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The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p

Titin is a large filamentous protein that forms a sarcomeric myofilament with a molecular spring region that develops force in stretched sarcomeres. The molecular spring has a complex make-up that includes the N2A element. This element largely consists of a 104-residue unique sequence (N2A-Us) flanked by immunoglobulin domains (I80 and I81). The N2A element is of interest because it assembles a signalosome with CARP (Cardiac Ankyrin Repeat Protein) as an important component; CARP both interacts with the N2A-Us and I81 and is highly upregulated in response to mechanical stress. The mechanical properties of the N2A element were studied using single-molecule force spectroscopy, including how these properties are affected by CARP and phosphorylation. Three protein constructs were made that consisted of 0, 1, or 2 N2A-Us elements with flanking I80 and I81 domains and with specific handles at their ends for study by atomic force microscopy (AFM). The N2A-Us behaved as an entropic spring with a persistence length (Lp) of ∼0.35 nm and contour length (Lc) of ∼39 nm. CARP increased the Lp of the N2A-Us and the unfolding force of the Ig domains; force clamp experiments showed that CARP reduced the Ig domain unfolding kinetics. These findings suggest that CARP might function as a molecular chaperone that protects I81 from unfolding when mechanical stress is high. The N2A-Us was found to be a PKA substrate, and phosphorylation was blocked by CARP. Mass spectrometry revealed a PKA phosphosite (Ser-9895 in NP_001254479.2) located at the border between the N2A-Us and I81. AFM studies showed that phosphorylation affected neither the Lp of the N2A-Us nor the Ig domain unfolding force (Funfold). Simulating the force-sarcomere length relation of a single titin molecule containing all spring elements showed that the compliance of the N2A-Us only slightly reduces passive force (1.4%) with an additional small reduction by CARP (0.3%). Thus, it is improbable that the compliance of the N2A element has a mechanical function per se. Instead, it is likely that this compliance has local effects on binding of signaling molecules and that it contributes thereby to strain- and phosphorylation- dependent mechano-signaling. published

The heart senses and responds to changes in mechanical loading conditions by initiating signaling events to produce structural and functional remodeling through a process known as mechanotransduction. Within cardiac myocytes, the giant protein titin has been implicated as a key player in the conversion of mechanical stimuli into downstream signaling events as part of the adaptive responses of mechanotransduction. The N2B element within the extensible I-band of titin has been proposed to be a stretch sensor and signaling hot spot important to titin’s role as a mechanosensor. To investigate the role of the N2B element in mechanotransduction, N2B knockout (N2BKO) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC) or volume overload by aortocaval fistula (ACF). Following 4 weeks TAC, WT mice respond with 37% LV hypertrophy (4.0 ± 0.1 mg/gBW for TAC vs 2.9 ± 0.1 mg/gBW for sham) compared to 51% (3.8 ± 0.1 mg/gBW for TAC vs 2.5 ± 0.1 mg/gBW for sham) in N2BKO. In contrast, N2BKO mice demonstrated a severe lack of a hypertrophic response following 1-week ACF with 26% (4.0 ± 0.1 mg/gBW for ACF vs 3.1 ± 0.1 mg/gBW for sham) LV hypertrophy in WT compared to -2% (2.8 ± 0.1 mg/gBW for ACF vs 2.8 ± 0.1 mg/gBW for sham) in N2BKO. Additionally, while there was no incidence of mortality in WT mice subjected to ACF, approximately 36% (5 of 14) of N2BKO mice died within 1 week following ACF. Western blot analysis of canonical hypertrophy signaling kinases revealed no differences between WT and N2BKO mice at baseline or 1 week post-ACF. Four-and-a half LIM domains 1 and 2 (FHL1 and FHL2) have previously been shown to localize to the N2B element and FHL1 has been linked to mechanotransduction in TAC. In N2BKO, FHL1 protein is expressed at levels comparable to WT in both TAC and ACF; however, FHL2 protein levels are reduced by ~98% compared to WT (1.00 ± 0.26 for WT vs 0.02 ± 0.01% for N2BKO). These data indicate that while the N2B element is not required for mechanotransduction in pressure overload of the LV, it is critical for mechanosensing and the adaptive hypertrophy response during volume overload. FHL2 is an attractive candidate as a link between titin’s N2B element and the hypertrophy response following ACF and further work will critical examine its involvement.

