Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1ß-evoked signaling in vascular smooth muscle cells and vascular inflammation.

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs in vitro proliferated and migrated less than WT SMCs in response to IL-1ß. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1ß induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using in vitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1ß-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1ß-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

A single phenylalanine residue in ß-arrestin2 critically regulates its binding to G protein-coupled receptors.

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of ß-arrestin2 (ß-arr2) and blocks its association with activated G protein-coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of ß-arr2-GPCR complexes. ß-arr2 Phe116Ala mutant has negligible effect on blunting ß2-adrenergic receptor-induced cAMP generation unlike ß-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6-activated forms of bovine ß-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of ß-arr2. Surprisingly, Phe116 is dispensable for the association of ß-arr2 with its non-GPCR partners. ß-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with ß-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent ß-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that ß-arr2 Phe116Ala interaction with activated ß2-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of ß-arr2, regulates the formation of ß-arr2-GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent ß-arr2 localization and trafficking.

The deubiquitinase ubiquitin-specific protease 20 is a positive modulator of myocardial ß1-adrenergic receptor expression and signaling.

Reversible ubiquitination of G protein-coupled receptors regulates their trafficking and signaling; whether deubiquitinases regulate myocardial ß1-adrenergic receptors (ß1ARs) is unknown. We report that ubiquitin-specific protease 20 (USP20) deubiquitinates and attenuates lysosomal trafficking of the ß1AR. ß1AR-induced phosphorylation of USP20 Ser-333 by protein kinase A-α (PKAα) was required for optimal USP20-mediated regulation of ß1AR lysosomal trafficking. Both phosphomimetic (S333D) and phosphorylation-impaired (S333A) USP20 possess intrinsic deubiquitinase activity equivalent to WT activity. However, unlike USP20 WT and S333D, the S333A mutant associated poorly with the ß1AR and failed to deubiquitinate the ß1AR. USP20-KO mice showed normal baseline systolic function but impaired ß1AR-induced contractility and relaxation. Dobutamine stimulation did not increase cAMP in USP20-KO left ventricles (LVs), whereas NKH477-induced adenylyl cyclase activity was equivalent to WT. The USP20 homolog USP33, which shares redundant roles with USP20, had no effect on ß1AR ubiquitination, but USP33 was up-regulated in USP20-KO hearts suggesting compensatory regulation. Myocardial ß1AR expression in USP20-KO was drastically reduced, whereas ß2AR expression was maintained as determined by radioligand binding in LV sarcolemmal membranes. Phospho-USP20 was significantly increased in LVs of wildtype (WT) mice after a 1-week catecholamine infusion and a 2-week chronic pressure overload induced by transverse aortic constriction (TAC). Phospho-USP20 was undetectable in ß1AR KO mice subjected to TAC, suggesting a role for USP20 phosphorylation in cardiac response to pressure overload. We conclude that USP20 regulates ß1AR signaling in vitro and in vivo Additionally, ß1AR-induced USP20 phosphorylation may serve as a feed-forward mechanism to stabilize ß1AR expression and signaling during pathological insults to the myocardium.

USP20 (Ubiquitin-Specific Protease 20) Inhibits TNF (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates Atherosclerosis.

Objective- Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results- To address this question, we used male Ldlr-/- mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1ß (interleukin-1ß). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions- USP20 attenuates TNF- and IL-1ß-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

Manifold roles of ß-arrestins in GPCR signaling elucidated with siRNA and CRISPR/Cas9.

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. ß-Arrestins (ßArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete ßArr1/2 and G proteins have cast doubt on the role of ß-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of ßArr1/2 and reconstitution with ßArr1/2 in three different parental and CRISPR-derived ßArr1/2 knockout HEK293 cell pairs to assess the effect of ßArr1/2 deletion on ERK1/2 activation by four Gs-coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for ßArr2 or ßArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For ß2 adrenergic receptors (ß2ARs) and ß1ARs, ßArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with ßArr1/2. Loss of desensitization and receptor internalization in CRISPR ßArr1/2 knockout cells caused ß2AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing ßArr1/2. These data suggest that ßArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from ßArr1/2- or G protein-deleted cells to GPCR behavior in native systems.

Mdm2 regulates cardiac contractility by inhibiting GRK2-mediated desensitization of ß-adrenergic receptor signaling.

