gap junction molecules, and exerts a functional effect on intercellular communication in the heart. Methods and Results We utilized a combination of microscopy, immunochemistry, patch clamp and optical mapping to assess the interactions between AnkG, plakophilin-2 (PKP2) and Connexin43 (Cx43). Co-immunoprecipitation studies from rat heart lysate demonstrated associations between the three molecules. Using siRNA technology we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Cx43, as well as a decrease in intercellular adhesion strength and electrical coupling. Regulation of AnkG and of Nav1.5 by PKP2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed. Conclusions These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc, at the intersection of three complexes often considered independent: the VGSC, gap junctions and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy; ARVC) are discussed.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure.

Rationale GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Non-technical summary The heartbeat depends on the spread of electrical waves through the cardiac muscle. If that spread becomes disturbed, arrhythmias and death may ensue. Patients with heart disease are predisposed to cardiac arrhythmias by unidentified mechanisms. Using both experiments and computer models we demonstrate that structural defects in the heart leading to contiguous areas of physical narrowing and expansion of the musculature can alter the spread of the waves, in such a way that some waves may return abnormally along the same narrow pathway as the original electrical wave (reflection), leading to extra beats and arrhythmia initiation. The possibility of reflection is enhanced when structural defects combine with alterations in the sodium channels responsible for the electrical waves, such as seen in inherited and acquired cardiac electrical diseases. Our results provide a novel mechanism whereby a substrate (structural heterogeneity) and a trigger (abnormal sodium channel activity) combine to promote life-threatening arrhythmia initiation.Abstract Patients with structural heart disease are predisposed to arrhythmias by incompletely understood mechanisms. We hypothesized that tissue expansions promote source-to-sink mismatch leading to early after-depolarizations (EADs) and reflection of impulses in monolayers of well-polarized neonatal rat ventricular cardiomyocytes. We traced electrical propagation optically in patterned monolayers consisting of two wide regions connected by a thin isthmus. Structural heterogeneities provided a substrate for EADs, retrograde propagation along the same pathway (reflection) and reentry initiation. Reflection always originated during the action potential (AP) plateau at the distal expansion. To determine whether increased sodium current (I Na ) would promote EADs, we employed adenoviral transfer of Nav1.5 (Ad-Nav1.5). Compared with uninfected and adenoviral expression of green fluorescent protein (Ad-GFP; viral control), Ad-Nav1.5 significantly increased Nav1.5 protein expression, peak and persistent I Na density, AP upstroke velocity, AP duration, conduction velocity and EAD incidence, as well as reflection incidence (29.2%, n = 48 vs. uninfected, 9.4%, n = 64; and Ad-GFP, 4.8%, n = 21). Likewise, the persistent I Na agonist veratridine (0.05-3 μM) prolonged the AP, leading to EADs and reflection. Reflection led to functional reentry distally and bigeminal and trigeminal rhythms proximally. Reflection was rare in the absence of structural heterogeneities. Computer simulations demonstrated the importance of persistent I Na in triggering reflection and predicted that the gradient between the depolarizing cells at the distal expansion and the repolarizing cells within the isthmus enabled retrograde flow of depolarizing electrotonic current to trigger EADs and D. S. Auerbach and K. R. Grz eda contributed equally to this work. reflection. A combination of a substrate (structural heterogeneity) and a trigger (increased persistent I Na and EADs) promotes reflection and a...

Increased abundance of GRK2 [G protein–coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.