Titin, the largest protein known, forms a giant filament in muscle where it spans the half sarcomere from Z disk to M band. Here we genetically targeted a stretch of 14 immunoglobulin-like and fibronectin type 3 domains that comprises the I-band/A-band (IA) junction and obtained a viable mouse model. Super-resolution optical microscopy (structured illumination microscopy, SIM) and electron microscopy were used to study the thick filament length and titin's molecular elasticity. SIM showed that the IA junction functionally belongs to the relatively stiff A-band region of titin. The stiffness of A-band titin was found to be high, relative to that of I-band titin (∼ 40-fold higher) but low, relative to that of the myosin-based thick filament (∼ 70-fold lower). Sarcomere stretch therefore results in movement of A-band titin with respect to the thick filament backbone, and this might constitute a novel length-sensing mechanism. Findings disproved that titin at the IA junction is crucial for thick filament length control, settling a long-standing hypothesis. SIM also showed that deleting the IA junction moves the attachment point of titin's spring region away from the Z disk, increasing the strain on titin's molecular spring elements. Functional studies from the cellular to ex vivo and in vivo left ventricular chamber levels showed that this causes diastolic dysfunction and other symptoms of heart failure with preserved ejection fraction (HFpEF). Thus, our work supports titin's important roles in diastolic function and disease of the heart.

Background— Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established. Methods and Results— We generated a mouse model in which 9 Ig-like domains (Ig3–Ig11) were deleted from the proximal tandem Ig segment of the spring region of titin (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, whereas novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the IG KO, and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the sole likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels, with no consistent changes in extracellular matrix composition or extracellular matrix–based passive stiffness, supporting a titin-based mechanism for in vivo diastolic dysfunction. Additionally, IG KO mice have a reduced exercise tolerance, a phenotype often associated with diastolic dysfunction. Conclusions— Increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance.

Background— The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. Methods and Results— Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean±SEM. RV diastolic stiffness (β) was significantly increased in PAH patients (PAH, 0.050±0.005 versus control, 0.029±0.003; P 2 versus control, 218±21 μm 2 ; P P 200%; P interaction P Conclusions— RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Increasing titin length in the heart through functional deletion of the RNA recognition motif (RRM) of RBM20 protein leads to a reduction of left ventricular (LV) chamber stiffness in sedentary mice. We tested the therapeutic effect of upregulating compliant titins by creating diastolic dysfunction in a mouse model with inducible RBM20 inhibition (MerCreMer;cRbm20ΔRRM). Under transcriptional control of an MHC6 promoter, RBM20 function of MerCreMer;cRbm20ΔRRM mice was inhibited in time- and cardiac-specific manners. Adult male MerCreMer;cRbm20ΔRRM mice had undergone transaortic constriction (TAC) surgery with Deoxycorticosterone acetate (DOCA) pellet implantation. Two weeks after TAC+DOCA surgery, mice developed LV hypertrophy and diastolic dysfunction as demonstrated by echocardiography. Four weeks after surgery, pressure volume analysis revealed that LV chamber stiffness was markedly increased in TAC+DOCA mice (140% increased versus sham). However, the TAC+DOCA mice with RBM20 inhibition developed less severe LV chamber stiffness (29% increased versus sham). Passive stiffness measurements showed a reduction of cardiomyocyte stiffness in TAC+DOCA mice with RBM20 inhibition. Inhibition of the RBM20-based splicing system results in expression of compliant mutant N2BA titin which consequently lead to reduction of cardiomyocyte stiffness and LV chamber stiffness in a mouse model with diastolic dysfunction.