The oncoprotein Mdm2 is a RING domain-containing E3 ubiquitin ligase that ubiquitinates G protein-coupled receptor kinase 2 (GRK2) and ß-arrestin2, thereby regulating ß-adrenergic receptor (ßAR) signaling and endocytosis. Previous studies showed that cardiac Mdm2 expression is critical for controlling p53-dependent apoptosis during early embryonic development, but the role of Mdm2 in the developed adult heart is unknown. We aimed to identify if Mdm2 affects ßAR signaling and cardiac function in adult mice. Using Mdm2/p53-KO mice, which survive for 9-12 months, we identified a critical and potentially novel role for Mdm2 in the adult mouse heart through its regulation of cardiac ß1AR signaling. While baseline cardiac function was mostly similar in both Mdm2/p53-KO and wild-type (WT) mice, isoproterenol-induced cardiac contractility in Mdm2/p53-KO was significantly blunted compared with WT mice. Isoproterenol increased cAMP in left ventricles of WT but not of Mdm2/p53-KO mice. Additionally, while basal and forskolin-induced calcium handling in isolated Mdm2/p53-KO and WT cardiomyocytes were equivalent, isoproterenol-induced calcium handling in Mdm2/p53-KO was impaired. Mdm2/p53-KO hearts expressed 2-fold more GRK2 than WT. GRK2 polyubiquitination via lysine-48 linkages was significantly reduced in Mdm2/p53-KO hearts. Tamoxifen-inducible cardiomyocyte-specific deletion of Mdm2 in adult mice also led to a significant increase in GRK2, and resulted in severely impaired cardiac function, high mortality, and no detectable ßAR responsiveness. Gene delivery of either Mdm2 or GRK2-CT in vivo using adeno-associated virus 9 (AAV9) effectively rescued ß1AR-induced cardiac contractility in Mdm2/p53-KO. These findings reveal a critical p53-independent physiological role of Mdm2 in adult hearts, namely, regulation of GRK2-mediated desensitization of ßAR signaling.

G Protein-Coupled Receptor Signaling Through ß-Arrestin-Dependent Mechanisms.

ß-arrestin1 (or arrestin2) and ß-arrestin2 (or arrestin3) are ubiquitously expressed cytosolic adaptor proteins that were originally discovered for their inhibitory role in G protein-coupled receptor (GPCR) signaling through heterotrimeric G proteins. However, further biochemical characterization revealed that ß-arrestins do not just "block" the activated GPCRs, but trigger endocytosis and kinase activation leading to specific signaling pathways that can be localized on endosomes. The signaling pathways initiated by ß-arrestins were also found to be independent of G protein activation by GPCRs. The discovery of ligands that blocked G protein activation but promoted ß-arrestin binding, or vice-versa, suggested the exciting possibility of selectively activating intracellular signaling pathways. In addition, it is becoming increasingly evident that ß-arrestin-dependent signaling is extremely diverse and provokes distinct cellular responses through different GPCRs even when the same effector kinase is involved. In this review, we summarize various signaling pathways mediated by ß-arrestins and highlight the physiologic effects of ß-arrestin-dependent signaling.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of ß-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation.

Toll-like receptor 4 (TLR4) promotes vascular inflammatory disorders such as neointimal hyperplasia and atherosclerosis. TLR4 triggers NFκB signaling through the ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6). TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin-specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein ß-arrestin2. Although ß-arrestin2 effects on TRAF6 suggest an anti-inflammatory role, physiologic ß-arrestin2 promotes inflammation in atherosclerosis and neointimal hyperplasia. We hypothesized that anti- and proinflammatory dimensions of ß-arrestin2 activity could be dictated by ß-arrestin2's ubiquitination status, which has been linked with its ability to scaffold and localize activated ERK1/2 to signalosomes. With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were ß-arrestin2-dependent. Generation of transgenic mice with smooth muscle cell-specific expression of either USP20 or its catalytically inactive mutant revealed anti-inflammatory effects of USP20in vivoandin vitro Carotid endothelial denudation showed that antagonizing smooth muscle cell USP20 activity increased NFκB activation and neointimal hyperplasia. We found that ß-arrestin2 ubiquitination was promoted by TLR4 and reversed by USP20. The association of USP20 with ß-arrestin2 was augmented when ß-arrestin2 ubiquitination was prevented and reduced when ß-arrestin2 ubiquitination was rendered constitutive. Constitutive ß-arrestin2 ubiquitination also augmented NFκB activation. We infer that pro- and anti-inflammatory activities of ß-arrestin2 are determined by ß-arrestin2 ubiquitination and that changes in USP20 expression and/or activity can therefore regulate inflammatory responses, at least in part, by defining the ubiquitination status of ß-arrestin2.

