Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes.

Serine/threonine protein phosphatase 5 (PP5) is ubiquitously expressed in eukaryotic cells; however, its function in cardiomyocytes is unknown. Under basal conditions, PP5 is autoinhibited, but enzymatic activity rises upon binding of specific factors, such as the chaperone Hsp90. Here we show that PP5 binds and dephosphorylates the elastic N2B-unique sequence (N2Bus) of titin in cardiomyocytes. Using various binding and phosphorylation tests, cell-culture manipulation, and transgenic mouse hearts, we demonstrate that PP5 associates with N2Bus in vitro and in sarcomeres and is antagonistic to several protein kinases, which phosphorylate N2Bus and lower titin-based passive tension. PP5 is pathologically elevated and likely contributes to hypo-phosphorylation of N2Bus in failing human hearts. Furthermore, Hsp90-activated PP5 interacts with components of a sarcomeric, N2Bus-associated, mechanosensor complex, and blocks mitogen-activated protein-kinase signaling in this complex. Our work establishes PP5 as a compartmentalized, well-controlled phosphatase in cardiomyocytes, which regulates titin properties and kinase signaling at the myofilaments

Sex-specific cardiac remodeling in early and advanced stages of hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most frequent genetic cardiac disease with a prevalence of 1:500 to 1:200. While most patients show obstructive HCM and a relatively stable clinical phenotype (stage II), a small group of patients progresses to end-stage HCM (stage IV) within a relatively brie

Analysis of turbulent transport properties in the near field of an isothermal free jet.

Hypertrophic cardiomyopathy (HCM) is the most frequent genetic cardiac disease with a prevalence of 1:500 to 1:200. While most patients show obstructive HCM and a relatively stable clinical phenotype (stage II), a small group of patients progresses to end-stage HCM (stage IV) within a relatively brief period. Previous research has shown sex-differences in stage II HCM with more diastolic dysfunction in female than in male patients. Moreover, female patients more often show progression to heart failure. Here we investigated if differences in functional and structural properties of the heart may underlie sex-differences in disease progression from stage II to stage IV HCM. Cardiac tissue from stage II and IV patients was obtained during myectomy (n = 54) and heart transplantation (n = 10), respectively. Isometric force was measured in membrane-permeabilized cardiomyocytes to define active and passive myofilament force development. Titin isoform composition was assessed using gel electrophoresis, and the amount of fibrosis and capillary density were determined with histology. In accordance with disease stage-dependent adverse cardiac remodeling end-stage patients showed a thinner interventricular septal wall and larger left ventricular and atrial diameters compared to stage II patients. Cardiomyocyte contractile properties and fibrosis were comparable between stage II and IV, while capillary density was significantly lower in stage IV compared to stage II. Women showed more adverse cellular remodeling compared to men at stage II, evident from more compliant titin, more fibrosis and lower capillary density. However, the disease stage-dependent reduction in capillary density was largest in men. In conclusion, the more severe cellular remodeling in female compared to male stage II patients suggests a more advanced disease stage at the time of myectomy in women. Changes in cardiomyocyte contractile properties do not explain the progression of stage II to stage IV, while reduced capillary density may underlie disease progression to end-stage heart failure

Cofilin and DNase I affect the conformation of the small domain of actin.

Cofilin binding induces an allosteric conformational change in subdomain 2 of actin, reducing the distance between probes attached to Gln-41 (subdomain 2) and Cys-374 (subdomain 1) from 34.4 to 31.4 A (pH 6.8) as demonstrated by fluorescence energy transfer spectroscopy. This effect was slightly less pronounced at pH 8.0. In contrast, binding of DNase I increased this distance (35.5 A), a change that was not pH-sensitive. Although DNase I-induced changes in the distance along the small domain of actin were modest, a significantly larger change (38.2 A) was observed when the ternary complex of cofilin-actin-DNase I was formed. Saturation binding of cofilin prevents pyrene fluorescence enhancement normally associated with actin polymerization. Changes in the emission and excitation spectra of pyrene-F actin in the presence of cofilin indicate that subdomain 1 (near Cys-374) assumes a G-like conformation. Thus, the enhancement of pyrene fluorescence does not correspond to the extent of actin polymerization in the presence of cofilin. The structural changes in G and F actin induced by these actin-binding proteins may be important for understanding the mechanism regulating the G-actin pool in cells