Familial hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere and may lead to hypertrophic, dilated, restrictive, and/or arrhythmogenic cardiomyopathy, congestive heart failure, or sudden cardiac death. We hypothesized that hearts from transgenic HCM mice harboring a mutant myosin heavy chain increase the energetic cost of contraction in a sex-specific manner. To do this, we assessed Ca2+sensitivity of tension and crossbridge kinetics in demembranated cardiac trabeculas from male and female wild-type (WT) and HCM hearts at an early time point (2 mo of age). We found a significant effect of sex on Ca2+sensitivity such that male, but not female, HCM mice displayed a decrease in Ca2+sensitivity compared with WT counterparts. The HCM transgene and sex significantly impacted the rate of force redevelopment by a rapid release-restretch protocol and tension cost by the ATPase-tension relationship. In each of these measures, HCM male trabeculas displayed a gain-of-function when compared with WT counterparts. In addition, cardiac remodeling measured by echocardiography, histology, morphometry, and posttranslational modifications demonstrated sex- and HCM-specific effects. In conclusion, female and male HCM mice display sex dimorphic crossbridge kinetics accompanied by sex- and HCM-dependent cardiac remodeling at the morphometric, histological, and cellular level.

Background: Left ventricular (LV) stiffening contributes to heart failure with preserved ejection fraction (HFpEF), a syndrome with no effective treatment options. Increasing the compliance of titin in the heart has become possible recently through inhibition of the splicing factor RNA binding motif-20. Here, we investigated the effects of increasing the compliance of titin in mice with diastolic dysfunction. Methods: Mice in which the RNA recognition motif (RRM) of one of the RNA binding motif-20 alleles was floxed and that expressed the MerCreMer transgene under control of the αMHC promoter (referred to as cRbm20 ΔRRM mice) were used. Mice underwent transverse aortic constriction (TAC) surgery and deoxycorticosterone acetate (DOCA) pellet implantation. RRM deletion in adult mice was triggered by injecting raloxifene ( cRbm20 ΔRRM -raloxifene), with dimethyl sulfoxide (DMSO)–injected mice ( cRbm20 ΔRRM -DMSO) as the control. Diastolic function was investigated with echocardiography and pressure-volume analysis; passive stiffness was studied in LV muscle strips and isolated cardiac myocytes before and after elimination of titin-based stiffness. Treadmill exercise performance was also studied. Titin isoform expression was evaluated with agarose gels. Results: cRbm20 ΔRRM -raloxifene mice expressed large titins in the hearts, called supercompliant titin (N2BAsc), which, within 3 weeks after raloxifene injection, made up ≈45% of total titin. TAC/DOCA cRbm20 ΔRRM -DMSO mice developed LV hypertrophy and a marked increase in LV chamber stiffness as shown by both pressure-volume analysis and echocardiography. LV chamber stiffness was normalized in TAC/DOCA cRbm20 ΔRRM -raloxifene mice that expressed N2BAsc. Passive stiffness measurements on muscle strips isolated from the LV free wall revealed that extracellular matrix stiffness was equally increased in both groups of TAC/DOCA mice ( cRbm20 ΔRRM -DMSO and cRbm20 ΔRRM -raloxifene). However, titin-based muscle stiffness was reduced in the mice that expressed N2BAsc (TAC/DOCA cRbm20 ΔRRM -raloxifene). Exercise testing demonstrated significant improvement in exercise tolerance in TAC/DOCA mice that expressed N2BAsc. Conclusions: Inhibition of the RNA binding motif-20–based titin splicing system upregulates compliant titins, which improves diastolic function and exercise tolerance in the TAC/DOCA model. Titin holds promise as a therapeutic target for heart failure with preserved ejection fraction.