Ubiquitin-Related Roles of ß-Arrestins in Endocytic Trafficking and Signal Transduction.

The non-visual arrestins, ß-arrestin1, and ß-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of ß-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of ß-arrestins in protein ubiquitination is evolutionarily conserved. ß-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of ß-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on ß-arrestin plays a critical role in stabilizing ß-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as ß-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc.

Calcium desensitizer catechin reverses diastolic dysfunction in mice with restrictive cardiomyopathy.

Diastolic dysfunction refers to an impaired relaxation and an abnormality in ventricular blood filling during diastole while systolic function is preserved. Cardiac myofibril hypersensitivity to Ca(2+) is a major factor that causes impaired relaxation of myocardial cells. The present study investigates the effect of the green tea extract catechins on myofibril calcium desensitization and restoration of diastolic function in a restrictive cardiomyopathy (RCM) mouse model with cardiac troponin mutations. Wild type (WT) and RCM mice were treated daily with catechin (epigallocatechin-3-gallate, EGCg, 50 mg/kg body weight) for 3 months. Echocardiography and cell based assays were performed to measure cardiac structure and flow-related variables including chamber dimensions, fraction shortening, trans-mitral flow patterns in the experimental mice. In addition, myocyte contractility and calcium dynamics were measured in WT and RCM cardiomyocytes treated in vitro with 5 µM EGCg. Our data indicated that RCM mice treated with EGCg showed an improved diastolic function while systolic function remained unchanged. At the cellular level, sarcomere relaxation and calcium decay were accelerated in RCM myocardial cells treated with EGCg. These results suggest that catechin is effective in reversing the impaired relaxation in RCM myocardial cells and rescuing the RCM mice with diastolic dysfunction.

Phosphorylation of the deubiquitinase USP20 by protein kinase A regulates post-endocytic trafficking of ß2 adrenergic receptors to autophagosomes during physiological stress.

Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated ß2 adrenergic receptors (ß2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated ß2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a ß2AR-dependent manner. Upon ß2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated ß2AR complex, and facilitates trafficking of the ubiquitinated ß2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the ß2AR to autophagosomes while promoting plasma membrane recycling of internalized ß2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis.

Dose-dependent diastolic dysfunction and early death in a mouse model with cardiac troponin mutations.

Our aim was to explore the dose-dependent diastolic dysfunction and the mechanisms of heart failure and early death in transgenic (TG) mice modeling human restrictive cardiomyopathy (RCM). The first RCM mouse model (cTnI(193His) mice) carrying cardiac troponin I (cTnI) R193H mutation (mouse cTnI R193H equals to human cTnI R192H) was generated several years ago in our laboratory. The RCM mice manifested a phenotype similar to that observed in RCM patients carrying the same cTnI mutation, i.e. enlarged atria and restricted ventricles. However, the causes of heart failure and early death observed in RCM mice remain unclear. In this study, we have produced RCM TG mice (cTnI(193His)-L, cTnI(193His)-M and cTnI(193His)-H) that express various levels of mutant cTnI in the heart. Histological examination and echocardiography were performed on these mice to monitor the time course of the disease development and heart failure. Our data demonstrate that cTnI mutation-caused diastolic dysfunction is dose-dependent. The key mechanism is myofibril hypersensitivity to Ca(2+) resulting in an impaired relaxation in the mutant cardiac myocytes. Prolonged relaxation time and delay of Ca(2+) decay observed in the mutant cardiac myocytes are correlated with the level of the mutant protein in the heart. Markedly enlarged atria due to the elevated end-diastolic pressure and myocardial ischemia are observed in the heart of the transgenic mice. In the mice with the highest level of the mutant protein, restricted ventricles and systolic dysfunction occur followed immediately by heart failure and early death. Diastolic dysfunction caused by R193H troponin I mutation is specific, showing a dose-dependent pattern. These mouse models are useful tools for the study of diastolic dysfunction. Impaired diastole can cause myocardial ischemia and fibrosis formation, resulting in the development of systolic dysfunction and heart failure with early death in the RCM mice with a high level of the mutant protein in the heart.