Thymosin β(4) Induces a Conformational Change in Actin Monomers

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin β(4) (tβ(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tβ(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 Å and decreases the distance between the purine base of bound ATP (ɛATP) and Lys-61 by 1.9 Å, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tβ(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tβ(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tβ(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tβ(4) and cofilin compete for actin binding. Tβ(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tβ(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tβ(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding

Mapping the phosphoinositide-binding site on chick cofilin explains how PIP2 regulates the cofilin-actin interaction

Cofilin plays a key role in the choreography of actin dynamics via its ability to sever actin filaments and increase the rate of monomer dissociation from pointed ends. The exact manner by which phosphoinositides bind to cofilin and inhibit its interaction with actin has proven difficult to ascertain. We determined the structure of chick cofilin and used NMR chemical shift mapping and structure-directed mutagenesis to unambiguously locate its recognition site for phosphoinositides (Pis). This structurally unique recognition site requires both the acyl chain and head group of the PI for a productive interaction, and it is not inhibited by phosphorylation of cofilin. We propose that the interaction of cofilin with membrane-bound Pis abrogates its binding to both actin and actin-interacting protein 1, and facilitates spatiotemporal regulation of cofilin activity

Contribution of Impaired Parasympathetic Activity to Right Ventricular Dysfunction and Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension

BACKGROUND: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). METHODS: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (alpha-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. RESULTS: Patients with PAH with lower RV ejection fraction (<41%) had a significantly reduced heart rate recovery in comparison with patients with higher RV ejection fraction. In PAH RV samples, alpha-7 nicotinic acetylcholine receptor was increased and acetylcholinesterase activity was reduced versus controls. No difference in muscarinic acetylcholine type 2 receptor expression was observed. Chronic PYR treatment in PH rats normalized the cardiovascular autonomic function, demonstrated by an increase in parasympathetic activity and baroreflex sensitivity. PYR improved survival, increased RV contractility, and reduced RV stiffness, RV hypertrophy, RV fibrosis, RV inflammation, and RV alpha-7 nicotinic acetylcholine receptor and muscarinic acetylcholine type 2 receptor expression, as well. Furthermore, PYR reduced pulmonary vascular resistance, RV afterload, and pulmonary vascular remodeling, which was associated with reduced local and systemic inflammation. CONCLUSIONS: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.Science Without Borders grant, Conselho Nacional de Desenvolvimento Cientifico e TecnologicoVICI grant from the Netherlands Organization for Scientific Research (NWO)Sao Paulo Research FoundationNetherlands CardioVascular Research Initiative grantVENI grant from NWOL'Oreal/UNESCO for Women in ScienceNetherlands Institute for Advanced Studies (NIAS)American Thoracic Society (ATS: Jerry Wojciechowski Memorial Pulmonary Hypertension Research Grant)European Respiratory SocietyInstitute for Cardiovascular Research (ICaR-VU)Vrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Pulmonol, Amsterdam, NetherlandsVrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Physiol, Amsterdam, NetherlandsVrije Univ Amsterdam Med Ctr, Amsterdam Cardiovasc Sci, Dept Cardiol, Amsterdam, NetherlandsLeiden Univ, Med Ctr, Dept Mol Cell Biol, Lab Expt Cardiol, Leiden, NetherlandsUniv Fed Sao Paulo, Inst Sci & Technol, Sao Paulo, BrazilUniv Sydney, Heart & Lung Transplant Unit, St Vincents Hosp, Sydney, NSW, AustraliaUniv Sydney, Bosch Inst, Sydney, NSW, AustraliaUniv Paris Saclay, Univ Paris Sud, Le Kremlin Bicetre, FranceINSERM, UMR S 999, Le Plessis Robinson, FranceUniv Fed Sao Paulo, Inst Sci & Technol, Sao Paulo, BrazilCNPq-Brasil-245849/2012-2FAPESP-2016/08263-9Netherlands CardioVascular Research Initiative grant: CVON 2012-08VENI grant from NWO: 916.14.099Web of Scienc