Increasing titin length in the heart through functional deletion of the RNA recognition motif (RRM) of RBM20 protein leads to a reduction of left ventricular (LV) chamber stiffness in sedentary mice. We tested the therapeutic effect of upregulating compliant titins by creating diastolic dysfunction in a mouse model with inducible RBM20 inhibition (MerCreMer;cRbm20ΔRRM). Under transcriptional control of an MHC6 promoter, RBM20 function of MerCreMer;cRbm20ΔRRM mice was inhibited in time- and cardiac-specific manners.Adult male MerCreMer;cRbm20ΔRRM mice had undergone transaortic constriction (TAC) surgery with Deoxycorticosterone acetate (DOCA) pellet implantation. Two weeks after TAC+DOCA surgery, mice developed LV hypertrophy and diastolic dysfunction as demonstrated by echocardiography. Four weeks after surgery, pressure volume analysis revealed that LV chamber stiffness was markedly increased in TAC+DOCA mice (140% increased versus sham). However, the TAC+DOCA mice with RBM20 inhibition developed less severe LV chamber stiffness (29% increased versus sham). Passive stiffness measurements showed a reduction of cardiomyocyte stiffness in TAC+DOCA mice with RBM20 inhibition.Inhibition of the RBM20-based splicing system results in expression of compliant mutant N2BA titin which consequently lead to a reduction of cardiomyocyte stiffness and LV chamber stiffness in a mouse model with diastolic dysfunction.

We investigated the cellular and molecular mechanisms of diastolic dysfunction in pure volume overload induced by aortocaval fistula (ACF) surgery in the mouse. Four weeks of volume overload resulted in significant biventricular hypertrophy; protein expression analysis in left ventricular (LV) tissue showed a marked decrease in titin's N2BA/N2B ratio with no change in phosphorylation of titin's spring region. Titin-based passive tensions were significantly increased; a result of the decreased N2BA/N2B ratio. Conscious echocardiography in ACF mice revealed eccentric remodeling and pressure volume analysis revealed systolic dysfunction: reductions in ejection fraction (EF), +dP/dt, and the slope of the end-systolic pressure volume relationships (ESPVR). ACF mice also had diastolic dysfunction: increased LV end-diastolic pressure and reduced relaxation rates. Additionally, a decrease in the slope of the end diastolic pressure volume relationship (EDPVR) was found. However, correcting for altered geometry of the LV normalized the change in EDPVR and revealed, in line with our skinned muscle data, increased myocardial stiffness in vivo. ACF mice also had increased expression of the signaling proteins FHL-1, FHL-2, and CARP that bind to titin's spring region suggesting that titin stiffening is important to the volume overload phenotype. To test this we investigated the effect of volume overload in the RBM20 heterozygous (HET) mouse model, which exhibits reduced titin stiffness. It was found that LV hypertrophy was attenuated and that LV eccentricity was exacerbated. We propose that pure volume overload induces an increase in titin stiffness that is beneficial and limits eccentric remodeling.

Background Right ventricular ( RV ) diastolic function is impaired in patients with pulmonary arterial hypertension ( PAH ). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca 2+ sensitivity underlie diastolic dysfunction in PAH . This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH . Methods and Results RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca 2+ /calmoduling‐dependent‐kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors ( P IIδ ‐mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamK IIδ had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca 2+ sensitivity is mediated by sarcomeric troponin I ( cTnI ) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors ( P cTnI phosphorylation (Ser22/23) ( P Ca 2+ ‐handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca 2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors ( P Conclusions Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca 2+ handling proteins contribute to RV diastolic dysfunction in PAH.