Insights into restrictive cardiomyopathy from clinical and animal studies.

Cardiomyopathies are diseases that primarily affect the myocardium, leading to serious cardiac dysfunction and heart failure. Out of the three major categories of cardiomyopathies (hypertrophic, dilated and restrictive), restrictive cardiomyopathy (RCM) is less common and also the least studied. However, the prognosis for RCM is poor as some patients dying in their childhood. The molecular mechanisms behind the disease development and progression are not very clear and the treatment of RCM is very difficult and often ineffective. In this article, we reviewed the recent progress in RCM research from the clinical studies and the translational studies done on diseased transgenic animal models. This will help for a better understanding of the mechanisms underlying the etiology and development of RCM and for the design of better treatments for the disease.

Insights into restrictive cardiomyopathy from clinical and animal studies / 老年心脏病学杂志（英文版）

Catdiomyopathies are diseases that primarily affect the myocardium,leading to serious cardiac dysfimction and heart failure.Out of the three major categories of candiomyopathies(hypertrophic,dilated and restrictive),restrictive cardiomyopathy(RCM)is less common and also the least studied However,the prognosis for RCM is poor as some patients dying in their childhood The molecular mechanisms behind the disease development and progression are not very clear and the treatment of RCM is very difficult and often ineffective.In this article,we reviewed the recent progress in RCM research from the clinical studies and the translational studies done on diseased transgenic animal models.This will help for a better understanding of tare mechanisms underlying the etiology and development of RCM and for the design of better treatments for the disease.

Deficiency of methionine sulfoxide reductase A causes cellular dysfunction and mitochondrial damage in cardiac myocytes under physical and oxidative stresses.

Methionine sulfoxide reductase A (MsrA) is an enzyme that reverses oxidation of methionine in proteins. Using a MsrA gene knockout (MsrA(-/-)) mouse model, we have investigated the role of MsrA in the heart. Our data indicate that cellular contractility and cardiac function are not significantly changed in MsrA(-/-) mice if the hearts are not stressed. However, the cellular contractility, when stressed using a higher stimulation frequency (2Hz), is significantly reduced in MsrA(-/-) cardiac myocytes. MsrA(-/-) cardiac myocytes also show a significant decrease in contractility after oxidative stress using H(2)O(2). Corresponding changes in Ca(2+) transients are observed in MsrA(-/-) cardiomyocytes treated with 2Hz stimulation or with H(2)O(2). Electron microscope analyses reveal a dramatic morphological change of mitochondria in MsrA(-/-) mouse hearts. Further biochemical measurements indicate that protein oxidation levels in MsrA(-/-) mouse hearts are significantly higher than those in wild type controls. Our study demonstrates that the lack of MsrA in cardiac myocytes reduces myocardial cell's capability against stress stimulations resulting in a cellular dysfunction in the heart.

Impaired relaxation is the main manifestation in transgenic mice expressing a restrictive cardiomyopathy mutation, R193H, in cardiac TnI.

Transgenic mice were generated to express a restrictive cardiomyopathy (RCM) human cardiac troponin I (cTnI) R192H mutation in the heart (cTnI(193His) mice). The objective of this study was to assess cardiac function during the development of diastolic dysfunction and to gain insight into the pathophysiological impact of the RCM cTnI mutation. Cardiac function and pathophysiological changes were monitored in cTnI193His mice and wild-type littermates for a period of 12 mo. It progressed gradually from abnormal relaxation to diastolic dysfunction characterized with high-resolution echocardiography by a reversed E-to-A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. At the age of 12 mo, cardiac output in cTnI(193His) mice was significantly declined, and some transgenic mice showed congestive heart failure. The negative impact of cTnI193His on ventricular contraction and relaxation was further demonstrated in isolated mouse working heart preparations. The main morphological change in cTnI193His myocytes was shortened cell length. Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO. The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation characterized morphologically by enlarged atria and restricted ventricles and functionally by diastolic dysfunction and diastolic heart failure. The results demonstrate a critical role of the COOH-terminal domain of cTnI in the diastolic function of cardiac muscle.