Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

New aspects in cardiac L-type Ca2+ channel regulation.

Cardiac excitation-contraction coupling is initiated with the influx of Ca2+ ions across the plasma membrane through voltage-gated L-type calcium channels. This process is tightly regulated by modulation of the channel open probability and channel localization. Protein kinase A (PKA) is found in close association with the channel and is one of the main regulators of its function. Whether this kinase is modulating the channel open probability by phosphorylation of key residues or via alternative mechanisms is unclear. This review summarizes recent findings regarding the PKA-mediated channel modulation and will highlight recently discovered regulatory mechanisms that are independent of PKA activity and involve protein-protein interactions and channel localization.

Disruptors of AKAP-Dependent Protein-Protein Interactions.

A-kinase anchoring proteins (AKAPs) are a family of multivalent scaffolding proteins. They engage in direct protein-protein interactions with protein kinases, kinase substrates and further signaling molecules. Each AKAP interacts with a specific set of protein interaction partners and such sets can vary between different cellular compartments and cells. Thus, AKAPs can coordinate signal transduction processes spatially and temporally in defined cellular environments. AKAP-dependent protein-protein interactions are involved in a plethora of physiological processes, including processes in the cardiovascular, nervous, and immune system. Dysregulation of AKAPs and their interactions is associated with or causes widespread diseases, for example, cardiac diseases such as heart failure. However, there are profound shortcomings in understanding functions of specific AKAP-dependent protein-protein interactions. In part, this is due to the lack of agents for specifically targeting defined protein-protein interactions. Peptidic and non-peptidic inhibitors are invaluable molecular tools for elucidating the functions of AKAP-dependent protein-protein interactions. In addition, such interaction disruptors may pave the way to new concepts for the treatment of diseases where AKAP-dependent protein-protein interactions constitute potential drug targets.Here we describe screening approaches for the identification of small molecule disruptors of AKAP-dependent protein-protein interactions. Examples include interactions of AKAP18 and protein kinase A (PKA) and of AKAP-Lbc and RhoA. We discuss a homogenous time-resolved fluorescence (HTRF) and an AlphaScreen® assay for small molecule library screening and human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) as a cell system for the characterization of identified hits.

Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP.

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

Equilibrium Bird Species Diversity in Atlantic Islands.

Half a century ago, MacArthur and Wilson proposed that the number of species on islands tends toward a dynamic equilibrium diversity around which species richness fluctuates [1]. The current prevailing view in island biogeography accepts the fundamentals of MacArthur and Wilson's theory [2] but questions whether their prediction of equilibrium can be fulfilled over evolutionary timescales, given the unpredictable and ever-changing nature of island geological and biotic features [3-7]. Here we conduct a complete molecular phylogenetic survey of the terrestrial bird species from four oceanic archipelagos that make up the diverse Macaronesian bioregion-the Azores, the Canary Islands, Cape Verde, and Madeira [8, 9]. We estimate the times at which birds colonized and speciated in the four archipelagos, including many previously unsampled endemic and non-endemic taxa and their closest continental relatives. We develop and fit a new multi-archipelago dynamic stochastic model to these data, explicitly incorporating information from 91 taxa, both extant and extinct. Remarkably, we find that all four archipelagos have independently achieved and maintained a dynamic equilibrium over millions of years. Biogeographical rates are homogeneous across archipelagos, except for the Canary Islands, which exhibit higher speciation and colonization. Our finding that the avian communities of the four Macaronesian archipelagos display an equilibrium diversity pattern indicates that a diversity plateau may be rapidly achieved on islands where rates of in situ radiation are low and extinction is high. This study reveals that equilibrium processes may be more prevalent than recently proposed, supporting MacArthur and Wilson's 50-year-old theory.