Background— Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography. Methods and Results— Compliant titins were upregulated through deletion of the RNA Recognition Motif of the splicing factor RBM20 ( Rbm20 ΔRRM mice). A genome-wide exon expression analysis and a candidate approach revealed that the phenotype is likely to be dominated by greatly increased lengths of titin’s spring elements. At both cardiomyocyte and left ventricular chamber levels, diastolic stiffness was reduced in heterozygous (+/−) Rbm20 ΔRRM mice with a further reduction in homozygous (−/−) mice at only the intact myocyte level. Fibrosis was present in only −/− Rbm20 ΔRRM hearts. The Frank-Starling Mechanism was reduced in a graded fashion in Rbm20 ΔRRM mice, at both the cardiomyocyte and left ventricular chamber levels. Exercise tests revealed an increase in exercise capacity in +/− mice. Conclusions— Titin is not only important in diastolic but also in systolic cardiac function. Upregulating compliant titins reduces diastolic chamber stiffness owing to the increased compliance of myocytes, but it depresses end-systolic elastance; under conditions of exercise, the beneficial effects on diastolic function dominate. Therapeutic manipulation of the RBM20-based splicing system might be able to minimize effects on fibrosis and systolic function while improving the diastolic function in patients with heart failure.

Exercise has beneficial effects on diastolic dysfunction but the underlying mechanisms are not well understood. Here we studied the effects of exercise on the elastic protein titin, an important determinant of diastolic stiffness, in both the left ventricle and the diaphragm. We used wild type mice and genetically engineered mice with HFpEF symptoms (IG KO mice), including diastolic dysfunction. In the diaphragm muscle, exercise increased the expression level of titin (increased titin:MHC ratio) which is expected to increase titin-based stiffness. This effect was absent in the LV. We also studied the constitutively expressed titin residues S11878 and S12022 that are known targets of CaMKIIδ and PKCα with increased phosphorylation resulting in an increase in titin-based passive stiffness. The phosphorylation level of S11878 was unchanged whereas S12022 responded to exercise with a reduction in the phosphorylation level in the LV and, interestingly, an increase in the diaphragm. These changes are expected to lower titin’s stiffness in the LV and increase stiffness in the diaphragm. We propose that these disparate effects reflect the unique physiological needs of the two tissue types and that both effects are beneficial.

Volume overload (VO) heart failure occurs due to pathologies such as mitral valve regurgitation, infarcts and ventricular septal defects, and leads to characteristic eccentric dilation. Changes in passive myocardial stiffness are not well understood and here we dissected the contribution of extracellular matrix (ECM)-based and titin-based stiffness to overall passive stiffness. Titin is a giant protein that regulates passive tension and hence diastolic function in the myocardium. In heart failure, differential splicing and phosphorylation events can occur that alter the stiffness of this protein and influence hemodynamics. Increased diastolic stiffness due to differential splicing and phosphorylation of titin has been observed in pressure overload hypertrophy; however, there are no published studies investigating whether pure LV VO during compensated heart failure leads to changes in titin based passive stiffness. We studied the role of titin in modulating diastolic function in VO induced by aortocaval fistula (ACF) in the mouse. ACF was induced in three-month-old male C57BL/6 animals and allowed to progress for 4 weeks. At 4 weeks, echocardiography confirmed the presence of eccentric dilation and decreased systolic function as expected. Tissues mechanics indicated an increase in titin, which correlated with a significant decrease in N2BA/N2B ratio. ECM based passive stiffness was unchanged in VO. In summary, VO causes an increase in titin based passive stiffness.

Diastolic dysfunction is a poorly understood but clinically pervasive syndrome. Titin is the main determinant of cellular passive stiffness. However, the physiological role that titin's tandem Ig segment plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction needs to be established by performing studies at a wide range of organizational levels (skinned cells to the in vivo LV chamber). We produced a novel KO model by deleting nine immunoglobulin(Ig)-like domains from the proximal tandem Ig segment (Ig 3-11) of titin's spring region (IG KO). Exon microarray analysis revealed no adaptations in titin splicing, while novel phospho-specific antibodies did not detect changes in titin phosphorylation. Passive myocyte stiffness was increased in the KO and immunoelectron microscopy revealed increased extension of the remaining titin spring segments as the likely underlying mechanism. Diastolic stiffness was increased at the tissue and organ levels; myocardial stiffness studies did not detect changes in extracellular matrix based passive stiffness, supporting a titin-based mechanism. IG KO mice have a reduced exercise tolerance and develop LV hypertrophy that is associated with a marked increase in expression of hypertrophy-associated four and a half LIM proteins (FHL). These findings suggest that titin-based stiffness plays a role in diastolic dysfunction and hypertrophic signaling and that increased titin-based passive stiffness is sufficient to cause diastolic dysfunction with exercise intolerance and LV hypertrophy.

The giant sarcomeric protein titin spans the length of the half sarcomere and consists of an I-band region that functions as a molecular spring and an A-band region. Here we use a mouse model in which a portion of titin near the IA junction has been removed (IA KO) resulting in diastolic dysfunction and increased total and titin-based passive tension. We test the hypothesis that exercise ameliorates increased titin-based stiffness. In this study, 3 month old male IA KO and WT mice were allowed free access to a running wheel for 28 days and distance, speed, and duration were recorded each night. After 28 days mice were sacrificed and the LV was used for skinned muscle mechanics, analysis gels, and western blots. WT and IA KO mice ran for the same duration but IA KO mice ran slower and therefore less distance than their WT counterparts (WT mice ran ∼6 km per night while IA KOs ran ∼4.5 km). Stiffness in both WT and IA KO mice was reduced after exercise; titin-based tension at a sarcomere length of 2.3 μm was reduced by ∼16% and ∼20% respectively. No changes in titin isoform ratios were observed but exercised mice showed hypo-phosphorylation of S26, a PKC site in titin's PEVK region. S26 phosphorylation is known to increase titin's stiffness so hypo-phosphorylation is consistent with the reduction in stiffness observed in exercised mice. We conclude that exercise improves diastolic dysfunction though modulation of titin phosphorylation.

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ. Fil: Hidalgo, Carlos G.. University Of Arizona; Estados Unidos Fil: Chung, Charles S.. University Of Arizona; Estados Unidos Fil: Saripalli, Chandra. University Of Arizona; Estados Unidos Fil: Methawasin, Mei. University Of Arizona; Estados Unidos Fil: Hutchinson, Kirk R.. University Of Arizona; Estados Unidos Fil: Tsaprailis, George. University Of Arizona; Estados Unidos Fil: Labeit, Siegfried. University of Heidelberg; Alemania Fil: Mattiazzi, Ramona Alicia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnológico la Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Médicas; Argentina Fil: Granzier, Henk L.. University Of Arizona; Estados Unidos

Rationale: Mechanisms underlying diastolic dysfunction need to be better understood. Objective: To study the role of titin in diastolic dysfunction using a mouse model of experimental heart failure induced by transverse aortic constriction. Methods and Results: Eight weeks after transverse aortic constriction surgery, mice were divided into heart failure (HF) and congestive heart failure (CHF) groups. Mechanical studies on skinned left ventricle myocardium measured total and titin-based and extracellular matrix-based passive stiffness. Total passive stiffness was increased in both HF and CHF mice, and this was attributable to increases in both extracellular matrix-based and titin-based passive stiffness, with titin being dominant. Protein expression and titin exon microarray analysis revealed increased expression of the more compliant N2BA isoform at the expense of the stiff N2B isoform in HF and CHF mice. These changes are predicted to lower titin-based stiffness. Because the stiffness of titin is also sensitive to titin phosphorylation by protein kinase A and protein kinase C, back phosphorylation and Western blot assays with novel phospho-specific antibodies were performed. HF and CHF mice showed hyperphosphorylation of protein kinase A sites and the proline glutamate valine lysine (PEVK) S26 protein kinase C sites, but hypophosphorylation of the PEVK S170 protein kinase C site. Protein phosphatase I abolished differences in phosphorylation levels and normalized titin-based passive stiffness levels between control and HF myocardium. Conclusion: Transverse aortic constriction-induced HF results in increased extracellular matrix-based and titin-based passive stiffness. Changes in titin splicing occur, which lower passive stiffness, but this effect is offset by hyperphosphorylation of residues in titin spring elements, particularly of PEVK S26. Thus, complex changes in titin occur that combined are a major factor in the increased passive myocardial stiffness in HF.